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cancercontroljournal.org Vol. 23, No. 2, April 2016 H. LEE MOFFITT CANCER CENTER & RESEARCH INSTITUTE, AN NCI COMPREHENSIVE CANCER CENTER Assessing Prognosis in Uveal Melanoma Zélia M. Corrêa, MD, PhD Therapeutic Options for Retinoblastoma Pia R. Mendoza, MD, and Hans E. Grossniklaus, MD Primary Vitreoretinal Lymphoma: Management of Isolated Ocular Disease Matthew T. Witmer, MD Management of Primary Acquired Melanosis, Nevus, and Conjunctival Melanoma Andrew Kao, MD, Armin Afshar, MD, Michele Bloomer, MD, et al Sebaceous Carcinoma of the Eyelid Carlos Prieto-Granada, MD, and Paul Rodriguez-Waitkus, MD, PhD Role of Vismodegib in the Management of Advanced Periocular Basal Cell Carcinoma Kyle F. Cox, MD, and Curtis E. Margo, MD Extranodal Marginal Zone B-cell Lymphoma of the Ocular Adnexa Jean Guffey Johnson, MD, Lauren A. Terpak, MS, Curtis E. Margo, MD, et al Ophthalmic Complications Related to Chemotherapy in Medically Complex Patients Lynn E. Harman, MD Cancer Control is included in Index Medicus/MEDLINE Jacksonville, FL Permit No. 4390 H LEE MOFFITT CANCER CENTER & RESEARCH INSTITUTE INC 12902 Magnolia Drive Tampa FL 33612-9416 PAID NON-PROFIT ORG U.S. POSTAGE Editorial Board Members Editor: Lodovico Balducci, MD Senior Member Program Leader, Senior Adult Oncology Program Moffitt Cancer Center Deputy Editor: Julio M. Pow-Sang, MD Senior Member Chair, Department of Genitourinary Oncology Director of Moffitt Robotics Program Moffitt Cancer Center Editor Emeritus: John Horton, MB, ChB Professor Emeritus of Medicine & Oncology Guest Editor: Richard D. Kim, MD Associate Member Gastrointestinal Oncology Bela Kis, MD, PhD Assistant Member Diagnostic Radiology Rami Komrokji, MD Associate Member Malignant Hematology Conor C. Lynch, PhD Assistant Member Tumor Biology Kristen J. Otto, MD Associate Member Head and Neck and Endocrine Oncology Michael A. Poch, MD Assistant Member Genitourinary Oncology Curtis E. Margo, MD Professor Departments of Pathology and Cell Biology and Ophthalmology Morsani College of Medicine University of South Florida Jeffery S. Russell, MD, PhD Assistant Member Endocrine Tumor Oncology Moffitt Cancer Center Journal Advisory Committee: Saïd M. Sebti, PhD Senior Member Drug Discovery Daniel A. Anaya, MD Associate Member Gastrointestinal Oncology Aliyah Baluch, MD Assistant Member Infectious Diseases Dung-Tsa Chen, PhD Associate Member Biostatistics Elizabeth M. Sagatys, MD Assistant Member Pathology - Clinical Bijal D. Shah, MD Assistant Member Malignant Hematology Production Team: Veronica Nemeth Editorial Coordinator Sherri Damlo Medical Copy Editor Diane McEnaney Graphic Design Consultant Associate Editor of Educational Projects John M. York, PharmD Akita Biomedical Consulting 1111 Bailey Drive Paso Robles, CA 93446 Phone: 805-238-2485 Fax: 949-203-6115 E-mail: [email protected] For Consumer and General Advertising Information: Veronica Nemeth Editorial Coordinator Cancer Control: Journal of the Moffitt Cancer Center 12902 Magnolia Drive – MBC-JRNL Tampa, FL 33612 Phone: 813-745-1348 Fax: 813-449-8680 E-mail: [email protected] Lubomir Sokol, MD, PhD Senior Member Hematology/Oncology Hatem H. Soliman, MD Assistant Member Breast Oncology Hey Sook Chon, MD Assistant Member Gynecological Oncology Jonathan R. Strosberg, MD Assistant Member Gastrointestinal Oncology Jasreman Dhillon, MD Assistant Member Pathology - Anatomic Jennifer Swank, PharmD Clinical Pharmacist Medical Oncology/Internal Medicine Jennifer S. Drukteinis, MD Associate Member Diagnostic Radiology Sarah W. Thirlwell, MSc, MSc(A), RN Nurse Director Supportive Care Medicine Program Clement K. Gwede, PhD Associate Member Health Outcomes and Behavior Eric M. Toloza, MD, PhD Assistant Member Thoracic Oncology Sarah E. Hoffe, MD Associate Member Radiation Oncology Nam D. Tran, MD Assistant Member Neuro-Oncology John V. Kiluk, MD Associate Member Breast Oncology Jonathan S. Zager, MD Senior Member Cutaneous Oncology Cancer Control is a member of the Medscape Publishers’ Circle®, an alliance of leading medical publishers whose content is featured on Medscape (www.medscape.com). Most issues and supplements of Cancer Control are available at cancercontroljournal.org Cancer Control: Journal of the Moffitt Cancer Center (ISSN 1073-2748) is published by H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. Telephone: 813-745-1348. Fax: 813-449-8680. E-mail: [email protected]. Internet address: cancercontroljournal.org. Cancer Control is included in Index Medicus ®/MEDLINE® and EMBASE®/ Excerpta Medica, Thomson Reuters Science Citation Index Expanded (SciSearch®) and Journal Citation Reports/Science Edition. Copyright 2016 by H. Lee Moffitt Cancer Center & Research Institute. All rights reserved. Cancer Control: Journal of the Moffitt Cancer Center is a peer-reviewed journal that is published to enhance the knowledge needed by professionals in oncology to help them minimize the impact of human malignancy. Each issue emphasizes a specific theme relating to the detection or management of cancer. The objectives of Cancer Control are to define the current state of cancer care, to integrate recently generated information with historical practice patterns, and to enlighten readers through critical reviews, commentaries, and analyses of recent research studies. Disclaimer: All articles published in this journal, including editorials and letters, represent the opinions of the author(s) and do not necessarily reflect the opinions of the editorial board, the H. Lee Moffitt Cancer Center & Research Institute, Inc, or the institutions with which the authors are affiliated unless clearly specified. The reader is advised to independently verify the effectiveness of all methods of treatment and the accuracy of all drug names, dosages, and schedules. Dosages and methods of administration of pharmaceutical products may not be those listed in the package insert and solely reflect the experience of the author(s) and/or clinical investigator(s). April 2016, Vol. 23, No. 2 Cancer Control 87 Table of Contents Editorial Are Ocular and Ocular Adnexal Cancers Overdiagnosed? Historical Perspectives on Diagnosis 90 Curtis E. Margo, MD Articles Assessing Prognosis in Uveal Melanoma 93 Zélia M. Corrêa, MD, PhD Therapeutic Options for Retinoblastoma 99 Pia R. Mendoza, MD, and Hans E. Grossniklaus, MD Primary Vitreoretinal Lymphoma: Management of Isolated Ocular Disease 110 Management of Primary Acquired Melanosis, Nevus, and Conjunctival Melanoma 117 Matthew T. Witmer, MD Andrew Kao, MD, Armin Afshar, MD, Michele Bloomer, MD, and Bertil Damato, MD, PhD Sebaceous Carcinoma of the Eyelid 126 Role of Vismodegib in the Management of Advanced Periocular Basal Cell Carcinoma 133 Carlos Prieto-Granada, MD, and Paul Rodriguez-Waitkus, MD, PhD Kyle F. Cox, MD, and Curtis E. Margo, MD Extranodal Marginal Zone B-cell Lymphoma of the Ocular Adnexa 140 Jean Guffey Johnson, MD, Lauren A. Terpak, MS, Curtis E. Margo, MD, and Reza Setoodeh, MD 88 Cancer Control April 2016, Vol. 23, No. 2 Table of Contents Articles, continued Ophthalmic Complications Related to Chemotherapy in Medically Complex Patients 150 Lynn E. Harman, MD Departments Disparities Report: Disparities Among Minority Women With Breast Cancer Living in Impoverished Areas of California 157 Infectious Disease Report: Bordetella pertussis Infection in Patients With Cancer 163 Sundus Haji-Jama, Kevin M. Gorey, PhD, Isaac N. Luginaah, PhD, Guangyong Zou, PhD, Caroline Hamm, MD, and Eric J. Holowaty, MD Abraham Yacoub, MD, Sowmya Nanjappa, MD, Tyler Janz, and John N. Greene, MD Pharmacy Report: Megestrol Acetate-Induced Adrenal Insufficiency 167 Pathology Report: Presacral Noncommunicating Enteric Duplication Cyst 170 Sowmya Nanjappa, MD, Christy Thai, PharmD, Sweta Shah, MD, and Matthew Snyder, PharmD Shabnam Seydafkan, MD, David Shibata, MD, Julian Sanchez, MD, Nam D. Tran, MD, Marino Leon, MD, and Domenico Coppola, MD Symposium Report: Second Annual Moffitt Anatomic Pathology Symposium 175 Ten Best Readings Relating to Ocular Oncology 187 About the art in this issue: Lynn E. Harman, MD, is an ophthalmologist at the University of South Florida Morsani College of Medicine and the James A. Haley Veterans Affairs Hospital in Tampa, Florida. She practices general ophthalmology and specializes in the diagnosis and treatment of glaucoma. Dr Harman is an amateur photographer who enjoys her hobby when traveling. For the works included in this issue, she used a high-dynamicrange imaging technique which expands the breadth of color and luminosity that is typically captured on a single digital image. Cover: Bernese Alps Articles: Virgin River Gorge Themes at the Shard Manhattan Chicago River Half Dome From Glacier Point Hohenschwangau Castle Los Angeles Point Reyes Beach North April 2016, Vol. 23, No. 2 Cancer Control 89 Editorial Are Ocular and Ocular Adnexal Cancers Overdiagnosed? Historical Perspectives on Diagnosis Applying the term overdiagnosis to cancer reflects a conceptual shift in thinking about cancer as a neoplastic proliferation based on histopathological criteria to a disease that, if left untreated, becomes symptomatic and results in morbidity or death.1-3 During the past decade, select cancers — correctly diagnosed histologically as cancer — would have gone undetected during a patient’s life (ie, remained clinically silent) if it were not for screening programs. The most robust evidence for overdiagnosis comes from randomized clinical trials of cancer screening and observational studies. For some cancers, the results of randomized trials have revealed no compensatory decrease in cause-specific mortality rates among a screened cohort of patients.1 Observational studies complement these findings, demonstrating a rapid rise in cancer diagnoses after screening programs are instituted without any long-term change in cancer-specific mortality rates.3 The notion that early detection will lead to early treatment and reduced rates of cancer-specific mortality is intuitively appealing. Although this ideal does occur with some cancers like colon and cervical cancer, this is not universally the case. In industrialized countries, the overdiagnosis phenomenon involves mostly prostate, breast, renal, and thyroid cancers.1 Given that these seemingly counterintuitive findings may be at odds with compelling anecdotes of success, the discussion about cancer screening has become controversial.4-7 Overdiagnosis arises because cancer screening uncovers a reservoir of indolent tumors that would have otherwise gone unnoticed throughout life. Early detection of either nonprogressive or slow-growing cancer gets caught up in this net, which is larger if precursor lesions and in situ neoplasia are factored into the screening process. For select patients, death from other causes will occur during the period of cancer inexpression. Because of tumor latencies and competing risks of mortality, some patients screened for cancer are subject to the costs and adverse events of treatment without any prospect of benefit. However, the conundrum of overdiagnosis involves applying this epidemiological perspective to individual patients. At the time of screening diagnosis, it may be impossible to distinguish persons with indolent, nonprogressive lesions from those with potentially lethal cancers. The problem of overdiagnosis has surfaced in ophthalmology before but not as the result 90 Cancer Control of epidemiological sleuthing and certainly not in the context of screening programs. Claims of overdiagnosis were raised because there was a sense of incongruity between the lesions observed under the microscope and the commensurate treatments. A few examples illustrating this phenomenon are offered. Beginning in the late 1930s, Reese8-11 solidified the idea of conjunctival melanosis as a precursor lesion to conjunctival melanoma. He distinguished it from congenital melanosis and termed it precancerous melanosis.8-11 Acknowledging that its progression to melanoma is often protracted, Reese8-11 estimated that approximately 17% of cases of precancerous melanosis become cancerous (ie, conjunctival melanoma) within 5 years and that conjunctiva melanoma had a mortality rate of at least 40%.Based on clinical and pathological observations, he favored orbital exenteration for some early stages of melanosis when it appeared flat.7,9,11 Few ophthalmologists were as influential in establishing practice guidelines as Reese, and few pathologists had much proficiency in interpreting findings on conjunctival biopsies as he did. By 1966, Zimmerman12 accumulated considerable experience with conjunctival melanosis treated by radical surgery. He grew concerned that orbital exenteration might not be justified for some cases of precancerous melanosis because, to him, the histological findings suggested low potential for aggressive growth or distant metastasis.12 He proposed a method for evaluating acquired melanosis based on conjunctival biopsy that stratified risk for melanoma progression.12 This strategy was formally presented by Zimmerman13 about a decade later. Folberg et al14,15 would later introduce new terminology for conjunctival intraepithelial melanocytic neoplasia (ie, primary acquired melanosis with and without atypia) and then test the prognostic capabilities of different histopathological variables. These studies tempered the role that radical surgery played in the management of acquired conjunctival melanosis by introducing a means of gauging the likelihood of progression.14,15 Jakobiec and Silbert16 perceived a similar challenge with the overdiagnosis of iris melanoma. They used a retrospective, clinicopathological study to test their hypothesis and then published their results in 1981 with the provocative title “Are Most Iris `Melanomas’ Really Nevi?:”16 Just as Zimmerman12,13 had emphasized that April 2016, Vol. 23, No. 2 the diagnosis of precancerous melanosis inflated the risk of cancer in the minds of clinicians, Jakobiec and Silbert16 believed taxonomy obstructing effective communication. Seldom have charges of cancer overdiagnosis been so unambiguously stated. The dilemma of how best to classify indolent, melanocytic tumors of the iris did not simply materialize. Inadequacies with the classification of melanocytic tumors of the uveal tract had been known since the 1960s, which is when it was realized that the original system developed in 1931 by Callender17 did not include nevus (Table).18-20 In 1978, McClean et al18,19 addressed this oversight by studying 105 spindle A melanomas of the choroid and ciliary body that had already been histologically diagnosed by expert consultants. After reviewing each case, they found that 15 cases (14.3%) of spindle A melanomas were actually cytologically benign nevi.18,19 Among this group, no evidence existed of spread or tumor-related death.18,19 Another 75 tumors (71.4%) were diagnosed as spindle cell melanomas based on cytological features.18,20 They consisted of mixtures of spindle A and B melanocytes, and they had a prognosis similar to those previously reported as spindle B melanoma.18,20 Another 15 tumors (14.3%) contained some proportion of epithelioid melanocytes and had a prognosis similar to a mixed-cell type.18 These cases were accordingly reclassified as mixed-cell type melanomas.18 The so-called “classic” spindle A melanocyte fell along a cytological spectrum, and it could be found in both nevi and spindle melanomas.20 The authors recommended that choroidal and ciliary body nevus be added to the spectrum of diagnoses and that the distinction between spindle A and B types of melanomas be dropped (see Table).18,20 The approach to testing the perception of the overdiagnoses of cancer and precancer that these investigators took was similar. Based on histopathological observations, they proposed improved means of predicting clinical behavior and then tested those algorithms against clinical outcomes retrospectively ob- tained. In each situation, pilot studies supported their premises of overdiagnosis. However, uncommon cancers with prolonged clinical courses are difficult to study. Nonetheless, the boundary between indolent and aggressive neoplasia examined by these investigators must always be scrutinized in order to improve diagnostic precision. These examples emphasize a critical difference between the overdetection of cancer, related to excessive cancers found in screening programs, and overdiagnosis, which applies to pathologically verified cancers (or in situ disease) of an indolent type.3 These overdiagnoses are not the result of misdiagnoses; rather, the diagnoses satisfied the standards for precancerous melanosis, iris melanoma, and spindle A choroidal and ciliary body melanoma that existed at the time. What was in dispute was whether the diagnostic categories were too broad, harboring lesions with little propensity for growth or metastatic spread and no risk of death. Epidemiological deduction provides a belated defense against overdiagnosis, but this safeguard is ill suited for uncommon cancers or ones not subject to screening programs like those in and around the eye. However, the legacy of overdiagnosis of ocular and ocular adnexal cancers may unconsciously linger in the minds of many clinicians, possibly explaining the intensity with which ocular oncologists have critiqued new systems of cancer staging.21 Ever since the seventh edition of the American Joint Committee on Cancer Staging System was published,21 a flurry of studies has assessed the updated algorithms.22-26 Although most have found that the modifications are based on sound evidence and are thusly improved, some already anticipate supplementing them with new technologies.27 Establishing the interface of benign and malignant and indolent and aggressive cancers has been the province of anatomical pathologists for a century, but this jurisdiction is yielding to molecular “pathologists” who have at their disposal the genetic makeup of tumors from which to work. The genetic signatures of primary ocular cancers (eg, retinoblastoma, uveal Table. — Histological Classification of Choroidal and Ciliary Body Melanomas Original Grouping17 Spindle A–type melanoma Modified Classification18-20 Comment Melanocytic nevus Callender did not consider benign category Spindle cell melanoma Not synonymous with nevus Spindle B–type melanoma 17 Spindles A and B–type melanocytes commonly found together Epithelioid-type melanoma Epithelioid type Worse prognosis Mixed spindle- and epithelioid-type melanoma Mixed spindle- and epithelioid-type melanoma Prognosis falls between spindle and epithelioid types of melanoma Fascicular pattern melanoma Spindle cell melanoma Similar outcome as spindle cell melanoma Necrotic melanoma Necrotic melanoma Tumors too necrotic to classify April 2016, Vol. 23, No. 2 Cancer Control 91 melanoma) may forecast risk with greater reliability and precision than before and also provide insights into targeted therapies.27,28 In conclusion, timely, accurate diagnosis and the ability to forecast the behavior of malignancy are the goals of clinical practice and are central themes running through papers in this issue of Cancer Control. Clinical decision-making in ocular oncology may continue to rely on histopathological parsing to minimize overdiagnosis, but predicting the biological behavior of cancer will increasingly depend on molecular-based strategies as we look toward the future. 23. Shinder R, Ivan D, Seigler D, et al. Feasibility of using American Joint Committee on Cancer Classification criteria for staging eyelid carcinomas. Orbit. 2011;30(5):202-207. 24. Sniegowski MC, Roberts D, Bakhoum M, et al. Ocular adnexal lymphoma: validation of American Joint Committee on Cancer seventh edition staging guidelines. Br J Ophthamol. 2014;98(9):1265-1260. 25. Graue GF, Finger PT, Maher E, et al. Ocular adnexal lymphoma staging and treatment: American Joint Committee on Cancer versus Ann Arbor. Eur J Ophthalmol. 2013;23(3):344-355. 26. Yousef YA, Al-Hussaini M, Mehyar M, et al. Predictive value of TNM classification, international classification, and Reese-Ellsworth staging of retinoblastoma for the likelihood of high-risk pathologic features. Retina. 2015;35(9):1863-1869. 27. Kapatai G, Brundler MA, Jenkinson H, et al. Gene expression profiling identifies different sub-types of retinoblastoma. Br J Cancer. 2013;109(2):512-525. 28. Onken DD, Worley LA, Tuscan MD, et al. An accurate, clinically feasible multi-gene expression assay for predicting metastasis in uveal melanoma. J Mol Diagn. 2010;12(4):461-468. Curtis E. Margo, MD Department of Pathology and Cell Biology Department of Ophthalmology University of South Florida Morsani College of Medicine Tampa, Florida [email protected] References 1. Welch HG, Black WC. Overdiagnosis in cancer. J Natl Cancer Inst. 2010;102(9):605-613. 2. Kramer BS.The science of early detection. Urol Oncol. 2004;22(4):344-347. 3. Bae J-M. Overdiagnosis: epidemiologic concept and estimation. Epidemiol Health. 2015;37:e2015004. 4. Barry MJ. Screening for prostate cancer--the controversy that refuses to die. N Engl J Med. 2009;360(13):1351-1354. 5. Wegwarth O, Gigerenzer G. Less or more: overdiagnosis and overtreatment: evaluation of what physicians tell their patients about screening harms. JAMA Intern Med. 2013;173(22):2086-2087. 6. Heleno B, Thomasen MF, Rodrigues DS, et al. Quantification of harms in cancer screening trials. literature review. BMJ. 2013:347;f5334. 7. Welch HG, Gorski DH, Albertsen PC. Trends in metastatic breast and prostate cancer – lessons in cancer dynamics. N Engl J Med. 2015;373(18):1685-1687. 8. Reese AB. Precancerous melanosis and diffuse malignant melanoma of the conjunctiva. Arch Ophthalmol. 1938;19:354-365. 9.Reese AB. Precancerous melanosis and the results malignant melanoma (cancerous melanoma) of conjunctiva and skin of lids. Arch Ophthalmol. 1943;29:737-747. 10. Reese AB. Precancerous and cancerous melanosis of the conjunctiva. Am J Ophthalmol. 1955;39:96-100. 11. Reese AB. Tumors of the Eye. Hagerstown, MD: Harper & Row; 1977: 250-254. 12. Zimmerman LE. Criteria for management of melanosis. Arch Ophthalmol. 1966;76:307-8. 13. Zimmerman LE. The histogenesis of conjunctival melanomas: the first Algernon B. In: Jakobiec FA, ed. Ocular and Adnexal Tumors. Birmingham, AL: Aesculapius; 1977: 600-631. 14. Folberg R, McLean I, Zimmerman LE. Primary acquired melanosis of the conjunctiva. Hum Pathol. 1985;16(2):129-135. 15. Folberg R, McLean IW. Primary acquired melanosis and melanoma of the conjunctiva: terminology, classification and biologic behavior. Human Pathol. 1986;17(7):652-654. 16. Jakobiec FA, Silbert G. Are most iris “melanomas” really nevi? A clinicopathologic study of 189 lesions. Arch Ophthalmol. 1981;99(12):2117-2132. 17. Callender GR. Malignant melanotic tumors of the eye: a study of histological types in 111 cases. Trans Am Acad Ophthalmol Otolaryngol. 1931;36:131-141. 18. McLean IW, Zimmerman LE, Evans RM. Reappraisal of Callender’s spindle A type of malignant melanoma of choroid and ciliary body. Am J Ophthalmol. 1978;86(4):557-564. 19. McLean IW. Prognostic features of uveal melanoma. Ophthalmol Clin North Am. 1995;8(1):143-153. 20. McLean IW, Foster WD, Zimmerman LE. Modifications of Callender’s classification of uveal melanoma at the Armed Forces Institute of Pathology. Am J Ophthalmol. 1983;96(4):502-509. 21. Edge S, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual. 7th ed. New York: Springer; 2010. 22. Yousef YA, Finger PT. Predictive value of the seventh edition American Joint Committee on Cancer staging system for conjunctival melanoma. Arch Ophthalmol. 2012;130(5):599-606. 92 Cancer Control April 2016, Vol. 23, No. 2 Genomic, analytical, and sequencing technologies are a critical step toward use of targeted therapies for select patients with high-risk uveal melanoma. Photo courtesy of Lynn E. Harman, MD. Virgin River Gorge. Assessing Prognosis in Uveal Melanoma Zélia M. Corrêa, MD, PhD Background: Because uveal melanoma is the most common primary malignant intraocular tumor in adults and carries a significant risk of metastases, which are mostly unresponsive to available systemic therapy, researchers have been searching for prognostic indicators to identify patients at increased risk for developing such metastasis. Methods: The purpose of this study is to describe recent advances in prognostic testing of patients with uveal melanoma and the impact of these advances on the management of uveal melanoma. The relevant, peerreviewed literature as extracted and then further reviewed for scientific content. Results: Demographic characteristics, clinical, and histopathological features alone are inadequate for predicting metastatic risk in individual patients with uveal melanoma. Some research has shown that cytogenetic abnormalities and principally transcriptomic features of tumor cells can independently predict high risk for uveal melanoma metastatic spread. Gene expression profiling of uveal melanoma cells may be accurate and biologically informative for molecular prognostication. Methods for detecting chromosomal gains and losses have predictive value but require additional clinical and cytological information. The latest step in the evolution of molecular testing has been the discovery of major driver mutations for possible use in targeted therapy. Conclusions: Assay validation, quality control, and interpretation of results are essential for the reliability and reproducibility of these tests. Although these prognostic tests have improved the ability to identify patients at increased risk for developing metastasis, their use has not changed the management of uveal melanoma. However, genomic, analytical, and sequencing technologies will provide a critical step toward useful targeted therapies for patients with high-risk uveal melanoma. Introduction Uveal melanoma is the most common primary, maligFrom the Department of Ophthalmology, College of Medicine, University of Cincinnati, Cincinnati, Ohio. Address correspondence to Zélia M. Corrêa, MD, PhD, Department of Ophthalmology, Medical Arts Building, 222 Piedmont Avenue, Suite 1500, Cincinnati, OH 45267. E-mail: [email protected] Submitted August 26, 2015; accepted October 30, 2015. This work was supported in part by an unrestricted grant from Research to Prevent Blindness (New York, New York), the Quest for Vision Fund and Ophthalmology Research and Education Fund of the University of Cincinnati (Cincinnati, Ohio), and the Mary Knight Asbury Chair endowment fund from the University of Cincinnati. April 2016, Vol. 23, No. 2 nant intraocular tumor in adults and carries a significant risk for metastasis that is typically unresponsive to available systemic therapy, so researchers have been searching for prognostic indicators to identify patients with an increased risk for developing such metastasis.1,2 Previously identified factors indicative of poor prognosis include older age at presentation, male sex, large tumor size (basal diameter and thickness), scleral invasion and/or extraocular extension, presence of ocular or oculodermal melanocytosis, ciliary body involvement, epithelioid cells, high values of the mean diameter of the 10 largest nucleoli, higher microvascular density, vascular mimicry patterns, tumor-infilCancer Control 93 trating lymphocytes and macrophages, high mitotic activity, high expression of insulin-like growth factor 1 receptor, and high expression of class 1/2 HLA.2-9 However, none of these factors are accurate enough to be used across the entire spectrum of uveal melanoma. The challenge clinicians and researchers face is the ability to accurately identify high-risk patients so as to further detect the (micro) metastasizing cells at an early phase — possibly a prerequisite for proper patient selection in future therapeutic interventions. Methods The purpose of this study is to describe the recent advances in prognostic testing of patients with uveal melanoma and how these advances are being applied in clinical practice. A search of the peer-reviewed literature was performed, and relevant articles were identified and carefully reviewed for scientific content and relevance. The author critically reviewed these publications and summarized the most up-to-date information related to prognostic testing for uveal melanoma. Several reports have been published about the clinical and histological factors associated with the development of metastasis in patients with uveal melanoma, and peer-reviewed publications discussing the prognosis of patients with uveal melanoma date as early as 1948.10 This review article will focus on publications since 1990, for which recent advances in prognostic testing of patients with uveal melanoma are reported when the implications of a gain or loss of specific chromosomes in these tumors were first realized. Results After relying for several decades on demographical, clinical, and histopathological features to provide a prognostic estimate for uveal melanoma, and after realizing our limited rate of accuracy for predicting outcomes in individual patients, the research focus shifted toward chromosomal and cytogenetic indicators of metastatic risk in uveal melanoma, following the trend of other cancers.11,12 Early studies of karyotype analysis used fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). Through karyotype analysis, Prescher et al13 discovered that monosomy 3 and increased copies of chromosome 8q were commonly found in uveal melanoma. Thus, the association between uveal melanoma with monosomy 3 and chromosome 8q gain and the development of metastasis has been known for a long time.14 The loss of 1 chromosome 3 was also proven to be part of a 2-step mutation mechanism for the inactivation of BAP1, a tumor suppressor gene.15 Concomitant loss of chromosomes 1p and 3 has a stronger correlation with metastasis.16 The association with chromo94 Cancer Control some 8q gain was also shown to be less significant than for monosomy of chromosome 3 and than either one of them separately.17 Gain of chromosome 8 or acquisition of an isochromosome 8q may be a later event in the setting of uveal melanoma and is seen in both low- and high-risk uveal melanoma.17 Gain of isochromosome 8q is more frequently associated with metastasis when it is accompanied by chromosome 8p loss (so-called isochromosome 8q).16 This frequently occurs in tumors that have lost a copy of chromosome 3, and it is considered an independent prognostic factor of progressive disease.17 Chromosome 6p abnormality was predictive of a more favorable outcome.18 Uveal melanomas with gain of chromosome 6p may represent a separate group of tumors with an alternative genetic pathway in carcinogenesis, because gain of chromosome 6p is frequently found in tumors with disomy 3.19,20 Other chromosomal abnormalities have also been detected in uveal melanoma; however, they often lead to contradictory results regarding the prognostic impact.21 Although FISH has been used to detect these abnormal chromosomal gains and losses, it is a low-resolution technique associated with many false-negative and false-positive results, so its prognostic utility has been limited.22,23 Schouten et al24 described the multiplex ligation–dependent probe amplification (MLPA) that can be used for detecting the relative quantities of as many as 40 different DNA sequences. Array CGH and MLPA have higher rates of accuracy than FISH but are limited by intratumoral genetic heterogeneity and an inability to detect isodisomy 3 (tumor cells are functionally monosomy 3 but have duplicated the diseased copy of the chromosome).23 Molecular classification based on gene-expression profiling (GEP) of posterior uveal melanoma has also been shown to be superior to FISH and array CGH for detecting monosomy 3 and other chromosomal abnormalities, as well as the clinicopathological prognostic factors for predicting metastasis in uveal melanoma.25,26 Multiplex Ligation–Dependent Probe Amplification MLPA is an assay that analyzes the gain and loss of chromosomal material.24 Through the reaction, denatured genomic DNA is mixed with probes for the specific target genes of interest. MLPA is sensitive and sequence specific for detecting changes in DNA copy numbers, detecting deletions and amplifications of single exons. MLPA can be performed on fresh, snap-frozen, formalin-fixed, paraffin-embedded tissue samples, although fresh and snap-frozen samples have been reported to yield more reliable results.27 Use of MLPA for posterior uveal melanoma tissue was described by a group of researchers led by Damato et al3 and Coupland et al.21 Their initial reApril 2016, Vol. 23, No. 2 search analyzed uveal melanoma tissue samples from 73 patients.3,21 MLPA can detect chromosomal abnormalities that correlated with metastatic death, most importantly loss of chromosome 3 and gain of chromosomal material on 8q.28 A larger subsequent study showed that losses of chromosomes 1p and 3 and gain of chromosome 8q correlated with increased rates of mortality, whereas gain in chromosome 6p correlated with improved rates of survival.29 MLPA also provided prognostic information related to chromosomal aberrations in patients with uveal melanomas, and the authors reported that their results correlated with the clinicopathological features of the tumors.29 However, although the results of MLPA provided accurate prognoses in this study, the modality did not offer discriminatory stratification of cases as robust as that seen with GEP testing.29 In addition, the estimation of metastatic risk using MLPA requires the clinician to order the test, carefully interpret its complex report, and add clinicopathological features to improve its rate of accuracy.30 The intratumor genetic heterogeneity of posterior uveal melanoma has also been studied with MLPA using formalin-fixed, paraffin-embedded tumor tissues.31 Researchers found that 25% of the studied tumors were homogeneous; consequently, they found that heterogeneity causes equivocal results on MLPA and may impair the rate of accuracy of their results.31 In retrospective studies, MLPA results reportedly correlate well with the development of metastasis and the survival rate of patients with posterior uveal melanoma; however, the test has not been prospectively validated in a multicenter clinical trial.4,28,30 This test is now commercially available. Gene Expression Profile GEP can be used for the rapid detection of the upregulation or down-regulation of select genes in a tissue sample.26 The technique involves isolating RNA from a tissue sample followed by its conversion to complementary DNA, whose targets are subsequently hybridized to gene chips, and microarray analysis is performed. Onken et al26 demonstrated that uveal melanomas could be divided into 2 distinct prognostic classes that predict a person’s death from metastasis. The authors initially identified 62 genes that showed distinct aberrant expression patterns in the studied samples.26 When the authors combined their findings with the clinical outcome of patients, the up-regulation or down-regulation of specific gene clusters identified by the GEP assay enabled this stratification scheme to predict metastatic risk.26 Class 1 tumors generally have the clinical and pathological features known to be associated with decreased metastatic risk, such as the presence of spindle cells, whereas class 2 tumors generally have more aggressive clinical and pathological features such as epithelioid cells.26 April 2016, Vol. 23, No. 2 Subsequently, a multicenter prospective study was conducted to develop and later validate a 15-gene polymerase chain reaction–based assay that could discern between class 1 and class 2 tumors.32,33 This multicenter validation ensured that this test would yield equally reliable results in different settings and that the results would strongly correlate with rates of survival and metastasis development.33 Since then, this test has become commercially available for routine use in clinical practice and has been adopted by several national and international centers.34 The assay examines the expression patterns of 12 class-discriminating genes identified by the previous analysis and 3 control genes shown to be unchanged in uveal melanomas.26 When compared with monosomy 3 and the clinicopathological features of the tumor, GEP demonstrated superior rates of accuracy at predicting the risk of metastatic disease in patients with uveal melanoma.25 Since the development of the assay, class 1 tumors have been further subdivided in classes 1A and B.34 The 5-year rates of metastatic risk have been estimated to be approximately 2% for class 1A tumors, 21% for class 1B tumors, and 72% for class 2 tumors.35 CGH, FISH, and MLPA provide static measurements of structural chromosomal changes, whereas GEP detects a dynamic RNA signature of the tumor microenvironment.16 This fact may explain why this test has been shown to be the most robust prognostic test for uveal melanoma available.32-34,36-38 Although subdividing class 1 tumors has been helpful in stratifying the very-low-risk patients (class 1A tumors) from the medium-risk patients (class 1B tumors), this system lacks discrimination compared with the prior system of 2 classes.39 PRAME was recently identified as the most significant predictor of metastasis in patients with class 1 tumors.40 Incorporating this biomarker should further improve the accuracy rate of the GEP test. However, several points must be considered regarding the GEP test, as well as any others looking at genetic and transcriptomic features of tumors. Most importantly, any tissue sample can be tested and will originate a result (even if with a low-confidence rate). Some authors have misinterpreted the purpose of GEP, erroneously employing it in nonmelanoma specimens.41,42 It is important to emphasize that MLPA and GEP are prognostic in nature and not diagnostic. Although some have claimed that use of GNAQ/GNA11 mutations ensures that a tumor is uveal melanoma, at least 15% of uveal melanomas do not have this mutation.43 Rather, the proper diagnostic approach of any tumor (including uveal melanoma) is by histopathology or cytopathology.36 Another important fact is that the GEP assay can be performed in paraffin-fixed tissue, but tumor-cell procurement for this test is best obtained by fine neeCancer Control 95 dle aspiration biopsy at the time of plaque implantation or immediately following enucleation of the eye containing the tumor.35 The tumor aspirate is flushed in an RNA-stabilizing buffer solution and then frozen until the time the assay is performed.33 An advantage of this test is that it requires a minimal amount of cells to generate a result. A single-center study of 159 tumoral samples obtained via biopsy showed that the obtained tumor aspirate was insufficient for GEP in a single case (0.6%) compared with 34 cases (21.9%) that had an insufficient aspirate for cytology diagnosis.36 In cases of very small uveal melanoma, a heterogeneous genetic make-up may limit this result (and likely any other test result) in samples yielded by needle aspiration in select patients.31,44 A study performed during the early development of the current 15-gene test — before it was commercially available — showed discordance of GEP classification in a few cases of very small uveal melanomas sampled at 2 distinct sites.44 Overall, discordant results were seen in 7.5% of the tested cases compared with up to 75% of tested cases for which MLPA findings showed tumor heterogeneity.31 That early study of GEP concluded that the tumor samples obtained via biopsy from a single site have a small likelihood of prognostic misclassification (and more likely in thinner tumors [< 3.5 mm in thickness]); therefore, the authors recommended taking this information into account when advising patients with smaller tumors about their prognosis.44 Newer genomic sequencing technologies have also allowed us a more rapid understanding of the molecular landscape of posterior uveal melanoma.45 In addition, the GEP assay (and other prognostic tests) for uveal melanoma have provided opportunities for early detection in patients at high risk of metastasis, thus significantly changing how patients with uveal melanoma are clinically monitored.35 Mutational Profiling Although it is not as useful in the prognostication of patients, detection of specific mutations in uveal melanoma has the potential to improve therapeutic options for metastatic disease in the future. Through a search for mutations in the oncogenic mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase pathway (ERK), Onken et al46 first identified the GNAQ mutation in approximately 50% of uveal melanoma samples. A mutation in GNAQ was detected in samples of posterior uveal melanoma at all of the stages of malignant progression, indicating that such a mutation may play a role in the initial development of the tumor.46 Mutations in GNA11 were also sufficient to induce metastases in a mouse model, so researchers concluded that mutations in GNAQ and GNA11 affect a critical oncogenic signaling cascade to affect the 96 Cancer Control metastatic potential of tumors.43 However, this finding has been questioned because GNAQ and GNA11 mutations in humans result in a benign nevus unless accompanied by further genetic events such as BAP1 mutation.15 GNA11 mutations were initially thought to be more aggressive than GNAQ due to their incidence being slightly higher in metastatic tumors.43 We now know this is because GNA11 mutations are slightly more common in ciliary body melanomas, which have a higher metastatic rate.43 Thus, it is more likely that GNA11 is not any more aggressive than GNAQ. Mutations in GNAQ and GNA11 are mutually exclusive and represent early or initiating events that constitutively activate the MAPK pathway.45 Although these mutations are sensitive to MAPK, protein kinase C, and Akt inhibitors, GNAQ and GNA11 remain difficult therapeutic targets.47 Researchers have demonstrated that the oncogenic activity of mutant GNAQ/GNA11 is mediated at least in part through YAP, which could have therapeutic potential.47,48 Furthermore, mutations in BAP1, SF3B1, and another driver mutation, EIF1AX, may be largely mutually exclusive, and they occur later in tumor progression.49 BAP1 mutations have been reported to be strongly associated with metastasis, class 2 GEP, and older age.50,51 Using multiple regression analysis, BAP1 mutations associated with EIF1AX mutations were also related to class 1 GEP and the absence of ciliary body involvement, whereas SF3B1 mutations are associated with a more favorable outcome and younger age.48-51 BAP1 mutations can arise in the germ line, leading to a newly described BAP1 familial cancer syndrome.45,52 Researchers and clinicians are realizing that, similar to other cancers, uveal melanomas comprise a heterogeneous group of diseases, each with distinct molecular features that might respond best to a specific therapeutic strategy.53 All of these recent discoveries have led to new trials to assess several classes of compounds, including MAPK/ERK inhibitors,54 protein kinase C,55 histone deacetylase inhibitors,56 and combination therapy,57in the adjuvant setting for high-risk patients and in the setting of advanced disseminated disease. The next generation of targeted therapy agents is also being introduced into the clinical setting to evaluate the efficacy of these molecular agents in patients with uveal melanoma.58 Conclusions Prognostic testing has become an important component in the evaluation of patients with uveal melanoma to assess metastatic risk. Although gene expression profiling (GEP) and chromosomal counting methods, such as multiplex ligation–dependent probe amplification, each provide different information about metastatic risk stratification, clinicians can now offer April 2016, Vol. 23, No. 2 patients important insight about their prognosis for metastases. Although the potential for tumor heterogeneity and biopsy sampling error must be further investigated, new developments such as the identification of PRAME expression via GEP may increase the rate of accuracy in small tumors tested by GEP. Although the therapeutic options available for the treatment of metastatic posterior uveal melanoma are in their infancy, patients continue to express their desire to have accurate prognostic information about their tumors in order to plan their lives and to consider enrollment in clinical trials for adjuvant therapy,59 several of which are now available (NCT02601378, NCT01551459, NCT01377025, NCT01413191). Many centers are also revising their surveillance protocols to better suit the needs of patients.60 Recent advances in genomic sequencing technologies have also increased our knowledge of the molecular landscape of uveal melanoma, thus leading to clinical trials using targeted molecules to treat metastatic uveal melanoma. Patients whose increased risk for developing metastasis is detected by genomic testing may wish to enroll in adjuvant treatment trials or they may wish to choose periodic surveillance testing for the earlier detection of asymptomatic metastatic disease. In addition, clinicians can now offer treatments that have the potential to extend the lives of patients with uveal melanoma.60 References 1. Augsburger JJ, Correa ZM, Shaikh AH. Effectiveness of treatments for metastatic uveal melanoma. Am J Ophthalmol. 2009;148(1):119-127. 2. Augsburger JJ, Gamel JW. Clinical prognostic factors in patients with posterior uveal malignant melanoma. Cancer. 1990;66(7):1596-1600. 3. Damato B, Eleuteri A, Taktak AF, et al. Estimating prognosis for survival after treatment of choroidal melanoma. Prog Retin Eye Res. 2011;30(5):285-295. 4. Damato BE, Heimann H, Kalirai H, et al. Age, survival predictors, and metastatic death in patients with choroidal melanoma: tentative evidence of a therapeutic effect on survival. JAMA Ophthalmol. 2014;132(5):605-613. 5. McLean IW, Ainbinder DJ, Gamel JW, et al. Choroidal-ciliary body melanoma. A multivariate survival analysis of tumor location. Ophthalmology. 1995;102(7):1060-1064. 6.Gamel JW, McCurdy JB, McLean IW. A comparison of prognostic covariates for uveal melanoma. Invest Ophthalmol Vis Sci. 1992;33(6):1919-1922. 7. Folberg R, Rummelt V, Parys-Van Ginderdeuren R, et al. The prognostic value of tumor blood vessel morphology in primary uveal melanoma. Ophthalmology. 1993;100(9):1389-1398. 8. Kivela T, Kujala E. Prognostication in eye cancer: the latest tumor, node, metastasis classification and beyond. Eye (Lond). 2013;27(2):243-252. 9. Toivonen P, Makitie T, Kujala E, et al. Microcirculation and tumorinfiltrating macrophages in choroidal and ciliary body melanoma and corresponding metastases. Invest Ophthalmol Vis Sci. 2004;45(1):1-6. 10. Benjamin B, Cumings JN, et al. Prognosis in uveal melanoma. Br J Ophthalmol. 1948;32(10):729-747. 11. Kerbel RS, Cornil I, Theodorescu D. Importance of orthotopic transplantation procedures in assessing the effects of transfected genes on human tumor growth and metastasis. Cancer Metastasis Rev. 1991;10(3):201-215. 12. Bowden GT, Schneider B, Domann R, et al. Oncogene activation and tumor suppressor gene inactivation during multistage mouse skin carcinogenesis. Cancer Res. 1994;54(7 Suppl):1882s-1885s. 13. Prescher G, Bornfeld N, Becher R. Nonrandom chromosomal abnormalities in primary uveal melanoma. J Natl Cancer Inst. 1990;82(22):1765-1769. 14. Prescher G, Bornfeld N, Hirche H, et al. Prognostic implications of monosomy 3 in uveal melanoma. Lancet. 1996;347(9010):1222-1225. 15. Harbour JW, Onken MD, Roberson ED, et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science. 2010;330(6009):1410-1413. 16. Ehlers JP, Worley L, Onken MD, et al. Integrative genomic analysis April 2016, Vol. 23, No. 2 of aneuploidy in uveal melanoma. Clin Cancer Res. 2008;14(1):115-122. 17. van den Bosch T, van Beek JG, Vaarwater J, et al. Higher percentage of FISH-determined monosomy 3 and 8q amplification in uveal melanoma cells relate to poor patient prognosis. Invest Ophthalmol Vis Sci. 2012;53(6):2668-2674. 18. White VA, Chambers JD, Courtright PD, et al. Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma. Cancer. 1998;83(2):354-359. 19. Aalto Y, Eriksson L, Seregard S, et al. Concomitant loss of chromosome 3 and whole arm losses and gains of chromosome 1, 6, or 8 in metastasizing primary uveal melanoma. Invest Ophthalmol Vis Sci. 2001;42(2):313-317. 20. Gordon KB, Thompson CT, Char DH, et al. Comparative genomic hybridization in the detection of DNA copy number abnormalities in uveal melanoma. Cancer Res. 1994;54(17):4764-4768. 21. Coupland SE, Lake SL, Zeschnigk M, et al. Molecular pathology of uveal melanoma. Eye (Lond). 2013;27(2):230-242. 22.White VA, McNeil BK, Horsman DE. Acquired homozygosity (isodisomy) of chromosome 3 in uveal melanoma. Cancer Genet Cytogenet. 1998;102(1):40-45. 23. Speicher MR, Prescher G, du Manoir S, et al. Chromosomal gains and losses in uveal melanomas detected by comparative genomic hybridization. Cancer Res. 1994;54(14):3817-3823. 24. Schouten JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 2002;30(12):e57. 25. Worley LA, Onken MD, Person E, et al. Transcriptomic versus chromosomal prognostic markers and clinical outcome in uveal melanoma. Clin Cancer Res. 2007;13(5):1466-1471. 26. Onken MD, Worley LA, Ehlers JP, et al. Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res. 2004;64(20):7205-7209. 27. Lake SL, Kalirai H, Dopierala J, et al. Comparison of formalin-fixed and snap-frozen samples analyzed by multiplex ligation-dependent probe amplification for prognostic testing in uveal melanoma. Invest Ophthalmol Vis Sci. 2012;53(6):2647-2652. 28. Damato B, Dopierala J, Klaasen A, et al. Multiplex ligation-dependent probe amplification of uveal melanoma: correlation with metastatic death. Invest Ophthalmol Vis Sci. 2009;50(7):3048-3055. 29. Lake SL, Damato BE, Dopierala J, et al. Multiplex ligation-dependent probe amplification analysis of uveal melanoma with extraocular extension demonstrates heterogeneity of gross chromosomal abnormalities. Invest Ophthalmol Vis Sci. 2011;52(8):5559-5564. 30. Damato B, Dopierala JA, Coupland SE. Genotypic profiling of 452 choroidal melanomas with multiplex ligation-dependent probe amplification. Clin Cancer Res. 2010;16(24):6083-6092. 31. Dopierala J, Damato BE, Lake SL, et al. Genetic heterogeneity in uveal melanoma assessed by multiplex ligation-dependent probe amplification. Invest Ophthalmol Vis Sci. 2010;51(10):4898-4905. 32. Onken MD, Worley LA, Tuscan MD, et al. An accurate, clinically feasible multi-gene expression assay for predicting metastasis in uveal melanoma. J Molec Diag. 2010;12(4):461-468. 33. Onken MD, Worley LA, Char DH, et al. Collaborative Ocular Oncology Group report number 1: prospective validation of a multi-gene prognostic assay in uveal melanoma. Ophthalmology. 2012;119(8):1596-1603. 34. Harbour JW, Chen R. The DecisionDx-UM gene expression profile test provides rsk stratification and individualized patient care in uveal melanoma. PLoS Curr. 2013;5:pii. 35. Harbour JW, Chao DL. A molecular revolution in uveal melanoma: implications for patient care and targeted therapy. Ophthalmology. 2014;121(6):1281-1288. 36. Correa ZM, Augsburger JJ. Sufficiency of FNAB aspirates of posterior uveal melanoma for cytologic versus GEP classification in 159 patients, and relative prognostic significance of these classifications. Graefes Arch Clin Exp Ophthalmol. 2014;252(1):131-135. 37. Gill HS, Char DH. Uveal melanoma prognostication: from lesion size and cell type to molecular class. Can J Ophthalmol. 2012;47(3):246-253. 38. Petrausch U, Martus P, Tonnies H, et al. Significance of gene expression analysis in uveal melanoma in comparison to standard risk factors for risk assessment of subsequent metastases. Eye (Lond). 2008;22(8):997-1007. 39. Field MG, Harbour JW. Recent developments in prognostic and predictive testing in uveal melanoma. Curr Opin Ophthalmol. 2014;25(3):234-239. 40.Field MG, Decatur CL, Harbour JW. PRAME as a biomarker for a new molecular subclass of uveal melanoma. Clin Cancer Res. 2016;22(5):1234-1242. 41. Seider MI, Stewart PJ, Mishra KK, et al. Uveal melanoma gene expression profile test result provided for uveal metastasis. Ophthalmic Surg Lasers Imaging Retina. 2014;45(5):441-442. 42. Klufas MA, Itty S, McCannel CA, et al. Variable results for uveal melanoma-specific gene expression profile prognostic test in choroidal metastasis. JAMA Ophthalmol. 2015;133(9):1073-1076. 43. Van Raamsdonk CD, Griewank KG, Crosby MB, et al. Mutations in Cancer Control 97 GNA11 in uveal melanoma. N Engl J Med. 2010;363(23):2191-2199. 44. Augsburger JJ, Correa ZM, Augsburger BD. Frequency and implications of discordant gene expression profile class in posterior uveal melanomas sampled by fine needle aspiration biopsy. Am J Ophthalmol. 2015;159(2):248-256. 45. Harbour JW. Genomic, prognostic, and cell-signaling advances in uveal melanoma. Am Soc Clin Oncol Educ Book. 2013:388-391. 46.Onken MD, Worley LA, Long MD, et al. Oncogenic mutations in GNAQ occur early in uveal melanoma. Invest Ophthalmol Vis Sci. 2008;49(12):5230-5234. 47. Field MG, Harbour JW. GNAQ/11 mutations in uveal melanoma: is YAP the key to targeted therapy? Cancer Cell. 2014;25(6):714-715. 48. Yu FX, Zhang K, Guan KL. YAP as oncotarget in uveal melanoma. Oncoscience. 2014;1(7):480-481. 49. Decatur CL, Ong E, Garg N, et al. Driver mutations in uveal melanoma: associations with gene expression profile and patient outcomes. JAMA Ophthalmol. 2016. Epub ahead of print. 50. Harbour JW, Roberson ED, Anbunathan H, et al. Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat Genet. 2013;45(2):133-135. 51. van Essen TH, van Pelt SI, Versluis M, et al. Prognostic parameters in uveal melanoma and their association with BAP1 expression. Br J Ophthalmol. 2014;98(12):1738-1743. 52. Njauw CN, Kim I, Piris A, et al. Germline BAP1 inactivation is preferentially associated with metastatic ocular melanoma and cutaneousocular melanoma families. PLoS One. 2012;7(4):e35295. 53. Patel M, Smyth E, Chapman PB, et al. Therapeutic implications of the emerging molecular biology of uveal melanoma. Clin Cancer Res. 2011;17(8):2087-2100. 54. Templeton IE, Musib L. MEK inhibitors beyond monotherapy: current and future development. Curr Opin Pharmacol. 2015;23:61-67. 55. Cerne JZ, Hartig SM, Hamilton MP, et al. Protein kinase C inhibitors sensitize GNAQ mutant uveal melanoma cells to ionizing radiation. Invest Ophthalmol Vis Sci. 2014;55(4):2130-2139. 56. Landreville S, Agapova OA, Matatall KA, et al. Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma. Clin Cancer Res. 2012;18(2):408-416. 57. Chen X, Wu Q, Tan L, et al. Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations. Oncogene. 2014;33(39):4724-4734. 58. Luke JJ, Triozzi PL, McKenna KC, et al. Biology of advanced uveal melanoma and next steps for clinical therapeutics. Pigment Cell Melanoma Res. 2015;28(2):135-147. 59. Cook SA, Damato B, Marshall E, et al. Psychological aspects of cytogenetic testing of uveal melanoma: preliminary findings and directions for future research. Eye. 2009;23(3):581-585. 60.Shoushtari AN, Carvajal RD. Treatment of uveal melanoma. Cancer Treat Res. 2016;167:281-293. 98 Cancer Control April 2016, Vol. 23, No. 2 Various forms of chemotherapy and targeted therapies have helped improve rates of survival and ocular salvage in retinoblastoma. Photo courtesy of Lynn E. Harman, MD. Themes at the Shard. Therapeutic Options for Retinoblastoma Pia R. Mendoza, MD, and Hans E. Grossniklaus, MD Background: Retinoblastoma is the most common primary intraocular malignancy in children. The management of retinoblastoma is complex and depends on several factors. Methods: This review provides an update on current and emerging therapeutic options for retinoblastoma. The medical literature was searched for articles relevant to the management of retinoblastoma. The results of prospective and retrospective studies on chemotherapy and focal therapy for retinoblastoma are summarized. Animal models for novel therapeutic agents are also discussed. Results: Treatment strategies for retinoblastoma involve intravenous chemoreduction, local administration routes of chemotherapy (eg, intra-arterial, intravitreal), focal therapy for tumor consolidation (eg, photocoagulation, thermotherapy, cryotherapy, plaque brachytherapy), external beam radiotherapy, and surgical enucleation. Emerging therapies include alternative chemotherapeutic agents, molecularly targeted therapies, and novel drug-delivery systems. Conclusion: In the past 10 years, the management strategy for retinoblastoma has significantly changed, shifting toward local chemotherapy and away from systemic chemotherapy. Innovations in the field of molecular biology and the development of targeted therapies have led to improvements in survival rates and ocular salvage for this disease. However, the need still exists to further assess the long-term effects of such directional changes in therapy. Introduction Retinoblastoma is the most common primary intraocular malignancy in children; its incidence rate is 1 in 14,000 to 20,000 livebirths.1 However, compared with other pediatric malignancies, retinoblastoma is rare, comprising 3% to 4% of all pediatric cancers.2 It is a tumor of the developing retina that arises from primitive retinal stem cells or cone precursor cells associated with inactivation of both alleles of the tumor suppresFrom the Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia. Address correspondence to Hans E. Grossniklaus, MD, 1365 Clifton Road, Northeast, BT428, Atlanta, GA 30322. E-mail: [email protected] Submitted October 14, 2015; accepted January 5, 2016. No significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. April 2016, Vol. 23, No. 2 sor gene RB1. Major advances, such as our understanding of the molecular biology of the tumor and the development of targeted therapy, have improved survival rates in developed countries.3 In the United States, the 5-year overall survival rate is 97%.4 Much of this success is due to the benefits of effective treatments, including chemotherapy, focal or consolidation therapy, external beam radiotherapy, and surgical enucleation (Table 1).5 With early diagnosis and aggressive, multimodal treatment strategies, near-complete cure rates are possible, and many patients retain functional vision in at least 1 eye. However, in developing nations, where access to health care is limited, retinoblastoma can cause blindness and death.6 Among the mainstay treatments for retinoblastoma are intravenous chemotherapy (involving agents associated with significant toxicities), surgical enucleation, external beam radiotherapy, or all 3 options, potenCancer Control 99 tially result in disfigurement and secConcepts and Principles of Table 1. — Therapeutic Options ondary malignancies. However, in reTreatment in Retinoblastoma cent years, an ongoing evolution has The goal of treatment in retinoblasChemotherapy taken place, where treatment is being toma is the elimination of the tumor Intravenous geared toward more targeted therapy while concurrently minimizing coland local medication delivery. Local lateral injury to other tissues. The Chemoreduction delivery increases exposure to intrapriorities in management are (1) preAdjuvant therapy ocular target areas, reduces overall venting metastasis, (2) reducing the Metastatic retinoblastoma systemic exposure and harmful late risk of long-term secondary tumors Intra-arterial effects, and improves effectiveness (eg, osteosarcoma, soft-tissue sarcoIntravitreal and rates of tolerability. The introma), (3) saving the eye, and (4) prePeriocular duction of intra-arterial and intravitserving vision.11 Caring for a patient Focal Therapy real chemotherapy has resulted in a with retinoblastoma must be a mulchange in treatment algorithms and tidisciplinary effort involving pediThermotherapy improved outcomes, with their posatric oncologists, ocular oncologists, Photocoagulation sibility of ocular salvage and mainteradiation oncologists, ocular patholCryotherapy nance of vision.7 ogists, and geneticists to optimize Plaque brachytherapy Preclinical models that simulate treatment outcomes. External Beam Radiotherapy human disease are needed for rare Treatment depends on tumor Enucleation childhood cancers such as retinoblasstage, laterality and number of tutoma because not enough patients mor foci (unifocal, unilateral, mulwith the disease exist for large-scale, tifocal, or bilateral), the localization multicenter clinical trials. Despite the lack of clinical and size of tumors within the eye, presence of vittrials, rates of survival and ocular preservation have reous seeding, age and health of the child, and the improved.7 In vitro studies and animal models remain family’s wishes.12 The type of management depends instrumental in understanding biological mechanisms on the tumor classification, which is based on 2 sysand assessing safety and effectiveness of the treatment tems: the International Classification of Retinoblasoptions for this disease.8-10 Examples of emerging thertoma13 used to predict success of chemoreduction14 apies under investigation for retinoblastoma are alterand the tumor, node, and metastasis classification native chemotherapeutic agents, molecularly targeted of the American Joint Committee on Cancer15 for tutherapies, and novel drug-delivery systems. mor staging (Table 2). Location of the tumor in relaTable 2. — International Classification and General Therapeutic Options for Retinoblastoma Eye Group General Feature Specific Feature (Presence of ≥ 1) Therapy A Small tumor away from fovea and optic disc Tumor ≤ 3 mm Laser photocoagulation Thermotherapy Cryotherapy Plaque brachytherapy B Larger tumor Macular Juxtapapillary Subretinal fluid Tumor > 3 mm Tumor located < 3 mm from fovea Tumor located ≤ 1.5 mm from optic disc Presence of subretinal fluid ≤ 3 mm from tumor margin Laser photocoagulation Thermotherapy Cryotherapy Plaque brachytherapy Intravenous/intra-arterial chemoreduction C Focal seeds Presence of subretinal, vitreous seeds, or both located ≤ 3 mm from main tumor Intra-arterial chemotherapy Intravitreal chemotherapy D Diffuse seeds Presence of subretinal, vitreous seeds, or both located > 3 mm from main tumor Intra-arterial chemotherapy Intravitreal chemotherapy Enucleation E Extensive tumor Tumor occupying > 50% of the globe Neovascular glaucoma Opaque media from hemorrhage in anterior chamber, vitreous, or subretinal space Invasion of postlaminar optic nerve, choroid (> 2 mm), sclera, orbit, anterior chamber Intra-arterial chemotherapy Enucleation Adjuvant intravenous chemotherapy if high-risk histopathological features present 100 Cancer Control April 2016, Vol. 23, No. 2 tion to the optic disc and macula is important in the clinical evaluation because it largely affects the visual prognosis. Involvement of certain tissues in and around the eye, including the optic nerve, choroid, iris, sclera, and orbit, increase the likelihood of tumor metastasis.16 In children, unilateral retinoblastoma is differently managed than bilateral tumors. In general, unilateral cases are the result of sporadic mutations, whereas bilateral cases are typically multifocal, hereditary, and occur in patients carrying a germline RB1 mutation, thus placing them at risk for other malignancies (eg, intracranial neuroblastic tumors [pinealoblastoma in trilateral retinoblastoma]). In general, unilateral tumors can be managed with focal therapy alone; chemoreduction, intra-arterial chemotherapy with focal therapy, or both; or enucleation. If genetic testing is not performed, then patients with unilateral disease require close follow-up of the unaffected eye to exclude bilateral involvement. Alternatively, treatment for bilateral tumors will depend on the extent of tumor in each eye. In asymmetric disease, an attempt is made to salvage the eye with less severe disease. One may consider giving patients systemic intravenous chemoreduction as initial therapy to treat both eyes as well as to prevent metastasis and associated secondary tumors or to use local (intra-arterial) chemotherapy with focal treatment.7,17,18 Initial chemotherapy in retinoblastoma can be given either systemically (intravenous) or locally (directly through the ophthalmic artery/intra-arterial). Chemoreduction is then usually followed by consolidation, which is achieved by focal therapy (eg, laser photocoagulation, thermotherapy, cryotherapy, plaque brachytherapy). Focal therapy may be employed as the only or initial therapy for small retinoblastoma tumors located away from the fovea and optic nerve (group A); it is often used for tumor consolidation in retinoblastoma following chemoreduction for more advanced (groups B–D) tumors. Surgical removal of the eye or enucleation is reserved for very advanced (group E) cases. Management Chemotherapy Intravenous: Systemic chemotherapy generally involves a combination 2-, 3-, or 4-drug regimen delivered through an intravenous catheter. Three classes of agents are commonly employed: DNA-crosslinking agents (carboplatin, cisplatin), DNA topoisomerase 2 inhibitors (etoposide, topotecan, teniposide) and Vinca alkaloids (vincristine; Fig 1). The most commonly used regimen is vincristine/etoposide/carboplatin.11 Chemoreduction via systemic chemotherapy was introduced as a management option for retinoblastoma in the mid-1990s following preliminary observations April 2016, Vol. 23, No. 2 that systemic chemotherapy delivered prior to external beam radiotherapy contributed to tumor control and ocular salvage.19 A subsequent study on eyes treated with chemoreduction combined with focal treatments (cryotherapy, thermotherapy, or plaque radiotherapy) demonstrated tumor regression and decreased need for additional external beam radiotherapy and enucleation.20 This was a major advancement in management because it was evident that satisfactory tumor control could be attained with systemic chemotherapy while saving the eye and avoiding the adverse events of external beam radiotherapy.20 Using the International Classification of Retinoblastoma, treatment success was found in 100% of group A eyes, 93% of group B eyes, 90% of group C eyes, and 48% group D eyes.14 This launched the so-called “systemic chemotherapy era” (1990s to 2006); however, with the advent of local routes of administration (intra-arterial and intravitreal), use of systemic chemotherapy has decreased in recent years.14 Intravenous chemotherapy is indicated as initial therapy for bilateral advanced disease in which attempts are made to salvage both eyes. Many centers use systemic chemotherapy in bilateral (germline) retinoblastoma for intraocular retinoblastoma control as well as to prevent metastasis, reduce the likelihood of the development of pineoblastoma, and to reduce the long-term risk of secondary cancers.17,18 Systemic chemotherapy is also utilized as adjuvant treatment to prevent metastasis following enucleation in patients with optic nerve invasion posterior to the lamina cribrosa, massive choroidal invasion, or any combination of optic nerve and choroidal invasion.21 The treatment regimens used include vincristine/etoposide/carboplatin, vincristine/doxorubicin/idarubicin/cyclophosphamide, or hybrid regimens, typically delivered monthly for 6 to 9 months.22 A major obstacle to use of intravenous chemotherapy in retinoblastoma is the blood–retina barrier, which limits the entry of some chemotherapeutic agents into the eye, thereby reducing their efficacy.23 Systemic chemotherapy for retinoblastoma is generally safe and effective, but it is not without adverse events, such as neurotoxicity, hyponatremia, nephrotoxicity, ototoxicity, and secondary leukemia. Treatment failure in the form of persistent vitreous seeds, subretinal seeds, and intraretinal tumors following therapy have been attributed to the inability of the treatment drugs to reach the tumor.24 Tumor resistance or unresponsiveness to chemotherapy is thought to occur more often in well-differentiated tumors, presumably because cells are not cycling, so they are less likely to respond to treatment modalities affecting cell division.25 Intra-Arterial: In the late 1980s, melphalan was found to be effective for the management of retinoblastoma in vitro.26 Yamane et al27 and Suzuki et al28 then began to treat patients with intracarotid artery melphalan, developing a technique to safely and effectively Cancer Control 101 Fig 1. — Chemotherapeutic agents used in retinoblastoma. Alkylating agents such as melphalan cause DNA intra–strand-linking and cross-linking, resulting in DNA damage and disruption of transcription. Topoisomerase inhibitors such as etoposide and topotecan cap free double-strand breaks in DNA, preventing DNA repair and apoptosis. Platinum-based agents such as carboplatin bind DNA bases, resulting in kinked DNA. Vinca alkaloids such as vincristine bind tubulin dimers and block mitotic spindle formation. Reprinted from Grossniklaus HE. Retinoblastoma. Fifty years of progress. The LXXI Edward Jackson Memorial Lecture. Am J Ophthalmol. 2014;158(5):875-891, with permission from Elsevier. cannulate the ophthalmic artery for chemotherapy infusion. In the United States, intra-arterial chemotherapy (IAC) was initially used by Abramson et al.29 IAC involves transfemoral catheterization with advancement into the ophthalmic artery, which is where melphalan, topotecan, or carboplatin is infused. The medication is slowly delivered for 30 minutes in a pulsatile fashion with care to not occlude the artery and to minimize reflux into the internal carotid artery. Most patients receive 3 monthly sessions. Melphalan is the most frequently used agent, with topotecan added if extensive vitreous seeding is present. Generally, IAC is employed for unilateral or nongermline retinoblastoma, recurrent tumors following previous intravenous chemotherapy or plaque radiotherapy, recurrent subretinal seeds involving at least 2 quadrants, recurrent vitreous seeds, or, if the patient wishes to avoid systemic therapy, enucleation can be considered if consent is obtained. In a 5-year experience with IAC, complete regression was achieved for solid tumors in 94%, for subretinal seeds in 95%, and for vitreous seeds in 87% of study patients.30 In recent 102 Cancer Control years, globe-salvage results (85%–94% globe salvage rate in group D tumors with primary IAC) have been reported in study patients with advanced retinoblastoma.30,31 In some centers, most group D eyes are primarily treated with IAC, and, on occasion, group D and E eyes are managed with combined intravenous chemotherapy followed by intra-arterial chemotherapy — particularly if the patient has advanced bilateral disease or disease in a single eye.7 Fair success has been reported in group E tumors with total retinal detachment cautiously managed with initial IAC.32,33 Treatment of more advanced disease with initial IAC has also not been shown to compromise survival rates.7 After adequate reduction is visualized and subretinal fluid is resolved, complete consolidation with either thermotherapy or cryotherapy and supplemental intravitreal chemotherapy after IAC are commonly performed. Consolidation is generally performed during the second or third cycle of chemoreduction. In infants younger than 3 months who weigh less than 7 kg, IAC is initially deferred to allow the femoral artery to grow before attempting catheterization. April 2016, Vol. 23, No. 2 In such cases, patients are treated with 1 to 2 doses of single-agent carboplatin as bridge therapy before definitive IAC.34 Reports of high rates of efficacy of IAC abound, but studies also exist on its associated ocular toxicities.35-37 Use of intra-arterial infusion of melphalan and carboplatin compromise the ocular vasculature in nonhuman primates and induces inflammation and leukostasis in human retinal endothelial cells.35,36 Other complications include vitreous hemorrhage, microemboli to the retina and choroid, myositis, eyelid edema, orbital congestion with resulting dysmotility, choroidal atrophy, optic atrophy, ophthalmic artery stenosis, and branch-retinal artery occlusion resulting in blindnesss.37 A major drawback to this therapy is that only skilled neurosurgeons and/or interventional radiologists at major cancer centers can treat patients using IAC.38 The therapy is also expensive.39 In developing countries, IAC is not available or is too costly, so enucleation as initial therapy is still a mainstay of treatment for advanced retinoblastoma.40 In addition, a study has shown that initial chemotherapy prior to enucleation for advanced retinoblastoma can mask pathological evidence of tumor extension, thus leading to reduced surveillance and the undertreatment of high-risk disease — thereby increasing the risk of metastatic death.41 Intravitreal: The major question encountered in the management of retinoblastoma is how to treat subretinal and vitreous seeds of tumor unresponsive to laser treatment, conventional chemoreduction therapy, or IAC.24 Treatment of vitreous seeds is challenging due to lack of vasculature in the vitreous. Another route of administration developed to address this problem is the intravitreal method, which involves injecting the therapeutic agent into the vitreous cavity of the eye through the pars plana under aseptic precautions. Although health care professionals were initially skeptical of injecting drugs through the sclera into the vitreous for fear of tumor spread along needle tracks, techniques for safe and effective intravitreal injections have been developed. Intraocular pressure is first lowered prior to the injection. A small-volume dose of melphalan, topotecan, or a combination of both is injected into the eye using a fine needle (30- or 32-gauge) and the needle is frozen with a cryoprobe as it is withdrawn from the eye to prevent tumor seeding.42 The first-line indication for intravitreal chemotherapy includes vitreous seeds refractory to standard therapy and recurrent vitreous seeds after previous therapy.43,44 In an analysis of 57 eyes with viable recurrent vitreous seeds, intravitreal melphalan led to eye salvage in 51% of eyes after 10 years of follow-up; no cases of extraocular extension were reported during that time.45 A systematic review showed that proper techApril 2016, Vol. 23, No. 2 nique does not increase the risk of tumor spread.46 Treatment of retinoblastoma with recurrent vitreous seeds has been reported to have a success rate of 83%.47 The number of injections depends on the response, and 6 injections delivered weekly or biweekly is recommended. Reported adverse events include transient vitreous hemorrhage, chorioretinal atrophy, and extraocular tumor spread.42,48,49 Melphalan is the most commonly used drug for intravitreal injections. Although it caused no systemic toxicity in humans or rabbits, melphalan did result in significant retinal toxicity at high doses and lower but safer doses only achieved incomplete tumor control.42,50 Periocular: Chemotherapeutic agents may be periocularly injected, either as subconjunctival or subtenon. Periocular injection enables transcleral drug delivery, using the large surface area of the sclera and its high permeability to small molecules without the danger of puncturing the globe.51 Periocular chemotherapy achieves levels within the vitreous rapidly and at levels 6 to 10 times higher than the intravenous route.52 In general, periocular injection of chemotherapeutic agents has been used for retinoblastoma control as an adjunct to systemic chemotherapy and, on occasion, to treat tumor recurrence. Either carboplatin or topotecan can be employed. The first-line indications for periocular chemotherapy are bilateral advanced group D or E eyes in which a higher local dose of chemotherapy is needed, treatment of vitreous seeds, and recurrent localized tumor. Subtenon chemotherapy using carboplatin can increase tumor control, especially if it is coupled with intravenous chemoreduction. Subtenon carboplatin showed initial favorable results as single therapy, but long-term follow-up revealed a high failure rate as initial treatment; therefore, it should be combined with other therapeutic approaches.53 Complications of periocular chemotherapy include orbital and eyelid edema, ecchymosis, orbital fat atrophy, muscle fibrosis leading to strabismus, and optic atrophy.53-55 Because of its significant toxic effects, periocular chemotherapy is rarely used in current practice. Focal Therapy Thermotherapy/Transpupillary Thermotherapy: This type of focal therapy uses an 810-nm diode laser directly on the tumor to heat it up to subcoagulation temperatures of 42 to 60 °C to induce a cytotoxic effect. It is used for local control and for small (< 4.5-mm base and 2.5-mm thickness) tumors posteriorly located to the equator of the globe. In cases of macular or juxtapapillary tumors, consolidation should be performed with caution so as to protect the optic disc and neurosensory papillomacular bundle. The tumor may be observed while on chemoreduction and, if unresponsive, foveal-sparing thermotherapy may be administered. In an analysis of 68 macular retinoblastomas treated with Cancer Control 103 chemoreduction, tumor recurrence occurred in 17% of those consolidated with foveal-sparing thermotherapy compared with 35% of those observed without consolidation.56 Complications of transpupillary thermotherapy include iris atrophy, focal cataracts, tumor seeding into the vitreous, and retinal fibrosis, transition, and vascular occlusion. Laser Photocoagulation: This type of therapy uses an 520-nm argon laser to coagulate the blood supply of the tumor by generating heat with temperatures in excess of 65 °C within the treatment spot. Direct photocoagulation to the tumor must be avoided. A second mechanism occurring around the ring of tissue beyond the laser spot is the increase in temperature to a thermotherapeutic range (45–60 oC). In this region, synergism exists with carboplatin, so this laser treatment is typically performed within 24 hours of the intravenous chemotherapy cycle. Small posterior tumors without seeding respond well to laser photocoagulation. It is not used for tumors impinging on the fovea, because of the risk of compromising a patient’s central vision. In such cases, some form of chemotherapy can be used to shrink the tumor prior to the start of definitive laser treatment. Photocoagulation is also used to treat tumor-associated retinal neovascularization. Complications include vitreous seeding if the laser power is too high, and retinal fibrosis, traction, and vascular occlusion. Cryotherapy: Cryotherapy involves a cryoprobe by which liquid nitrogen is delivered and directly applied to the outer surface of the sclera adjacent to the tumor. It is suitable for small (< 3.5-mm base and < 2-mm thickness) tumors anteriorly located to the equator of the globe. Complications of cryotherapy include retinal tears and detachment, proliferative vitreoretinopathy, and chorioretinal atrophy. Plaque Brachytherapy: This type of treatment refers to implantation of radioactive material on the sclera over the base of the tumor. Tissue absorption of ionizing radiation causes DNA damage and cell death. Because retinoblastoma has a high rate of proliferating cells, it is quite radiosensitive. Brachytherapy is used for solitary, medium-sized tumors (6–15 mm and > 3 mm from the optic disc or fovea) and, if little subretinal fluid is present, plaque radiotherapy can generally be used to achieve tumor control. Iodine-125 and ruthenium-106 isotopes are the most common source of radiation currently used in brachytherapy for retinoblastoma to deliver 40 to 45 Gy to the apex of the tumor. In addition to treatment for retinoblastoma, ocular brachytherapy is commonly used for uveal melanoma, in which a clinician uses a similar surgical technique of plaque application of suturing to the episcleral surface but uses a lower dose of radiation. Complications of plaque brachytherapy include radiation retinopathy and optic neuropathy.18 104 Cancer Control Consolidation alone may be used in group A and B tumors. Bilateral cases with small, extrafoveal tumors can be managed with focal treatments alone. In asymmetric disease, a single eye can be managed with focal techniques and the other with enucleation or intra-arterial chemotherapy with or without systemic chemotherapy.7 The desired end point is tumor regression in the form of a completely calcified tumor (type 1 regression) or a flat chorioretinal scar (type 4 regression). Enucleation Enucleation remains a favored approach in cases of extensive retinoblastoma with buphthalmos, neovascular glaucoma, aqueous seeding, and transcleral extension (advanced group E eyes) — particularly for unilateral cases with no likelihood of functional vision.6 Surgery involves careful removal of the eye with minimal globe trauma to prevent tumor seeding into the orbit, and then excising a long section of optic nerve for proper histopathological evaluation.57 Following enucleation, an orbital implant is placed to provide the socket with enough volume and allow the 4 rectus muscles to attach for motility. Immediately after enucleation, fresh tissue should be harvested on a separate tray for genetic studies. A corollary to enucleation is the adequate histopathological evaluation of globe pathology, because retinoblastoma with histological features signifying a high risk of metastasis requires adjuvant intravenous chemotherapy.58 High-risk features include choroidal invasion, retrolaminar optic nerve invasion, scleral and orbital invasion, and anterior chamber invasion. An analysis of 1,020 study patients with high-risk features after enucleation showed that 24% of study volunteers at high risk developed metastasis if not treated with adjuvant chemotherapy, whereas 4% of these study patients had metastasis if treated with chemotherapy.59 The regimen for adjuvant chemotherapy is vincristine/etoposide/carboplatin for 4 to 6 cycles. External Beam Radiotherapy External beam radiotherapy can be used to treat eyes unresponsive to other treatments, those with a tumor located at the optic nerve resection margin (36 Gy to orbit and 10 Gy boost to chiasm), and as treatment of extraocular retinoblastoma such as those extending through the sclera, orbit, or intracranially (40–45 Gy).18 External beam radiotherapy is rarely used and employed only when necessary because of its subsequent risks. It is known to cause midfacial hypoplasia in young patients and increase the risk of development of soft-tissue sarcomas, brain tumors, osteosarcomas, and other cancers.60 In patients with bilateral disease, the incidence of secondary malignancy at 50 years from diagnosis of retinoblastoma is 21% in patients who were not treated vs 38% for those who were treated with raApril 2016, Vol. 23, No. 2 diation.61-63 If possible, any radiation (including radiography, computed tomography, and external beam) should be avoided in individuals with heritable retinoblastoma to minimize their lifetime risk of developing secondary cancers. Extraocular Therapy Although their incidence is rare in developed countries, cases of extraocular disease are occasionally seen in neglected or inadequately treated patients.64,65 Tumors may directly extend through emissary vessels of the sclera into the orbital soft tissue surrounding the eye. Treatment of orbital retinoblastoma includes systemic chemotherapy and external beam radiotherapy (40–45 Gy) with a rate of cure between 60% and 85%.18 Treatment may also extend to the optic nerve into the brain and meninges with subsequent seeding of spinal fluid. Most recurrences occur in the central nervous system and are associated with a low cure rate; regimens proven to be effective include vincristine, cyclophosphamide, doxorubicin, and platinum as well as epipodophyllotoxin-based drugs.66 No preclinical or clinical evidence supports the use of intrathecal chemotherapy. Craniospinal irradiation using 25 to 35 Gy is sometimes provided. Retinoblastoma may also hematogenously spread to the bone marrow, bones, lungs, or liver. For metastatic disease, multiple-agent, high-dose chemotherapy and autologous hematopoietic stem cell transplantation can be administered as rescue therapy.67 The Children’s Oncology Group developed a prospective, multi-institutional, international trial for metastatic retinoblastoma, and data will be available in early 2016; that study hopes to define differences in site of metastasis and outcome.68 Emerging Therapies New developments in the treatment of retinoblastoma are geared toward an ideal system that is affordable and can easily be used by ophthalmologists or ocular oncologists — even those in developing nations — and is locally effective to the main tumor as well as tumor seeds, causes minimal complications, and can achieve a sustained treatment effect to retinoblastoma without spreading tumor cells. A sustained treatment effect is desired because, even when compounds do reach the target site, rapid clearance from the eye often results in short intraocular residence times. In addition, many patients with retinoblastoma are young children who oftentimes require sedation to receive intraocular therapy — sustained drug-release mechanisms will enable continuous medication dosing. Preclinical models that accurately reflect human disease are needed for rare childhood cancers such as retinoblastoma because not enough patients exist for large-scale multicenter clinical trials.8 Despite this lack of clinical trials, rates of patient survival and ocular April 2016, Vol. 23, No. 2 preservation have improved.7 Animal models remain instrumental in understanding biological mechanisms and designing targeted therapies for this disease.8-10 Novel treatment options being explored in vitro and in vivo include alternative chemotherapeutic agents, molecularly targeted therapies, gene therapy, localized delivery, and the sustained-release of broad-spectrum chemotherapy agents. Alternative Chemotherapeutic Agents Topotecan, a topoisomerase inhibitor that works in the G0 phase of the cell cycle, may be used as a single agent or in combination with other agents for the treatment of retinoblastoma. Subconjunctival, sustained-release topotecan has been shown to decrease tumor burden in a transgenic mouse model of retinoblastoma.69 In a similar fashion, periocular injections of topotecan in fibrin sealant in humans have been shown to achieve retinoblastoma tumor volume reduction.70 Topotecan can also reduce retinoblastoma tumor burden when given systemically,71 via IAC,72 or via intravitreal injections.43 When compared with standard chemotherapeutic agents used to treat retinoblastoma, topotecan avoids the retinal toxicity of melphalan, the nephrotoxicity of carboplatin, and the hematotoxicity of etoposide.73 Topotecan is a good candidate drug for intravitreal injection because it has minimal retinal toxicity and achieves maximum concentration levels 50 times higher than systemic exposure.73,74 Our laboratory has shown that 20-µg topotecan can provide sustained retinoblastoma cell-line killing in vitro,75 similar to the clinically demonstrated retinoblastoma cell-line killing ability with 20-µg intravitreal topotecan.43 Anthracyclines are a class of chemotherapeutic drugs that inhibit topoisomerase and intercalate DNA. They are used to treat several cancers and are active against retinoblastoma; however, because of their limited blood–brain barrier penetration, their use is typically limited to extraocular and metastatic retinoblastoma. The anthracycline idarubicin has been shown to be effective against retinoblastoma tumor cells in bone marrow (response rate, 60%), but sufficient concentrations have not been achieved at target sites (eg, central nervous system).76 A study to improve intraocular exposure of the anthracycline doxorubicin while also reducing systemic exposure to the drug was successful in maintaining sustained levels of doxorubicin after the intravitreal injection of the drug encapsulated in polyhydroxybutyrate microspheres in rabbits.77 Molecularly Targeted Therapy RB1 was the first tumor suppressor gene identified and cloned, yet no effective, molecularly targeted cure currently exists for retinoblastoma.78 This could perhaps Cancer Control 105 be attributed to the variety of unknown secondary mutations and differential expression of other genes aside from RB1 involved in tumorigenesis, as well as questions on the cellular origin of retinoblastoma.79,80 Significant progress has been made in our understanding of tumor biology, leading to the discovery and development of small molecules for the treatment of retinoblastoma, including novel, targeted therapeutics such as inhibitors of the MDMX–p53 response (nutlin-3a), histone deacetylase (HDAC) inhibitors, and spleen tyrosine kinase (SYK) inhibitors.9,81 Nutlin-3 is an imidazole analogue that plays a role in the activation of p53, a tumor suppressor protein. It is a small molecule inhibitor of MDM2 and MDMX and prevents the association of both these proteins with p53. Studies have shown that nutlin-3 restores the p53 pathway in retinoblastoma cells lacking both retinoblastoma protein and p53 activity, thus inducing p53-mediated apoptosis.82 Subconjunctival injection of nutlin-3 in mouse models of retinoblastoma resulted in reduced tumor burden when used in combination with topotecan, demonstrating a synergistic effect.83 Nutlin-3 is being studied in phase 1 clinical trials for the treatment of retinoblastoma.82,83 Although it is not expressed in normal human retinas, SYK was found to be upregulated in retinoblastoma tumor samples and has been shown to be required for retinoblastoma cell survival.84 Inhibition of SYK with BAY-61-3606 and R406 caused cell death in retinoblastoma, and an in vivo study of orthotopic, xenograft mice showed that subconjunctival BAY-61-3606 was effective at blocking retinoblastoma cell proliferation.84 In addition, studies of SYK inhibition have also implicated several downstream signaling molecules as mediators of SYK survival, including the B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2) family of proteins.85 Myeloid cell leukemia 1, a member of the BCL2 family, is a potential target because it is upregulated in retinoblastoma and is being developed as a therapy for other cancers.86 Another class of targeted therapies in phase 1 clinical trials are the inhibitors of HDAC.87 Cells with elevated E2F1 activity are uniquely sensitive to inhibitors of HDAC through the overexpression of proapoptotic factors. Cells lacking retinoblastoma protein have increased E2F1 activity, and retinoblastoma-derived cell lines have demonstrated particular sensitivity to apoptosis induced from inhibitors of HDAC.88 In a preclinical trial, inhibitors of HDAC slowed the growth of retinoblastoma-derived tumors in both transgenic and xenograft murine models of retinoblastoma, with minimal off-target effects, suggesting that inhibitors of HDAC may specifically inhibit the proliferation of retinoblastoma tumor cells and, thus, have lower systemic toxicities relative to chemotherapeutic agents.89 106 Cancer Control Gene Therapy Suicide gene therapy is based on the introduction of a viral or bacterial gene into tumor cells, thus allowing the conversion of a nontoxic compound into a lethal drug to kill the tumor cells. In a phase 1 study, intravitreal injections of an adenovirus vector containing HSVtk followed by treatment with ganciclovir were shown to be safe and effective against vitreous seeds.90 Although these results were promising, it may be unlikely that this type of gene therapy could be used as first-line treatment for retinoblastoma but rather its use may be better suited as an adjunct to standard therapy for the treatment of refractory vitreous seeds.90 Local Drug-Delivery Systems Nanotechnology-based drug-delivery systems for the treatment of cancer have significantly evolved during the past decade by enabling options for site-specific delivery and increased bioavailability.91 Investigations have been conducted on the potential use of different materials as intraocular drug carriers, such as biodegradable polyesters, dendrimers, liposomes, mesoporous silica, and gold nanoparticles.91 These particles can be modified to target specific cells, and they can be designed to ensure sustained drug release to increase the therapeutic effectiveness of the drug molecules they contain.92 Nanoparticle-based systems designed for the treatment of retinoblastoma have improved rates of drug delivery to the posterior segment of the eye and have increased the intravitreal half-life of chemotherapy agents, thus highlighting their potential in treatment of this cancer.93 The intravitreal delivery of doxorubicin-loaded, polyester-based microspheres in the ocular tissue from rabbits exhibited reduced rates of toxicity to surrounding normal structures.77 Dendrimer nanoparticles containing carboplatin significantly reduced tumor volume compared with free carboplatin in a retinoblastoma mouse model.94 Polylactic glycolic acid nanoparticles developed to deliver doxorubicin95 and etoposide96 were tested on Y79 retinoblastoma cell lines and may be potential candidates for sustained drug-release models. Gold-based nanoparticles have unique physical properties, including a strong ability to absorb near-infrared light, which enables the photothermal destruction of cancer cells.97 Light-activated drug release can be attained using gold nanoparticles conjugated with chemotherapeutic agents.98 For example, gold nanocages are encapsulated with a smart polymer, wherein the nanocages absorb light converted to heat, thus causing the collapse of the polymer and the subsequent release of doxorubicin.99 The light-responsiveness of gold and other photosensitive nanocarriers infers considerable potential for ophthalmic conditions such as retinoblastoma because of the regular use of lasers in the treatment of retinal disease April 2016, Vol. 23, No. 2 and the added benefit of facilitating the controlled timing and location of delivery of therapeutic agents. Gold nanoparticles can also cross the blood–retina barrier without significant rates of cytotoxicity.100 The intravitreal injection of gold-tethered liposomes and viral-like nanoparticles containing topotecan is being investigated in a rabbit model of retinoblastoma that develops vitreous seeds.55,101 Another injectable agent being studied as a delivery system of chemotherapeutic agents for the treatment of retinoblastoma is the biodegradable carrier fibrin glue.102 Carboplatin103 and topotecan69 released from fibrin depots have both been shown to retain their bioactivity against retinoblastoma cells in culture. Carboplatin released from a fibrin depot was also shown to decrease tumor burden in a transgenic murine model of retinoblastoma cells.104 In another report, fibrin sealants sustained delivery of carboplatin in ocular tissues compared with carboplatin in plain solution, which clears rapidly in vivo.105 Promising results have been reported for topotecan combined with fibrin in clinical studies.71,106 roidal and retinal concentrations targeted toward the elimination of subretinal seeds and the prevention of choroidal invasion, with minimal biodistribution and toxicity to surrounding tissue. Our laboratory is investigating the suprachoroidal and intravitreal injection of topotecan using microneedles in a rabbit model of retinoblastoma (Fig 2).5 Conclusions Management of retinoblastoma is individualized according to patient characteristics and preferences, with the main goals being to save the patient’s life and to preserve useful vision, if possible. Therapeutic options for retinoblastoma have rapidly evolved in recent years, with a paradigm shift in standard treatment protocols toward the targeted delivery of chemotherapeutic agents. Increased use of intra-arterial and intravitreal types of chemotherapy with focal consolidation, along with the reduced use of systemic chemotherapy, enucleation, and external beam radiotherapy, has contributed to improved rates of survival and globe salvage while also minimizing unwanted treatment-related adverse events. However, these approaches are not without complications, and a need still exists to weigh the risks and benefits of treatment and to evaluate their long-term effects. New therapies being investigated include novel carriers and routes for local drug delivery as well as molecularly targeted therapies. Our under- Suprachoroidal Injection Suprachoroidal injection with microneedles is a route of targeted drug delivery to the choroid and retina that poses minimal risk of extraocular tumoral spread, because the vitreous and subretinal spaces are not entered. In a rabbit model, bevacizumab was determined to be A B safe and effective when injected into the suprachoroidal space; high concentrations were found in the posterior segment tissues.107 Microneedles are 0.8- to 1.0-mm long, 32-gauge needles with a short bevel that penetrate through the sclera into the supraciliary space. When the microneedle injects compounds into the supraciliary space, these C D compounds posteriorly spread into the suprachoroidal space.108 In phase 1 clinical trials, microneedles for suprachoroidal injections have been used to inject triamcinolone into the suprachoroidal space in patients with uveitis and macular edema following retinal venous occlusion (NCT02303184, NCT01789320). The suprachoroidal delivery of Fig 2A–D. — Intravitreal injection of topotecan in a rabbit model of retinoblastoma. (A) Vitreous seeds (asterisks) form in this retinoblastoma rabbit model. (B) A 32-gauge needle is inserted into the chemotherapeutic agents is a pars plana. (C) The needle (arrows) is inserted into its hub and topotecan is injected. (D) Vitreous promising approach for appliseeds have disappeared after 3 weekly injections of 20 µg topotecan. cations in retinoblastoma, and, Reprinted from Grossniklaus HE. Retinoblastoma. Fifty years of progress. The LXXI Edward Jackson ideally, will result in high choMemorial Lecture. Am J Ophthalmol. 2014;158(5):875-891, with permission from Elsevier. April 2016, Vol. 23, No. 2 Cancer Control 107 standing of tumor biology and how to develop safe and effective therapy continues to progress, thus helping to ensure that retinoblastoma will be a curable childhood cancer worldwide in the future. References 1. Broaddus E, Topham A, Singh AD. Incidence of retinoblastoma in the USA: 1975-2004. Br J Ophthalmol. 2009;93(1):21-23. 2. Parkin DM, Stiller CA, Draper GJ, et al. The international incidence of childhood cancer. Int J Cancer. 1988;42(4):511-520. 3. Tamboli D, Topham A, Singh N, et al. Retinoblastoma: a SEER dataset evaluation for treatment patterns, survival, and second malignant neoplasms. Am J Ophthalmol. 2015;160(5):953-958. 4. Broaddus E, Topham A, Singh AD. Survival with retinoblastoma in the USA: 1975-2004. Br J Ophthalmol. 2009;93(1):24-27. 5. Grossniklaus HE. Retinoblastoma. Fifty years of progress. The LXXI Edward Jackson Memorial Lecture. Am J Ophthalmol. 2014;158(5):875-891. 6. Canturk S, Qaddoumi I, Khetan V et al. Survival of retinoblastoma in less-developed countries impact of socioeconomic and health-related indicators. Br J Ophthalmol. 2010;94(11):1432-1436. 7. Abramson DH, Shields CL, Munier FL, et al. Treatment of retinoblastoma in 2015: agreement and disagreement. JAMA Ophthalmol. 2015;133(11):1341-1347. 8. Mendoza PR, Grossniklaus HE. The biology of retinoblastoma. Prog Mol Biol Transl Sci. 2015;134:503-516. 9.Pritchard EM, Dyer MA, Guy RK. Progress in small molecule therapeutics for the treatment of retinoblastoma. Mini Rev Med Chem. 2016;16(6):430-454. 10. Chévez-Barrios P, Hurwitz MY, Louie K et al. Metastatic and nonmetastatic models of retinoblastoma. Am J Pathol. 2000;157(4):1405-1412. 11. Shields CL, Lally SE, Leahey AM, et al. Targeted retinoblastoma management: when to use intravenous, intra-arterial, periocular, and intravitreal chemotherapy. Curr Opin Ophthalmol. 2014;25(5):374-385. 12. Shields CL, Shields JA. Retinoblastoma management: advances in enucleation, intravenous chemoreduction, and intra-arterial chemotherapy. Curr Opin Ophthalmol. 2010;21(3):203-212. 13. Linn Murphree A. Intraocular retinoblastoma: the case for a new group classification. Ophthalmol Clin North Am. 2005;18(1):41-53. 14. Shields CL, Mashayekhi A, Au AK, et al. The International classification of retinoblastoma predicts chemoreduction success. Ophthalmology. 2006;113(12):2276-2280. 15. Edge S, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual. 7th ed. New York: Springer; 2010. 16. Kaliki S, Shields CL, Rojanaporn D, et al. High-risk retinoblastoma based on international classification of retinoblastoma: analysis of 519 enucleated eyes. Ophthalmology. 2013;120(5):997-1003. 17. Ramasubramanian A, Kytasty C, Meadows AT, et al. Incidence of pineal gland cyst and pineoblastoma in children with retinoblastoma during the chemoreduction era. Am J Ophthalmol. 2013;156(4):825-829. 18. Rodriguez-Galindo C, Chantada GL, Haik BG, et al. Treatment of retinoblastoma: current status and future perspectives. Curr Treat Options Neurol. 2007;9(4):294-307. 19. Kingston JE, Hungerford JL, Madreperla SA, et al. Results of combined chemotherapy and radiotherapy for advanced intraocular retinoblastoma. Arch Ophthalmol. 1996;114(11):1339-1343. 20. Shields CL, Honavar SG, Meadows AT, et al. Chemoreduction plus focal therapy for retinoblastoma: factors predictive of need for treatment with external beam radiotherapy or enucleation. Am J Ophthalmol. 2002;133(5):657-664. 21. Sullivan EM, Wilson MW, Billups CA, et al. Pathologic risk-based adjuvant chemotherapy for unilateral retinoblastoma following enucleation. J Pediatr Hematol Oncol. 2014;36(6):e335-340. 22. Chantada GL, Dunkel IJ, de Davila MT, et al. Retinoblastoma patients with high risk ocular pathological features: who needs adjuvant therapy? Br J Ophthalmol. 2004;88(8):1069-1073. 23. Short BG. Safety evaluation of ocular drug delivery formulations: techniques and practical considerations. Toxicol Pathol. 2008;36(1):49-62. 24. Hwang CK, Aaberg TM Jr, Chevez-Barrios P, et al. Residual intraretinal retinoblastoma after chemoreduction failure. Arch Ophthalmol. 2012;130(2):246-248. 25. Demirci H, Eagle RC Jr, Shields CL, et al. Histopathologic findings in eyes with retinoblastoma treated only with chemoreduction. Arch Ophthalmol. 2003;121(8):1125-1131. 26. Inomata M, Kaneko A. Chemosensitivity profiles of primary and cultured human retinoblastoma cells in a human tumor clonogenic assay. Jpn J Cancer Res. 1987;78(8):858-868. 27. Yamane T, Kaneko A, Mohri M. The technique of ophthalmic arterial infusion therapy for patients with intraocular retinoblastoma. Int J Clin Oncol. 2004;9(2):69-73. 28. Suzuki S, Yamane T, Mohri M, et al. Selective ophthalmic arterial 108 Cancer Control injection therapy for intraocular retinoblastoma: the long-term prognosis. Ophthalmology. 2011;118(10):2081-2087. 29. Abramson DH, Dunkel IJ, Brodie SE, et al. A phase I/II study of direct intraarterial (ophthalmic artery) chemotherapy with melphalan for intraocular retinoblastoma initial results. Ophthalmology. 2008;115(8):1398-1404. 30. Shields CL, Manjandavida FP, Lally SE, et al. Intra-arterial chemotherapy for retinoblastoma in 70 eyes: outcomes based on the international classification of retinoblastoma. Ophthalmology. 2014;121(7):1453-1460. 31. Abramson DH, Daniels AB, Marr BP, et al. Intra-arterial chemotherapy (ophthalmic artery chemosurgery) for group D retinoblastoma. PLoS One. 2016;11(1):e0146582. 32. Shields CL, Kaliki S, Shah SU, et al. Effect of intraarterial chemotherapy on retinoblastoma-induced retinal detachment. Retina. 2012;32(4):799-804. 33. Palioura S, Gobin YP, Brodie SE, et al. Ophthalmic artery chemosurgery for the management of retinoblastoma in eyes with extensive (>50%) retinal detachment. Pediatr Blood Cancer. 2012;59(5):859-864. 34. Gobin YP, Dunkel IJ, Marr BP, et al. Combined, sequential intravenous and intra-arterial chemotherapy (bridge chemotherapy) for young infants with retinoblastoma. PLoS One. 2012;7(9):e44322. 35. Tse BC, Steinle JJ, Johnson D, et al. Superselective intraophthalmic artery chemotherapy in a nonhuman primate model: histopathologic findings. JAMA Ophthalmol. 2013;131(7):903-911. 36. Steinle JJ, Zhang Q, Thompson KE, et al. Intra-ophthalmic artery chemotherapy triggers vascular toxicity through endothelial cell inflammation and leukostasis. Invest Ophthalmol Vis Sci. 2012;53(4):2439-2445. 37. Vajzovic LM, Murray TG, Aziz-Sultan MA, et al. Supraselective intra-arterial chemotherapy: evaluation of treatment-related complications in advanced retinoblastoma. Clin Ophthalmol. 2011;5:171-176. 38. Jabbour P, Chalouhi N, Tjoumakaris S, et al. Pearls and pitfalls of intraarterial chemotherapy for retinoblastoma. J Neurosurg Pediatr. 2012;10(3):175-181. 39. Aziz HA, Lasenna CE, Vigoda M, et al. Retinoblastoma treatment burden and economic cost: impact of age at diagnosis and selection of primary therapy. Clin Ophthalmol. 2012;6:1601-1606. 40. Zanaty M, Barros G, Chalouhi N. Update on intra-arterial chemotherapy for retinoblastoma. Sci World J. 2014;2014:869604. 41. Zhao J, Dimaras H, Massey C, et al. Pre-enucleation chemotherapy for eyes severely affected by retinoblastoma masks risk of tumor extension and increases death from metastasis. J Clin Oncol. 2011;29(7):845-851. 42. Munier FL, Soliman S, Moulin AP, et al. Profiling safety of intravitreal injections for retinoblastoma using an anti-reflux procedure and sterilisation of the needle track. Br J Ophthalmol. 2012;96(8):1084-1087. 43. Ghassemi F, Shields CL, Ghadimi H, et al. Combined intravitreal melphalan and topotecan for refractory or recurrent vitreous seeding from retinoblastoma. JAMA Ophthalmol. 2014;132(8):936-941. 44.Ghassemi F, Shields CL. Intravitreal melphalan for refractory or recurrent vitreous seeding from retinoblastoma. Arch Ophthalmol. 2012;130(10):1268-1271. 45. Kaneko A, Suzuki S. Eye-preservation treatment of retinoblastoma with vitreous seeding. Jpn J Clin Oncol. 2003;33(12):601-607. 46. Smith SJ, Smith BD. Evaluating the risk of extraocular tumour spread following intravitreal injection therapy for retinoblastoma: a systematic review. Br J Ophthalmol. 2013;97(10):1231-1236. 47. Munier FL, Gaillard MC, Balmer A, et al. Intravitreal chemotherapy for vitreous seeding in retinoblastoma: Recent advances and perspectives. Saudi J Ophthalmol. 2013;27(3):147-150. 48. Seregard S, Singh AD. Retinoblastoma: direct chemotherapeutic drug delivery into the vitreous cavity. Br J Ophthalmol. 2012;96(4):473-474. 49. Suzuki S, Aihara Y, Fujiwara M, et al. Intravitreal injection of melphalan for intraocular retinoblastoma. Jpn J Ophthalmol. 2015;59(3):164-172. 50. Francis JH, Schaiquevich P, Buitrago E, et al. Local and systemic toxicity of intravitreal melphalan for vitreous seeding in retinoblastoma: a preclinical and clinical study. Ophthalmology. 2014;121(9):1810-1817. 51. Geroski DH, Edelhauser HF. Transscleral drug delivery for posterior segment disease. Adv Drug Deliv Rev. 2001;52(1):37-48. 52. Hayden BC, Jockovich ME, Murray TG, et al. Pharmacokinetics of systemic versus focal Carboplatin chemotherapy in the rabbit eye: possible implication in the treatment of retinoblastoma. Invest Ophthalmol Vis Sci. 2004;45(10):3644-3649. 53. Abramson DH, Frank CM, Dunkel IJ. A phase I/II study of subconjunctival carboplatin for intraocular retinoblastoma. Ophthalmology. 1999;106(10):1947-1950. 54. Mulvihill A, Budning A, Jay V, et al. Ocular motility changes after subtenon carboplatin chemotherapy for retinoblastoma. Arch Ophthalmol. 2003;121(8):1120-1124. 55. Schmack I, Hubbard GB, Kang SJ, et al. Ischemic necrosis and atrophy of the optic nerve after periocular carboplatin injection for intraocular retinoblastoma. Am J Ophthalmol. 2006;142(2):310-315. 56. Shields CL, Mashayekhi A, Cater J, et al. Macular retinoblastoma managed with chemoreduction: analysis of tumor control with or without adjuvant thermotherapy in 68 tumors. Arch Ophthalmol. 2005;123(6):765-773. 57. Schiedler V, Dubovy SR, Murray TG. Snare technique for enucleation of eyes with advanced retinoblastoma. Arch Ophthalmol. 2007;125(5):680-683. April 2016, Vol. 23, No. 2 58. Sastre X, Chantada GL, Doz F, et al. Proceedings of the consensus meetings from the International Retinoblastoma Staging Working Group on the pathology guidelines for the examination of enucleated eyes and evaluation of prognostic risk factors in retinoblastoma. Arch Pathol Lab Med. 2009;133(8):1199-1202. 59. Honavar SG, Singh AD, Shields CL, et al. Postenucleation adjuvant therapy in high-risk retinoblastoma. Arch Ophthalmol. 2002;120(7):923-931. 60. Imhof SM, Mourits MP, Hofman P, et al. Quantification of orbital and mid-facial growth retardation after megavoltage external beam irradiation in children with retinoblastoma. Ophthalmology. 1996;103(2):263-268. 61.Wong FL, Boice JD Jr, Abramson DH, et al. Cancer incidence after retinoblastoma. Radiation dose and sarcoma risk. JAMA. 1997;278(15):1262-1267. 62. Marees T, Moll AC, Imhof SM, et al. Risk of second malignancies in survivors of retinoblastoma: more than 40 years of follow-up. J Natl Cancer Inst. 2008;100(24):1771-1779. 63. Wong JR, Morton LM, Tucker MA, et al. Risk of subsequent malignant neoplasms in long-term hereditary retinoblastoma survivors after chemotherapy and radiotherapy. J Clin Oncol. 2014;32(29):3284-3290. 64. Ali MJ, Honavar SG, Reddy VA. Orbital retinoblastoma: present status and future challenges - a review. Saudi J Ophthalmol. 2011;25(2):159-167. 65. Truong B, Green AL, Friedrich P, et al. Ethnic, racial, and socioeconomic disparities in retinoblastoma. JAMA Pediatr. 2015;169(12):1096-1104. 66. Radhakrishnan V, Kashyap S, Pushker N, et al. Outcome, pathologic findings, and compliance in orbital retinoblastoma (International Retinoblastoma Staging System stage III) treated with neoadjuvant chemotherapy: a prospective study. Ophthalmology. 2012;119(7):1470-1477. 67. Dunkel IJ, Chan HS, Jubran R, et al. High-dose chemotherapy with autologous hematopoietic stem cell rescue for stage 4B retinoblastoma. Pediatr Blood Cancer. 2010;55(1):149-152. 68. Children’s Oncology Group website. ARET0321: phase III study for children diagnosed with extra-ocular retinoblastoma. https://childrensoncologygroup.org/index.php/aret0321. Accessed April 4, 2016. 69. Tsui JY, Dalgard C, Van Quill KR, et al. Subconjunctival topotecan in fibrin sealant in the treatment of transgenic murine retinoblastoma. Invest Ophthalmol Vis Sci. 2008;49(2):490-496. 70. Mallipatna AC, Dimaras H, Chan HS, et al. Periocular topotecan for intraocular retinoblastoma. Arch Ophthalmol. 2011;129(6):738-745. 71. Chantada GL, Fandino AC, Casak SJ, et al. Activity of topotecan in retinoblastoma. Ophthalmic Genet. 2004;25(1):37-43. 72. Francis JH, Gobin YP, Dunkel IJ, et al. Carboplatin +/- topotecan ophthalmic artery chemosurgery for intraocular retinoblastoma. PLoS One. 2013;8(8):e72441. 73. Buitrago E, Del Sole MJ, Torbidoni A, et al. Ocular and systemic toxicity of intravitreal topotecan in rabbits for potential treatment of retinoblastoma. Exp Eye Res. 2013;108:103-109. 74. Buitrago E, Hocht C, Chantada G, et al. Pharmacokinetic analysis of topotecan after intra-vitreal injection. Implications for retinoblastoma treatment. Exp Eye Res. 2010;91(1):9-14. 75. Jijelava K, Kompella U, Durazo S, et al. Evaluation of the efficacy of topotecan Au-tethered liposomes and AU-011 for the treatment of retinoblastoma in vitro. Invest Ophthamol Visual Sci. 2014. http://iovs.arvojournals.org/article.aspx?articleid=2268458. Accessed March 29, 2016. 76. Chantada GL, Fandino A, Mato G, et al. Phase II window of idarubicin in children with extraocular retinoblastoma. J Clin Oncol. 1999;17(6):1847-1850. 77. Hu T, Le Q, Wu Z, et al. Determination of doxorubicin in rabbit ocular tissues and pharmacokinetics after intravitreal injection of a single dose of doxorubicin-loaded poly-beta-hydroxybutyrate microspheres. J Pharm Biomed Anal. 2007;43(1):263-269. 78. Friend SH, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986;323(6089):643-646. 79. Corson TW, Gallie BL. One hit, two hits, three hits, more? Genomic changes in the development of retinoblastoma. Genes Chromosomes Cancer. 2007;46(7):617-634. 80. Kapatai G, Brundler MA, Jenkinson H, et al. Gene expression profiling identifies different sub-types of retinoblastoma. Br J Cancer. 2013;109(2):512-525. 81. Sachdeva UM, O’Brien JM. Understanding pRb: toward the necessary development of targeted treatments for retinoblastoma. J Clin Invest. 2012;122(2):425-434. 82. Laurie NA, Donovan SL, Shih CS, et al. Inactivation of the p53 pathway in retinoblastoma. Nature. 2006;444(7115):61-66. 83.Brennan RC, Federico S, Bradley C, et al. Targeting the p53 pathway in retinoblastoma with subconjunctival Nutlin-3a. Cancer Res. 2011;71(12):4205-4213. 84. Zhang J, Benavente CA, McEvoy J, et al. A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature. 2012;481(7381):329-334. 85. Gobessi S, Laurenti L, Longo PG, et al. Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells. Leukemia. 2009;23(4):686-697. 86. Azmi AS, Wang Z, Philip PA, et al. Emerging Bcl-2 inhibitors for the treatment of cancer. Expert Opin Emerg Drugs. 2011;16(1):59-70. 87. Falkenberg KJ, Johnstone RW. Histone deacetylases and their inApril 2016, Vol. 23, No. 2 hibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov. 2014;13(9):673-691. 88. Zhao Y, Tan J, Zhuang L, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim. Proc Natl Acad Sci U S A. 2005;102(44):16090-10695. 89. Dalgard CL, Van Quill KR, O’Brien JM. Evaluation of the in vitro and in vivo antitumor activity of histone deacetylase inhibitors for the therapy of retinoblastoma. Clin Cancer Res. 2008;14(10):3113-3123. 90. Chevez-Barrios P, Chintagumpala M, Mieler W, et al. Response of retinoblastoma with vitreous tumor seeding to adenovirus-mediated delivery of thymidine kinase followed by ganciclovir. J Clin Oncol. 2005;23(31):7927-7935. 91. Bertrand N, Wu J, Xu X,et al. Cancer nanotechnology: the impact of passive and active targeting in the era of modern cancer biology. Adv Drug Deliv Rev. 2014;66:2-25. 92. Salmaso S, Caliceti P. Stealth properties to improve therapeutic efficacy of drug nanocarriers. J Drug Deliv. 2013;2013:374252. 93. Bhavsar D, Subramanian K, Sethuraman S, et al. Management of retinoblastoma: opportunities and challenges. Drug Deliv. 2015:1-9. 94. Kang SJ, Durairaj C, Kompella UB, et al. Subconjunctival nanoparticle carboplatin in the treatment of murine retinoblastoma. Arch Ophthalmol. 2009;127(8):1043-1047. 95. Boddu SH, Jwala J, Chowdhury MR, et al. In vitro evaluation of a targeted and sustained release system for retinoblastoma cells using doxorubicin as a model drug. J Ocul Pharmacol Ther. 2010;26(5):459-468. 96. Mitra M, Dilnawaz F, Misra R, et al. Toxicogenomics of nanoparticulate delivery of etoposide: potential impact on nanotechnology in retinoblastoma therapy. Cancer Nanotechnol. 2011;2(1-6):21-36. 97. Choi WI, Sahu A, Kim YH, et al. Photothermal cancer therapy and imaging based on gold nanorods. Ann Biomed Eng. 2012;40(2):534-546. 98. Menon JU, Jadeja P, Tambe P, et al. Nanomaterials for photo-based diagnostic and therapeutic applications. Theranostics. 2013;3(3):152-166. 99. Yavuz MS, Cheng Y, Chen J, et al. Gold nanocages covered by smart polymers for controlled release with near-infrared light. Nat Mater. 2009;8(12):935-939. 100.Kim JH, Kim JH, Kim KW, et al. Intravenously administered gold nanoparticles pass through the blood-retinal barrier depending on the particle size, and induce no retinal toxicity. Nanotechnology. 2009;20(50):505101. 101. Kang SJ, Grossniklaus HE. Rabbit model of retinoblastoma. J Biomed Biotechnol. 2011;2011:394730. 102. Martin NE, Kim JW, Abramson DH. Fibrin sealant for retinoblastoma: where are we? J Ocul Pharmacol Ther. 2008;24(5):433-438. 103. Gorodetsky R, Peylan-Ramu N, Reshef A, et al. Interactions of carboplatin with fibrin(ogen), implications for local slow release chemotherapy. J Control Release. 2005;102(1):235-245. 104. Van Quill KR, Dioguardi PK, Tong CT, et al. Subconjunctival carboplatin in fibrin sealant in the treatment of transgenic murine retinoblastoma. Ophthalmology. 2005;112(6):1151-1158. 105.Simpson AE, Gilbert JA, Rudnick DE, et al. Transscleral diffusion of carboplatin: an in vitro and in vivo study. Arch Ophthalmol. 2002;120(8):1069-1074. 106. Yousef YA, Halliday W, Chan HS, et al. No ocular motility complications after subtenon topotecan with fibrin sealant for retinoblastoma. Can J Ophthalmol. 2013;48(6):524-528. 107. Patel SR, Berezovsky DE, McCarey BE, et al. Targeted administration into the suprachoroidal space using a microneedle for drug delivery to the posterior segment of the eye. Invest Ophthalmol Vis Sci. 2012;53(8):4433-4441. 108. Kim YC, Oh KH, Edelhauser HF, et al. Formulation to target delivery to the ciliary body and choroid via the suprachoroidal space of the eye using microneedles. Eur J Pharm Biopharm. 2015;95(part B):398-406. Cancer Control 109 Treatment regimens for primary vitreoretinal lymphoma continue to evolve. Photo courtesy of Lynn E. Harman, MD. Manhattan. Primary Vitreoretinal Lymphoma: Management of Isolated Ocular Disease Matthew T. Witmer MD Background: The prognosis for patients with primary vitreoretinal is dismal. The close association of primary vitreoretinal lymphoma with primary central nervous system lymphoma is responsible for high rates of mortality. Traditional treatments consist of systemic chemotherapy and whole-brain radiotherapy. The optimal approach for the treatment of isolated primary vitreoretinal lymphoma is unclear. Methods: A review of the relevant medical and scientific literature was performed, focusing on the clinical features of primary vitreoretinal lymphoma and the progress made in the management of isolated ocular disease. Results: Ocular treatment options for primary vitreoretinal lymphoma have recently expanded with the addition of intravitreal chemotherapeutic agents and localized radiation. Based on several retrospective reports, a general shift has been made toward local therapy (eg, orbital radiotherapy, intravitreal chemotherapy) for ocular disease. No prospective, randomized clinical trials yet exist to guide therapy. Conclusions: Optimal treatment regimens for isolated primary vitreoretinal lymphoma continue to evolve. Further investigations into novel therapies and protocols are needed to decrease recurrence rates, reduce or prevent central nervous system involvement, and improve rates of overall survival. Introduction Primary vitreoretinal lymphoma (formerly known as primary intraocular lymphoma or reticulum cell sarcoma) continues to have a poor prognosis.1,2 Between 65% and 90% of patients with primary vitreoretinal lymphoma will develop primary central nervous system (CNS) lymphoma, a disease that ultimately results in a high rate of mortality.1,2 Several different treatment modalities can be used for these patients, each with varying degrees of success. Due to the rarity of From the Retina Centers of Washington, Rockville, Maryland. Address correspondence to Matthew T. Witmer, MD, 15020 Shady Grove Road, Suite 302, Rockville, MD 20850. E-mail: [email protected] Submitted July 13, 2015; accepted November 27, 2015. No significant relationship exists between the author and the companies/organizations whose products or services may be referenced in this article. 110 Cancer Control primary vitreoretinal lymphoma, no prospective, randomized controlled trials exist to guide therapeutic decisions. Consequently, no consensus exists regarding the optimal treatment regimen. Pathogenesis Primary vitreoretinal lymphoma is most often (> 95% of the time) diffuse large B-cell lymphoma, which is a type of high-grade malignancy.3 The malignant cells are typically found in the vitreous, retina, or optic nerve head; however, the anatomical site of origin in many cases is unknown, as spillover into multiple compartments of the eye can occur early. Some have suggested a solitary, systemic, primary source of the malignant cells followed by intraocular metastases,4 whereas others have suggested that the malignancy has multiple, independent sites of origin (eg, eye, CNS) that can occasionally develop simultaneously.5 Infiltration of malignant lymphoApril 2016, Vol. 23, No. 2 cytes into the eye or brain from the systemic circulation may be due to the ability of a malignant clone of cells, which expresses specific cell-surface molecules, to selectively home in to CNS tissue.6 Signaling molecules, such as chemokines, may direct these cells to the CNS.7 This theory is supported by evidence of tumor clones related to primary CNS lymphoma found in the blood and bone marrow of affected patients.8 The CNS may provide an attractive microenvironment for these cells to colonize. Close association exists between primary vitreoretinal lymphoma and primary CNS lymphoma. Approximately 20% of patients with primary CNS lymphoma develop lymphoma in the eye, and as many as 90% of patients with primary vitreoretinal lymphoma develop CNS disease.6 The similarities between primary vitreoretinal lymphoma and primary CNS lymphoma include a unique affinity for CNS tissue (whereby this rarely appears outside the CNS), a worse prognosis than most extranodal types of non-Hodgkin lymphoma (NHL), and a high response rate to methotrexate.6 Historically, the treatment strategies for the 2 malignancies overlapped due to these similarities and the frequency with which they clinically coexisted. Although they are pathologically similar in appearance, evidence suggests that primary vitreoretinal lymphoma differs from systemic diffuse large B-cell lymphoma in its origin from mature B cells due to differing frequency of expressed immunoglobulins.9 epithelium (RPE) lesions, or a combination of both. The sub-RPE lesions may appear as large RPE detachments with classic, creamy, or yellow colors. Others have described punctate or spiculated sub-RPE deposits, also referred to as a leopard-spot pattern of pigmentation.2 In more advanced cases, malignant cells can seed the anterior chamber, where they can settle in the angle or accumulate on the surface of the iris or lens. Less often, the tumor infiltrates the optic nerve head, where it presents as a mass lesion or results in ocular ischemia by interfering with the posterior circulation of the eye. Diagnosis Diagnosis can be difficult owing to its insidious onset, tendency to simulate other conditions, and its relative rarity.6,10 It is estimated that approximately 380 cases are diagnosed in the United States each year.6 The median age of diagnosis is 60 years and immunosuppression increases the risk of developing the condition.11,12 Delay in diagnosis, often confounded with prior diagnoses and treatments for uveitis, is common. In a large series, the median time to diagnosis was 6 months.10 However, delays in diagnosis as long as 24 months are not unusual.13,14 The diagnosis of primary vitreoretinal lymphoma is usually based on a cytological examination of a vitreous specimen on biopsy, supplemented with findings from immunohistochemistry, flow cytometry, gene rearrangement analysis, and assays for key cytokines. Vitreous Biopsy Definitive diagnosis of primary vitreoretinal lymphoma relies on vitreous biopsy. The decision to perform vitreous biopsy is often made after treatment for chronic progressive uveitis has failed. An anterior chamber tap for levels of interleukin (IL) 10 has been proposed to screen patients for vitreous biopsy.16 However, this approach is controversial because the performance profile of the laboratory study (eg, positive and negative predictive probabilities, rates of sensitivity and specificity) is unclear.16 The findings of the vitreous samples of patients with primary vitreoretinal lymphoma often show evidence of apoptosis, cellular necrosis, scavenging macrophages, and reactive inflammatory cells.17 It is typically recommended that corticosteroids be withheld prior to vitreous biopsy because they may alter cellular morphology.18 Clinical Examination Primary vitreoretinal lymphoma may present as a collection of nonclumped vitreous cells visible upon slit lamp examination. The vitreous gel may appear hazy due to dispersed vitreous cells. When primary vitreoretinal lymphoma affects the retina, it can present with grey- to cream-colored retinal infiltrates (often found along retinal arterioles), sub-retinal pigment Cytology Although cytology (or histology) remains the gold standard of care, diagnostic sensitivity is not absolute due to sampling phenomenon, problems with cellular preservation, and difficulties with interpretation.19 Frequently, specimens are obtained with too few cells or cells that are poorly preserved. Under ideal circumstances, malignant B cells appear unambiguously ab- April 2016, Vol. 23, No. 2 Ocular Imaging Findings on autofluorescent imaging will vary according to the location of the lesions. If lymphoma cells accumulate under the RPE, then the RPE may have hyperfluoresecence. However, if the retinal deposits are anterior to the RPE, then they appear to have hypofluorescence due to blocking. Spectral-domain optical coherence tomography can be used to demonstrate an intraretinal tumor, which may extend through the full-thickness retina, toward the RPE. Multiple, punctate, sub-RPE deposits have also been described.15 Tumor deposits on fluorescein angiography will typically have hypofluorescence on early and late frames. However, leakage may be present from retinal veins and staining of retinal arterioles.15 Cancer Control 111 normal with large, irregular nuclei, scanty cytoplasm, and prominent nucleoli (Figs 1 and 2).4,6 Retinal, biopsy, choroidal biopsy, or both are less commonly performed than vitreous biopsy because this procedure has a higher risk of ocular and visual morbidity. Intraocular Cytokine Assay B-cell primary vitreoretinal lymphoma typically presents with elevated IL-10 levels in the eye.16 Lymphoma cells are thought to produce this cytokine, which acts as a growth factor. This is in contrast to IL-6, which is more commonly produced by inflammatory cells and is characteristic of uveitis. Vitreous IL-10 levels, in combination with the IL-10:IL-6 ratio, have been used as a diagnostic test for lymphoma, although they do not have a 100% sensitivity rate. 20 Additional studies have verified the high accuracy rate of cytokine profiling, but they have also confirmed that negative testing does not exclude the disease.19,21 Fig 1. — Finding on vitreous biopsy of primary vitreoretinal lymphoma. The tissue was concentrated and sectioned from a cell block. The section stained with hematoxylin and eosin shows degenerating, moderately large lymphocytes. Many cells are necrotic. Nuclei vary in size and shape, and their nuclear chromatin is hyperchromatic. Photo courtesy of Curtis Margo, MD. Immunohistochemistry With samples of adequate size, immunohistochemistry studies for cluster designation20 and κ and λ light chain restriction can confirm B-cell monoclonality. However, demonstration of surface light chain on lymphocytes fixed in formalin can be technically challenging. Flow Cytometry Flow cytometry is well suited for the detection of cell-surface markers if the sample is sufficiently cellular. The ratio of κ:λ light chains may be useful to confirm a monoclonal expansion of lymphocytes. Polymerase Chain Reaction Monoclonal gene rearrangements in IGH (B-cell lymphoma) or TRG (T-cell lymphoma) are strongly inter-related with malignancy.21,22 A technique known as microdissection can be employed to obtain abnormal cells for analysis.21 Treatment No currently agreed upon therapeutic strategy exists for primary vitreoretinal lymphoma without CNS involvement (ie, isolated eye disease). Because no large comparative clinical series, or randomized, masked, clinical trials exist, the optimal treatment of primary vitreoretinal lymphoma relies on clinical judgment. Treatment regimens include local therapy, systemic therapy, or a combination of both. However, it is unclear whether any of the available treatments of isolated ocular primary vitreoretinal lymphoma increase rates of overall survival.23 Local therapy consists of intravitreal chemotherapy and ocular radiotherapy. Systemic therapy includes intravenous chemotherapy, 112 Cancer Control Fig 2. — Magnified image of a histological specimen from an eye with primary vitreoretinal lymphoma. The arrow denotes Bruch membrane, anterior to which malignant cells have invaded the retina. Photo courtesy of Curtis Margo, MD. intrathecal chemotherapy, whole-brain radiotherapy, and peripheral blood stem cell transplantation. Despite available treatments, which may produce adequate initial responses, recurrence is common, often involving the CNS.10 The goals of therapy include eradication of local disease with prevention of local and CNS recurrences as well as minimizing adverse events. Primary Central Nervous System Lymphoma Many of the current treatment strategies for primary vitreoretinal lymphoma are based on established treatments of primary CNS lymphoma due to their high coincidence and presumed shared pathogenesis. Several of the current treatments for primary vitreoretinal lymApril 2016, Vol. 23, No. 2 phoma first demonstrated their effectiveness in the treatment of primary CNS lymphoma. Based on data from older studies in which the option of solely supportive care was more common, untreated primary CNS lymphoma was found to have an extremely poor prognosis (average survival rate < 3 months).12,24 The survival rate from primary CNS lymphoma is significantly worse in patients with AIDS.12 Initial success in the treatment of primary CNS lymphoma has been achieved through whole brain irradiation, but disease invariably recurs; the median survival rate is approximately 12 months and the local recurrence rate is nearly 90%.25 In 1 report, the 2-year survival rate was 28%.25 Treatment of NHL metastatic to the brain with high-dose methotrexate alone was introduced in 1977.26 The subsequent use of high-dose methotrexate alone in patients with primary CNS lymphoma produced a response rate between 38% and 74% and a median overall survival rate of about 2 years.27-29 Through the use of multiple chemotherapeutic agents, including methotrexate, response rates for primary CNS lymphoma have increased to 71%, with a median overall survival rate of 50 months.30 The success of these therapies in primary CNS lymphoma led to their application in primary vitreoretinal lymphoma. with primary CNS lymphoma or primary vitreoretinal lymphoma found a 100% response rate when patients were treated with intravenous methotrexate, vincristine, and thiotepa and intrathecal methotrexate and arabinofuranosyl cytidine in 21-day cycles without radiation.34 However, recurrence rates were high: 66% of cases recurred within 4.5 years.34 Experience with systemic chemotherapy and ASCT is limited.35 One trial demonstrated longer rates of progression-free survival and overall survival in patients with relapsed or refractory primary CNS lymphoma administered ASCT (in combination with high-dose chemotherapy) compared with those patients who did not receive it.36 Ten of the 43 study patients (23%) had intraocular involvement.36 Several challenges have arisen due to the direct application of these systemic treatments for primary CNS lymphoma to the management of primary vitreoretinal lymphoma. One in particular is the blood–ocular barrier, which limits access of systemically administered medication to the eye. Another is the vitreous: It is normally avascular and will limit chemical diffusion of specific agents depending on their physical properties. The vitreous is a prime tissue that harbors lymphoma. When given systemically, high-dose methotrexate may achieve tumoricidal levels in the anterior chamber for up to 74 hours but at inadequate levels in the vitreous fluid.37,38 Systemic Chemotherapy Systemic therapy for primary vitreoretinal lymphoma without CNS involvement can include intravenous and intrathecal chemotherapy and whole-brain radiotherapy. The preferred strategy varies by clinical center.2 Systemic chemotherapy regimens for primary vitreoretinal lymphoma have included a single agent, multiple agents, and chemotherapeutic agents plus autologous stem cell transplantation (ASCT). Prior single-agent treatment regimens have included high-dose arabinofuranosyl cytidine, high-dose methotrexate, ifosfamide, and trofosfamide with varying degrees of success.31-33 Prior studies with intravenous high-dose methotrexate for the treatment of primary CNS lymphoma with concurrent primary vitreoretinal lymphoma achieved response rates that approached 100% in the brain, despite lower response rates of 78% in the eye.29,32 One trial with either ifosfamide or trofosfamide found a 100% response rate with a mean progression-free survival rate of 18 months and a median overall survival rate of 32 months.33 Comparing the efficacy of systemic chemotherapy alone for primary vitreoretinal lymphoma is confounded by the fact that many earlier studies included initial or simultaneous treatment with radiation; intravenous, intrathecal, and intravitreal chemotherapy; ASCT; or all therapies combined. One series of 14 study patients Local Ocular Disease Local therapy for primary vitreoretinal lymphoma has included intravitreal chemotherapy and orbital radiotherapy. Often times, local treatment of ocular disease is initiated because health care professionals may be hesitant to treat a patient systemically unless evidence of CNS disease is present.2 Ocular lymphoma cells are highly sensitive to radiation.39 However, this treatment has not significantly increased the overall survival rate of these patients.40-42 Local response rates are generally high, but recurrence is common. When the tumor reappears, additional radiation is considered hazardous due to the cumulative effects of the radiation on the retina and optic nerve. The complications of orbital radiation include dry eyes, cataract, radiation retinopathy, and optic neuropathy. Although cataracts are surgically treatable, radiation injury to the retina and optic nerve is often permanent. Radiation to the orbits usually involves 35 to 40 Gy delivered in 14 to 15 separate doses, including both eyes in the treatment field. Experience with intravitreal chemotherapy for primary vitreoretinal lymphoma has included methotrexate and rituximab. Intravitreal methotrexate for intraocular lymphoma is typically administered in a 400-µg dose in 0.1 uL, every 2 weeks as induction therapy, followed by consolidation and maintenance therapy.5,43,44 April 2016, Vol. 23, No. 2 Cancer Control 113 When administered intravitreally, this dose maintains therapeutic levels in the eye for at least 5 days.45 The adverse events of intravitreal methotrexate include epithelial keratopathy, cataract, retinopathy, and hypotony (ie, low intraocular pressure). Treated patients may also be at risk of tachyphylaxis with recurrent injections.6,43-46 Epithelial keratopathy, which has been reported in up to 58% of eyes,46 may be reduced by paracentesis before the injection.47 No double-masked, randomized clinical trials exist for use of intravitreal methotrexate. The largest reported series consists of 44 eyes.5 In that series, remission was achieved after a mean of 6.4 injections (Table).5,43-46,48-51 The major shortcoming of intravitreal therapy is that it fails to prevent CNS relapse or disease in the contralateral eye. Intravitreal rituximab has demonstrated efficacy and has the ability to penetrate full-thickness retina.52 It is typically administered on a weekly basis as 1 mg in a 0.1-mL dose.48 Experience with the medication has revealed a high response rate (≤ 100%), but it also has a high recurrence rate when treatment is discontinued (≤ 55% within 3 months).49 Recurrences typically occur within 3 months following treatment discontinuation.49 Additional courses of rituximab may cause disease remission; however, the effectiveness of the drug may diminish.49 Grossly visible retinal lesions may resolve with rituximab. Rituximab has been reported to cause clinical remission in some patients unresponsive to methotrexate.49 Adverse events of rituximab include vitritis, elevated intraocular pressure (60%), and iridocyclitis, with mutton-fat keratic precipitates (35%).49 Despite resolution of ocular lesions with rituximab, 69% of patients in a single trial developed CNS lymphoma.49 Rituximab has a half-life of about 5 days in the eye and exerts tumoricidal effects for 3 to 4 weeks.53,54 Combination intravitreal methotrexate/rituximab has also been used with some benefit and may potentially decrease the adverse events seen with single-agent therapy.48 A collaborative study of patients with primary vitreoretinal lymphoma without clinical or radiographic evidence of CNS involvement from 16 centers (including 9 study patients [11%] with lymphoma in the spinal fluid) found that local therapy did not increase the risk of brain relapse compared with systemic therapy.10 This retrospective series of 83 immunocompetent study patients with primary vitreoretinal lymphoma from 7 countries evaluated 16 different treatment regimens (3 local and 16 extensive).10 The median progression-free survival rate was 29.6 months and the overall median survival rate was 58 months; this rate was not impacted by the therapeutic regimen.10 Relapse rates were high (56%); the median time to relapse was 19 months.10 The site of relapse (eg, eyes, brain, other sites) was also not affected by the treatment regimen, with most (92%) patients experiencing relapse in the brain or eyes.10 Another collaborative review lasting 20 years and covering 78 study patients with primary vitreoretinal lymphoma without signs of primary CNS lymphoma from 17 centers found that use of systemic chemotherapy was not associated with a reduced rate of CNS lym- Table. — Local Therapy With Intravitreal Chemotherapy Study Intravitreal Treatment No. of Cases of Primary Vitreoretinal Lymphoma No. of Cases of Primary Lymphoma of the CNS No. of Eyes de Smet45 Methotrexate/thiotepa 1 1 Fishburne 43 Methotrexate 4 2 Frenkel5 Methotrexate 26 Hashida49 Rituximab Itty48 Kitzmann 50 Ohguro 51 Sen 44 Smith46 Ocular Response, % (n) No. of Injections Until Remission No. of Eye Relapses 1 100 (1) 6 (3rd week) 0 18 7 100 (7) 6.7 (mean) 0 13.1 (mean) 13 44 100 (40) with all data 6.4 (mean) 0 21 (median) 13 0 20 100 (20) 4 (mean) 11/20 (55%) 24.7 (mean) Rituximab 1 0 2 100 (2) 6 (mean) 2/2 (100%) 11 Rituximab 3 3 5 100 (5) 2 (mean) 0 3.6 (median) Rituximab 2 1 3 100 (3) 1.67 (mean) 0 2 (mean) Methotrexate 1 1 1 100 (1), then tachyphylaxis 3, followed by tachyphylaxis 1 (100%) 48 Methotrexate 16 16 26 100 (22) 100 (4) after pars plana vitrectomy 8.5 (median) 6/26 (23%) 18.5 (median) Follow-Up, mo CNS = central nervous system. 114 Cancer Control April 2016, Vol. 23, No. 2 phoma, but it was associated with more severe adverse events compared with those seen with local treatment (ocular radiotherapy, ocular chemotherapy, or both).55 Combination Therapy Combination therapy consists of local and systemic therapy. Some institutions recommend systemic treatment with high-dose methotrexate plus adjuvant local radiotherapy,56,57 whereas other centers prefer intravitreal chemotherapy with methotrexate or rituximab rather than radiotherapy.5,48 One retrospective series of 221 immunocompetent patients with primary CNS lymphoma and primary vitreoretinal lymphoma found that those who received dedicated ocular therapy in combination with systemic treatment had increased progression-free survival rates compared with those not receiving dedicated ocular therapy.58 However, no difference was seen in overall survival rates, and the study did not contain a control group and involved a variety of treatments.58 Those who did not receive dedicated ocular therapy were not at increased risk of developing ocular recurrence.58 Recommendations No universally agreed-upon therapeutic strategy exists for primary vitreoretinal lymphoma without CNS involvement, although a common (but not standard) approach involves systemic therapy for patients with CNS disease and local therapy for those with primary vitreoretinal lymphoma confined to the eye. Recommendations from the International Primary Central Nervous System Lymphoma Collaborative Group and the British Neuro-Oncology Society differ.6,18 For the treatment of ocular-only primary vitreoretinal lymphoma, the Primary Central Nervous System Lymphoma Collaborative Group recommends local therapy for unilateral disease and local therapy, systemic chemotherapy, or both for those with bilateral disease.6 Orbital radiotherapy may be the preferred local therapy in patients with bilateral disease, whereas intravitreal chemotherapy with or without orbital radiotherapy is preferred for unilateral disease.6 In patients with a prior history of radiotherapy, intravitreal chemotherapy is recommended.6 By contrast, the British Neuro-Oncology Society recommends systemic chemotherapy with high-dose methotrexate and ocular radiotherapy to both globes for isolated primary vitreoretinal lymphoma.18 The British Neuro-Oncology Society recognized that intravitreal methotrexate was an effective treatment option for isolated recurrences.18 Conclusions The search for the optimal treatment for primary vitreoretinal lymphoma without central nervous system involvement presents a daunting challenge. At the crux April 2016, Vol. 23, No. 2 of the dilemma is whether local treatment will be effective given the high risk of central nervous system disease and whether early systemic treatment has the potential to improve survival. The differential treatment response of primary vitreoretinal lymphoma may be related to different molecular genetic subtypes of the malignancy, which is a hypothesis that requires further study.3 Lack of a superior treatment strategy despite a decade of collective experience suggests that any major impact on reducing relapse rates in central nervous system and improving outcomes awaits future innovative therapeutic approaches. References 1. Mochizuki M, Singh AD. Epidemiology and clinical features of intraocular lymphoma. Ocul Immunol Inflamm. 2009;17(2):69-72. 2. Davis JL. Intraocular lymphoma: a clinical perspective. Eye (Lond). 2013;27(2):153-162. 3.Coupland SE. Molecular pathology of lymphoma. Eye (Lond). 2013;27(2):180-189. 4. Coupland SE, Damato B. Understanding intraocular lymphomas. Clin Experiment Ophthalmol. 2008;36(6):564-578. 5. Frenkel S, Hendler K, Siegal T, et al. Intravitreal methotrexate for treating vitreoretinal lymphoma: 10 years of experience. Br J Ophthalmol. 2008;92(3):383-388. 6. Chan CC, Rubenstein JL, Coupland SE, et al. Primary vitreoretinal lymphoma: a report from an International Primary Central Nervous System Lymphoma Collaborative Group symposium. Oncologist. 2011;16(11):1589-1599. 7. Falkenhagen KM, Braziel RM, Fraunfelder FW, et al. B-Cells in ocular adnexal lymphoproliferative lesions express B-cell attracting chemokine 1 (CXCL13). Am J Ophthalmol. 2005;140(2):335-337. 8. McCann KJ, Ashton-Key M, Smith K, et al. Primary central nervous system lymphoma: tumor-related clones exist in the blood and bone marrow with evidence for separate development. Blood. 2009;113(19):4677-4680. 9. Coupland SE, Loddenkemper C, Smith JR, et al. Expression of immunoglobulin transcription factors in primary intraocular lymphoma and primary central nervous system lymphoma. Invest Ophthalmol Vis Sci. 2005;46(11):3957-3964. 10. Grimm SA, Pulido JS, Jahnke K, et al. Primary intraocular lymphoma: an International Primary Central Nervous System Lymphoma Collaborative Group Report. Ann Oncol. 2007;18(11):1851-1855. 11. Behin A, Hoang-Xuan K, Carpentier AF, et al. Primary brain tumours in adults. Lancet. 2003;361(9354):323-331. 12. Coté TR, Manns A, Hardy CR, et al; AIDS/Cancer Study Group. Epidemiology of brain lymphoma among people with or without acquired immunodeficiency syndrome. J Natl Cancer Inst. 1996;88(10):675-679. 13. Hoffman PM, McKelvie P, Hall AJ, et al. Intraocular lymphoma: a series of 14 patients with clinicopathological features and treatment outcomes. Eye (Lond). 2003;17(4):513-521. 14. Jahnke K, Korfel A, Komm J, et al. Intraocular lymphoma 2000– 2005: results of a retrospective multicentre trial. Graefes Arch Clin Exp Ophthalmol. 2006;244(6):663-669. 15. Fardeau C, Lee CP, Merle-Béral H, et al. Retinal fluorescein, indocyanine green angiography, and optic coherence tomography in non-Hodgkin primary intraocular lymphoma. Am J Ophthalmol. 2009;147(5):886894, 894.e881. 16. Cassoux N, Giron A, Bodaghi B, et al. IL-10 measurement in aqueous humor for screening patients with suspicion of primary intraocular lymphoma. Invest Ophthalmol Vis Sci. 2007;48(7):3253-3259. 17. Coupland SE. Vitreous biopsy: specimen preparation and interpretation. Monogr Clin Cytol. 2012;21:61-71. 18. British Neuro-Oncology Society; National Cancer Action Team. Rare Brain and CNS Tumours Guidelines: Guidelines on the Diagnosis and Management of Adult Pineal Area Tumours. Published June 2011. http://www.bnos.org.uk/wp-content/uploads/2015/08/Guidelines-on-the -Diagnosis-and-Management.pdf. Accessed April 6, 2016. 19. Kimura K, Usui Y, Goto H; Group JILS. Clinical features and diagnostic significance of the intraocular fluid of 217 patients with intraocular lymphoma. Jpn J Ophthalmol. 2012;56(4):383-389. 20. Sugita S, Takase H, Sugamoto Y, et al. Diagnosis of intraocular lymphoma by polymerase chain reaction analysis and cytokine profiling of the vitreous fluid. Jpn J Ophthalmol. 2009;53(3):209-214. 21. Wang Y, Shen D, Wang VM, et al. Molecular biomarkers for the diagnosis of primary vitreoretinal lymphoma. Int J Mol Sci. 2011;12(9):5684-5697. 22. Shen DF, Zhuang Z, LeHoang P, et al. Utility of microdissection and polymerase chain reaction for the detection of immunoglobulin gene rearCancer Control 115 rangement and translocation in primary intraocular lymphoma. Ophthalmology. 1998;105(9):1664-1669. 23. Hormigo A, Abrey L, Heinemann MH, et al. Ocular presentation of primary central nervous system lymphoma: diagnosis and treatment. Br J Haematol. 2004;126(2):202-208. 24. Jellinger K, Radaskiewicz TH, Slowik F. Primary malignant lymphomas of the central nervous system in man. Acta Neuropathol Suppl. 1975;(suppl 6):95-102. 25. Nelson DF, Martz KL, Bonner H, et al. Non-Hodgkin’s lymphoma of the brain: can high dose, large volume radiation therapy improve survival? Report on a prospective trial by the Radiation Therapy Oncology Group (RTOG): RTOG 8315. Int J Radiat Oncol Biol Phys. 1992;23(1):9-17. 26. Skarin AT, Zuckerman KS, Pitman SW, et al. High-dose methotrexate with folinic acid in the treatment of advanced non-Hodgkin lymphoma including CNS involvement. Blood. 1977;50(6):1039-1047. 27. Herrlinger U, Schabet M, Brugger W, et al. German Cancer Society Neuro-Oncology Working Group NOA-03 multicenter trial of single-agent high-dose methotrexate for primary central nervous system lymphoma. Ann Neurol. 2002;51(2):247-252. 28. Herrlinger U, Küker W, Uhl M, et al. NOA-03 trial of high-dose methotrexate in primary central nervous system lymphoma: final report. Ann Neurol. 2005;57(6):843-847. 29. Batchelor T, Carson K, O’Neill A, et al. Treatment of primary CNS lymphoma with methotrexate and deferred radiotherapy: a report of NABTT 96-07. J Clin Oncol. 2003;21(6):1044-1049. 30. Pels H, Schmidt-Wolf IG, Glasmacher A, et al. Primary central nervous system lymphoma: results of a pilot and phase II study of systemic and intraventricular chemotherapy with deferred radiotherapy. J Clin Oncol. 2003;21(24):4489-4495. 31. Baumann MA, Ritch PS, Hande KR, et al. Treatment of intraocular lymphoma with high-dose Ara-C. Cancer. 1986;57(7):1273-1275. 32. Batchelor TT, Kolak G, Ciordia R, et al. High-dose methotrexate for intraocular lymphoma. Clin Cancer Res. 2003;9(2):711-715. 33. Jahnke K, Thiel E, Bechrakis NE, et al. Ifosfamide or trofosfamide in patients with intraocular lymphoma. J Neurooncol. 2009;93(2):213-217. 34. Sandor V, Stark-Vancs V, Pearson D, et al. Phase II trial of chemotherapy alone for primary CNS and intraocular lymphoma. J Clin Oncol. 1998;16(9):3000-3006. 35. Soussain C, Merle-Béral H, Reux I, et al. A single-center study of 11 patients with intraocular lymphoma treated with conventional chemotherapy followed by high-dose chemotherapy and autologous bone marrow transplantation in 5 cases. Leuk Lymphoma. 1996;23(3-4):339-345. 36. Soussain C, Hoang-Xuan K, Taillandier L, et al. Intensive chemotherapy followed by hematopoietic stem-cell rescue for refractory and recurrent primary CNS and intraocular lymphoma: Société Française de Greffe de Moëlle Osseuse-Thérapie Cellulaire. J Clin Oncol. 2008;26(15):2512-2518. 37. de Smet MD, Stark-Vancs V, Kohler DR, et al. Intraocular levels of methotrexate after intravenous administration. Am J Ophthalmol. 1996;121(4):442-444. 38. Henson JW, Yang J, Batchelor T. Intraocular methotrexate level after high-dose intravenous infusion. J Clin Oncol. 1999;17(4):1329. 39. Margolis L, Fraser R, Lichter A, et al. The role of radiation therapy in the management of ocular reticulum cell sarcoma. Cancer. 1980;45(4):688-692. 40. Fine HA, Mayer RJ. Primary central nervous system lymphoma. Ann Intern Med. 1993;119(11):1093-1104. 41. Ferreri AJ, Blay JY, Reni M, et al. Relevance of intraocular involvement in the management of primary central nervous system lymphomas. Ann Oncol. 2002;13(4):531-538. 42. Schlegel U, Schmidt-Wolf IG, Deckert M. Primary CNS lymphoma: clinical presentation, pathological classification, molecular pathogenesis and treatment. J Neurol Sci. 2000;181(1-2):1-12. 43. Fishburne BC, Wilson DJ, Rosenbaum JT, et al. Intravitreal methotrexate as an adjunctive treatment of intraocular lymphoma. Arch Ophthalmol. 1997;115(9):1152-1156. 44. Sen HN, Chan CC, Byrnes G, et al. Intravitreal methotrexate resistance in a patient with primary intraocular lymphoma. Ocul Immunol Inflamm. 2008;16(1):29-33. 45. de Smet MD, Vancs VS, Kohler D, et al. Intravitreal chemotherapy for the treatment of recurrent intraocular lymphoma. Br J Ophthalmol. 1999;83(4):448-451. 46. Smith JR, Rosenbaum JT, Wilson DJ, et al. Role of intravitreal methotrexate in the management of primary central nervous system lymphoma with ocular involvement. Ophthalmology. 2002;109(9):1709-1716. 47. Hardwig PW, Pulido JS, Bakri SJ. The safety of intraocular methotrexate in silicone-filled eyes. Retina. 2008;28(8):1082-1086. 48.Itty S, Pulido JS. Rituximab for intraocular lymphoma. Retina. 2009;29(2):129-132. 49. Hashida N, Ohguro N, Nishida K. Efficacy and complications of intravitreal rituximab injection for treating primary vitreoretinal lymphoma. Transl Vis Sci Technol. 2012;1(3):1. 50. Kitzmann AS, Pulido JS, Mohney BG, et al. Intraocular use of rituximab. Eye (Lond). 2007;21(12):1524-1527. 51. Ohguro N, Hashida N, Tano Y. Effect of intravitreous rituximab in116 Cancer Control jections in patients with recurrent ocular lesions associated with central nervous system lymphoma. Arch Ophthalmol. 2008;126(7):1002-1003. 52. Pulido JS, Bakri SJ, Valyi-Nagy T, et al. Rituximab penetrates fullthickness retina in contrast to tissue plasminogen activator control. Retina. 2007;27(8):1071-1073. 53. Kim H, Csaky KG, Chan CC, et al. The pharmacokinetics of rituximab following an intravitreal injection. Exp Eye Res. 2006;82(5) 760-766. 54. Chu LC, Eberhart CG, Grossman SA, et al. Epigenetic silencing of multiple genes in primary CNS lymphoma. Int J Cancer. 2006;119(10):2487-2491. 55. Riemens A, Bromberg J, Touitou V, et al. Treatment strategies in primary vitreoretinal lymphoma: a 17-center European collaborative study. JAMA Ophthalmol. 2015;133(2):191-197. 56. Berenbom A, Davila RM, Lin HS, et l. Treatment outcomes for primary intraocular lymphoma: implications for external beam radiotherapy. Eye (Lond). 2007;21(9):1198-1201. 57. Stefanovic A, Davis J, Murray T, et al. Treatment of isolated primary intraocular lymphoma with high-dose methotrexate-based chemotherapy and binocular radiation therapy: a single-institution experience. Br J Haematol. 2010;151(1):103-106. 58. Grimm SA, McCannel CA, Omuro AM, et al. Primary CNS lymphoma with intraocular involvement: International PCNSL Collaborative Group Report. Neurology. 2008;71(17):1355-13601. April 2016, Vol. 23, No. 2 Many patients with conjunctival melanoma experience suboptimal outcomes because of iatrogenic tumor seeding, inadequate local tumor control, and surgical morbidity. Photo courtesy of Lynn E. Harman, MD. Chicago River. Management of Primary Acquired Melanosis, Nevus, and Conjunctival Melanoma Andrew Kao, MD, Armin Afshar, MD, Michele Bloomer, MD, and Bertil Damato, MD, PhD Background: The management of conjunctival melanoma is difficult because of the rarity of the disease, confusing terminology, high rates of local tumor recurrence, controversies regarding treatment, a poor evidence base, unreliable prognostication, and significant mortality rates. Methods: The medical literature was reviewed, focusing on treatment and management options for conjunctival melanoma. Recent trends and developments were summarized with respect to terminology, local treatment, histology, genetic analysis, prognostication, and systemic treatment, highlighting the scope for research and possible improvements in patient care. Results: Histopathological diagnostic terminology for primary acquired melanosis is being superseded by more explicit terminology, thus differentiating hypermelanosis from conjunctival melanocytic intraepithelial neoplasia. Topical chemotherapy and increased use of adjunctive radiotherapy have helped improve rates of local tumor control. Use of exenteration has become rare. Regional and systemic metastases are common in patients with nonbulbar conjunctival melanoma, although long-term survivors with metastases are growing in number. Prognostication is mainly based on tumor size and location, but histological and genetic data into multivariate analyses will soon be incorporated. The role of sentinel lymph-node biopsy continues to be controversial. Chemotherapy for metastatic disease is being superseded by targeted therapy based on genetic abnormalities such as BRAF mutations. Conclusions: The management of conjunctival melanoma requires expert care from an experienced, multidisciplinary team. The goal of therapy is to provide good local tumor control with minimal morbidity, high-quality pathology, and adequate psychological support. Maximizing patient enrollment in multicenter clinical trials is likely to strengthen evidence-based decision-making. Introduction Conjunctival melanoma is a rare but potentially From the Department of Ophthalmology, University of California, San Francisco, California. Address correspondence to Bertil Damato, MD, PhD, Department of Ophthalmology, 8 Koret Way, San Francisco, CA 94143. E-mail: [email protected] Submitted August 15, 2015; accepted November 12, 2015. No significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. April 2016, Vol. 23, No. 2 sight- and life-threatening malignancy of the eye.1,2 It arises from melanocytes of the basal layer of the conjunctival epithelium and comprises approximately 2% to 7% of ocular melanomas.1-3 The incidence is approximately 0.24 to 0.8 cases per 1 million and is increasing.1,2 Risk factors for conjunctival melanoma are not well understood because of the rarity of the disease and lack of large population-based studies.1,2 The disease is most common in whites and rare in Asian/Pacific Islanders.2 Most studies have not shown any sex predilection.2 Conjunctival melanoma has a Cancer Control 117 strong association with primary acquired melanosis (PAM) and conjunctival nevi. Conjunctival melanoma tends to locally recur, seed to distant parts of the conjunctiva, and systemically metastasize to regional lymph nodes. Once metastatic disease has occurred, outcomes are often fatal. Classification of Conjunctival Pigmented Lesions Melanocytic lesions of the conjunctiva are generally classified as PAM, nevus, and melanoma (Fig 1). The term PAM tends to be both clinically and histologically used. Although this term is adequate for clinical findings, it is not ideal for histological diagnosis because it encompasses a wide range of conditions, from benign to malignant, that include complexion melanosis and PAM with and without atypia. Furthermore, PAM with atypia tends to be categorized as mild, moderate, or severe, and is often inconsistently used. To improve the rate of precision in the reporting of histological findings, Damato and Coupland4 recommend distinguishing between hypermelanosis and conjunctival melanocytic intraepithelial neoplasia. In their classification, hypermelanosis refers to the overproduction of melanin from a normal population of melanocytes, as occurs with complexion/racial melanosis.4 To make classification more consistent, Damato and Coupland4 have created a system for scoring the grade of malignancy from 0 to 10 according to pattern of melanocytic pro- liferation, extent of vertical spread, and degree of cellular atypia (Table).4 Conjunctival melanocytic intraepithelial neoplasia with a score of 0 would correspond with PAM without atypia and would indicate an overpopulation of melanocytes showing no cellular atypia and confined to the basal layer of the epithelium. Higher scores correspond to PAM with atypia, indicating cellular atypia of melanocytes with invasion into the more superficial layers of the epithelium but without breaching the basement membrane of the epithelium. No consensus exists as to how severe PAM/conjunctival melanocytic intraepithelial neoplasia with atypia should be classified prior to being labeled as melanoma in situ. Damato and Coupland4 suggest designating any atypia with a score exceeding 5 as in situ, which corresponds to confluent proliferation of atypical melanocytes involving more than 50% of the thickness of the epithelium. This designation might improve communication between the pathologist and clinician, resulting in fewer delays of treatment due to vagaries in classification. Multicenter studies are needed to determine whether this 10-point scoring system improves objectivity and clinical care compared with simply categorizing disease as mild, moderate, and severe. Conventionally, conjunctival melanoma implies invasion of the substantia propria, even if the word invasive is omitted. We believe that it would be safe practice to distinguish between melanoma in situ and A B C D E F Fig 1A–F. — Clinical features of pigmented conjunctival lesions. (A) Complexion melanosis appearing as diffuse conjunctival pigmentation in a pigmented individual. (B) Primary acquired melanosis manifesting as a discrete patch of conjunctival pigment. (C) Nevus shown as a slightly elevated pigmented lesion with intraepithelial cysts. (D) Invasive melanoma demonstrated as a pigmented nodule arising from a patch of conjunctival melanosis. (E) Invasive melanoma of the palpebral conjunctiva. (F) Invasive melanoma with locally disseminated disease, due to prior biopsy at an outside institution, without use of adjuvant therapy. 118 Cancer Control April 2016, Vol. 23, No. 2 Table. — Coupland–Damato Scoring System for Conjunctival Melanocytic Intraepithelial Neoplasia Feature Melanocytic pattern Vertical spread of melanocytes with atypia Cellular atypia Total Description Score No melanocytic neoplasia (± hypermelanosis) 0 Basal/lentiginous proliferation or 1 Pagetoid spread (± basal/lentiginous) or 2 Nonconfluent nests (± basal/lentiginous pagetoid) or 3 Confluent nests (± basal/lentiginous pagetoid) 4 Basal layer of epithelium 0 < 50% epithelial thickness 1 50%–90% epithelial thickness 2 Total replacement of epithelium 3 Nuclei < basal squamous cells 0 Nuclei ≥ basal squamous cells 1 Cytoplasm < basal squamous cells 0 Cytoplasm ≥ basal squamous cells 1 Nucleolus and mitoses absent 0 Nucleolus ± mitoses present 1 (Must be ≤ 10 points) Length of specimen invaded by most severe disease, % Changes extend to or near lateral resection edge(s) Yes/No Invasive melanoma (if yes, report elsewhere) Yes/No From Damato B, Coupland SE. Conjunctival melanoma and melanosis: a reappraisal of terminology, classification and staging. Clin Exp Ophthalmol. 2008;36(8):786-795. ed, or amelanotic; unifocal or multifocal; and with or without adjacent melanosis.2,6 Amelanotic melanomas can be white, yellow, pink, or red in color. Feeder vessels are usually present. Hemorrhage is rare. The tumor surface does not keratinize. Most tumors are located in the bulbar conjunctiva, usually involving the limbus.2 Less commonly, tumors may present on the palpebral or fornical conjunctiva, plica semilunaris, or caruncle.2 Regional lymph nodes may be enlarged if regional metastasis has occurred.7 Differential Diagnosis Primary acquired melanosis requires incisional biopsy to distinguish diffuse invasive melanoma and melanoma in situ from hypermelanosis and conjunctival melanocytic intraepithelial neoplasia without atypia (ie, PAM without atypia). Congenital ocular melanocytosis is slate-grey in color rather than brown and involves the sclera alone; the overlying conjunctiva is transparent. Conjunctival nevi tend to have multiple cysts and are not associated with feeder vessels, except when occurring in children. Squamous cell carcinoma is usually amelanotic but can be deeply pigmented, especially in dark-skinned individuals. It usually presents as frosty keratinization, which does not occur with melanoma. Typically, lymphoma has a salmon-pink color, is located in the plica and fornices, and is bilateral. Rare mimicking lesions can include pinguecula, pterygium, pyogenic granuloma, deposition of pigmented foreign material, extraocular spread of uveal melanoma, metastasis from cutaneous melanoma, staphyloma, subconjunctival hematoma, foreign body, and hematic cyst.8 Clinical Examination invasive melanoma when discussing pathology. In the presence of invasive melanoma, it can be difficult or impossible to histologically distinguish between primary conjunctival melanocytic intraepithelial neoplasia that caused invasive melanoma and pagetoid spread from the invasive tumor. Damato and Coupland4 suggest that PAM is limited to clinical findings and that histological reports should distinguish between hypermelanosis and melanocytic intraepithelial neoplasia, numerically scoring the degree of malignancy instead of using vague terms such as mild, moderate, and severe. Clinical Features Melanoma in situ presents as 1 or more brown patches developing anywhere in the conjunctiva, almost always unilaterally, and becoming more extensive over time.5 Melanocytic proliferation can become amelanotic, especially after unsuccessful treatment. Invasive conjunctival melanoma can be nodular, diffuse, or mixed; deeply pigmented, lightly pigmentApril 2016, Vol. 23, No. 2 Examination of the entire conjunctiva must include the superior fornix and superior palpebral regions, either after double eversion of the upper eyelid with a Desmarres retractor or by pinching and lifting the upper lid away from the bulbar conjunctiva while performing inspection with a binocular indirect ophthalmoscope and a 20-D lens. The preauricular, postauricular parotid, submandibular, and cervical lymph nodes are routinely palpated.7 Imaging Color drawings are valuable for defining tumor size and extent; Fig 2 can be used as a template for this purpose. In addition, if performed correctly, color photography with adequate retraction of the eyelids is useful for documenting surface features and tumor margins. Anterior segment ocular coherence tomography uses light waves to obtain cross-sectional images of various eye structures, with an axial resolution of up to 5 μm.9,10 PAM/conjunctival melanocytic intraepithelial Cancer Control 119 neoplasia without atypia appears as normal-thickness epithelium, with strong hyper-reflectivity of the basal epithelium, correlating with the basal layer of melanocytes seen on histopathology.9,10 Nevi are hyperreflective, well circumscribed, and often have visible cysts, with appearances varying according to whether the lesion is junctional, compound, or subepithelial.10 Invasive melanoma reveals subepithelial disease that casts a significant shadow (Fig 3).10 Ultrasonographic Fig 2. — Template for drawing conjunctival tumors that shows the entire conjunctival surface divided into regions (cornea, limbus, bulbar conjunctiva, fornix, plica, caruncle, palpebral conjunctiva, eyelid skin) and sectors. A B biomicroscopy using transducers between 50 and 100 MHz can measure the thickness of conjunctival melanomas with reasonable accuracy and may identify tumors greater than 2 mm in thickness so that sentinel lymph-node biopsy can be planned before tumor excision.11 High-frequency ultrasonography can be useful for detecting or excluding suspected underlying uveal melanoma. Diagnosis Histology Histology by a trained pathologist is required to differentiate between hypermelanosis, conjunctival melanocytic intraepithelial neoplasia, in situ melanoma, invasive melanoma, and other pathology. Bleaching of specimens is often required to examine cell morphology. Features of atypical melanocytes include nuclear pleomorphism, prominent nucleoli, increased eosinophilic cytoplasm, and mitotic figures. Hypermelanosis is characterized by increased melanin granules within normal melanocytes in the basal epithelium. Conjunctival melanocytic intraepithelial neoplasia with a score of 0 consists of an increased number of melanocytes without atypical features (Fig 4A). Conjunctival melanocytic intraepithelial neoplasia with higher scores is identified by intraepithelial growth of atypical melanocytes, extending up to full thickness, but with no invasion through to the epithelial basement membrane (Fig 4B). Invasive melanoma is defined by atypical melanocytes that have invaded the basement membrane into the substantia propria (Fig 4C). Histological assessment has been improved due to the development of immunohistochemistry. Markers for melanocytes include S100, human melanoma black 45, SRY-box containing gene 10, and Melan-A. A red chromogen is useful in the presence of a heavily pigmented lesion. C Fig 3A–C. — Ocular coherence tomographical features of conjunctival melanoma. (A) Slit lamp photograph of a raised, reddish, nonpigmented conjunctival lesion suspected to be squamous neoplasia. (B) Ultra high-resolution optical coherence tomography in the area of the black dotted arrow disclosed a subepithelial lesion (a) beneath a normal thickness epithelium (b). The lesion was casting a significant shadow (c) on the underlying tissue. Foci of epithelial cleavage (d) and moderate hyper-reflectivity of the epithelium were noted. (C) Histopathological examination confirmed the diagnosis of melanoma (hematoxylin and eosin stain, ×200). Reprinted from Shousha MA, Karp CL, Canto AP, et al. Diagnosis of ocular surface lesions using ultra-high-resolution optical coherence tomography. Ophthalmology. 2013;120(5):883-891. © May 2013, with permission from Elsevier. 120 Cancer Control April 2016, Vol. 23, No. 2 A B C Fig 4A–C. — Histology of pigmented conjunctival lesions (hematoxylin and eosin stain, ×400). (A) Conjunctival melanocytic intraepithelial neoplasia without atypia with an increased number of melanocytes within the basal epithelium without atypical features. (B) Melanoma in situ with atypical melanocytes and invasion of superficial layers of epithelium, but without progression through the epithelial basement membrane. (C) Invasive melanoma with highly atypical melanocytes that fill the epithelium, invading the underlying substantia propria. Biopsy Incisional biopsy of nodular melanoma can cause tumor seeding, so it should be performed for PAM alone; otherwise, excisional biopsy is preferred when possible. The specimen should be placed on a stiff card or filter paper to prevent scrolling so that oblique histological sections are avoided. Care must be taken to avoid crush artifact. Testing for BRAF mutations should be considered at this stage if concern exists for metastatic disease potentially treatable with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors. When a patient is referred to an oncology center after biopsy, we routinely arrange for our ophthalmic pathologist to review the histology results. geons not following the “no touch” technique or who may not understand the importance of using fresh instruments for closure to avoid seeding.12 After witnessing recurrences in distant parts of the conjunctiva in such cases, Damato and Coupland12 routinely administer adjunctive topical chemotherapy to all such patients irrespective of histological findings. Although data are insufficient for statistical evaluation of this approach, clinical impressions have been positive.2 Treatment Adjuvant Therapy Cryotherapy: Historically, cryotherapy was the only treatment for PAM/conjunctival melanocytic intraepithelial neoplasia with atypia; in modern times, its use is advocated by some as adjunctive therapy following local excision. It is delivered using the double or triple freeze-thaw technique.2 Jakobiec et al13 recommend using a liquid nitrogen probe and a subconjunctival thermocouple to ensure that a temperature of –20 °C is achieved; however, Damato and Coupland14 reported an unacceptable tumor recurrence rate in addition to complications such as uveitis, hypotony, macular edema, and squamous metaplasia of the conjunctiva. This treatment has largely been superseded by topical chemotherapy. We now perform cryotherapy for residual or recurrent intraepithelial disease alone after at least 2 courses of topical chemotherapy. Topical Chemotherapy: This type of treatment is administered using mitomycin C (MMC), because other agents such as fluorouracil are less effective. The standard protocol is to prescribe drops 4 times per day for 2 weeks, repeating the treatment after a 2-week rest period. We prefer a 1-week course of topical chemotherapy, repeated every 4 weeks for 4 cycles, because doing so causes less pain and inflammation as well Surgical Excision The conventional approach to treatment is to excise nodular conjunctival melanomas with wide safety margins and lamellar scleral dissection, to ensure deep and lateral clearance, and to administer adjunctive cryotherapy as a precaution.1 Such extensive surgery often requires amniotic membrane grafting, which can result in significant morbidity (eg, corneal scarring, scleral translucency). Furthermore, significant rates of local tumor recurrence have been reported, sometimes exceeding 30%.1,7,12 Growing circumstantial evidence suggests that such local recurrence is associated with higher rates of mortality.12 For at least 15 years, the approach was to excise the tumor with minimal surgical clearance, perform no lamellar scleral excision, and provide no adjunctive cryotherapy, instead administering adjunctive radiotherapy for all cases with invasive melanoma, irrespective of histological evidence of clearance.8 In addition, adjunctive topical chemotherapy is warranted if adjacent intraepithelial neoplasia is present, whether this consists of primary disease or secondary pagetoid spread.8 Many patients are referred to an oncology center after undergoing incisional biopsy or excision by surApril 2016, Vol. 23, No. 2 Primary Exenteration Primary exenteration has been abandoned as a primary treatment for PAM.5 Such extensive surgery is now performed for advanced invasive melanoma alone. Cancer Control 121 as improved patient compliance. Topical steroids are helpful in reducing the inflammation, and our preference is to use 0.1% fluorometholone because it does not penetrate the globe. No consensus exists regarding the ideal dose of MMC. We prescribe 0.03% MMC because we found that lower concentrations were associated with local treatment failure, whereas higher doses caused limbal stem cell failure in select patients. Some authors advocate punctual occlusion during topical chemotherapy2; however, we do not favor this practice, especially with patients who have tumor recurrence in the nasolacrimal system. When administering topical chemotherapy, the patient must be provided with disposable gloves, petroleum jelly to protect the eyelid skin, a disposal container (to be returned to the hospital with all soiled materials), and detailed oral and written instructions on the storage and administration of the MMC drops. Radiotherapy: Radiotherapy can consist of brachytherapy, which uses isotopes such as strontium, ruthenium, or iodine, or teletherapy, which uses photons or a proton beam (Fig 5). Adjunctive radiotherapy after local excision was reported by Collins15 in 1918 and later by Lederman16 in 1958 but it has since fell into disuse. Damato and Coupland12 reported high rates of local tumor control with adjunctive ruthenium plaque radiotherapy, delivering a dose of 100 Gy to a depth of 1 mm, which involves suturing the plaque over the target area and removing it after 1 day with topical anesthesia. They found this to be less labor intensive than strontium brachytherapy, which requires delivery for several days.12 Iodine plaques are more bulky, so they are more likely to be uncomfortable for the patient; however, radiotherapy can be tailored to each individual by adjusting the number and distribution of the iodine seeds. Such tailoring of the radiation dose is easier with proton beam radiotherapy, which does not require surgery but may be inconvenient for the patient because of increased travel time. Proton beam radiotherapy may be useful for lesions in the nonbulbar conjunctiva, which cannot easily be treated with brachytherapy.17 Damato18 has also achieved good re- A B sults with radiotherapy alone in select patients with unresectable tumors. Topical Interferon Immunotherapy: This type of treatment is a relatively recent addition to the therapeutic armamentarium. Although interferon α-2b is commonly used to treat ocular surface squamous neoplasia and conjunctival papillomas, data are limited on its use for conjunctival melanoma.19,20 No consensus exists regarding the most appropriate dosing regimen. Several case series have shown it to be effective for the treatment of conjunctival melanoma and conjunctival melanocytic intraepithelial neoplasia with good rates of tolerance by patients2; however, further studies are required to examine its efficacy and safety data in patients with pigmented conjunctival lesions. Outcomes Loss of Vision Loss of can occur as a result of keratitis following tumor excision, cryotherapy, or topical chemotherapy; hypotony and macular edema can occur following bulbar cryotherapy; and cataracts can occur following radiotherapy. Patient-Reported Outcomes Outcomes reported by patients such as grittiness, discharge, epiphora, and concerns about appearance are common, but, to our knowledge, they have not yet been analyzed in a published study. Local Recurrence The rate of local recurrence of PAM/conjunctival melanocytic intraepithelial neoplasia has been reported to be 27%.21 The reported rate of progression to invasive melanoma following treatment for PAM/conjunctival melanocytic intraepithelial neoplasia ranges from 13% to 46%.21,22 Recurrent invasive melanoma has been reported in 8% to 62% of study patients at 5 years and in approximately 38% to 52% of study patients at 10 years.1,7,12,23 Factors associated with lower rates of local recurrence include extent of initial disease, the “no touch” surgical technique, and adjunctive therapy (particularly radiotherapy).12 Rarely, tumor recurrence can intraocularly occur, particularly if the Bowman membrane has been surgically disrupted and if adjunctive radiotherapy has not been administered.24 Fig 5A–B. — Conjunctival melanoma (A) at presentation and (B) after excision with adjunctive proton beam radiotherapy and topical chemotherapy. 122 Cancer Control Secondary Exenteration Secondary exenteration was previously reported in 13% of study patients.25 It has now become rare due to improved rates of local tumor control.7,23,25,26 April 2016, Vol. 23, No. 2 Metastatic Disease Metastasis can be regional, systemic, or both. Regional metastases develop by lymphatic spread and involve the parotid, preauricular, postauricular, and submandibular nodes.7 Systemic metastases can occur by lymphatic or hematogenous spread. Approximately 20% to 30% of patients develop regional metastases; of these, 50% subsequently develop systemic metastatic disease.27,28 Conversely, up to 25% of patients develop systemic metastases alone without any clinical evidence of regional metastases.7 Treatment for metastatic disease is the same as for cutaneous melanoma and includes cytotoxic chemotherapy, immunotherapy, and molecularly targeted therapy.29 The 10-year mortality rate from conjunctiva melanoma ranges from 13% to 38%.7,12 Prognostication Ocular Predictors Ocular predictors of regional and systemic metastatic disease — hence mortality — include large basal tumor diameter, increased tumor thickness, and nonbulbar conjunctival involvement (particularly the caruncular tumor location).14 The tumor, node, and metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC)30 stages conjunctival melanomas as: T(is) (intraepithelial melanoma), T1 (bulbar), T2 (nonbulbar, including palpebral, forniceal, or caruncular), T3 (invasion into globe, eyelid, nasolacrimal system, orbit, or sinus), and T4 (invasion into brain). Two studies have validated the current AJCC staging criteria, and both have demonstrated that higher-staged tumors have a higher risk of local recurrence, local and distant metastasis, and death.30-32 The current staging system is currently being revised, for example, to describe the circumferential tumor extent in quadrants irrespective of whether the tumor crosses the horizontal or vertical meridians.4,33 Regional Nodal Involvement Regional nodal involvement is a known predictor of systemic metastasis and this has stimulated interest in sentinel lymph-node biopsy.27,28,34-38 It involves injecting radionuclide (technetium 99m sulfur colloid) into the subconjunctival space surrounding the tumor, followed by imaging of the afferent lymphatic system, and then excising any suspicious lymph nodes, intraoperatively identifying them using a handheld γ probe. The excised nodes are serially “bread loafed” in 1- to 2-mm slices and examined for malignant cells using immunohistochemistry. Sentinel lymph-node biopsy has been advocated in patients with tumors in a nonlimbal location, histological ulceration, and with a tumor thickness of at least 2 mm.34 This procedure is controversial because of its unproven value in predicting metastasis and prolonging survival.36 Furthermore, April 2016, Vol. 23, No. 2 lymph-node resection can result in complications, including scarring and facial nerve (VII) palsy. Histological Predictors Histological predictors of metastasis include tumor thickness exceeding 2 mm, surface ulceration, epithelioid cytomorphology, high mitotic count, and microsatellites, as well as vascular invasion or lymphatic invasion.7,39-43 Genetic Predictors Genetic predictors of metastasis have not been identified, although BRAF, CDKN1A, RUNX2, MLH1, TIMP2, MGMT, and ECHS1 have been reported.8 BRAF V600E mutation occurs in about 50% of cases of conjunctival melanomas.44 Approximately 50% of such melanomas respond to systemic BRAF inhibitors, so routine testing for this mutation occurs in some centers.45 Further Studies Diagnosis In theory, hope exists for the external validation and refinement of the Damato–Coupland scoring system for conjunctival melanocytic intraepithelial neoplasia4; however, in practice, the system is limited by the rarity of invasive melanoma in such cases, because most patients now receive treatment. High-resolution ocular coherence tomography is now available, so this imaging modality could be used to detect various grades of conjunctival melanocytic intraepithelial neoplasia, not only at presentation but also following topical chemotherapy.10 Such imaging could reduce the need for conjunctival biopsies. Treatment In view of the morbidity caused by topical MMC, we avoid such treatment in patients with conjunctival melanocytic intraepithelial neoplasia who have a score of 3 or below, instead preferring long-term surveillance for such cases. It would be useful to determine how many such patients develop more severe conjunctival melanocytic intraepithelial neoplasia, invasive melanoma, or both. With regard to invasive disease, future randomized trials should study extensive resection and cryotherapy compared with minimalist resection with radiotherapy and topical chemotherapy. Such studies should measure both patient-reported and clinical outcomes (ie, local tumor control, ocular conservation, vision, metastasis-free survival rates). Instruments for measuring patient-reported outcomes must be validated in the setting of conjunctival melanoma. Prognostication Survival estimates for patients with choroidal melanomas have been improved by mathematical methods that combine the AJCC staging system with histological Cancer Control 123 grade of malignancy and genetic tumor type.29,30,46 Similar prognostication methods should be applied to patients with conjunctival melanoma. Further evaluation of sentinel lymph-node biopsy is also warranted.27,34-38 The search for genetic predictors should continue so that different subgroups of conjunctival melanoma are identified, similar to that seen in uveal melanoma.47 It would be useful to understand why nonbulbar conjunctival melanomas are associated poor rates of survival — that is, whether this is because of genetic differences (possibly related to sunlight exposure) or stromal influences (eg, lymphatic density). Systemic Adjuvant Therapy In patients known to be at high risk for metastasis, it would be worth undertaking randomized trials of systemic chemotherapy or immunotherapy, similar to those in progress for cutaneous melanoma. Because of the rarity of conjunctival melanoma, it may be possible to enroll patients with conjunctival melanoma in such trials, subsequently performing subgroup analyses. Treatment for Metastatic Disease Similar to those of systemic adjuvant therapy, clinical trials evaluating treatment for clinical metastases are limited by the rarity of conjunctival melanomas, so investigators should explore the feasibility of enrolling these patients in trials studying cutaneous melanoma. Future Directions Many patients with conjunctival melanoma experience suboptimal outcomes because of iatrogenic tumor seeding, inadequate local tumor control, and surgical morbidity. The published evidence base is poor, so a need exists for clinical trials and basic science research. Because of the rarity of conjunctival melanoma, multicenter studies are needed. Such collaboration is becoming easier with the development in electronic medical records, automatic extraction of data, and the collaboration of ocular oncology working groups and organizations. Progress should also be encouraged by the formation of patient advocacy groups, which can help raise awareness of the importance of holistic care, emphasizing the need for doctor–patient communication, adequate patient education, informed consent, effective emotional support, and, if necessary, referral to psychology. The greatest improvement in patient care will occur when ocular oncologists are supported by a skilled multidisciplinary team and when biopsy or treatment at nonspecialty centers is no longer considered acceptable. References 1. Lim LA, Madigan MC, Conway RM. Conjunctival melanoma: a review of conceptual and treatment advances. Clin Ophthalmol. 2013;6:521-531. 2. Wong JR, Nanji AA, Galor A, et al. Management of conjunctival malignant melanoma: a review and update. Expert Rev Ophthalmol. 2014;9(3):185-204. 124 Cancer Control 3. Damato BE, Coupland SE. Ocular melanoma. Saudi J Ophthalmol. 2012;26(2):137-144. 4. Damato B, Coupland SE. Conjunctival melanoma and melanosis: a reappraisal of terminology, classification and staging. Clin Exp Ophthalmol. 2008;36(8):786-795. 5. Reese AB. Precancerous melanosis and malignant melanoma of the conjunctiva and skin of the lids. Trans Am Ophthalmol Soc. 1942;40:224-232. 6. Shields CL, Shields JA. Conjunctival primary acquired melanosis and melanoma: tales, fairy tales, and facts. Ophthal Plast Reconstr Surg. 2009;25(3):167-172. 7. Shildkrot Y, Wilson MW. Conjunctival melanoma: pitfalls and dilemmas in management. Curr Opin Ophthalmol. 2010;21(5):380-386. 8. Kenawy N, Lake SL, Coupland SE, et al. Conjunctival melanoma and melanocytic intra-epithelial neoplasia. Eye (Lond). 2013;27(2):142-152. 9. Nanji AA, Sayyad FE, Galor A, et al. High-resolution optical coherence tomography as an adjunctive tool in the diagnosis of corneal and conjunctival pathology. Ocul Surf. 2015;13(3):226-235. 10. Shousha MA, Karp CL, Canto AP, et al. Diagnosis of ocular surface lesions using ultra-high-resolution optical coherence tomography. Ophthalmology. 2013;120(5):883-891. 11. Ho VH, Prager TC, Diwan H, et al. Ultrasound biomicroscopy for estimation of tumor thickness for conjunctival melanoma. J Clin Ultrasound. 2007;35(9):533-537. 12. Damato B, Coupland SE. An audit of conjunctival melanoma treatment in Liverpool. Eye (Lond). 2009;23(4):801-809. 13. Jakobiec FA, Brownstein S, Wilkinson RD, et al. Combined surgery and cryotherapy for diffuse malignant melanoma of the conjunctiva. Arch Ophthalmol. 1980;98(8):1390-1396. 14. Damato B, Coupland SE. Management of conjunctival melanoma. Expert Rev Anticancer Ther. 2009;9(9):1227-1239. 15. Collins ET. Case of epibulbar sarcoma cured by removal and the application of radium. T Ophthal Soc UK. 1918;38;125-127. 16. Lederman M. Radiotherapy of cancerous and precancerous melanosis. T Ophthal Soc UK. 1958:LXXVIII;147-164. 17. Wuestemeyer H, Sauerwein W, Meller D, et al. Proton radiotherapy as an alternative to exenteration in the management of extended conjunctival melanoma. Graefes Arch Clin Exp Ophthalmol. 2006;244(4):438-446. 18. Damato B. Melanotic conjunctival lesions. In: Damato B. Ocular Tumours: Diagnosis and Treatment. Oxford, UK: Butterworth-Heinemann; 2000:21-29. 19. Finger PT, Sedeek RW, Chin KJ. Topical interferon alfa in the treatment of conjunctival melanoma and primary acquired melanosis complex. Am J Ophthalmol. 2008;145(1):124-129. 20.Herold TR, Hintschich C. Interferon alpha for the treatment of melanocytic conjunctival lesions. Graefes Arch Clin Exp Ophthalmol. 2010;248(1):111-115. 21. Shields JA, Shields CL, Mashayekhi A, et al. Primary acquired melanosis of the conjunctiva: experience with 311 eyes. Trans Am Ophthalmol Soc. 2007;105:61-72. 22. Folberg R, McLean IW, Zimmerman LE. Conjunctival melanosis and melanoma. Ophthalmology. 1984;91(6):673-678. 23. Shields CL, Shields JA, Gündüz K, et al. Conjunctival melanoma: risk factors for recurrence, exenteration, metastasis, and death in 150 consecutive patients. Arch Ophthalmol. 2000;118(11):1497-1507. 24. Sandinha T, Russell H, Kemp E, et al. Malignant melanoma of the conjunctiva with intraocular extension: a clinicopathological study of three cases. Graefes Arch Clin Exp Ophthalmol. 2007;245(3):431-436. 25. Shields JA, Shields CL, Gündüz K, et al. Clinical features predictive of orbital exenteration for conjunctival melanoma. Ophthal Plast Reconstr Surg. 2000;16(3):173-178. 26. Paridaens AD, McCartney AC, Minassian DC, et al. Orbital exenteration in 95 cases of primary conjunctival malignant melanoma. Br J Ophthalmol. 1994;78(7):520-528. 27. Esmaeli B. Regional lymph node assessment for conjunctival melanoma: sentinel lymph node biopsy and positron emission tomography. Br J Ophthalmol. 2008;92(4):443-445. 28. Tuomaala S, Kivelä T. Metastatic pattern and survival in disseminated conjunctival melanoma: implications for sentinel lymph node biopsy. Ophthalmology. 2004;111(4):816-821. 29. Bhatia S, Tykodi SS, Lee SM, et al. Systemic therapy of metastatic melanoma: on the road to cure. Oncology (Williston Park). 2015;29(2):126-135. 30. Edge SB, Byrd DR, Compton CC, et al, ed. AJCC Cancer Staging Manual. 7th ed. New York: Springer; 2010. 31. Shields CL, Kaliki S, Al-Dahmash SA, et al. American Joint Committee on Cancer (AJCC) clinical classification predicts conjunctival melanoma outcomes. Ophthal Plast Reconstr Surg. 2012;28(5):313-323. 32. Yousef YA, Finger PT. Predictive value of the seventh edition American Joint Committee on Cancer staging system for conjunctival melanoma. Arch Ophthalmol. 2012;130(5):599-606. 33. Damato B, Coupland SE. Clinical mapping of conjunctival melanomas. Br J Ophthalmol. 2008;92(11):1545-1549. 34. Cohen VM, Tsimpida M, Hungerford JL, et al. Prospective study of sentinel lymph node biopsy for conjunctival melanoma. Br J Ophthalmol. April 2016, Vol. 23, No. 2 2013;97(12):1525-1529. 35. Pfeiffer ML, Savar A, Esmaeli B. Sentinel lymph node biopsy for eyelid and conjunctival tumors: what have we learned in the past decade? Ophthal Plast Reconstr Surg. 2013;29(1):57-62. 36. Mendoza PR, Grossniklaus HE. Sentinel lymph node biopsy for eyelid and conjunctival tumors: what is the evidence? Int Ophthalmol Clin. 2015;55(1):123-136. 37. Tuomaala S, Kivelä T. Sentinel lymph node biopsy guidelines for conjunctival melanoma. Melanoma Res. 2008;18(3):235. 38.Schwarz KA, Davison SP, Crane AE. Sentinel lymph node biopsy in the setting of conjunctival melanoma. Plast Reconstr Surg. 2008;121(4):212e-213e. 39. Savar A, Esmaeli B, Ho H, et al. Conjunctival melanoma: localregional control rates, and impact of high-risk histopathologic features. J Cutan Pathol. 2011;38(1):18-24. 40. Missotten GS, Keijser S, De Keizer RJet al. Conjunctival melanoma in the Netherlands: a nationwide study. Invest Ophthalmol Vis Sci. 2005;46(1):75-82. 41. Esmaeli B, Roberts D, Ross M, et al. Histologic features of conjunctival melanoma predictive of metastasis and death (an American Ophthalmological thesis). Trans Am Ophthalmol Soc. 2012;110:64-73. 42. Sheng X, Li S, Chi Z, et al. Prognostic factors for conjunctival melanoma: a study in ethnic Chinese patients. Br J Ophthalmol. 2015;99(7):990-996. 43. Heindl LM, Hofmann-Rummelt C, Adler W, et al. Prognostic significance of tumor-associated lymphangiogenesis in malignant melanomas of the conjunctiva. Ophthalmology. 2011;118(12):2351-2360. 44. Lake SL, Jmor F, Dopierala J, et al. Multiplex ligation-dependent probe amplification of conjunctival melanoma reveals common BRAF V600E gene mutation and gene copy number changes. Invest Ophthalmol Vis Sci. 2011;52(8):5598-5604. 45.Blum ES, Yang J, Komatsubara KM, et al. Clinical management of uveal and conjunctival melanoma. Oncology (Williston Park). 2016;30(1):29-32, 34-43, 48. 46. Damato B, Eleuteri A, Taktak AF, et al. Estimating prognosis for survival after treatment of choroidal melanoma. Prog Retin Eye Res. 2011;30(5):285-295. 47. Bagger M, Andersen MT, Andersen KK, et al. The prognostic effect of American Joint Committee on Cancer staging and genetic status in patients with choroidal and ciliary body melanoma. Invest Ophthalmol Vis Sci. 2014;56(1):438-444. April 2016, Vol. 23, No. 2 Cancer Control 125 Helpful immunostains for the differential diagnosis of PSC include EMA, Ber-Ep4, AR, and adipophilin. Photo courtesy of Lynn E. Harman, MD. Half Dome From Glacier Point. Sebaceous Carcinoma of the Eyelid Carlos Prieto-Granada, MD, and Paul Rodriguez-Waitkus, MD, PhD Background: Periocular sebaceous carcinoma (PSC) is a rare but aggressive neoplasm that tends to clinically and histopathologically mimic other conditions. PSC can be challenging to diagnose using histomorphology alone given its overlap with 2 more common tumors that occur in this area (basal cell carcinoma [BCC] and squamous cell carcinoma [SCC]). Use of immunohistochemistry can help resolve this differential diagnosis. Methods: A review of the literature was performed, focusing on the epidemiology, morphology, and immunohistochemical features of PSC. Results: The most useful immunostains in the differential diagnosis of PSC are epithelial membrane antigen, Ber-Ep4, androgen receptor (AR), and adipophilin. To discern PSC from BCC, one should use EMA, Ber-Ep4, AR, and adipophilin, whereas discerning PSC from SCC can be achieved by evaluating AR and adipophilin. In addition, p53 and ERBB2 (formally known as HER2/neu) are other potentially useful immunohistochemical markers for the differential diagnosis of PSC. Conclusions: Use of new immunohistochemical techniques, as well as the elucidation of molecular alterations, such as the presence of ERBB2 amplification, will advance our understanding of PSC. Introduction Cutaneous sebaceous carcinoma is a rare disease that makes up an estimated 0.05% of all cutaneous malignancies.1,2 However, because the eyelid and periocular regions harbor a high density of sebaceous glands either associated with eyelashes (Zeis) or the tarsal plate (meibomian), it is not surprising that, when considering all cutaneous sebaceous carcinomas, up to 40% of these tumors arise in this particular anatomical site.3 In the United States, sebaceous carcinoma represents From the Departments of Dermatology & Cutaneous Surgery (CPG, PR-W) and Pathology & Cell Biology (PR-W), Morsani College of Medicine, University of South Florida, Tampa, Florida. Address correspondence to Carlos Prieto-Granada, MD, University of South Florida, 13330 USF Laurel Drive, Fourth floor, Tampa, FL 33612. E-mail: [email protected] Submitted August 15, 2015; accepted December 17, 2015. No significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. 126 Cancer Control 5% of all malignant neoplasms arising in the periocular region, followed basal cell carcinoma (BCC), which makes up 90% of eyelid malignancies.4-6 In general, periocular sebaceous carcinoma (PSC) presents in the elderly (average of 70 years of age).1-3 Predisposing factors, such as immunosuppression, prior history of radiotherapy to the head and neck, and Muir–Torre syndrome, occur in a minority of patients.7 PSC can clinically present as a diffuse thickening of the eyelid, often exhibiting inflammatory features. More advanced cases are characterized as a discrete, palpebral mass. The clinical presentation of early-stage PSC has the potential of mimicking non-neoplastic lesions, notably chalazion, keratoconjunctivitis, and other inflammatory, non-neoplastic processes.5,8,9 Thus, those with PSC are at an increased risk for delayed diagnosis. Historically, a diagnosis of PSC was associated with a poor prognosis and a mortality rate that reached 50%.10,11 However, with increased disease awareness and the introduction of better diagnostic April 2016, Vol. 23, No. 2 techniques, rates of survival have improved (mortality rates of 2%–11%).4,11,12 Early identification is crucial and relies on a high level of clinical suspicion, together with an accurate histopathological diagnosis that often depends on small or limited specimens on biopsy. Histomorphology When examining the invasive component of PSC at low magnification, 4 basic morphological patterns can be recognized: trabecular, lobular, papillary, and BCC-like (Figs 1 and 2).7 Similar to BCC, a mixture of 2 or more of these patterns can be seen in a single lesion. However, all of the classic features of BCC are not found, such as peripheral palisading, tumor–stromal cleft artifacts, and mucinous/myxoid spindle cell stroma. The trabecular pattern of PSC has a tendency to reveal areas of central comedo-like necrosis. An in situ component of PSC may be encountered, either as a standalone lesion as well as adjacent or overlying an invasive lesion (see Figs 1C–F). The intraepithelial lesions are often bowenoid in appearance and can exhibit exuberant pagetoid spread (see Figs 1D and 1E). They can also radially spread by undermining the normal conjunctival epithelium (see Fig 1F). Keratinization is not usually a feature of PSC. The presence of changing keratinization should prompt the health care professional to consider squamous cell carcinoma (SCC) with sebaceous differentiation rather than PSC.7 In a similar fashion to cutaneous sebaceous carcinoma, cytologically PSC tumors are composed of 3 basic cell types: basaloid, sebaceous/vacuolated cells, and intermediate forms. These cell types are usually intermixed at varying ratios, which will determine the level of differentiation of a given tumor, among other factors (see Figs 2D, 2E, and 2G). Broadly speaking, the percentage of basaloid cells will be inversely correlated with the level of differentiation, which ranges from welldifferentiated tumors to poorly differentiated and anaplastic/de-differentiated tumors. Another factor to consider when grading a lesion is the architecture of the tumor. Well-differentiated PSC tumors are often nodular, whereas poorly differentiated tumors are more infiltrative. Taking into account all of these features, including grading, architecture, presence or absence of an in situ component/pagetoid spread, as well as presence of spread to distant sites, the following 4-tiered classification has been proposed13: PSC in situ, low-grade, high-grade infiltrating PSC with or without pagetoid spread, and PSC with extraocular/extracutaneous spread, including distant metastases. In addition to its ability to clinically mimic other lesions, PSC can also overlap the histopathology of several benign and malignant entities encountered in the periocular region. Among the benign processes, the health care professional should consider sebaceous adenoma,14 follicular adnexal tumors (eg, desmoplastic trichoepithe- A B C D E F Fig 1A–F. Composite demonstrating different morphological patterns of PSC. (A) Basal cell carcinoma–like type of configuration. H & E, ×40. (B) Complete with superficial ulceration with the presence of vacuolated cells. H & E, ×200. (C, D) Usual presentation of PSC with extensive intraepidermal involvement exhibiting pagetoid spread. A subjacent main tumor mass is present with tumor cells expanding into the epidermis through the follicular structures. C: H & E, ×20. D: H & E, ×100 (E) High-power imaging shows the nuclear features of PSC: vesicular chromatin with prominent yet small nucleoli. H & E, ×200. (F) The radial extension of PSC cells through normal epithelium reveals conjunctival mucosa undermined by tumor cells. H & E, ×40. H & E = hematoxylin and eosin, PSC = periocular sebaceous carcinoma. April 2016, Vol. 23, No. 2 Cancer Control 127 A B C D E F G H I Fig 2A–I. — Panel exhibiting immunohistochemical staining patterns of 3 markers useful for the differential diagnosis of PSC. (A–C) Epithelial membrane antigen often exhibits patchy but intense membranous and cytoplasmic positivity in PSC. An internal control is present and represented by normal sebaceous glands (C, arrow). A: H & E, ×20. B: H & E, ×20. C: H & E, ×200. (A, D, E) Although this case exhibits a basal cell carcinoma–like pattern (D and E: H & E, ×200), (F) Ber-Ep4 is completely negative. F: H & E, ×200. An internal control is present in the germinative portion of an adjacent follicular structure (arrow). (G) The presence of intermediate cells with lipid vacuoles (arrows) is highlighted with adipophilin. G: H & E, ×400. (H, I) A “vacuolar-type” staining pattern is seen. H: H & E, ×200. I: H & E, ×400. H & E = hematoxylin and eosin, PSC = periocular sebaceous carcinoma. lioma),15 trichoadenoma,16 and squamous papilloma.17-19 When considering malignancies, the 2 main possibilities are BCC — particularly with sebaceous differentiation — and SCC with sebaceous differentiation (rare). The latter also includes a basaloid variant. A rare but plausible differential diagnostic possibility — particularly for poorly differentiated types of PSC — is Merkel cell carcinoma, which is a malignant neoplasm with a predilection to arise in the head and neck area.20,21 When confronted with in situ PSC with abundant pagetoid spread, other pagetoid malignancies such as pagetoid SCC in situ, melanoma in situ, and intraepidermal Merkel cell carcinoma should be considered. Morphologically, the nuclear features in PSC tumor cells have a distinctive shape — namely, they are round, oval, or indented — and contain fine, inkdrop, or smudgy chromatin with 1 or 2 small nucleoli (see Figs 1E and 2G).7 These features are not found in 128 Cancer Control Merkel cell carcinoma, which have washed-out, neuroendocrine-type chromatin. By contrast, melanoma cells often display large nuclei with vesicular chromatin and macronucleoli. In a proportion of poorly differentiated types of PSC (approximately 23%–77%), morphological assessment will be hampered by the presence of overlapping features with other entities included the differential diagnosis.4,22,23 In such situations, immunohistochemical stains may be helpful. Immunohistochemistry Among the malignant tumors that arise in the periocular region, the 3 most common are BCC, squamous cell carcinoma (SCC), and PSC.5,6 Several immunohistochemical markers are useful in the differential diagnosis, including epithelial antigens, as are hormonal markers and other markers highlighting fat vacuole production. Immunohistochemical stains will also reApril 2016, Vol. 23, No. 2 flect subjacent molecular alterations. The different expression profiles using the most useful markers for PSC, BCC, and SCC are summarized in the Table. Table. — Three Commonly Used Immunohistochemical Markers for the Differential Diagnosis of Periocular Sebaceous Carcinoma Type of Carcinoma Epithelial Membrane Antigen Ber-Ep4 Androgen Receptor Adipophilin p53 ERBB2 Periocular sebaceous + −/+ + + + + − + +/− − −/+ − + − − − +/− − Basal cell Epithelial Markers Squamous cell Epithelial antigens are tools that can be used to help in the differential diagnosis of PSC. The 2 epithelial markers deemed to be most useful in the differential diagnosis of PSC, BCC, and SCC are epithelial membrane antigen (EMA) and Ber-Ep4.7,24 Epithelial Membrane Antigen: Also known as mucin 1, cluster designation 227, and cancer antigen 15-3 (particularly when detected in serum), EMA is a 75-kDa transmembrane glycoprotein coded by the 1q21 locus. It is commonly used as an epithelial marker in conjunction with cytokeratins. In normal skin and conjunctiva, EMA is expressed in glandular structures, including sebocytes (see Fig 2C, arrow), as well as in squamous epithelium, but not in follicular structures.25 EMA exhibits a membranous and cytoplasmic signal (see Figs 2B and 2C) and is prominently expressed in the majority of cases of PSC and SCC, while being predominantly negative in BCC.7,24 Ber-Ep4: Ber-Ep4 is a monoclonal antibody directed toward the epithelial cell adhesion molecule, a transmembrane glycoprotein coded by the 2p21 locus and normally expressed at the basolateral membrane of cells by the majority of epithelial tissues, except in adult squamous epithelium and by some specific epithelium cell types (eg, hepatocytes).26,27 This marker is expressed in the secretory portion of eccrine glands and follicular germinative cells in normal skin and eyelids (see Fig 2F, arrow).28 This antibody exhibits a predominant membranous pattern of staining; it is most useful in distinguishing BCC from PSC and SCC because it is frequently positive for Ber-Ep4 (70%–100%).24,28 By contrast, PSC and SCC are negative for Ber-Ep4 in 74% to 94% and 100% of cases, respectively (see Fig 2F).24,28 Cytokeratin: Intermediate filaments known as cytokeratins are expressed in carcinomas (and rarely in a few mesenchymal tumors). Because cytokeratin is also expressed in other entities included in the differential diagnosis of PSC, expression of CAM 5.2 (a cytokeratin cocktail) is not particularly useful to resolve the differential diagnosis for PSC.29 Cytokeratin 7 is expressed in a higher percentage in PSC cases (88%) compared with BCC cases (28%); this is especially true in SCC cases (9%), so it could be used as an adjunctive tool.24 In addition, the expression of cytokeratin 19 may be helpful in distinguishing sebaceous carcinomas (17% focally positive) from BCC (64% strongly positive and 14% focally positive).30 However, given the ubiquity of cytokeratins April 2016, Vol. 23, No. 2 in epithelial malignancies, the ability of cytokeratins to discriminate among the common tumors around the eye is limited. Thomsen–Friedenreich (T) and its Precursor (Tn) Antigen: These markers are composed of Oglycosylated, mucin-type complex carbohydrates and are — in addition to the glycophorin protein system — part of the MNS blood group system. They are expressed in carcinomas from a visceral origin while being hidden from the immune system via the addition of covalently linked carbohydrates and sialic acid in the majority of normal tissues, with the exception of mature sebocytes, the luminal aspect of the sweat glands, and in the intercellular junctions of the spinous layer of the epidermis.31,32 A small study specifically testing the immunohistochemical expression of the Thomsen– Friedenreich (T) antigen found a strong expression of this marker in the majority of sebaceous carcinoma cases (n = 8) tested as well as in the sebaceous adenoma (n = 15) and sebaceoma (n = 9) samples, while being negative in the all but 1 of the tested BCC cases (n = 13); only 1 case of BCC was positive for sebaceous differentiation.13,32 Although no follow-up studies were found in the literature regarding this marker, one could consider incorporating T-antigen immunohistochemistry to resolve this differential diagnosis. Lipid-Processing Markers Sebaceous carcinomas are related to the production of lipids (mainly triglycerides). Historically, the health care professional was only able to rely on the often cumbersome histochemical demonstration of lipids utilizing Oil red O or Sudan black IV, inconvenient stains that required a frozen tissue sample and were associated with both poor rates of sensitivity (40%) and stain fading over time.33 However, another group of immunohistochemical markers demonstrating lipid production are now available. This group of markers is composed of 3 lipid droplet–associated proteins comprised of the so-called perilipin, adipophilin, and TIP47 group (also known as perilipins 1, 2, and 3, respectively). These molecules are associated with the phospholipid monolayer membrane surrounding the triglyceride core of the intracytoplasmic lipid droplets and are related in the formation, maintenance, modifiCancer Control 129 cation, and involution of such structures.34 All of these markers appear specific for lipid droplets in normal tissues as well as in a variety of malignant neoplasms with lipogenic features.28,35-38 Although adipophilin appears to be the most sensitive marker of the group, perilipin is considered to have superior rates of specificity, and both immunostains have proven to be of particular utility in the differential diagnosis of PSC.7,23,25,33,39,40 Adipophilin: Also known as adipocyte differentiation–related protein and perilipin 2, adipophilin is a lipid vesicle–associated protein coded by a gene in 9p22.1. This marker is expressed in normal sebocytes. However, when using this stain, the health care professional must be aware of its different staining patterns, including its diffuse cytoplasmic signal pattern, which is often encountered in well-differentiated areas of PSC tumors; its vacuolar, vesicular-rimming pattern (see Figs 2G and 2I); or the presence of both of these, thus highlighting the intermediate cells of a PSC tumor; and a fine particulate, granular, or dusty pattern (considered nonspecific and could be found in other tumors [eg, SCC, BCC]). The health care professional must remember the poorly differentiated areas of PSC tumors will often be lipid-poor and basaloid cell-rich; thus, they may be predominantly negative or weakly positive for adipophilin in an overall patchy pattern.7,23,25,33,39 Adipophilin can be useful when differentiating PSC that is predominantly immunoreactive, showing either vacuolar or diffuse patterns, or is predomiA nantly negative or weakly positive with a finely granular pattern in the setting of BCC and SCC. Perilipin: Perilipin is a transmembranous protein related to lipid vesicles coded by the 15q26 locus that is expressed in sebocytes. Perilipin appears to be more specific but less sensitive than adipophilin.33,39 The different patterns of staining are similar to those of adipophilin, and the B health care professional could use this marker in cases for which adipophilin was unsatisfactory. Androgen Receptor Androgen receptor (AR) is coded by Xq12 and forms part of a family of nuclear-located, steroid hormone transcription factors/ receptors that bind testosterone and dihydrotestosterone. In normal skin and conjunctiva, AR is predominantly expressed in follicular and adnexal structures, especially in the sebaceous 130 Cancer Control glands.25,41,42 Because it is a transcription factor and a hormone receptor from the steroid class, its immunohistochemical signal is predominantly nuclear (see Fig 3). This marker can be of particularly help when differentiating between PSC (predominantly positive, 33%–83%), SCC (predominantly negative, 90%–100%), and BCC (occasionally negative, 50%–86%).27,28 Underlying Mutations/Genetic Aberrations p53 Protein: The p53 protein is a transcription factor coded by TP53 (17p13.1), which induces apoptosis when DNA damage occurs. Approximately 67% of PSC tumors will harbor missense or nonsense TP53 mutations that are not of the ultraviolet (UV) signature type (unrelated to UV damage).43 Patients with germline TP53 mutations (Li–Fraumeni syndrome) frequently develop PSC, along with other malignancies that characterize the condition.44 Because the gene mutation rate is quite high in the setting of PSC, many of these tumors (40%–72%) will show an overexpression (accumulation) of p53 protein on immunohistochemistry translated by a strong nuclear signal.7,45 By contrast, this pattern of p53 staining occurs in fewer than 20% of BCC cases and 50% to 60% in SCC cases.7,46,47 Thus, some have advocated for use of p53 immunohistochemistry as a diagnostic aid in the differential diagnosis of PSC.7 Both the presence of TP53 mutations and C Fig 3A–C. — Pattern of immunohistochemical staining with androgen receptor. (A) In this case, periocular sebaceous carcinoma exhibited a striking bowenoid appearance predominantly composed of basaloid cells. H & E, ×40. (B, C) Androgen-receptor immunohistochemical stain demonstrated diffuse and strong nuclear positivity in the tumor cells with a weak cytoplasmic blush. B: H & E, ×100. C: H & E, ×200. H & E = hematoxylin and eosin. April 2016, Vol. 23, No. 2 the consequent p53 overexpression are more frequently encountered in PSC than extraocular sebaceous carcinoma and are inversely correlated with the presence of alterations of the mismatched repair (MMR) proteins and Muir–Torre syndrome.45 Mismatched Repair Proteins: Assessing the expression of MMR proteins with immunohistochemistry for mutL homolog 1, mutS homolog 2, mutS homolog 6, and PMS1 homolog 2 (MLH1, MSH2, MSH6 and PMS2, MMR system components) in cutaneous neoplasms — particularly in sebaceous adenoma — is becoming an easy and convenient way to screen for Muir–Torre syndrome, a variant of the Lynch syndrome spectrum.48-50 However, by contrast to the extraocular forms of sebaceous neoplasms, most PSC tumors appear to be unrelated to loss of MMR expression. Therefore, PSC should be considered as a poor “index lesion” to screen for Muir–Torre syndrome.45,51,52 Nevertheless, periocular sebaceous adenoma appears to follow the same trend as extraocular sebaceous neoplasms in terms of its relationship with Muir–Torre syndrome.53 ERBB2 (formally known as HER2/neu): PSC has been shown to exhibit 2+ and 3+ patterns of ERBB2 (formally known as HER2/neu) immunohistochemical positivity in up to 82% of cases, with 75% of these cases showing ERBB2 amplifications by fluorescence in situ hybridization.50 Based on the results of a study reporting on a series of 14 cases, these findings have diagnostic value because both ERBB2 immunoreactivity of more than 1+ and gene amplification are infrequently found in mimickers and they provide a possible therapeutic avenue for PSC.50,54 Adhesion Protein Expression and Epithelial Mesenchymal Transition–Related Markers: Similar to other types of aggressive carcinomas, such as lobular carcinoma of the breast, PSC (in particular, poorly differentiated PSC) has been shown to disrupt the intercellular and cell stroma adhesion machinery with loss or diminished membranous E-cadherin and beta-catenin protein expression in 53% to 83% and 56% to 61% of cases, respectively.55,56 Loss or diminished expression of these markers was correlated with the presence of the epigenetic promoter methylation of CDH1 (16q22.1).55 Similarly, PSC also appears to overexpress ZEB2 in up to 68% of cases.57 ZEB2 is a transcription factor gene closely related to the epithelial to mesenchymal transition phenomenon that also interacts with CDH1-producing gene repression and the subsequent decrease of E-cadherin expression. The epithelial to mesenchymal transition is an embryonic process to which poorly differentiated neoplasms regress to enhance their invasiveness and metastatic potential by increased cell migration. The presence of the immunohistochemical overexpression of ZEB2, as well as loss of E-cadherin expression, and CDH1 promoter methylation in the setApril 2016, Vol. 23, No. 2 ting of PSC have been correlated with poor rates of survival.55,57 Loss, diminution, and aberrant E-cadherin expression as well as CDH1 promoter methylation58 can also be found in both BCC59-62 and SCC,61,63 so these findings preclude use of ZEB2 as a diagnostic marker in this setting. Conclusions Several markers are differentially expressed in periocular sebaceous carcinoma (PSC) compared with lesions in the differential diagnosis. A panel of immunohistochemical stains is recommended because different combinations of immunoprofiles characterize these entities, of which epithelial membrane antigen (EMA), Ber-Ep4, androgen receptor (AR), and adipophilin appear to be the most useful. An optimal stain panel to discriminate PSC from basal cell carcinoma (BCC) is presented by EMA, Ber-Ep4, AR, and adipophilin, while distinguishing PSC from squamous cell carcinoma (SCC) can be achieved by exploring the expressions of AR and adipophilin. The 3 most typical immunophenotypes are the following: (1) PSC is positive for EMA, AR, and adipophilin but negative for Ber-Ep4, (2) SCC shows a predominant immunoprofile of EMA positivity but is negative for AR, Ber-Ep42, and adipophilin, and (3) BCC is predominantly negative for EMA and adipophilin but positive for Ber-Ep4. Two other potential immunohistochemical markers that may be useful in the differential diagnosis are p53 and HER2/neu, both of which will be predominantly expressed in PSC. Mismatched repair protein immunohistochemical expression aberrations appear to be absent in the majority of cases of PSC, because they appear to harbor TP53 mutations. PSC as an index case to screen of Muir–Torre syndrome is currently not recommended. The introduction of new immunohistochemical stains for PSC and the expansion of our knowledge regarding the underlying molecular aberrations of PSC, such as the presence of ERBB2 amplifications, represent an exciting new era in understanding of this entity. These advancements could facilitate the early detection of PSC and possibly result in improved patient outcomes. References 1. Anderson HL, Joseph AK. A pilot feasibility study of a rare skin tumor database. Dermatol Surg. 2007;33(6):693-696. 2. Kyllo RL, Brady KL, Hurst EA. 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Ruizeveld de Winter JA, Trapman J, Vermey M, et al. Androgen receptor expression in human tissues: an immunohistochemical study. J Histochem Cytochem. 1991;39(7):927-936. 42. Asadi-Amoli F, Khoshnevis F, Haeri H, et al. Comparative examination of androgen receptor reactivity for differential diagnosis of sebaceous carcinoma from squamous cell and basal cell carcinoma. Am J Clin Pathol. 2010;134(1):22-26. 43. Kiyosaki K, Nakada C, Hijiya N, et al. Analysis of p53 mutations and the expression of p53 and p21WAF1/CIP1 protein in 15 cases of sebaceous carcinoma of the eyelid. Invest Ophthalmol Vis Sci. 2010;51(1):7-11. 44. Baumuller S, Herwig MC, Mangold E, et al. Sebaceous gland carcinoma of the eyelid masquerading as a cutaneous horn in Li--Fraumeni syndrome. Br J Ophthalmol. 2011;95(10):1470, 1478. 45. Shalin SC, Sakharpe A, Lyle S, et al. p53 staining correlates with tumor type and location in sebaceous neoplasms. Am J Dermatopathol. 2012;34(2):129-135; quiz 136-128. 46. 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Abnormal immunoreactivity of the E-cadherin/catenin (alpha-, beta-, and gamma-) complex in premalignant and malignant non-melanocytic skin tumours. J Pathol. 2002;196(2):154-162. 62. Papanikolaou S, Bravou V, Gyftopoulos K, et al. ILK expression in human basal cell carcinoma correlates with epithelial-mesenchymal transition markers and tumour invasion. Histopathology. 2010;56(6):799-809. 63. Koseki S, Aoki T, Ansai S, et al. An immunohistochemical study of E-cadherin expression in human squamous cell carcinoma of the skin: relationship between decreased expression of E-cadherin in the primary lesion and regional lymph node metastasis. J Dermatol. 1999;26(7):416-422. April 2016, Vol. 23, No. 2 The role that vismodegib will play in advanced periocular basal cell carcinoma depends on its postmarketing results. Photo courtesy of Lynn E. Harman, MD. Hohenschwangau Castle. Role of Vismodegib in the Management of Advanced Periocular Basal Cell Carcinoma Kyle F. Cox, MD, and Curtis E. Margo, MD Background: Vismodegib is the first selective hedgehog pathway inhibitor approved to treat locally advanced and metastatic basal cell carcinoma (BCC). Limited information is available concerning its role in managing advanced BCC around the eye. Methods: The medical literature was searched for cases of nonsyndromic periocular BCC treated with vismodegib. Clinical information was abstracted and analyzed. In addition, a review of the pharmacology of vismodegib, including general effectiveness and safety, was conducted. Results: Thirty study patients with nonsyndromic periocular BCC treated with vismodegib were found in the literature. Vismodegib was used in 3 ways: medical therapy, adjuvant therapy prior to surgery or radiotherapy, and treatment of positive surgical margins. Complete regression was reported in 9 study patients (30%), with follow-up visits after therapy averaging fewer than 5 months. Four study participants developed squamous cell carcinoma while receiving treatment. Conclusions: Too few cases exist to draw any conclusions on the role that vismodegib might play in the management of periocular BCC. In addition, long-term follow-up data are not yet available. Although the objective response rate of advanced BCC is impressive in study patients receiving vismodegib, well-controlled clinical studies are needed to determine whether vismodegib has any impact on survival or quality of life. Introduction In general, periocular basal cell carcinoma (BCC) is usually treated with surgical excision and, when indicated, microscopic control of margins. Typically, recurrence rates are low and functional outcomes are good; however, some tumors because of their size, location, From the Departments of Ophthalmology (KFC, CEM) and Pathology and Cell Biology (CEM), University of South Florida Morsani College of Medicine, Tampa, Florida. Address correspondence to Kyle F. Cox, MD, Department of Ophthalmology, Morsani College of Medicine, MDC Box 21, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612. E-mail: [email protected] Submitted July 13, 2015; accepted January 5, 2016. No significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. April 2016, Vol. 23, No. 2 or lack of therapeutic response are inoperable.1 In such cases, the rates of continued morbidity are high and the rates of cure are low.1 Vismodegib is a selective hedgehog pathway inhibitor. It was approved by the US Food and Drug Administration (FDA) in 2012 and the European Medicines Agency in 2013 for the treatment of locally advanced and metastatic types of BCC.2-4 Given the rarity of these conditions and lack of treatment options, approval was based on noncomparative clinical series of study participants. Phase 1/2 trials showed favorable primary end point responses for both locally advanced and metastatic BCC.5 In a phase 2 study, 43% of patients with locally advanced BCC showed partial or complete responses, whereas 30% of those with metastatic tumor responded to treatment.6 Cancer Control 133 Since the US and European approvals of vismodegib,2-4 case reports and small clinical series have appeared in the literature reflecting how the therapy is used in clinical practice. Some of these reports involve periocular (or ocular adnexal) BCC.7-13 Use of vismodegib for BCC around the eye poses unique challenges and opportunities because conjunctival and orbital involvement may result in blindness and death.1 Hedgehog Pathway The hedgehog pathway is a signaling system that helps regulate cell growth and development. Initially described in Drosophila, the hedgehog pathway controls a range of cellular activities in invertebrates and vertebrates, starting during embryogenesis. Among vertebrates, the hedgehog genes encode polypeptides that, after modification by cholesterol, attach to cell surfaces where they affect signaling. The functional receptors for hedgehog are 2 transmembrane proteins called patched 1 and smoothened. The discovery that aberrant activity of the pathway was linked to several human tumors spurred investigation into drugs that could alter various components of the signaling sequence.14 Interest into the therapeutic potential of hedgehog pathway inhibitors was stimulated further when researchers tied the signaling system to diverse functions such as angiogenesis and potentially metastatic spread.15 BCC is driven by mutations in 1 or more hedgehog genes.16 Typically, these alterations take the form of a loss-of-function mutation in PTCH1 or an activating mutation in SMO. Nevoid basal cell carcinoma syndrome (also called Gorlin–Goltz syndrome) is caused by a germline mutation in PTCH1. The mechanism or mechanisms by which PTCH1 mutations eventually alter downstream transcription factors is an area of active investigation.16 Pharmacology Vismodegib is the first hedgehog inhibitor approved for the treatment of BCC.17 It appears to exert its clinical effect by binding to the smoothened receptor protein, preventing nuclear localization of transcription factors that target gene induction.14,18 This in turn can promote cellular proliferation, inhibit apoptosis, and further prolong cell survival through mechanisms such as angiogenesis. Compared with other small molecules capable of blocking this receptor, the pharmacokinetic profile of vismodegib was considered better suited for human use.19 Approximately one-third of a 150-mg oral dose of vismodegib (the recommended daily dose) is absorbed; more than 99% is bound to plasma proteins (mostly albumin).20 Vismodegib is synthetically derived and not extensively metabolized; more than 80% is excreted in feces.20,21 Metabolic oxidation in the liver is incompletely understood.20 134 Cancer Control After continuous daily dosing, the elimination halflife of vismodegib is 4 days, although the half-life of a single dose can be up to 12 days.20-22 Its bioavailability is nonlinear, decreasing with increasing strength and frequency of administration.20,23 Studies Phase 1 Although no dose-limiting toxicities were found among an initial cohort of patients enrolled in an open-label phase 1 trial, daily doses higher than 150 mg demonstrated no substantially greater steady-state plasma concentration.24 Once the maximum tolerated dose was determined to be 150 mg/day, enrollment of the phase 1 study expanded to include 68 patients with advanced/metastatic BCC and other solid tumors.25 In this study, tumor response was only seen among participants with BCC and medulloblastoma.25 Six participants (9%) experienced life-threatening or disabling adverse events and 20 participants (28%) experienced severe adverse events; however, the majority of adverse events experienced were mild to moderate in nature (eg, muscles spasms, fatigue, alopecia, dysgenesia, diminished appetite).25 Those with BCC had an overall response rate of 55%, stable disease was seen in 33%, and the median duration of response was 12.8 months (range, 3.7–26.4 months).25 Five deaths (7%) were reported.25 Phase 2 Two phase 2 clinical trials followed, one of which was a dual-cohort study of 96 volunteers with locally advanced/metastatic BCC; the other was a randomized trial involving 41 study patients with nevoid basal cell carcinoma syndrome.6,26 The objective response rate for 63 study patients with locally advanced disease was 43%; of those participants, 13 (21%) had a complete response.6 The median duration of response was 7.6 months (range, 1–12.9 months), and 34 of the 63 specimens (54%) obtained via biopsy showed no residual tumor.6 The median duration of drug exposure was approximately 10 months.6 Anatomical locations of individual tumors were not available, so analysis of advanced periocular BCC could not be performed. Each study patient in the dual cohort trial experienced 1 or more adverse event, and 12% of those resulted in treatment discontinuation.6 Serious adverse events occurred in 26 study patients (27%); of those, 7 (7%) of those events were grade 5 and fatal.6 The clinical context of the fatalities were incompletely presented, but the authors found no drug-related culpability because all 7 volunteers had preexisting comorbidities.6 Vismodegib displayed similar rates of effectiveness in reducing BCC in persons with nevoid basal cell carApril 2016, Vol. 23, No. 2 cinoma syndrome.6,26 Objective tumor shrinkage was seen in 65% of treated study patients compared with 11% in those receiving placebo (P = .003), but the rate of adverse events led to treatment discontinuation in more than 50% of volunteers (14/26).26 A 12-month update of a phase 2 trial found a 4.7% increase in objective response rate for study patients with advanced BCC (from 42.9% to 47.6%).27 The median duration of response in these patients also increased from 7.7 to 9.5 months, and no new safety concerns were reported.27 An interim analysis of an international, open-label trial with safety as its primary end point used data from 499 study volunteers with advanced/metastatic BCC.28 Treatment was discontinued in 400 volunteers (80%), 180 (36%) experienced adverse events, 70 (14%) had disease progression, and 51 (10%) requested treatment be stopped.28 Overall response rate for those with advanced BCC was 66.7% (302/453); of the 302 cases of clinical responses, 153 of those were complete responses.28 Deaths were reported in 31 study volunteers, 21 of which were considered to be due to a treatment-related adverse event.28 An expanded access, open-label multicenter study of 150 mg vismodegib daily in 119 participants with advanced BCC demonstrated an objective response rate of 46% in those with locally advanced disease and 31% in those with metastatic BCC.17 Inclusion criteria was a tumor diameter of at least 1 cm and either confirmation of an inoperable tumor or a contraindication to surgery.17 Study patients were followed for 6.5 months17; during that time, the rate of adverse events paralleled those previously reported.6 Four grade 4 treatmentemergent adverse events and 2 treatment-related deaths were reported.17 Treatment Results The outcomes of periocular tumors are difficult to identify because site-specific characteristics are not always available in postmarketing clinical series of BCC. In cases that can be identified as periocular, vismodegib has been used as neoadjuvant therapy prior to surgery and radiotherapy and as treatment for positive surgical margins.7-13,17,28,29 We were able to find 30 patients for whom sufficient clinical information could be abstracted for analysis (Table 1).7-13 Vismodegib was reasonably effective in a series of 7 study patients with advanced periocular BCC not amenable to surgery or radiotherapy whose largest tumor, on average, was 3.4 cm in size.7 None had metastatic disease, but 4 study patients had orbital involvement.7 All study patients had BCC that had previously failed controlled surgical resection.7 The average duration of treatment with vismodegib was 11 weeks (range, 4–16 weeks), and the mean duration of followup was 7.3 months (range, 5–10 months).7 Two study April 2016, Vol. 23, No. 2 patients (29%) demonstrated complete regression, 2 (29%) had more than 50% regression, 2 (29%) had less than 50% regression, and 1 (14%) had disease progression.7 Six (86%) reported experiencing adverse events similar to those previously documented (eg, alopecia, muscle cramps).6,7,24-26 Two study patients also developed squamous cell carcinoma (SCC).7 Demirci et al8 described the results of a study involving 8 patients with periocular and orbital BCC, one of whom had nevoid basal cell carcinoma syndrome. Of the 7 nonsyndromic study patients, 5 received vismodegib alone for treatment.8 Four obtained partial response, and, at the time of publication, all 4 were still taking vismodegib.8 One study patient with complete response was followed for 3 months.8 In another study patient, vismodegib was used as adjuvant therapy for involved surgical margins following surgery and in another study patient as neoadjuvant therapy prior to surgery.8 In both of these roles, tumor response was deemed to be complete during observation periods of 11 and 16 months, respectively.8 Adverse events were characterized as comparable in severity and frequency as those seen in earlier studies.6,7,24-26 Data from 1 patient in this series were also published in a separate report.7 In a series of 13 study patients with high-risk BCC for whom vismodegib was used as neoadjuvant therapy prior to surgery, 2 tumors were located around the eye.9 One tumor with a surface area of 2.8 cm2 had an 86% reduction in its size prior to surgery, and the resected tissue showed histological cure.9 The second tumor with a surface area of 2.0 cm2 regressed 5% and, when resected, it was still histologically present.9 Vismodegib has also been used as adjuvant therapy prior to radiotherapy in a man with recurrent BCC of the medial canthus and lower eyelid.10 After receiving 150 mg/day vismodegib for 2 months, cross-sectional measurements of his tumor decreased from 6.5 × 7.4 mm to 6.3 × 5.6 mm prior to radiotherapy.10 Following radiotherapy, he was deemed to be disease free at 12 months by magnetic resonance imaging.10 Two other cases of periocular BCC treated with vismodegib have been reported because the study patients developed SCC (keratoacanthoma types) within 2 and 7 weeks of starting therapy.11 Both secondary cancers arose in previously documented normal skin.11 Meaningful clinical follow-up beyond the context of the new tumors was not provided.11 A man aged 84 years with left upper eyelid and orbital BBC was treated with vismodegib and had complete response in 3 months; however, the tumor recurred and progressed after 9 months.12 Vismodegib was stopped at 18 months and orbital exenteration was performed the next month.12 The case was published as an example of secondary resistance. A retrospective, interventional case series includCancer Control 135 Table 1. — Study Patients With Nonsyndromic Periocular Basal Cell Carcinoma Treated With Vismodegib Study Age, y Sex Orbital Involvement TNM Stage Duration of Therapy, mo Response Intervention After Vismodegib Follow-Up After Treatment, mo Comment Aasi11 53 M N NA 1.75 Complete — 0 New case of SCC Ally9 Demirci8 Gill7 Ozgur 13 100 W N NA Unknown “Regression” — Unknown New case of SCC 39 W N NA 3.5 Partial Surgery 11 — 100 W N NA 4 Complete Surgery 20 — 60 M Y T3bN0M0 8 Complete — 11 Used to treat positive surgical margin 60 M Y T4N0M0 5 Partial — 0 — 63 W Y T3bN0M0 7 Partial — 0 — 73 M N T3aN0M0 9 Complete — 3 — 79 M Y T3bN0M0 13 Partial — 0 — 79 M a Y T3bN0N0 5 Complete Surgery 16 Adjuvant prior to surgery 86 M Y T3bN0M0 7 Partial — 0 — — New case of SCC 43 M N NA 1 Complete — 10 53 60 M M N N NA NA 1.75 2.5 Complete Partial — — 8 6 75 M Y NA 4 Partial — 8 — 75 W Y NA 4 Progression — 8 — 91 M Y NA 2.25 Partial — 6 — — 101 W Y NA 4 Partial — 5 New case of SCC 51 W N T4N2cM1 9 Partial — 0 — 56 M N T3bN0M0 18 Complete — 0 — 58 M Y T3bN1M0 7 Partial — 0 — 64 M N T3bN0M0 9 Partial — 0 — 65 M N T4N1M1 18 Progression — 2 Died of disease 69 W N T3bN0M0 10 Partial — 0 Lost to follow-up 70 M Y T3bN0M0 11 Stable — 0 — 76 M N T2N1M1 13 Stable — 0 — 84 M N T3bN0M0 12 Complete — 0 — 86 M N T3bN0M0 11 Partial — 13 Recurrence after 3 mo Papastepfanous12 84 M Y NA 18 Partial Exenteration 0 Recurrence after initial response Pollom10 70 M Y NA 2 Partial RT 12 Adjuvant prior to RT Average 7.6 4.8 Data reported through December 2015. All study patients were white. from this study patient reported in another publication. M = man, N = no, NA = not applicable, RT = radiotherapy, SCC = squamous cell carcinoma, TNM = tumor, node, metastasis, W = woman, Y = yes. a Data ed 10 study patients with locally advanced BCC, 4 of whom had metastatic disease.13 Of the 6 study patients without metastasis, 2 had a complete response.13 Five would have needed exenteration but instead avoided surgery with vismodegib treatment, although, at the time of reporting, the follow-up period was short (or none at all) after stopping vismodegib.13 One study 136 Cancer Control patient died of progressive disease.13 The profile of reported adverse events was similar to that previously described.6,13,24-26 It is worth noting that, of the 30 cases of advanced eyelid and periocular BCC treated with vismodegib to date, the average follow-up period after stopping vismodegib is 4.8 months (see Table 1).7-13 April 2016, Vol. 23, No. 2 Inoperable and Locally Advanced Disease The role that vismodegib might play in the clinical treatment of inoperable and locally advanced BCC depends on the perception of what these terms actually mean. Because these conditions have no consensus definition, a panel sponsored by Roche (Basel, Switzerland) of oncologists, dermatologists, and radiation oncologists helped to establish the guidelines for these terms.30 Although the panel addressed the management of periocular BCC, no member of the group was an ocular oncologist or oculoplastic surgeon.30 The proposed guidelines of inoperability for use of vismodegib was based on the tumor, node, and metastasis staging classification of the American Joint Committee of Cancer for eyelid carcinoma (Table 2).30,31 Consensus was defined as majority opinion of the 9-member panel.30 Tumors greater than 2 cm in size that do not invade adjacent ocular or orbital structures (T3a) should be first considered for radiotherapy.27 The panel also recommended that eyelid tumors of stages T3a (invasive), T3b, and T4 not appropriate for radical local therapy be assessed for possible systemic therapy with vismodegib.30 The panel also noted that additional factors must be considered in determining the suitability of medical therapy, including general medical health, severe disfigurement, and loss of function.30 However, the panel’s recommendations were not ideal because guidelines for inoperable tumors defer to a judgment of what constitutes “not appropriate for radical local therapy.”30 Any guidelines recommending vismodegib as alternative therapy to surgery must weigh the limited knowledge of vismodegib against its long-term outcomes. For example, locally advanced BCC involving the orbit would require surgical exenteration (stage T3b) for cure.30 In an otherwise healthy patient, this surgery should confer long-term, tumor-free survival, albeit with a significant cosmetic cost. However, medical treatment with vismodegib offers an approximately 30% likelihood of complete response and an unknown risk of recurrence beyond 1 year.28 In addition, the long-term behavior of regressed BCC once vismodegib is discontinued is unknown. compounds that overcome vismodegib resistance in vitro are under development for clinical use.36 Information is limited about the drug–drug interactions of vismodegib, and the safety of vismodegib in persons with renal and hepatic dysfunction is unknown.4,5 A man aged 72 years treated with vismodegib developed cholestatic hepatic dysfunction 1 month after beginning treatment.37 The patient self-medicated with aspirin and naproxen because of vismodegib-related myalgias, suggesting a drug interaction may have been involved.37 Ventarola and Silverstein 38 searched the FDA Adverse Events Reporting System from January 2012 through January 2013 for cases of vismodegib hepatic toxicity, and they found 65 cases of vismodegib-associated reactions, the most frequent of which was gastrointestinal distress (34%) followed by liver toxicity (23%). In 4 cases, vismodegib was considered the sole agent implicated in the hepatotoxic event.38 However, Ventarola and Silverstein 38 Table 2. — Tumor Staging for Carcinoma of the Eyelid Description Comment TX Stage Primary tumor cannot be assessed — T0 No evidence of primary tumor T1 Tumor ≤ 5 mm in greatest dimension and Does not invade tarsus or margin of eyelid Average horizontal length of adult eyelid: ~ 3 cm T2a Tumor > 5 mm but ≤ 10 mm in greatest dimension or Any tumor than invades tarsus or involves margin of eyelid Average vertical length of upper tarsus: 10–12 mm Average vertical length of lower tarsus: ~ 4 mm T2b Tumor > 10 mm but ≤ 20 mm in greatest dimension or Tumor involves entire thickness of eyelid April 2016, Vol. 23, No. 2 — T3a Tumor > 20 mm in greatest dimension or Any tumor than invades adjacent ocular or orbital structures or Any tumor with perineural tumor invasion Adjacent ocular structures include bulbar conjunctiva Anterior orbit begins at orbital septum T3b Complete resection of tumor requires enucleation, exenteration, or bone resection Exenteration involves removal of orbital contents, including eye T4 Tumor unresectable due to extensive invasion of ocular, orbital, or craniofacial structures or brain Other Observations Four cases of secondary SCC were reported among the 19 patients treated for periocular BCC.7,11 Rapidly growing SCC has also been observed in persons receiving treatment with vismodegib for BCC located elsewhere.32,33 Some types of BCC do not respond to vismodegib, and some of these failures may reflect mutational resistance, which is a new area of investigation likely to gain momentum during the postmarketing period.18,34,35 At least 1 case of periocular BCC with secondary resistance has been described.12 Novel — — Adapted from Edge S, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual. 7th ed. New York: Springer; 2010. Republished with permission of Springer; permission conveyed through Copyright Clearance Center, Inc. Cancer Control 137 stressed that inclusion in the FDA database does not mean the relationship is causal. Cost The cost of vismodegib will depend on factors such as geographical area and markup of intermediate providers.39,40 When made available, the cost of standard daily treatment in clinical studies has ranged from $7,500 to 9,000 per month.9,20 In a single case series of 12 study patients treated for periocular BCC, 2 (17%) discontinued treatment because of cost.13 Alternative Therapies Vismodegib was approved by the FDA and European Medicines Agency for the treatment of locally advanced and metastatic BCC without the benefit of a randomized clinical trial because locally advanced and metastatic BCC are uncommon and evidence of effective alternative therapies is lacking.2,4 Historically, patients may have been treated with agents like cisplatinum, which, although not curative, induce a transient reduction in tumor size.41,42 Some investigators have used cisplatinum as neoadjuvant therapy to reduce the tumor size of periocular BCC prior to surgery,41 whereas others have used cisplatinum/doxorubicin to treat advanced BCC around the eye with only short-term benefit.42,43 However, experience with these chemotherapeutic agents is limited in the setting of periocular BCC, and use of these agents has been associated with serious adverse events.41-44 Treatment for Operable Disease Although no study has examined use of vismodegib for operable BCC around eye, a phase 2 multicenter, open-label trial has studied its use at all skin sites.3 This nonrandomized trial studied 74 volunteers but was unable to achieve its predefined primary efficacy end point of histological clearance.3 In addition, of the 37 study patients deemed to have complete clinical regression (clearance), 12 (32%) had histological evidence of residual BCC.3 Thus, these results may influence the design of future clinical trials studying operable and inoperable tumors. Conclusions Vismodegib may be an important treatment option to patients with locally advanced basal cell carcinoma (BCC) around the eye. However, the exact role it will play has not been established. The reviewed literature suggests that many patients with locally advanced periocular BCC will completely or partially respond when treated with daily 150 mg vismodegib, but their duration of response is unknown because follow-up times have been for periods less than 10 months. Use of vismodegib outside of a clinical trial has expanded to include the neoadjuvant settings for sur138 Cancer Control gery and radiotherapy.7,8,10 However, because phase 2 studies of locally advanced and metastatic BCC are not placebo-controlled trials and follow-up has been limited, it is unclear whether vismodegib prolongs survival rates or whether it reduces serious rates of morbidity.45 Given that most patients with locally advanced periocular BCC developed the condition over 5 to 15 years and that clinical follow-up after treatment is brief, judging the role that vismodegib might play in management may be premature.1 When vismodegib is used to shrink surgical margins, it is uncertain whether these margins will remain tumor free over time, particularly after the medication is discontinued. A report from the US Veterans Affairs concluded in 2013 that the net clinical benefit of vismodegib treatment was minimal and that its use had a low likelihood of benefit as well as a low risk of harm.4 Vismodegib is not well tolerated in study patients: Approximately 40% are unable to continue therapy due to its adverse events.4 Reports of hepatotoxicity, particularly in persons taking other medications that may interact with vismodegib, have also been reported.6,37,38 More information is also needed on the safety of vismodegib in patients with renal and hepatic impairment. Whether any serious, long-term complications are associated with its use is unknown, and no evidence exists to suggest that vismodegib improves rates of survival. Ever since vismodegib was first approved for clinical use, case reports and small clinical series have appeared in the literature. These studies suffer from short-term follow-up, lack of control groups, and potentially publication bias, because complications and therapeutic misadventures tend to be under-reported after drugs receive market approval.46 Before the therapeutic potential of vismodegib can be realized, larger studies with appropriate control populations and long-term follow-up periods are necessary. References 1. Margo CE, Waltz K. Basal cell carcinoma of the eyelid and periocular skin. Surv Ophthalmol. 1993;38(2):169-192. 2. Axelson M, Liu K, Jiang X, et al. U.S. Food and Drug Administration approval: vismodegib for recurrent, locally advanced, or metastatic basal cell carcinoma. Clin Cancer Res. 2013;19(9):2289-2293. 3. Sofen H, Gross KG, Goldberg LH, et al. A phase II, multicenter, open-label, 3-cohort trial evaluating the efficacy and safety of vismodegib in operable basal cell carcinoma. J Am Acad Dermatol. 2015;73(1):99-105.e1. 4. US Department of Veterans Affairs (VA) Pharmacy Benefits Management Services; Medical Advisory Panel; VIAN Pharmacist Executive. Vismodegib (Erivedge) National Drug Monograph. Washington, DC: US VA; 2013. www.pbm.va.gov/clinicalguidance/drugmonographs/vismodegib_drug _monograph.docx. Accessed March 22, 2016. 5. Erdem GU, Sendur MA, Ozdemir NY, et al. A comprehensive review of the role of Hedgehog pathway and vismodegib in the management of basal cell carcinoma. Curr Med Res Opin. 2015;31(4):743-756. 6. Sekulic A, Migden MR, Oro AE, et al. Efficacy and safety of vismodegib in advanced basal cell carcinoma. N Engl J Med. 2012;366(23):2171-2179. 7. Gill HS, Moscato EE, Chang AL, et al. Vismodegib for periocular and orbital basal cell carcinoma. JAMA Ophthalmol. 2013;131(12):1591-1594. 8. Demirci H, Worden F, Nelson CC, et al. Efficacy of vismodegib (Erivedge) for basal cell carcinoma involving the orbit and periocular area. Ophthal Plast Reconstr Surg. 2015;31(6):463-466. April 2016, Vol. 23, No. 2 9. Ally MS, Aasi S, Wysong A, et al. An investigator-initiated openlabel clinical trial of vismodegib as a neoadjuvant to surgery for high risk basal cell carcinoma. J Am Acad Dermatol. 2014;71(5):904-911.e1. 10. Pollom EL, Bui TT, Chang ALS, et al. Concurrent vismodegib and radiotherapy for recurrent, advanced basal cell carcinoma. JAMA Dermatol. 2015;151(9):998-1001. 11. Aasi S, Silkiss R, Tang JY, et al. New onset of keratoacanthomas after vismodegib treatment for locally advanced basal cell carcinomas: a report of 2 cases. JAMA Dermatol. 2013;149(2):242-243. 12. Papastefanous VP, René C. Secondary resistance to vismodegib after initial successful treatment of extensive recurrent periocular basal cell carcinoma with orbital invasion. Ophthal Plast Reconstr Surg. 2015. Epub ahead of print. 13. Ozgur OK, Yin V, Chou E, et al. Hedgehog pathway inhibition for locally advanced periocular basal cell carcinoma and basal cell nevus syndrome. Am J Ophthalmol. 2015;160(2):220-227.e2. 14. Gould SE, Low JA, Marsters JC Jr, et al. Discovery and preclinical development of vismodegib. Expert Opin Drug Discov. 2014;9(8):969-684. 15. Xin M. Hedgehog inhibitors: a patent review (2013 - present). Expert Opin Therap Pat. 2015;25(5):549-565. 16. Geeraert P, Williams JS, Brownell I. Targeting the hedgehog pathway to treat basal cell carcinoma. J Drugs Dermatol. 2013;12(5):519-523. 17. Chang AL, Solomon JA, Hainsworth JD, et al. Expanded access study of patients with advanced basal cell carcinoma treated with the Hedgehog pathway inhibitor, vismodegib. J Am Acad Dermatol. 2014;70(1):60-69. 18. Ridky TW, Cotsarelis G. Vismodegib resistance in basal cell carcinoma: not a smooth fit. Cancer Cells. 2015;27(3):315-316. 19.Robarge KD, Brunton SA, Castanedo GM, et al. GDC-0449: a potent inhibitor of the hedgehog pathway. Bioorg Med Chem Lett. 2009;19(19):5576-5581. 20. Proctor AE, Thompson LA, O’Bryant CL. Vismodegib: an inhibitor of the hedgehog signaling pathway in the treatment of basal cell carcinoma. Ann Pharmcother. 2014;48(1):99-106. 21. Graham RA, Lum BL, Morrison G, et al. A single dose mass balance study of the Hedgehog pathway inhibitor vismodegib (GDC-0449) in humans using accelerator mass spectrometry. Drug Metab Dispos. 2011;39(8):1460-1467. 22. Graham RA, Hop CE, Borin MT, et al. Single and multiple dose intravenous and oral pharmacokinetics of the hedgehog pathway inhibitor vismodegib in healthy female subjects. Br J Clin Pharmacol. 2012;74(5):788-796. 23. Vismodegib [package insert]. South San Francisco, CA: Genentech; 2012. 24.Von Hoff DD, LoRusso PM, Rudin CM, et al. Inhibition of hedgehog pathway in advanced basal cell carcinoma. N Engl J Med. 2009;361(12);1164-1172. 25. LoRusso PM, Rudin CM, Reddy JC, et al. Phase 1 trial of Hedgehog pathway inhibitor vismodegib (GDC-0449) in patients with refractory, locally advanced or metastatic solid tumors. Clin Cancer Res. 2011;17(8):2502-2511. 26. Tang JY, Mackay-Wiggans JM, Aszterbaum M, et al. Inhibiting the hedgehog pathway in patients with basal cell nevus syndrome. N Engl J Med. 2012;366(23):2180-2188. 27. Sekulic A, Migden MR, Lewis K, et al; ERIVANCE BCC Investigators. Pivotal ERIVANCE basal cell carcinoma (BCC) study: 12-month update of efficacy and safety of vismodegib in advanced BCC. J Am Acad Dermatol. 2015;72(6):1021-1026. 28. Basset-Sequin N, Hauschild A, Grob JJ, et al. Vismodegib in patients with advanced basal cell carcinoma (STEVIE): a pre-planned interim analysis of an international, open-label trial. Lancet Oncol. 2015;16(6):729-736. 29.Kahana A, Worden FP, Elner VM. Vismodegib as eye-sparing adjuvant treatment for orbital basal cell carcinoma. JAMA Ophthalmol. 2013;131(10):1364-1366. 30. Peris K, Licitra L, Ascierto PA, et al. Identifying locally advanced basal cell carcinoma eligible for treatment with vismodegib: an expert panel consensus. Future Oncology. 2015;11(4):703-712. 31. Edge S, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual. 7th ed. New York: Springer; 2010: 523-530. 32. Poulalhon N, Dalle S, Balme B, Thomas L. Fast-growing cutaneous squamous cell carcinoma in a patient treated with vismodegib. Dermatology. 2015;230(2):101-104. 33. Saintes C, Saint-Jean M, Brocard A, et al. Development of squamous cell carcinoma into basal cell carcinoma under treatment with vismodegib. J Eur Acad Dermatol Venereol. 2015;29(5):1006-1009. 34. Atwood SX, Li M, Lee A, et al. GLI activation by atypical protein kinase C i/ʎ regulates the growth of basal cell carcinomas. Nature. 2013;28:494(7438):484-488. 35.Sharpe HJ, Pau G, Dijkgraaf GJ, et al. Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma. Cancer Cell. 2015;9(3):27:327-341. 36. Zhao J, Quan H, Zie C, Lou L. NL-103, a novel dual-targeted inhibitor of histone deacetylases and hedgehog pathway, effectively overcomes vismodegib resistance conferred by Smo mutations. Pharmacol Res Perspect. 2014;2(3):e00043. 37. Ash MM, Jolly PS. Cholestatic hepatic injury associated with vismodegib, aspirin, naproxen use: a case study and review of vismodegib April 2016, Vol. 23, No. 2 safety. Int J Dermatol. 2015;54(3):370-374. 38. Ventarola DJ, Silverstein DI. Vismodegib-associated hepatotoxicity: a potential side effect detected in postmarketing surveillance. J Am Acad Dermatol. 2014;71(2):397-398. 39. Zhang Y, Baicker K, Newhouse JP. Geographic variation in Medicare drug spending. N Engl J Med. 2010;363(5):405-409. 40. Acosta A, Ciapponi A, Aaserud M, et al. Pharmaceutical policies: effects of reference pricing, other pricing and purchasing policies. Cochrane Database Syst Rev. 2014;10:CD005959. 41. Morley M, Finger PT, Perlin M, et al. Cis-platinum for ocular basal cell carcinoma. Br J Ophthalmol. 1991;75(7):407-410. 42. Luxenberg MN, Guthrie TH Jr. Chemotherapies of eyelid and periorbital tumors. Trans Am Ophthalmol Soc. 1985;83:162-180. 43. Luxenberg MN, Gutherie TH Jr. Chemotherapy of basal cell and squamous cell carcinoma of the eyelids and periorbital tissues. Ophthalmology. 1986;93(4):504-510. 44. Smith CM, Reynard AM, eds. Textbook of Pharmacology. Philadelphia, PA: WB Saunders; 1992. 45. [No authors listed]. Vismodegib (ERIVEDGE) in basal cell carcinoma: too many unknowns. Prescrire Int. 2015;24(156):11-14. 46. French DD, Margo CE, Campbell RR. Enhancing post marketing surveillance: continuing challenges. Br J Clin Pharmacol. 2015;80(4):615-617. Cancer Control 139 Evidence is insufficient to recommend chemotherapy, immunotherapy, or antibiotics as initial treatment for localized extranodal marginal zone B-cell lymphoma of the ocular adnexa. Photo courtesy of Lynn E. Harman, MD. Los Angeles. Extranodal Marginal Zone B-cell Lymphoma of the Ocular Adnexa Jean Guffey Johnson, MD, Lauren A. Terpak, MS, Curtis E. Margo, MD, and Reza Setoodeh, MD Background: Low-grade B-cell lymphomas located around the eye present unique challenges in diagnosis and treatment. Extranodal marginal zone B-cell lymphoma is the most common lymphoma of the ocular adnexa (conjunctiva, orbit, lacrimal gland, and eyelid). Methods: A systematic search of the relevant literature was performed. Material pertinent to the diagnosis, prognosis, pathogenesis, and treatment of extranodal marginal zone B-cell lymphoma of the ocular adnexa was identified, reviewed, and analyzed, focusing on management strategies for primary localized disease. Results: The primary cause of extranodal marginal zone B-cell lymphoma of the ocular adnexa remains elusive, although an infectious agent is suspected. Radiotherapy is the most common initial treatment for localized disease. Initial treatment with chemotherapy, immunotherapy, and antibiotics has shown promising results, but the number of series is limited and controlled trials do not exist. Conclusions: Although the long-term outcome of localized extranodal marginal zone B-cell lymphoma of the ocular adnexa is good, optimal treatment remains a goal. The variation in rates of local and systemic relapse among treated stage 1E tumors suggests that critical factors affecting outcomes are not fully understood. Radiotherapy is the standard of care; at this time, the evidence is insufficient to recommend chemotherapy, immunotherapy, or antibiotics for initial treatment of extranodal marginal zone B-cell lymphoma localized to the ocular adnexa. Well-controlled comparative studies are needed. Introduction Lymphomas are the most common malignancy of the From the Department of Pathology (JGJ, RS), James Haley Veterans Hospital, Departments of Ophthalmology and Pathology and Cell Biology (CEM), Morsani College of Medicine (LAT), University of South Florida, Tampa, Florida. Address correspondence to Curtis E. Margo, MD, Department of Ophthalmology, MDC Box 21, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612. E-mail: [email protected] Submitted July 27, 2015; accepted October 5, 2015. No significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. The authors discuss unlabeled/unapproved uses of doxycycline and clarithromycin for extranodal marginal zone B-cell lymphoma of the ocular adnexa. 140 Cancer Control orbit and lacrimal gland, ranking third behind squamous cell carcinoma and melanoma among the malignancies of the conjunctiva.1-3 The eyelid is an uncommon site of primary lymphoma and is more often secondarily involved when tumors spread from the conjunctiva or orbit.4,5 The incidence of ocular adnexal lymphoma has increased in recent decades, primarily due to an increase in extranodal marginal zone B-cell lymphoma of the mucosal-associated lymphoid tissue (MALT) type.1,4,6,7 Most types of lymphoma have been reported in periocular tissues, of which 95% or more are B cell in origin; extranodal marginal zone B-cell lymphoma is the most frequent, making up approximately 70% of cases, followed by follicular lymphoma and diffuse large B-cell lymphoma.5,8-15 Extranodal April 2016, Vol. 23, No. 2 marginal zone B-cell lymphoma of the ocular adnexa shares similar features with extranodal marginal zone B-cell lymphoma located elsewhere, including general morphology and immunophenotype, and, presumptively, pathogenesis. Extranodal marginal zone B-cell lymphoma of the ocular adnexa is a presumed antigendriven neoplasm based on the model of gastric extranodal marginal zone B-cell lymphoma. The putative antigenic stimulus (or stimuli) is a subject of investigation. We provide a general overview of extranodal marginal zone B-cell lymphoma of the ocular adnexa, emphasizing key similarities and differences in biological behavior and management with other MALT-type lymphomas in different locations. Because most cases of extranodal marginal zone B-cell lymphoma of the ocular adnexa present as localized disease, this subset of patients with lymphoma will be the focus of clinical management in this article. Therapeutic studies were included if the researchers examined single treatment protocols for localized extranodal marginal zone B-cell lymphoma of the ocular adnexa (Ann Arbor stage 1E). To avoid omitting studies containing valuable clinical information on the treatment of localized extranodal marginal zone B-cell lymphoma, inclusion criteria allowed series with up to 15% of cases with disease located at other sites as long as outcomes were not collectively reported. Diagnosis The histological features of extranodal marginal zone B-cell lymphoma are similar to those of MALT-type lymphomas in general. Neoplastic lymphocytes consist of varying combinations of small cells resembling centrocytes, plasmacytoid lymphocytes, and monocytoid B cells, with fewer numbers of scattered large cells resembling centroblasts or immunoblasts. This heterogeneous population of cells is usually found among reactive follicles, some of which may eventually be overrun by lymphoma cells (follicular colonization). Plasma cells are prominent in some cases, and intranuclear pseudoinclusions (Dutcher bodies) are sometimes noted. Characteristic infiltration of the epithelium by neoplastic lymphocytes (lymphoepithelial lesion) is uncommon, and, when observed, it is found in the lacrimal gland and conjunctiva but not the orbit.16 However, collections of atypical lymphocytes in conjunctival epithelium and lacrimal ducts similar to lymphoepithelial lesions are not specific for extranodal marginal zone B-cell lymphoma. They have also been described in reactive processes of the conjunctiva and lacrimal gland.16 Monocytoid cells with abundant pale cytoplasm are observed less often in extranodal marginal zone B-cell lymphoma of the ocular adnexa than other sites.11 Transformation to diffuse large B-cell lymphoma should be considered when solid or sheet-like proliferations of large cells are encounApril 2016, Vol. 23, No. 2 tered. These larger cells are usually positive for B-cell chronic lymphocytic leukemia/lymphoma (BCL) 6 and sometimes cluster designation (CD) 10.17 Given the morphological overlap between reactive lymphoid hyperplasia and extranodal marginal zone B-cell lymphoma, ancillary tests are often used to enhance diagnostic certainty. Extranodal marginal zone B-cell lymphomas are positive for CD20, BCL2, paired box 5 (PAX5), and CD79A but typically do not express CD5, CD10, or CD23.16 Fewer than 5% of cases express CD5.11-14,16 In the setting of CD5 positivity, the differential diagnosis might include mantle cell lymphoma (positive for cyclin D1, t[11;14] present) and small lymphocytic lymphoma (positive for CD23).14 CD43 expression is less common in extranodal marginal zone B-cell lymphoma of the ocular adnexa compared with the salivary glands (12%–25% vs 70%).11,18 Lymphoma cells typically express immunoglobulin (Ig) M and are IgD negative; on average, the proliferation index is 15% by Ki-67 immunostaining.19 The immunohistochemical demonstration of light-chain restriction using formalin-fixed, paraffinembedded tissue is challenging because detection of cytoplasmic light chains in nonplasmacytic proliferations is prone to false-negative testing.20-22 An alternative approach to demonstrating light-chain restriction is flow cytometry, which requires fresh tissue.23,24 Flow cytometry may also be useful in confirming immunophenotype, particularly when the size of a specimen is limited.23,24 Recurrent structural genetic abnormalities have been identified in MALT-type lymphomas, and the frequency of these alterations vary, depending on anatomical site.25 Although it is not specific, trisomy of chromosome 3 is the most commonly reported cytogenetic finding among MALT-type lymphomas.25 Among the common sites of MALT lymphomas, t(14;18)(q32;q21) involving IgH and MALT1 is the most frequently seen in the ocular adnexal MALTomas.26 This translocation has been reported in about 25% of cases in some series.26 Clonality of B cells can be documented by polymerase chain reaction (PCR) of Ig heavy chain.27,28 More centers are employing combinations of immunohistochemistry, flow cytometry, and PCR to diagnosis low-grade lymphoma and relying less on morphology alone.29 Clinical Staging The approach to staging ocular adnexal lymphoma is similar to that for lymphoma in general, and typically includes thorough clinical and laboratory examinations with bone marrow biopsy.30 Although computed tomography (CT) or magnetic resonance imaging (MRI) with contrast is valuable to determining the extent of local disease of the orbit, eyelid, and paranasal sinuses, positron emission tomography (PET) may be superior for the initial staging of ocular adnexal lymCancer Control 141 phoma.31 When compared with CT, use of PET has upstaged a majority of patients with ocular adnexal lymphoma.32,33 Evidence suggests that subclinical involvement of the eye can occur in persons with periocular lymphoma.34,35 In a study of ocular adnexal lymphomas, 25 study patients with bilateral involvement had uveal thickening with ocular B-scan ultrasonography.34 (Intraocular disease might have been missed if staging were based on CT alone.34) Although uveal involvement with extranodal marginal zone B-cell lymphoma has been previously reported, few studies have documented how often it goes unrecognized when patients present with conjunctival and orbital disease.34,35 How uveal involvement might affect prognosis is also unclear. Roughly two-thirds of ocular adnexal lymphomas are stage 1E (localized extranodal tumors) using the Ann Arbor system.8,36,37 This disproportionate accumulation of cases in a single stage limits the discriminatory potential to forecast outcomes. Other shortcomings exist to the Ann Arbor system for ocular adnexal lymphoma, including the inability to take into account multicentric and bilateral tumors, extent of localized tissue involvement, and precise anatomical location around the eye.38 Application of the tumor, node, metastasis (TNM)–based system incorporating information on extent of disease within anatomical compartments of the ocular adnexa, bilateral nature, and information on regional lymph-node involvement may better predict outcome (Table 1).39-42 An initial study showed that tumor stage may more thoroughly describe the extent of disease, although it does not yet predict rate of relapse or survival.41 Stages N1 to N4 and M1 are associated with worse rates of survival.41 In a multicenter study using the TNM system, any prognostic utility ascribed to size and location of ocular adnexal lymphoma was eclipsed by histological classification and type of treatment.42 The capability of the TNM system to predict worse outcomes appears to reside in its ability to identify bilateral disease and to delineate nodal and metastatic involvement at the time of presentation.39,40 The full potential of the TNM system may not be realized until it is modified to include additional biomarkers and when 142 Cancer Control Table 1. — TNM Staging for Lymphoma of the Ocular Adnexa Clinical Stage TX Primary Tumor Pathological Stage Lymphoma extent not specified TX T0 No evidence of lymphoma T0 T1 Lymphoma involving the conjunctiva alone without orbital involvement T1 T1a Bulbar conjunctiva only T1a T1b Palpebral conjunctiva ± fornix ± caruncle T1b T1c Extensive conjunctival involvement, both bulbar and nonbulbar T1c T2 Lymphoma with orbital involvement ± any conjunctival involvement T2 T2a Anterior orbital involvement (± conjunctival involvement) but no lacrimal gland involvement T2a T2b Anterior orbital involvement (± conjunctival involvement) plus lacrimal involvement T2b T2c Posterior orbital involvement (± conjunctival involvement ± anterior orbit involvement and ± any extraocular muscle involvement) T2c T2d Nasolacrimal drainage system involvement (± conjunctival involvement but not including nasopharynx) T2d T3 Lymphoma with preseptal eyelid involvement ± orbital involvement ± any conjunctival involvement T3 T4 Orbital adnexal lymphoma extending beyond orbit to adjacent structures such as bone and brain T4 T4a Involvement of nasopharynx T4a T4b Osseous involvement (including periosteum) T4b T4c Involvement of maxillofacial, ethmoidal, and/or frontal sinuses T4c T4d Intracranial spread T4d Regional Lymph Nodes NX Regional lymph nodes cannot be assessed NX N0 No evidence of lymph node involvement N0 N1 Involvement of ipsilateral regional lymph nodes (preauricular, parotid, submandibular, and cervical) N1 N2 Involvement of contralateral or bilateral regional lymph nodes N2 N3 Involvement of peripheral lymph nodes not draining; ocular adnexal regional N3 N4 Involvement of central lymph nodes N4 M0 No evidence of involvement of other extranodal sites (no pathologic M0; use clinical M to complete stage group) M0 M1a Noncontiguous involvement of tissues or organs external to the ocular adnexa (eg, parotid glands, submandibular gland, lung, liver, spleen, kidney, breast) M1a M1b Lymphoma involvement of the bone marrow M1b M1c Both M1a and M1b M1c Distant Metastasis TNM = tumor, node, metastasis. Adapted from Edge S, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual. 7th ed. New York: Springer; 2010. Republished with permission of Springer; permission conveyed through Copyright Clearance Center, Inc. April 2016, Vol. 23, No. 2 new imaging technologies can be used to help provide superior metrics in terms of tumor location and volume. Pathogenesis Infectious agents have been suspected in the etiology of extranodal marginal zone B-cell lymphoma of the ocular adnexa based on the causative role that Helicobacter pylori infection plays in gastric MALT-type lymphoma.43 The progression of H pylori–induced chronic gastritis to extranodal marginal zone B-cell lymphoma is comprehensible both in terms of light microscopy and molecular changes.43 Unlike malignant transformation of lymphocytes through the direct infection of lymphotropic viruses like Epstein–Barr virus or human T-cell leukemia virus type 1, H pylori indirectly acts by inducing chronic inflammation. This antigen-driven process, coupled with other oncogenic events, can lead to the emergence of antigen-independent lymphocyte proliferation. Chlamydia psittaci The search for putative infectious agents for periocular lymphoma has resulted in conflicting results.44-47 Using PCR, an Italian study found that 80% of patients with extranodal marginal zone B-cell lymphoma of the ocular adnexa were exposed to Chlamydia psittaci.46 However, similar investigations from other regions of the world could not duplicate these results.44,47 In a review of 11 international studies, the overall rate of Chlamydia positivity in extranodal marginal zone B-cell lymphoma of the ocular adnexa was 23%, with the vast majority of all positive cases (90%) occurring in 3 countries.47 In a survey of 423 cases of extranodal marginal zone B-cell lymphoma of the ocular adnexa, the detection rate of Chlamydia ranged from 0% to 87%, with seemingly inconsistent geographic variation.44 To control for spurious laboratory results, investigators used a single standardized procedure for Chlamydia DNA detection among 142 cases of extranodal marginal zone B-cell lymphoma of the ocular adnexa.45 The results confirmed a variation in prevalence (eg, 11% in southern China, 47% in Germany); the overall prevalence of C psittaci DNA was 22%, which was more than twice the control group (10%; P = .04).45 However, based on historical prevalence, this result was not much greater than that established for non-neoplastic disorders of the orbit.44 Helicobacter pylori The causal association between H pylori infection and gastric extranodal marginal zone B-cell lymphoma is well established, so investigators have begun searching for a link between the bacterium and extranodal marginal zone B-cell lymphoma of the ocular adnexa.48,49 Chan et al48 described H pylori DNA in 4 of April 2016, Vol. 23, No. 2 5 cases of conjunctival extranodal marginal zone B-cell lymphomas using PCR amplification and Southern blot hybridization. The DNA of H pylori was not found in normal conjunctiva from the same study patients.48 The same group examined 8 study patients with orbital extranodal marginal zone B-cell lymphoma and found 1 study patient with the genomic fingerprints of H pylori.49 C pneumoniae (but not C psittaci or C trachomatis) was identified in another.49 Lee et al 5 0 identi f ied DNA of H pylor i i n 15 of 15 cases of extranodal marginal zone B-cell lymphoma of the conjunctiva that they examined; no such discovery was made in the control group (n = 8). These findings contrast with another study involving 13 cases of extranodal marginal zone B-cell lymphoma of the conjunctiva in which H pylori DNA could not be detected in any tumor (either using immunohistochemistry or PCR).51 The prevalence of gastric H pylori infection at the time of initial diagnosis of extranodal marginal zone B-cell lymphoma varies between where the primary site of lymphoma is located.52 For example, the prevalence rate of infection was found to be 45% (37 of 83 cases) in extranodal marginal zone B-cell lymphoma of the ocular adnexa compared with 25% (25 of 101 cases) in extranodal marginal zone B-cell lymphoma located elsewhere (but not the stomach).52 The rate of prevalence was 12% (18 of 156) among control cases without lymphoma.52 These findings led investigators to propose another pathogenic mechanism involving the attraction of circulating lymphomatous cells to the ophthalmic mucosa: Once around the eye, the lymphocytes would then be transformed under the influence of additional mitogenic stimuli.52 This study and the proposed hypothesis has generated need for further investigation.52,53 Other Infectious Agents An international study investigating the presence of viral DNA for herpes simplex virus types 1 and 2 and adenovirus types 8 and 19 in persons with extranodal marginal zone B-cell lymphoma of the ocular adnexa found no association.45 Although the conceptual model of an infectious etiology is appealing, consistent and reproducible evidence supporting any specific pathogen has been elusive.54,55 Autoimmunity The possibility of an autoantigenic stimulus is supported by the association of extranodal marginal zone B-cell lymphoma of the ocular adnexa with various autoimmune disorders (eg, Sjögren disease, Hashimoto disease, IgG4-related disorder).56 The connection between Sjögren disease and lymphoma has been known since the 1970s.57 Although Sjögren disease conveys a 14-fold greater risk of B-cell lymphoma, the cause for Cancer Control 143 this susceptibility is unclear.57 Several cases of extranodal marginal zone B-cell lymphoma of the ocular adnexa have been reported in persons with IgG4-related disease — an association that provides little additional insight into pathogenesis.58 Treatment A comprehensive review of treatment options for extranodal marginal zone B-cell lymphoma of the ocular adnexa is beyond the scope of this review. Most therapeutic studies have consisted of retrospective case series.30,58-79 Comparing clinical outcomes in these nonrandomized trials is perilous for several reasons. Clinical series of ocular adnexal lymphoma published prior to 2000 often lumped extranodal marginal zone B-cell lymphoma together with other low-grade lymphomas and atypical lymphoid hyperplasia.5 Even large studies involving 2 or more treatment arms had limited statistical power to exclude small but meaningful differences in outcome.5,8,15,30 The success of local therapy is often confounded by supplemental treatment given to partial responders because clinical outcomes are collectively reported.5,8,15,30 In addition, short-term studies (< 5 years) may also not adequately reflect the biological potential of the disease, particularly in terms of rates of local and systemic recurrence, and tumor-related mortality. Radiotherapy Typically, radiotherapy results in a high rate of local control that ranges from 85% to 100%, even in the presence of systemic disease.59,60,62,64,73,75,76,79-83 Since 2000, a total of 9 studies have examined the outcome of radiotherapy for localized extranodal marginal zone B-cell lymphoma of the ocular adnexa (Table 2).64,70,73,79-84 Some included small numbers of study patients with systemic disease, and clinical outcomes were measured in ways that prohibit uniform summarization. The majority of the 503 study patients with stage 1E disease achieved local control.64,70,73,79-84 When reported, complete response (CR) rates ranged from 52% to 93%, whereas the proportion of 5-year, systemic-free relapse rates usually exceeded 90%.64,70,73,79-84 Overall, systemic relapses occurred in 31 study patients (6.2%), ranging from a low rate of 2.2% (median follow-up of 32 months) to a high rate of 16.8% (median follow-up of 5.9 years).64,70,73,79-84 However, these results must be interpreted with caution because the methods and rigor used to detect recurrent disease differed and the protocols employed to treat partial response and recurrence varied. The different types of second-line therapies for incomplete localized responses confound the direct comparison of clinical series, particularly in terms of rates of systemic recurrence and tumor-related deaths. 144 Cancer Control Chemotherapy Use of chemotherapy for local ocular adnexal disease has been studied in 2 centers (n = 54); of the patients studied, 52 had stage 1E disease (Table 3).61,67-69 Among 21 study patients (19 [90%] with stage 1E) treated with cyclophosphamide, vincristine, and prednisolone, CR was reported in 16 (76%), local relapse occurred in 5, and systemic relapse occurred in 2; no deaths were reported during a median follow-up time of 55 months.68 The other trial studied chlorambucil in 33 participants with stage 1E extranodal marginal zone B-cell lymphoma.69 During a median follow-up of 26 months, CR was reported in 26, local recurrence in 1, and systemic relapse in 3; 1 tumor-related death was noted.69 Proponents of initial chemotherapy state that this treatment option eliminates local complications from radiotherapy, such as dry eyes, cataracts, and skin irritation, and is associated with fewer serious complications.68 Direct comparison with radiotherapy for stage 1E disease is methodologically problematic, because some reports include small proportions of study patients with more advanced disease.68 The collective rate of systemic relapse for radiotherapy is 6.2%, which is less than that currently reported for chemotherapy (9.3%; see Tables 2 and 3).61,64,69,70,73,79-84 Immunotherapy Rituximab, a chimeric mouse antihuman CD20 monoclonal antibody, has been used as treatment for lymphoma localized to the ocular adnexa.74 Two reports involving 12 study patients with localized extranodal marginal zone B-cell lymphoma have been published (see Table 3).61,67-69 In one study, 11 eyes of 10 study patients with ocular adnexal lymphoma were treated with systemic rituximab.61 CR was reported in 5 (56%; median follow-up of 31 months) without systemic or ocular adverse events, and no tumor-related deaths were reported.61 The remaining study patients required radiotherapy.61 Another report involved 5 study patients with extranodal marginal zone B-cell lymphoma of the ocular adnexa, 3 of whom had stage 1E disease.67 Four of the 5 study patients who initially responded to treatment locally relapsed.67 To date, rituximab has been reported in relatively few patients with extranodal marginal zone B-cell lymphoma localized to the ocular adnexa, with seemingly high rates of inadequate periocular response and local recurrence (see Table 3).61,67-69 The median follow-up times (< 31 months) are too short to assess the role of rituximab as therapy for local disease at this time.61,67-69 Antibiotic Therapy Because some cases of extranodal marginal zone B-cell lymphoma of the ocular adnexa have been associated with C psittaci infection, treatment with doxycycline has been attempted.47 A meta-analysis of 4 treatment April 2016, Vol. 23, No. 2 Table 2. — Radiotherapy for Localized Extranodal Marginal Zone B-Cell Lymphoma of the Ocular Adnexaa Study Location Stage Cases of Systemic Relapse Local Relapseb Goda80 89 Conjunctiva: 59 Lacrimal gland: 20 Orbit: 10 1E: 89 Hashimoto70 78 Conjunctiva: 37 Orbit: 29 Lacrimal gland: 12 1E: 78 Le84 31 Conjunctiva: 21 Orbit: 10 1E: 31 Bilateral: 4 None Nam73 66 Conjunctiva: 29 Orbit: 20 Lacrimal gland: 7 Eyelid: 10 1E: 66 Bilateral: 14 Son82 46 Conjunctiva: 37 Eyelid: 3 Orbit: 6 Suh83 48 Tran81 15 Other Outcome Measure No. of TumorRelated Deaths 7-y relapse-free survival: 64% 3 66 mo 5-y PFS: 86% 10-y OS: 95.3% None 5 5.9 y (range, 0.75–20.3) 100% local control 1 Conjunctiva: 2 Orbit: 0 Lacrimal gland: 2 Eyelid: 2 3 50 mo (range, 12–114) 5-y systemic relapse-free survival: 95.4% None 1E: 46 Bilateral: 3 1 1 32 mo (range, 3–114) CR with 5-y relapse-free survival: 93% None “Orbit” 1E: 46 Bilateral: 4 IV: 2 3 None 39–72 mo 10-y relapsefree survival: 93% 1c 24 Conjunctiva: 16 Orbit: 3 Lacrimal gland: 7 Eyelid: 1 1E: 22 Bilateral: 3 IV: 2 2 1 41 mo (range, 5–137) PFS: 90% 5-y OS: 100%d 1 case of high-grade lymphoma Uno64 50 Conjunctiva: 29 Orbit: 17 Eyelid: 2 Lacrimal gland: 2 1E: 50 Bilateral: 7 3 3 46 mo (range, 10–140) CR: 26 PR: 20 No response: 4 1 Woolf 79 71 + 10 lowgrade lymphomas Orbit: 56 Conjunctiva: 14 Lacrimal gland: 11 Eyelid: 4 1E: 81 Bilateral: 4 None Distant: 3 Contralateral lacrimal gland (local): 1 4.4 y (range, 0.2–10.4) 100% local controle None 503 7 Median Follow-Up 5.9 y Totalf a No. of Study Patients 499 6 (1.2%) g Includes series since 2000 with 85% or more localized cases of stage 1E disease. bLocal relapse defined as any portion of ocular adnexa, including the contralateral side, of initial cTumor death occurred in 1 study patient with stage IV disease. dHigh-grade lymphoma death occurred at 71 mo. e Of the 81 original study patients, 71 (88%) had extranodal marginal zone B-cell lymphoma. f Data not sufficiently detailed to provide totals for local and systemic relapses. gPercentage based on study patients with stage 1E disease. unilateral disease. CR = complete response, OS = overall survival, PFS = progression-free survival, PR = partial response. trials taking place prior to 2006 totaling 42 study patients found that 20 responded to oral antibiotics, 20 remained stable, and 2 progressed.47 Although CR was reported in 8 study patients, objective responses with radiography or slit lamp photography were available in 3.47 Since 2006, researchers have initiated 3 trials to study the effect of doxycycline on the management April 2016, Vol. 23, No. 2 of predominantly primary extranodal marginal zone B-cell lymphoma localized to the ocular adnexa in 151 study patients (Table 4).76,78,85-87 Because the rate of tumor response (ie, reduction in size) differs following treatment with doxycycline and radiotherapy or chemotherapy, it is difficult to directly measure and compare clinical outcomes.78,85,87 One study reported a 2-year failure-free survival rate of 67%,85 whereas anCancer Control 145 Table 3. — Chemotherapy or Immunotherapy for Localized Extranodal Marginal Zone Lymphoma of the Ocular Adnexaa Study Treatment No. of Study Patients Ben Simon69 Chlorambucil 33 Orbit: 10 Lacrimal gland: 8 Conjunctiva: 7 Eyelid: 6 Combination: 2 Song68 Cyclophosphamide Vincristine Prednisolone 21 Conjunctiva: 6 Orbit: 8 Eyelid: 5 Lacrimal gland: 2 Adnexa (general): 2 Ferreri67 Rituximab 5 Conjunctiva: 2 Orbital: 1 Tuncer 61 Rituximab 9 Conjunctiva: 5 Bilateral: 1 Orbit: 4 Cases of Local Relapseb Cases of Systemic Relapse 1E: All 1 3 26 (range, 8–62) CR: 26 1 1E: 19 Bilateral: 2 IIE: 2 5 2 58 (range, 6–163) CR: 16 PR: 5 None 1E: 3 IV: 2 4 1 23 (range, 2–4) 1E: All 6 None 31 (range, 10–61) Location Stage Median FollowUp, mo Other Outcome Measures No. of TumorRelated Deaths Chemotherapy Immunotherapy aIncludes series with ≤ 2 study patients with disease beyond the orbit. bLocal relapse defined as any portion of ocular adnexa, including the contralateral CR = complete response, PR = partial response. None CR: 5 PR: 4 None side, of initial unilateral disease. Table 4. — Antibiotic Therapy or Observation for Extranodal Marginal Zone Lymphoma of the Ocular Adnexaa Study Treatment No. of Study Patients Ferreri85 Doxycycline 27 Conjunctiva: 14 Orbit: 13 1E: 24 IIE: 3 Bilateral: 5 Unable to determine local from systemic relapses Ferreri87 Doxycycline 34 Conjunctiva: 23 Orbit: 14 Lacrimal gland: 5 Conjunctiva/orbit: 5 1E: All 14 failures Unable to delineate local from systemic Han78 Doxycycline 90 Conjunctiva: 74 Orbit: 12 Eyelid: 3 Lacrimal gland: 2 T1N0M0: 62 T2N0–2 M0: 22 T3N0–2 M0: 5 T4N0M0: 1 Matsuo None other than biopsy 8 Tanimoto86 None other than biopsyc 36 Location Stage Cases of Cases Median of Local Systemic Follow-Up Relapseb Relapse Other Outcome Measures No. of TumorRelated Deaths 21 mo (range, 4–204) Overall response: 64% 2-y failure-free survival: 67% None 37 mo (range, 15–62) 5-y PFS: 55% None 40.5 mo (range, 8–85) 5-y PFS: 61% None 5.4 y (range, 1–11) 7 spontaneously regressed None 10.5 y (range, 0.7–16.7) OS: 5-y: 94% 10-y: 94% 15-y: 71% Antibiotic Therapy 31 6 Observation Without Treatment 76 c Conjunctiva: 8 1E: All Conjunctiva: 15 Orbit: 19 Lacrimal gland: 2 1E: 36 Bilateral: 5 None None 15 2 aIncludes series with ≥ 8 5% localized stage 1E extranodal marginal zone B-cell lymphoma since 2000. bLocal relapse defined as any portion of the ocular adnexa, including the contralateral side, of initial unilateral c Eight of 13 study patients who declined treatment and are the focus of this review. 2 disease. OS = overall survival, PFS = progression-free survival. 146 Cancer Control April 2016, Vol. 23, No. 2 other series reported that the failure-free survival rate after 5 years was 55%.87 Progressive disease was treated with a variety of traditional protocols, and no tumor-related deaths were reported (see Table 4).76,78,85-87 In that study, patients were excluded from the trial if they had large tumors or tumors demonstrating rapid growth.87 Because criteria describing large tumor size and rapid growth were not provided, an exclusion bias of unknown clinical importance complicates any interpretation of these results.87 Long-term follow-up in 1 study of 90 patients initially treated with doxycycline had mixed results.78 Patients enrolled in the clinical trial received twice-daily oral 100 mg doxycycline for either 3 or 6 weeks and were followed for a mean time of 40.5 months.78 A total of 61% had no progression after 5 years, and 34 study patients who failed doxycycline therapy were successfully managed with chemotherapy, radiotherapy, or both.78 No tumor-related deaths were reported; systemic relapse was reported in 6 study patients.78 Pretreatment exposure to C psittaci was not determined. “Failed to progress” reports are difficult to compare to studies whose benchmark outcomes are either complete or partial regression, and this is particularly true for cases of lymphoma with an indolent course. Lacking or incomplete knowledge of a history of chlamydial infection among patients enrolled in antibiotic studies further complicates interpretation, because the global variation in C psittaci infection rate should affect the response rate to doxycycline. Future trials of antibiotic treatment may be more efficient if their enrollment is appropriately limited to patients with documented exposure to C psittaci. A 6-month trial of a second-line therapeutic agent, clarithromycin, was reported in a trial of 7 study volunteers who had extranodal marginal zone B-cell lymphoma that failed to respond to doxycycline.88 Partial response was reported in 4 study patients and stable disease for varying periods of time was reported in 3; however, all study patients experienced disease progression (average time to progression, 16 months).88 Observation Given the indolent nature of extranodal marginal zone B-cell lymphoma of the ocular adnexa, some investigators have wondered whether observation may be a viable option, particularly in persons of advanced age or with other comorbidities that may limit survival.76,86 Two groups reported on the long-term follow-up data of 44 study patients with localized disease (5 bilateral); the median follow-up periods were 5.4 years76 and 10.5 years.86 Twenty-five (57%) study patients did not require treatment throughout the course of the study, and 2 succumbed to progressive lymphoma.76,86 A total of 17 study patients (39%) progressed; transformaApril 2016, Vol. 23, No. 2 tion to high-grade lymphoma occurred in 1 case.76,86 The majority of tumors arose in conjunctiva (23 [52%]), 7 of which spontaneously regressed.76,86 Therefore, the study authors concluded that watchful waiting might be an acceptable option in select patients with unilateral extranodal marginal zone B-cell lymphoma of the ocular adnexa.86 Assessment of Clinical Outcome Comparing the treatment of primary lymphoma localized to the ocular adnexa is difficult because some series combine different histological types of lymphoma, some researchers do not separately analyze for anatomical subgroups (eg, conjunctiva, orbit), and some report clinical outcomes differently. In addition, not all studies employ standardized intervals for followup or methods for monitoring outcome (eg, CT, MRI, PET/CT, PET). Few provide information on primary tumor size, and most employ different salvage therapies for partial responders. Tumor size is an important parameter to monitor because the rapidity and completeness with which a tumor shrinks may correlate with durability of response.89 However, the speed at which tumor size is reduced may be of less importance in cases of low-grade lymphoma compared with other neoplasms, and it may be of less value in terms of assessing antibiotic effectiveness. Use of tumor diameter — not volume — as an outcome variable for extranodal marginal zone B-cell lymphoma of the ocular adnexa reflects how difficult it is to obtain reliable volumetric data on periocular tumors in general. Measuring the volume or planar dimensions of periocular tumors presents unique challenges. For example, oftentimes, lymphomas of the ocular adnexa are irregularly shaped and conform to the contours of the eye and orbital walls. It might be valuable to apply the Response Evaluation Criteria in Solid Tumors (RECIST) and RECIST 1.1 to these lesions, but these criteria have not been independently validated in the context of low-grade lymphoma of the ocular adnexa.89 One of the few studies that addressed the role of quantitative, monitored outcomes in the setting of extranodal marginal zone B-cell lymphoma of the ocular adnexa following radiotherapy was performed by Jung et al.90 These researchers found that the maximum tumor diameter decreased by 50% of its initial size after 4.7 months of treatment.90 The authors emphasize that partial and total responses are usually poorly defined in most clinical series — both in terms of tumor-specific size reductions and in follow-up intervals after treatment.90 Conclusions Extranodal marginal zone B-cell lymphoma of the ocular adnexa can arise in the setting of reactive lymphoid Cancer Control 147 hyperplasia, but the underlying cause of this presumed antigen-driven process is unclear. Given the epidemiological results to date, a more complex interplay of putative infectious agents and immune response is possible. In terms of treatment options for primary localized extranodal marginal zone B-cell lymphoma of the ocular adnexa, radiotherapy provides good local control and is associated with relatively low systemic spread. Studies assessing the effectiveness of other initial treatment options for localized disease (eg, chemotherapy, immunotherapy, antibiotics) are limited by several weaknesses, including small study size, enrollment biases, and imbalanced mixtures of anatomical locations (ie, lacrimal gland, conjunctiva, eyelid, orbit). The evidence is insufficient at this time to recommend an alternative to radiotherapy for primary localized extranodal marginal zone B-cell lymphoma of the ocular adnexa. As more optimal therapies are sought and randomized clinical trials remain unlikely, future investigations might consider use of methodologies to volumetrically measure tumor response, to establish standardized protocols for initial evaluation and follow-up, and to use equivalent rescue protocols for partial responses so that different clinical series can be compared against one another. References 1. Margo CE, Mulla ZD. Malignant tumors of the orbit: analysis of the Florida Cancer Registry. Ophthalmology. 1998;105(1):185-190. 2. Bonavolontà G, Strianese D, Grassi P, et al. An analysis of 2,480 space-occupying lesions of the orbit from 1976 to 2011. Ophthal Plast Reconstr Surg. 2013;29(7):79-86. 3. Shields JA, Shields CL, Scartozzi R. Survey of 1264 patients with orbital tumors and simulating lesions: the 2002 Montgomery Lecture, part 1. Ophthalmology. 2004;111(5):997-1008. 4. Margo CE, Mulla ZD. 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Jung SK, Paik JS, Jung SE, et al. Suggestion of response evaluation criteria in patients with ocular adnexal mucosa-associated lymphoid tissue lymphoma (OAML). Am Hematol. 2015;94(7):1185-1193. Cancer Control 149 Recognizing ocular complications of chemotherapy before they result in irreversible injury involves taking a clinical history and performing a basic eye examination. Photo courtesy of Lynn E. Harman, MD. Point Reyes Beach North. Ophthalmic Complications Related to Chemotherapy in Medically Complex Patients Lynn E. Harman, MD Background: Systemic cancer therapies cause a variety of ophthalmic complications. Mitigating harmful adverse events involves screening patients at risk for ocular injury and vision loss. Methods: A review of the relevant literature on the ophthalmic complications of cancer therapy was used to formulate an approach to screening patients for serious complications presenting at a nonophthalmic specialty center. Results: Rarely, ocular complications of cancer therapy can occur. Establishing a causal association for any given agent is complicated because many treatment-related adverse events result in symptoms and ocular findings indistinguishable from primary eye disorders. Conclusions: Recognizing potentially serious ocular complications of cancer therapy before they result in irreversible injury starts with taking a relevant clinical history and performing a basic eye examination, including assessments of visual acuity and fields. Given the wide range of treatment-related adverse events and the challenges of diagnosis, the screening process plays an important role in expediting referral to an ophthalmologic specialist. Introduction The majority of published research about the ocular complications of cancer therapy reviews the adverse events of individual chemotherapies and targeted therapies.1-7 Cataloging these adverse events can be approached in several ways. One method is anatomical, in which the therapeutic agents are listed according to the parts of the eye or ocular adnexa they affect (eg, conFrom the Department of Ophthalmology, University of South Florida Morsani College of Medicine, and Section of Ophthalmology, James Haley Veterans Affairs Hospital, Tampa, Florida. Address correspondence to Lynn E. Harman, MD, James Haley Veterans Affairs Hospital, Eye Clinic, 13000 Bruce B. Downs Boulevard, Tampa, FL 33612. E-mail: [email protected] Submitted July 14, 2015; accepted October 29, 2015. No significant relationship exists between the author and the companies/organizations whose products or services may be referenced in this article. 150 Cancer Control junctiva, lens, retina),1 whereas another method lists therapies according to the type of injury they cause (eg, punctate keratitis, sterile uveitis, macular edema).4 It is also possible to describe ocular toxicities and adverse events by pharmacological group (eg, plant alkaloids, antimetabolites, nitrosoureas) or by individual agent.2,3 Such surveys are invaluable when attempting to establish whether causal links exist between a particular ocular condition and a therapeutic agent; however, these inventories do not address clinical questions about how to manage the onset of a suspected event. Uncertainty sometimes exists about the seriousness of an ocular symptom or a new finding on an eye examination if it occurs in patients receiving chemotherapy. For those who are hospitalized or too ill to be seen at an ophthalmology center, the situation can be disconcerting. Few adverse drug reactions are characteristic enough to diagnose with absolute certainty on April 2016, Vol. 23, No. 2 an abbreviated examination, and confirming whether an adverse event is drug related with a drug rechallenge is usually not an option. Consultative input is desirable even when it may be logistically difficult. In this article, the term ocular is inclusively used to describe the eye, visual pathway, and ocular adnexa. More than 60 distinct ocular-related adverse events have been associated with systemic chemotherapies and targeted cancer therapies.1-7 These events vary from finding asymptomatic deposits in the retina to cataracts, cranial nerve palsies, to keratoconjunctivitis sicca. Most of these complications have clinical features that overlap with acquired diseases.1,4,5 Treatment-related adverse events range from being selflimited conditions to permanent vision loss.1,4,5 The diversity of these complications speaks to the variety of molecular and cellular mechanisms involved. Thus, this review will focus on evaluating ocular complaints occurring in the context of medically complex treatment and when it is difficult for patients to be seen at a specialty eye clinic. This paper will also provide a general approach for screening patients outside the setting of an eye clinic in order to expedite consultation with an eye specialist and reduce unnecessary referrals. It will also address when to anticipate, and possibly preemptively reduce, some common ocular-related adverse events associated with select chemotherapy agents. Given the vastness of the topic, this paper will not include complications of radiotherapy, therapies for primary cancers of the eye and ocular adnexa, or the ocular manifestations of graft-vs-host disease. Although selected examples of adverse events are cited to emphasize elements of the ocular history and eye examination, they are not meant to represent a comprehensive directory of complications. Incidence The proportion of patients who experience a serious adverse event from chemotherapy is low. Most of these complications are considered rare or reported only as single case reports.1-7 A 2006 comprehensive review found that 57 different ocular-related (ie, eye, visual pathway, ocular adnexal) complications could be attributed to cytotoxic chemotherapies (Table 1).4 A total of 38% of drug-specific complications were deemed to be rare and another 46% were identified through case reports.4 A total of 15% of drug-specific complications were considered to be somewhat common and 1.4% were noted to be common.4 The low overall proportion of adverse events comes with a clinical caveat1-7: The event rate is probably lower than anticipated for “incidental” (or unrelated) findings on routine eye examinations. Common conditions such as uncorrected presbyopia, refractive errors, dry eyes, chronic blepharitis, and age-related cataracts can April 2016, Vol. 23, No. 2 Table 1. — Somewhat Commonly Occurring Adverse Events of Cytotoxic Chemotherapies Adverse Event Drug Route of Administration Arteriovenous shunts Carmustine (central nervous system) Intra-arterial Blurred vision Busulfan Intravenous 5-fluorouracil Intravenous 5-fluorouracil Intravenous Deoxycoformycin Intravenous Corneal opacity Cytosine arabinoside Intravenous Cranial nerve palsies Plant alkaloids Intravenous Epiphora 5-fluorouracila Intravenous Eye pain 5-fluorouracil Intravenous Focal demyelination of the optic nerve Carmustine/cisplatin Intra-arterial Foreign body sensation Cytosine arabinoside Intravenous 5-fluorouracil Intravenous Keratitis Cytosine arabinosidea Intravenous Conjunctivitis 5-fluorouracil Intravenous Keratoconjunctivitis sicca Cyclophosphamide Intravenous Busulfan Intravenous Macular pigment Cisplatin Intravenous Papilledema Carmustine Intra-arterial Periorbital edema 5-fluorouracil Intravenous Methotrexate Intravenous Photophobia 5-fluorouracil Intravenous Ptosis Plant alkaloids Intravenous Retinal arterial narrowing Carmustine Intra-arterial Retinal hemorrhages Carmustine Intra-arterial aConsidered common. Adapted from Schmid KE, Kornek GV, Scheithauer W, et al. Update on ocular complications of systemic cancer chemotherapy. Surv Ophthalmol. 2006;51(1):19-40, with permission from Elsevier. all cause symptoms that could be mistakenly attributed to chemotherapy if the clinician has not taken a careful clinical history and performed a basic eye examination. Clinicians who do not specialize in ophthalmology are not typically expected to perform a complete eye examination, nor are they usually familiar with the nuances of minor, pre-existing eye conditions. This emphasizes the importance of a clinical history and basic tests of visual function (with corrective lenses) to screen for potentially serious problems. Attributing an ocular complication to a therapeutic agent is a process of exclusion. Some of the most challenging diagnoses in ophthalmology arise in the context of cancer treatment; for example, the distinction between sterile drug-related uveitis and infectious endophthalmitis (typically when a patient is immunocompromised), as well as the distinction between Cancer Control 151 drug-related cranial nerve palsy and metastatic cancer or an age-related vascular event. Complaints The ophthalmic complications of systemic cancer therapy can cause some of the same symptoms seen with primary eye diseases. They can include vision loss (central, peripheral [or both], night blindness), abnormal visual perceptions (distorted vision, smoky vision, double vision, floaters), and all degrees of eye and periocular pain, among other issues.1-7 The close temporal relationship of new-onset eye complaints with the initiation of cancer therapy provides presumptive evidence of a causal link. This chronological correlation, along with the cessation of symptoms after stopping the offending agent, may be the most compelling evidence of causation. Vision Loss/Impairment Patients who have difficulty describing their visual loss should be queried as to whether the loss is unilateral or bilateral, whether it occurs with or without pain, and whether it was preceded by or associated with other symptoms. If it was transient, then the clinician should ask the patient how long it lasted. Asking the patient to describe the visual experience that surrounded the sensory phenomenon of vision loss may also be helpful in sorting through diagnostic possibilities. Table 2 lists clinical inferences typically correlated with different types of complaints associated with vision loss or impairment. Double Vision Acute onset of binocular diplopia requires a neurological evaluation, whereas monocular diplopia does not. Monocular diplopia is typically caused by an underlying refractive problem. Patients may report double vision as being a shadowing or ghosting of images due to improperly corrected refractive error. Patients may not be aware whether they can see 2 images with both eyes open or with a single eye. Because such a distinction is critical, the clinician must directly question the patient to confirm and test for true binocular diplopia in all fields of gaze. If diplopia disappears when either eye is occluded (ensuring vision is tested in the direction of symptomatic double vision), then double vision is binocular. New-onset binocular diplopia necessitates a neurological evaluation.8-15 Examples of chemotherapy agents associated with cranial nerve palsies or double vision are listed in Table 3.4,5,8-18 Pain Characteristics of ocular and periocular pain can provide clues to an underlying disease process. Dry eyes and mild forms of keratitis may be described as produc152 Cancer Control ing a “sandy feeling” or foreign-body sensation. Generalized mild eye discomfort, heavy eyelids, and brow ache associated with near visual tasks are features of asthenopia, which may be secondary to use of outdated prescription glasses or need for reading glasses or bifocals. Onset of sudden, sharp, stabbing pain with intense sensitivity to light suggests a corneal epithelial erosion or corneal abrasion. Typically, 1 drop of topical anesthetic quickly relieves such pain. A deep boring pain exacerbated by eye movements is suggestive of scleritis. Optic neuritis can also produce pain or discomfort with eye movements, but it is usually not as severe as scleritis. Severe, unilateral, ocular and periocular pain progressing for minutes to hours and associated with ipsilateral blurred vision or halos around lights is a typical presentation of acute glaucoma. Two symptoms of acute Table 2. — Clinical Inferences Associated With Symptoms or Features of Visual Loss Symptom Clinical Inference Colored halos around lights Corneal edema from elevated intraocular pressure Decreased vision in dim illumination Impaired dark adaption Vitamin A deficiency Distortion of straight lines Macular dysfunction due to edema or blood beneath or within the retina Floaters, unilateral or bilateral Blood or inflammatory cells in vitreous Age-related collapse, condensation of vitreous gel; with photophobia and eye pain suggests inflammation Loss of side or peripheral vision Branch artery occlusion Cerebrovascular accident Retinal detachment Pain on eye movement Optic neuritis Orbital inflammation Scleritis Painless “spot” in center of vision Macular dysfunction Optic neuropathy Photophobia, tender eye Acute glaucoma Keratitis Uveitis Severe lancet-like pain, blurred vision Corneal abrasion Corneal erosion Keratitis Sudden, catastrophic vision loss Vascular occlusion of retina or optic nerve Transient obscurations of vision lasting seconds, usually bilateral Increased intracranial pressure Transient visual loss lasting minutes, binocular Cardiac arrhythmia Posterior circulation event Orthostatic hypotension Transient visual loss lasting minutes, monocular Anterior circulation event Amaurosis fugax April 2016, Vol. 23, No. 2 glaucoma, periocular pain and headache, are sometimes associated with nausea and vomiting. Terms and phrases frequently used to characterize pain associated with specific ocular conditions are listed in Table 4. Table 3. — Chemotherapeutic Agents Associated With Cranial Nerve Palsies or Diplopiaa Complication Select Example Route of Administration Bilateral lateral rectus palsy Cytosine arabinoside and mitoxantrone15 Intravenous Cavernous sinus syndrome Cisplatin14 Intra-arterial Cranial nerve palsy Vinca alkaloids10,11 Intravenous Diplopia Chlorambucil5 Intravenous Disturbance in oculomotor function 5-fluorouracil12 Intravenous Fibrosis of extraocular muscles Carmustine Nitrosoureas8,13 Intra-arterial Intravenous Internuclear ophthalmoplegia Methotrexate Nitrosoureas8,9 Intra-arterial Intrathecal Oculomotor nerve palsy Interferon α5 Intravenous Also refer to references 4 and 16 to 18 for a more thorough discussion on neuro-ophthalmological complications of chemotherapy. Table 4. — Descriptions of Ocular and Periocular Pain Description Clinical Inferencea Deep boring eye pain worse with eye movement Scleritis Foreign body sensation Grit or sand in eyes Dry eyes Mild keratitis Mild to moderate pain with eye movement and vision loss Optic neuritis Moderate to severe eye pain worse with light exposure Uveitis Ocular and periocular discomfort associated with near visual tasks Lid heaviness Tired eyes Brow ache Asthenopia Eye strain Does not suggest serious underlying disease Ocular and periocular pain severity increasing minutes to hours with progressive vision loss Acute glaucoma Ocular pain in presence of bright light (photophobia) After exclusion of known causes (eg, keratitis, uveitis) consider “essential” or idiopathic photophobia Sudden severe stabbing in 1 eye, relieved when eye is shut Recurrent erosion or corneal abrasion Pain relief with topical anesthetic Erosions more common in dry eyes or eyes with corneal edema a Clinical Other Symptoms Excessive tearing is a common complaint among middle-aged and older individuals with dry eyes (and dry eye with chronic blepharitis) due to reflex tearing, and it is often associated with a mild foreign body sensation. Exacerbations can be triggered by air conditioning, low humidity, or use of over-the-counter medications (eg, antihistamines). If the symptoms are not relieved by treatment with artificial tears, then other causes of epiphora should be considered. A variety of chemotherapy agents has been associated with excessive tearing, although the mechanisms triggering excessive or overflow lacrimation differ (Table 5).4,5,7,19-27 Excessive tearing also accompanies disorders of the ocular surface such as conjunctivitis and keratitis. In these situations, findings on an eye examination will usually indicate that the corneal surface lacks its normal luster or that the eye is inflamed. Because mild photophobia is a common chronic problem, particularly in certain environmental settings, it is important for the clinician to exclude the possibility of a pre-existing condition through a careful history. Keratitis and uveitis also commonly cause photophobia, and these diagnoses must be excluded before idiopathic (or drug-related) photophobia is a considered. Doing so requires a slit lamp inspection of Table 5. — Clinical Inferences Associated With Excessive Tearing Relevant History and Finding Clinical Inference Potential Drug Implication Excessive tearing associated with mass in medial canthal region Consider mechanical obstruction of nasallacrimal outflow Swelling in medial canthal region may or may not show signs of inflammation No drug implications In context of cancer treatment, consider infection or metastatic tumor New-onset tearing associated with mild red eyes, ocular discomfort, or photophobia Consider secondary to ocular surface disorder (keratitis or conjunctivitis) Diagnosis of drugrelated keratitis or conjunctivitis is process of exclusion 5-fluorouracil19,20 Capecitabine5,21 Cytosine arabinoside23-25 Deoxycoformycin4 Chorambucil4 Docetaxel26,27 Pre-existing symptoms associated with itching, or past “problems” with eyelids Consider ocular allergies, trichiasis, or abnormalities of eyelid position Same medications that exacerbate dry eyes can worsen pre-existing allergy symptoms Pre-existing symptoms worse in certain environments like air-conditioned rooms or under fans Mild foreign body sensation Reflex tearing due to dry eyes Often exacerbated by antihistamines and other common medications conditions do not always produce stereotypic pain complaints. April 2016, Vol. 23, No. 2 Cancer Control 153 the corneal surface (with fluorescein stain) and a magnified examination of the aqueous humor and vitreous cavity for cells and protein transudate. Presence of photophobia associated with ocular inflammation typically requires referral to an eye specialist. Eye Examination Patients unable to be promptly evaluated in a specialty ophthalmology clinic or onsite by an ophthalmologist should have a reliable assessment of visual function (central and peripheral vision) and a basic eye examination, which involves a modicum of basic equipment (Fig). Central vision can be tested using a near visual acuity card held at 40 cm. If patients are older than 40 years of age, near vision usually requires bifocals (for persons using distance correction) or separate near-reading glasses (unless near sighted). The clinician must individually test each eye. If prescription glasses are unavailable, then the patient’s uncorrected vision should be obtained and then tested again using over-the-counter readers (+1.25 to +3.00 D). For lens powers greater than +2.50 D, the near visual acuity card may need to be held closer than 40 cm for best focus. Confrontation visual field testing is designed to detect gross field defects. The patient should be situated approximately 1 m from the clinician at equal eye level. The clinician then presents 1 or 2 fingers in each quadrant of the visual field. While the patient occludes 1 eye and maintains central fixation with the other, the clinician asks the patient to count the clinician’s fingers, randomly shifting his or her fingers to include each sector of the visual field. In patients complaining of diplopia, the clinician must confirm their visual experience as binocular (ie, with both eyes open in each field of gaze). Testing should then be repeated, with the patient closing each eye so that the clinician can document diplopia that abates. Typically, moderate and large deviations in ocular alignment can be detected on casual inspection; however, small misalignments are difficult to identify, and they may require special examination techniques. The Amsler grid is used to screen for macular disease, and standard color plates (Hardy–Rand– Ritter or Ishihara) are valuable for the screening of optic neuropathies. These sensory tests are available in physical prints but are also available online (Amsler grid: http://development.aao.org/eyecare /conditions/macular-degeneration/amsler.cfm; color testing: http://colorvisiontesting.com). Both tests are presented individually to each eye and take less than 1 minute to complete. Inspection of periocular tissues, eyelids, and the front surface of the eyes with good illumination are often sufficient to detect clinically important inflammation during a basic eye examination. Abnormalities of 154 Cancer Control Fig. — Basic equipment for visual sensory testing includes an Amsler grid (top left), Ishihara color plates (bottom right), and a near vision card (bottom left). Visual stimuli are separately presented to each eye, making sure 1 eye is occluded. Over-the-counter +2.50 glasses (focal length 40 cm) may be needed for patients older than 40 years who have no reading glasses or bifocals without myopia. the cornea can often be appreciated without magnification because the tissue lacks its normal luster and transparency. If available, 1 drop of sterile fluorescein dye will stain epithelial defects and enhance them when using a cobalt blue light filter. A penlight can detect aberrations in the normal red light reflex seen through the pupil. Deviations are easier for the clinician to appreciate when they are unilateral and the patient’s opposite eye serves as a control. Although fundamentals of a pupil examination (eg, size, shape, reactivity to light, presence or absence of a relative afferent pupillary defect) are beyond the scope of this article, characterizing pupil function is helpful when localizing injury to the visual pathway and for prioritizing special studies. A penlight may also be useful for detecting small lesions of the eyelid such as petechial hemorrhages or abnormal position, but gross swelling or erythema of the eyelids can often be seen best under general illumination. New-onset proptosis is a rare complication of chemotherapy.4,14 In a patient with cancer who is receiving chemotherapy, new-onset proptosis is more likely to be secondary to hemorrhage (bleeding diathesis or infection) or a rapidly expanding tumor.28-30 Regardless of the cause, new-onset proptosis requires prompt evaluation. Subtle displacement of the eye in the orbit can be appreciated by viewing the patient from several feet away, looking for differences in symmetry and the relative amounts of visible sclera. Proptosis can be mimicked by enophthalmos of the opposite eye (eg, due to metastatic scirrhous breast carcinoma) or by lid ptosis. An exception to the penlight detection of important April 2016, Vol. 23, No. 2 ocular inflammation can occur with uveitis, in which signs of external inflammation may be subtle. In this situation, symptoms of pain and photophobia, newonset floaters, or evidence of decreased vision may be clues to more serious problems. Clinicians not specializing in ophthalmology are not typically expected to pursue an eye examination beyond these tests because doing so usually requires use of special equipment (slit lamp, tonometer, ophthalmoscope). Anticipated Complications Few complications occur with sufficient regularity that some authorities recommend preventive measures to mitigate their occurrence (Table 6).4,5,7,19-27,31-37 Examples of such complications include keratitis caused by cytarabine, ocular irritation from high-dose methotrexate, and canalicular and nasolacrimal duct stenosis after taking docetaxel.19-25,27 Optimal strategies are not universally agreed upon, but most offer some benefit.31,33,35,37 Logistical Dilemma Onset of new visual symptoms or other ocular complaints in patients receiving cancer treatment could represent an opportunistic infection, metastatic disease, or the coincidental occurrence or exacerbation of an unrelated eye disease. Some ocular or visual complaints arising in medically complex cases or in high-intensity medical care settings are neither vision-threatening nor have long-term adverse consequences. Most ocular-related complications of chemotherapy are rare,1-7 so a clinical history and eye Table 6. — Preemptive Strategies for Common Ocular-Related Adverse Events of Chemotherapy Adverse Event Strategya 5-fluorouracil Conjunctivitis (noninfectious) Cool compresses Topical methylcellulose drops Topical corticosteroids 4,37 Cyclophosphamide Dry eyes Artificial tear treatment4,7 Cytosine arabinoside Keratitis Topical corticosteroid drops started prior to treatment and tapered33-36 Topical artificial tears 33-36 Agent Topical 2-deoxycytine drops prior to therapy 33,36 Docetaxel Canalicular fibrosis Secondary epiphora Temporary intubation of lacrimal puncta in patients with early symptoms 31 Methotrexate Keratitis Photophobia Artificial tear treatment 32 aThe references cited discuss the relative merits of therapy and alternative strategies. April 2016, Vol. 23, No. 2 examination are usually required to distinguish treatment-related adverse events from common disorders of the eye, as well as to differentiate potentially serious from relatively unimportant problems. However, when patients cannot be examined at an eye clinic in a timely manner because of extenuating circumstances, screening for serious eye complications must be performed by their primary care physicians and oncologists. Conclusions The potential ocular complications with the greatest likelihood of causing adverse outcomes present with demonstrable declines in visual function (eg, loss of central vision, loss of peripheral vision), symptoms and/or signs of oculomotor nerve palsy (eg, diplopia, loss of ocular alignment related to cranial nerves III, IV, or VI), or abnormalities detectable on penlight examination (eg, loss of red reflex; inflammation or swelling of the eyelids, conjunctiva, sclera; proptosis).1-7,16-18 Correlating symptoms and basic clinical findings with previously reported treatment-related adverse events is the first step in identifying possible causative drugs.1-7,16-18 Because causal associations with drug exposures usually involve the exclusion of other disease processes, it is likely that an ophthalmologic specialist will need to be involved in the diagnostic evaluation; however, the urgency of the consultation will be dictated by the results of screening. For patients with suspected ocular complications related to cancer therapy who cannot be quickly seen at a specialty eye clinic, care must be expedited through communication with an ophthalmologist and include relevant ocular history findings, results of vision tests, and findings from the external eye examination. References 1. Imperia PS, Lazarus HM, Lass JH. Ocular complications of systemic cancer chemotherapy. Surv Ophthalmol. 1989;34(3):209-230. 2. Renouf DJ, Velazquez-Martin JP, Simpson R, et al. Ocular toxicity of targeted therapies. J Clin Oncol. 2012;30(26):3277-3286. 3. Liu CY, Francis JH, Brodie SE, et al. Retinal toxicities of cancer therapy drugs. Biologics, small molecule inhibitors, and chemotherapies. Retina. 2014;34(7):1261-1280. 4. Schmid KE, Kornek GV, Scheithauer W, Binder S. Update on ocular complications of systemic cancer chemotherapy. Surv Ophthalmol. 2006;51(1)19-40. 5. Al-Tweigeri T, Nabholtz, JM, Mackey JR. Ocular toxicity and cancer chemotherapy: a review. Cancer. 1996;78(7):1359-1373. 6. Kheir WJ, Sniegowski MC, El-Sawy T, et al. Ophthalmic complications of targeted cancer therapy and recently recognized ophthalmic complications of traditional chemotherapy. Surv Ophthalmol. 2014;59(5):493-502. 7. Hazin R, Abuzetun JY, Daoud YJ, et al. Ocular complications of cancer therapy: a primer for the ophthalmologist treating cancer patients. Curr Opin Ophthalmol. 2009;20(4):308-317. 8. Lepore FE, Nissenblatt MJ. Bilateral internuclear ophthalmoplegia after intrathecal chemotherapy and cranial irradiation. Am J Ophthlamol. 1981;92(6):851-853. 9. Kapp J, Vance R, Parker LJ, et al. Limitations of high dose intraarterial 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy for malignant gliomas. Neurosurgery. 1982;10(6):715-719. 10. Albert DM, Wong VG, Henderson ES. Ocular complications of vincristine therapy. Arch Ophthalmol. 1967;78(6):709-713. 11. Toker E, Yenice O, Oğüt MS. Isolated abducens nerve palsy induced by vincristine therapy. J AAPOS. 2004;8(1):69-71. 12.Bixenman WW, Nicholis JV, Warwick OH. Oculomotor disturCancer Control 155 bances associated with 5-flurouracil chemotherapy. Am J Ophthalmol. 1977;83(6):789-793. 13. Greenberg HS, Ensminger WD, Chandler WF, et al Intra-arterial BCNU chemotherapy for treatment of malignant gliomas of the central nervous system. J Neurosurg. 1984;61(3):423-429. 14. Margo CE. Murtagh FR. Ocular and orbital toxicity after intracarotid cisplatin therapy. Am J Ophthalmol. 1993;116(4):508-509. 15. Ventura GJ, Keating MJ, Castellanos AM, et al. Reversible bilateral lateral rectus muscle palsy associated with high-dose cytosine arabinoside and mitoxantrone therapy. Cancer. 1986;58(8):1633-1635. 16.Singh P, Singh A. Ocular adverse effects of anti-cancer chemotherapy and targeted therapy. J Can Therap Res. Published 2012. h t t p : / / w w w. h o a j o n l i n e . c o m / j o u r n a l s / p d f / 2 0 4 9 -7 9 6 2 -1- 5 . p d f . Accessed March 10, 2016. 17. Nolan CP, DeAngelis LM, Neurologic complications of chemotherapy and radiation therapy. Continuum. 2015;21(2):429-451. 18. Sioka C, Kyritsis AP. Central and peripheral nervous system toxicity of common chemotherapeutic agents. Cancer Chemother Pharmacol. 2009;63(5):761-767. 19. Agarwal MR, Esmaeli B. Burnstine MA. Squamous metaplasia of the canaliculi associated with 5-fluorouracil: a clinicopathologic case report. Ophthalmology. 2002;109(12):2359-2361. 20. Brink HM, Beex LV. Punctal and canalicular stenosis associated with systemic fluorouracil therapy. Doc Ophthalmol. 1995;90(1):1-6. 21. Fraunfelder FT, Meyer SM. Ocular toxicity of antineoplastic agents. Ophthalmology. 1983;90(1):1-3. 22. Kende G, Sirkin SR, Thomas PR, et al. Blurring of vision: a previously undescribed complication of cyclophosphamide therapy. Cancer. 1979;44(1):69-71. 23. Barletta JP, Fanous MM, Margo CE. Corneal and conjunctival toxicity with low-dose cytosine arabinoside. Am J Ophthalmol. 1992;113(5):587‑588. 24. Ritch PS, Hansen RM, Heuer DK. Ocular toxicity from high-dose cytosine arabinoside. Cancer. 1983;51(3):430-432. 25. Hopen G, Mondino BJ, Johnson BL, et al. Corneal toxicity with systemic cytarabine. Am J Ophthalmol. 1981;91(4):500-504. 26. Esmaeli B, Burnstine MA, Ahmadi MA, et al. Docetaxel-induced histologic changes in the lacrimal sac and the nasal mucosa. Ophthal Plast Reconstr Surg. 2003;19(4):305-308. 27. Esmaeli B, Hidaji L, Adinin RB, et al. Blockage of the lacrimal drainage apparatus as a side effect of docetaxel therapy. Cancer. 2003;98(3);504-507. 28. Barahimi B, Patel A, Bilyk JR. Orbital metastasis mimicking subperiosteal abscess. Orbit. 2010;29(3):165-167. 29. McNab AA. Nontraumatic orbital hemorrhage. Surv Ophthalmol. 2014;59(2)168-184. 30. Valenzuela AA, Archibald CW, Fleming B, et al. Orbital metastasis: clinical features, management and outcome. Orbit. 2009;28(2-3):153-159. 31. Leyssens B, Wildiers H, Lobelle JP, et al. A double-blind randomized phase II study on the efficacy of topical eye treatment in the prevention of docetaxel-induced dacryostenosis. Ann Oncol. 2010;21(2):419-423. 32. Doroshow JH, Locker GY, Gaasterland DE, et al. Ocular irritation from high dose methotrexate therapy: pharmacokinetics of drug in the tear film Cancer. 1981;48(10):2158-2162. 33. Lass JH, Lazarus HM, Reed MD, et al. Topical corticosteroid therapy for corneal toxicity from systemically administered cytarabine. Am J Ophthalmol. 1982;94(5):617-621. 34. Stentoft J. The toxicity of cytarabine. Drug Saf. 1990;5(1):7-27. 35. Higa GM, Gockerman JP, Hunt AL, et al. The use of prophylactic eye drops during high-dose cytosine arabinoside therapy. Cancer. 1991;68(8):1691-1693. 36. Lazarus HM, Hartnett, Reed MD, et al. Comparison of the prophylactic effects of 2-deoxycytidine and prednisolone for high-dose intravenous cytarabine-induced keratitis. Am J Ophthalmol. 1987;105(5):476-480. 37. Loprinzi CL, Wender DB, Veeder MH, et al. Inhibition of 5-fluorouracil-induced ocular irritation by ocular ice packs. Cancer. 1994;74(3):945-948. 156 Cancer Control April 2016, Vol. 23, No. 2 Disparities Report Disparities Among Minority Women With Breast Cancer Living in Impoverished Areas of California Sundus Haji-Jama, Kevin M. Gorey, PhD, Isaac N. Luginaah, PhD, Guangyong Zou, PhD, Caroline Hamm, MD, and Eric J. Holowaty, MD Background: Interaction effects of poverty and health care insurance coverage on overall survival rates of breast cancer among women of color and non-Hispanic white women were explored. Methods: We analyzed California registry data for 2,024 women of color (black, Hispanic, Asian, Pacific Islander, American Indian, or other ethnicity) and 4,276 non-Hispanic white women (Anglo-European ancestries and no Hispanic-Latin ethnic backgrounds) diagnosed with breast cancer between the years 1996 and 2000 who were then followed until 2011. The 2000 US census categorized rates of neighborhood poverty. Health care insurance coverage was either private, Medicare, Medicaid, or none. Cox regression was used to model rates of survival. Results: A 3-way interaction between ethnicity, health care insurance coverage, and poverty was observed. Women of color inadequately insured and living in poor or near-poor neighborhoods in California were the most disadvantaged. Women of color adequately insured and who lived in such neighborhoods in California were also disadvantaged. The incomes of such women of color were typically lower than the incomes of non-Hispanic white women. Conclusions: Women of color with or without insurance coverage are disadvantaged in poor and near-poor neighborhoods of California. Such women may be less able to bare the indirect, direct, or uncovered costs of health care for breast cancer treatment. Background Prognoses are excellent among women with breast cancer diagnosed early and treated in a timely manner with evidence-based surgical and adjuvant care.1 The vast majority of such women will survive for 5 to 10 years or more with a high quality of life, but racial and ethFrom the Dalla Lana School of Public Health (SH-J, EJH), University of Toronto, Toronto, Ontario, Canada, School of Social Work (KMG), University of Windsor, Windsor, Ontario, Canada, Departments of Geography (INL), Epidemiology and Biostatistics (GZ), and Oncology (CH), School of Medicine and Dentistry (CH), Western University, London, Ontario, Canada, Robarts Research Institute (GZ), London, Ontario, Canada, and the Department of Oncology (CH), Windsor Regional Cancer Center, Windsor, Ontario, Canada. Address correspondence to Kevin M. Gorey, PhD, 401 Sunset Avenue, Windsor Ontario, Canada, N9B 3P4. E-mail: [email protected] Submitted August 12, 2015; accepted January 19, 2016. This study was supported in part with funds from Canadian Institutes of Health Research grant no. 67161-2. This study was also supported by the California Department of Public Health; National Cancer Institute’s Surveillance, Epidemiology and End Results Program contracts HHSN261201000140C, HHSN261201000035C, and HHSN261201000034C; and the Centers for Disease Control and Prevention’s National Program of Cancer Registries under agreement U58DP003862-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The ideas and opinions expressed herein are those of the authors, and endorsement by the State of California, the Department of Public Health, the National Cancer Institute, and the Centers for Disease Control and Prevention or their contractors and subcontractors is not intended or should be inferred. April 2016, Vol. 23, No. 2 nic disparities persist.1 Findings from systematic reviews have found consistent disadvantages in breast cancer screening, diagnosis, treatment, and survival rates in the United States among ethnic minority women of color compared with non-Hispanic white women.2-13 NonHispanic white women have Anglo-European ancestries and no Hispanic-Latin ethnic backgrounds. Women of color represent a diverse population — defined as black, Hispanic, Asian, Pacific Islander, American Indian, or other minority ethnicity — and certain subpopulations of Asian and Hispanic American women even seem to be advantaged on access to breast cancer care and survival.14,15 However, ethnic minority women of color who live in poverty or are inadequately insured tend to be more alike than higher income women of color and they also tend to be disadvantaged on cancer care compared with non-Hispanic white women.14,15 This field of research may also be limited by its focus on the main effects of ethnicity, rates of income, and health care insurance coverage. Access to cancer care as well as rates of survival may be affected by diverse sociodemographic and economic factors, possibly in complex ways.16,17 For example, a 3-way interaction of ethnicity, health care insurance coverage, and poverty has been observed among patients with colon cancer, indicating that the multiplicative disadvantage of being inadequately insured and living in impoverished areas was worse for African Americans than for non-Hispanic Cancer Control 157 white Americans.16 Furthermore, such disadvantages may be greater for women than for men.17-20 Because select groups of African Americans and women who live in impoverished areas have fewer capital reserves than their non-Hispanic white American counterparts, researchers have suggested that these vulnerable groups may be less able to absorb the indirect, direct, or uncovered costs of cancer care.21 This suggestion led us to hypothesize a 3-way interaction of ethnicity, health care insurance coverage, and poverty. Furthermore, we hypothesize that the interaction will operate such that the survival disadvantage of women of color with breast cancer compared with non-Hispanic white women with breast cancer will be greatest in places where the economic divide between them is greatest. Methods Women diagnosed with breast cancer between 1996 and 2000 were randomly selected from 3 socioeconomic strata of the California Cancer Registry and followed until 2011. Cancer data were joined via US census tracts to the 2000 US census with strata based on federal poverty criteria defined as extremely poor (≥ 30% households poor), poor (5%–29%), and near-poor neighborhoods (< 5% poor).22-24 Based on previous analyses, primary health care insurance coverage was defined as adequate (private or Medicare) or inadequate (Medicaid or none).15,16,18-20 Oversampling of women living in poverty seemed to be associated with oversampling of women of color. Approximately one-third of this sample was women of color (n = 2,024), defined as being black, Hispanic, Asian, Pacific Islander, American Indian, or other ethnicity; the other two-thirds were non-Hispanic white women (n = 4,276). Within the study population, women of color were represented as black (28%), Hispanic (49%), Asian, Pacific Islander, American Indian (21%), or other minority ethnicity (2%). None of the ethnic minority subsamples significantly differed from each other with regard to rates of low income or inadequate health care insurance coverage. We used an age- and tumor grade–adjusted Cox regression model to explore hypotheses about the interacting effects of ethnicity, health care insurance coverage, and level of poverty on the predictive outcomes of 7-year overall survival (OS) rates (SPSS Statistics for Windows, v22.0, IBM, Armonk, New York).25 Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated. Prevalence estimates were used to describe our study population, and survival rates aided interpretation of the observed 3-way interaction. Prevalence estimates and rates per 100 participants were directly adjusted for age and grade using the study population as the standard population and reported as percentages. Standardized prevalence ratios, rate ratios, or rate differences were then used to assess the practical 158 Cancer Control significance of discrete comparisons with chi-square test–based 95% CIs. The median test was used for continuous comparisons of skewed distributions.26 Other details have been previously reported elsewhere.27,28 The study was reviewed and cleared by the University of Windsor research ethics board. Results Study Sample Table 1 displays study sample descriptions. Women of color were significantly younger than the non-Hispanic white women. They were also more likely to have high-grade, poorly or undifferentiated tumors than non-Hispanic white women. Women of color (56%) were also more than twice as likely as non-Hispanic white women (22%) to live in poor neighborhoods (adjusted prevalence ratio = 2.63; 95% CI, 2.46–2.81), and they were nearly twice as likely to be inadequately in- Table 1. — Sociodemographic and Clinical Characteristics of Women With Breast Cancer at Diagnosis: Unadjusted Percentage Distributions Variable Non-Hispanic White Women n Women of Color % n % Age,a y 25–44 458 10.7 446 22.0 45–54 874 20.4 512 25.3 55–64 907 21.2 437 21.6 65–74 1,021 23.9 354 17.5 ≥ 75 1,016 23.8 275 13.6 <5 1,763 41.2 337 16.6 5–29 1,552 36.3 548 27.1 ≥ 30 961 22.5 1139 56.3 Private 2,390 55.9 986 48.7 Medicare 1,298 30.4 476 23.5 Medicaid 167 3.9 298 14.7 Uninsured 421 9.8 264 13.0 Node negative 2,942 68.8 1227 60.6 Node positive 1,334 31.2 797 39.4 Well differentiated 1,052 24.6 279 13.8 Moderately differentiated 1,843 43.1 753 37.2 Poorly or undifferentiated 1,381 32.3 992 49.0 Neighborhood poverty, % Primary health insurers Summary stage Tumor grade All categorical ethnic group differences were statistically significant (chi-square test; P < .001). a Non-Hispanic white women (M = 62.9; SD = 14.1) vs women of color (M = 56.9; SD = 14.3); 1-way analysis of variance = 246.01; P < .001. M = mean, SD = standard deviation. April 2016, Vol. 23, No. 2 sured compared with nonHispanic white women (ie, uninsured or Medicaid insured; 28% vs 14%; adjusted prevalence ratio = 1.84; 95% CI, 1.71–2.05). Therefore, further analyses were adjusted for age and grade while testing the effects of ethnicity, health care insurance coverage, and poverty. Table 3. — Description of 3-Way Interaction of Ethnicity, Health Insurance, and Poverty on 7-Year Survival Rates Among Women With Breast Cancer Ethnic Group Living Within a Location No. of Cases of Breast Cancer Rate, % Rate Ratioa 95% CI Difference in Rate of Survival, % 0.90–0.99 4.4 Poor or near-poor neighborhood and adequately insured Non-Hispanic white women Women of color 2,922 76.7 1.00 714 72.3 0.94 Poor or near-poor neighborhood and inadequately insured Interaction of Ethnicity Non-Hispanic white women 393 68.6 1.00 by Health Insurance Women of color 171 54.5 0.79 0.69–0.91 14.1 Coverage and Poverty Extremely poor neighborhood Table 2 displays the survival analysis. Consistent with Non-Hispanic white women 961 64.1 1.00 findings from previous reWomen of color 1,139 62.0 0.97 0.91–1.03 2.1 search,14-20 having adequate All rates were adjusted for age and tumor grade. a A rate ratio of 1.00 was the baseline. health care insurance covCI = confidence interval. erage predicted rates of OS while living in poverty and being a woman of color Women of color and non-Hispanic white women living predicted rates of mortality. The women of color in our study were twice as likely to die within 7 years of being in extremely poor neighborhoods did not significantly diagnosed with breast cancer than were non-Hispanic differ on rates of OS, and the effect of health care insurance coverage did not differ by ethnicity. Typical or white women (HR = 2.28). Significant 2-way interactions median annual household incomes of women of color of ethnicity with adequate health care insurance cover($24,050) and non-Hispanic white women ($25,150) were age and poverty as well as a significant 3-way interacalso similar among those living in high poverty places. tion were observed (P = .047). Among women living in lower poverty, poor, or The 3-way interaction of ethnicity, health care insurnear-poor neighborhoods, women of color with priance coverage, and level of poverty is depicted in Table 3. vate or Medicare insurance coverage were modestly disadvantaged on rates of OS compared with their counterparts (4% rate difference to 6% rate ratio differTable 2. — Cox Regression Effects of Ethnicity, Health Insurance, and Poverty on 7-Year Mortality Rates ential). Among these women, median incomes of womAmong Women With Breast Cancer en of color ($61,700) and non-Hispanic white women ($68,725) differed by more than $7,000 (P < .05). In the Predictor Variable HR 95% CI same poor or near-poor neighborhoods, women of colMain Effecta or inadequately insured by Medicaid or those without Ethnicity (non-Hispanic white women) health care insurance coverage had an OS disadvanWomen of color 2.28 1.43–3.63 tage when compared with similar non-Hispanic white Health insurer (uninsured or Medicaid) women (14% rate difference to 21% rate ratio differenMedicare or private insurance 0.71 0.60–0.85 tial). The incomes of these women of color ($46,425) were typically about $15,000 lower than those of Neighborhood poverty (near poor) non-Hispanic white women ($61,000; P < .05). The Poor 1.34 1.17–1.53 OS disadvantage among women of color was greatest Extremely poor 1.90 1.63–2.23 among those living in places where the economic diInteraction Effect vide between women of color and non-Hispanic white Ethnicity by health insurer 0.66 0.51–0.85 women was greatest. Ethnicity by poverty 0.58 0.35–0.96 Ethnicity by health insurer by poverty 1.33 1.00–1.75 All effects were adjusted for all other effects in the regression model as well as for age and tumor grade. a Baseline. CI = confidence interval, HR = hazard ratio. April 2016, Vol. 23, No. 2 Adjunct Interpretive Findings Women of color living in poor and near-poor neighborhoods were less likely to be diagnosed early with node-negative disease (rate ratio = 0.95; 95% CI, 0.90–1.00). When breast-conservation surgery was Cancer Control 159 the most indicated mode of therapy, women of color were less likely than their non-Hispanic white women counterparts to receive it (rate ratio = 0.94; 95% CI, 0.88–1.00) or breast reconstruction (rate ratio = 0.44; 95% CI, 0.32–0.60). Women of color were significantly less likely to receive all adjuvant therapies when they were the most indicated: radiotherapy (rate ratio = 0.91; 95% CI, 0.84–0.99), chemotherapy (rate ratio = 0.91; 95% CI, 0.83–0.99), or hormone therapy (rate ratio = 0.87; 95% CI, 0.78–0.97). Women of color were also more likely to experience long waits for initial surgery (≥ 90 days after diagnosis; rate ratio = 1.48; 95% CI, 1.15–1.91) and radiotherapy (≥ 120 postoperative days; rate ratio = 1.28; 95% CI, 1.03–1.59). When these factors were added to the HR model of OS, the interactions involving ethnicity as well as the main effect of ethnicity no longer entered the model. Discussion The results of this study suggest that an interaction exists between ethnicity, health care insurance coverage, and poverty on rates of OS among a cohort of women with breast cancer living in select areas of California. Our data were able to produce 3 central findings across these 3 socioeconomic strata. Among women of color and non-Hispanic white women living in extremely poor neighborhoods — in areas where at least 30% of the households were poor — the rates of OS did not significantly vary, and this finding is similar to a previous report.23 Regardless of ethnicity, women living in extremely poor neighborhoods appear to be have similar cancer care and OS disadvantages.28 The largest rate of OS based on ethnicity was seen among those who were uninsured or Medicaid-insured and who also lived in neighborhoods where poverty was less prevalent. Women of color with breast cancer living in such poor or nearpoor neighborhoods were 21% less likely to survive than their non-Hispanic white women counterparts. Living in poor or near-poor neighborhoods proved to have the greatest effect on ethnicity and income. The difference among women of color and non-Hispanic white women in annual income was $14,575. Thus, it may be possible that uninsured women of color are less able to bare the uncovered costs of care due to a possible inability to cover out-of-pocket expenses; in addition, women of color covered by Medicaid were also at a disadvantage.29 In the same lower-poverty neighborhoods, women of color with private health care insurance or those with Medicare were 6% less likely to survive than were similarly insured non-Hispanic white women. On average, income among these women of color was $7,025 less than that of non-Hispanic white women. Women of color with private health care insurance coverage may have been more likely to be covered by so-called “bronze plans” 160 Cancer Control with high deductibles, whereas women of color with Medicare coverage may be less able to purchase necessary “Medigap” coverage.30,31 Disadvantages among women of color may also exist in relation to diagnostic and therapeutic care for breast cancer due to the possible inadequacy of their incomes and health care insurance coverage. That is to say that the effects we observed in this study may not be racial or ethnic effects per se; rather, they may be socioeconomic effects. This inference could be interpreted to mean that race or ethnicity does not matter in this instance. However, we think not for the following reasons. Our findings are similar to those of other studies, which may have observed only the tip of the proverbial socioeconomic iceberg — these disparities may be the result of structural inequalities not only in health care, but in education, employment, housing, and banking.5-10,32-34 For example, compared with non-Hispanic white women, women of color are more likely to live in poverty, to live in deeper poverty, and be less wealthy.21,35,36 Lacking capital reserves seems to further disadvantage women of color in many ways, including compounding their inability to purchase adequate health care insurance coverage for breast cancer care. Race/ethnicity still seems to matter very much in American health care.37 Limitations We focused on OS rates rather than cancer-specific rates of survival. Although vital status and survival duration are accurate in cancer registries, the underlying cause of death probably is not.38-40 In addition, the underlying cause of many deaths not coded as being a cancer-related death can be directly associated with lack of treatment or with treatment-related complications.41 Therefore, we believe that OS has a higher rate of accuracy and is a better practical indicator of policy and of clinical significance. Our findings could be confounded by comorbid differences between women of color and non-Hispanic white women. The California Cancer Registry did not code comorbidities known to be associated with socioeconomic factors and breast cancer survival.42 However, women of color and non-Hispanic white women with similar tumors were compared through mathematical modeling, matching them to cancer virulence proxy, grade, and on 2 correlates of other chronic diseases (age, poverty). Therefore, the 2 groups are quite similar, making comorbid alternative explanations unlikely. Our findings about women of color living in California may not be generalizable to all such women in the United States. Our sample of women of color was composed of even more diverse subsamples, some of which were quite small. The majority of the Hispanic participants were Mexican American (83%) and the remainder had diverse Central or South American or CaApril 2016, Vol. 23, No. 2 ribbean heritages. Asian American participants were Filipino (31%), Chinese (20%), Japanese (15%), Korean (6%), Vietnamese (6%), East Indian or Pakistani American (5%), or of another Asian heritage. The consistency and significance, both statistical and practical, of our findings, along with our explanation of a coherent health care insurance theory, bode for convergence. That is, it seems likely that these findings apply to most women of color, particularly those with inadequate incomes and health care insurance coverage. This study was exploratory. Its findings are best thought of as screened hypotheses. We recommend that investigators systematically replicate these analyses with much more powerful ethnic group subsamples. Narrative study of the experiences of each ethnic group would also substantially advance our practical knowledge. Conclusions Women of color living in poor and near-poor neighborhoods of California are disadvantaged in terms of breast cancer care. It is those neighborhoods where they may be less able than non-Hispanic white women to bare the indirect, direct, or uncovered costs of care. Intersecting structural barriers may exist between high-quality care for women of color, those who live in poverty, and those who are uninsured or underinsured. Thus, US policy makers ought to be cognizant of these factors as they consider future reforms of health care. Acknowledgments: We acknowledge the administrative and logistical assistance of Kurt Snipes of the Cancer Surveillance and Research Branch, California Department of Public Health. We also acknowledge the research and technical assistance of Arti ParikhPatel of the California Cancer Registry and Madhan Balagurusamy, Daniel Edelstein, and Nancy Richter of the University of Windsor. References 1. DeSantis C, Ma J, Bryan L, Jemal A. Breast cancer statistics, 2013. CA Cancer J Clin. 2014;64(1):52-62. 2. Consedine NS, Tuck NL, Ragin CR, et al. Beyond the black box: a systematic review of breast, prostate, colorectal, and cervical screening among native and immigrant African-descent Caribbean populations. J Immigr Minor Health. 2015;17(3):905-924. 3. Jones CEL, Maben J, Jack RH, et al. A systematic review of barriers to early presentation and diagnosis with breast cancer among black women. BMJ Open. 2014;4:4076. 4. Alexandraki I, Mooradian AD. Barriers related to mammography use for breast cancer screening among minority women. J Natl Med Assoc. 2010;102(3):206-218. 5. Lee HY, Vang S. Barriers to cancer screening in among Americans: the influence of health care accessibility, culture, and cancer literacy. J Commun Health. 2010;35(3):302-314. 6. Purc-Stephenson RJ, Gorey KM. Lower adherence to screening mammography guidelines among ethnic minority women in America: a meta-analytic review. Prev Med. 2008;46(6):479-488. 7. Wells KJ, Roetzheim RG. Health disparities in receipt of screening mammography in Latinas: a critical review of recent literature. Cancer Control. 2007;14(4):369-379. 8. Wu TY, Bancroft J, Guthrie B. An integrative review on breast cancer screening practice and correlates among Chinese, Korean, Filipino, and Asian April 2016, Vol. 23, No. 2 Indian American women. Health Care Women Int. 2005;26(3):225-246. 9. Shavers VL, Brown ML. Racial and ethnic disparities in the receipt of cancer treatment. J Natl Cancer Inst. 2002;94(5):334-357. 10. Wen KY, Fang CY, Ma GX. Breast cancer experience and survivorship among Asian Americans: a systematic review. J Cancer Surviv. 2014;8(1):94-107. 11. Lopez-Class M, Gomez-Duarte J, Graves K, et al. A contextual approach to understanding breast cancer survivorship among Latinas. Psychooncology. 2012;21(2):115-124. 12. McKenzie F, Jeffreys M. Do lifestyle or social factors explain ethnic/ racial inequalities in breast cancer survival? Epidemiol Rev. 2009;31:52-66. 13. Gerend MA, Pai M. Social determinants of black-white disparities in breast cancer mortality: a review. Cancer Epidemiol Biomarkers Prev. 2008;17(11):2913-2923. 14. Haji-Jama S, Gorey KM, Luginaah IN, et al. Health insurance mediation of the Mexican American non-Hispanic white disparity on early breast cancer diagnosis. SpringerPlus. 2013;2:285. 15. Richter NL, Gorey KM, Haji-Jama S, et al. Care and survival of Mexican American women with node negative breast cancer: historical cohort evidence of health insurance and barrio advantages. J Immigr Minor Health. 2015;17(3):652-659. 16. Gorey KM, Haji-Jama S, Bartfay E, et al. Lack of access to chemotherapy for colon cancer: multiplicative disadvantage of being extremely poor, inadequately insured and African American. BMC Health Serv Res. 2014;14:133. 17. Levitz NR, Haji-Jama S, Munro T, et al. Multiplicative disadvantage of being an unmarried and inadequately insured woman living in poverty with colon cancer: Historical cohort in California. BMC Womens Health. 2015;15:8. 18. Gorey KM, Luginaah IN, Holowaty EJ, et al. Mediation of the effects of living in extremely poor neighborhoods by health insurance: Breast cancer care and survival in California, 1996 to 2011. Int J Equity Health. 2013;12:6. 19. Gorey KM, Richter NL, Luginaah IN, et al. Breast cancer among women living in poverty: Better care in Canada than in the United States. Soc Work Res. 2015;39(2):107-118. 20. Gorey KM, Luginaah IN, Bartfay E, et al. Better colon cancer care for extremely poor Canadian women compared with American women. Health Soc Work. 2013;38(4):240-248. 21. Shapiro TM. The Hidden Cost of Being African American: How Wealth Perpetuates Inequality. New York: Oxford University Press; 2004. 22. US Census Bureau. 2000 Census of Population and Housing in California: Summary Tape File 3 [CD-ROM]. Washington, DC: US Government Printing Office; 2002. 23. Wilson WJ. The Truly Disadvantaged: The Inner City, The Underclass, and Public Policy. 2nd ed. Chicago, IL: University of Chicago Press; 2012. 24. Jargowsky PA. Stunning progress, hidden problems: The dramatic decline of concentrated poverty in the 1990s. In: Berube A, Katz B, Lang RE, eds. Redefining Urban and Suburban America: Evidence from Census 2000. Washington, DC: Brookings Institution Press; 2005: 137-171. 25. Vittinghoff E, Glidden DV, Shiboski SC, et al. Regression Methods in Biostatistics: Linear, Logistic, Survival, and Repeated Measures Models. 2nd ed. New York: Springer; 2012. 26. Corder GW, Foreman DI. Nonparametric Statistics for Non-Statisticians: A Step-by-Step Approach. Hoboken, NJ: Wiley; 2009. 27. Gorey KM, Luginaah IN, Bartfay E, et al. Effects of socioeconomic status on colon cancer treatment accessibility and survival in Toronto, Ontario, and San Francisco, California, 1996 to 2006. Am J Public Health. 2011;101(1):112-119. 28. Gorey KM, Luginaah IN, Hamm C, et al. Breast cancer care in Canada and the United States: ecological comparisons of extremely impoverished and affluent urban neighborhoods. Health Place. 2010;16(1):156-163. 29.Smolen JR, Thorpe JR, Jr, Bowie JV, et al. Health insurance and chronic conditions in low income urban whites. J Urban Health. 2014;91(4):637-647. 30.Magge H, Cabral HJ, Kazis LE, et al. Prevalence and predictors of underinsurance among low-income adults. J Gen Intern Med. 2013;28(9):1136-1142. 31. Wharam JF, Ross-Degnan D, Rosenthal MB. The ACA and high-deductible insurance—strategies for sharpening a blunt instrument. New Engl J Med. 2013;369(16):1481-1484. 32. Maar M, Burchell A, Little J, et al. A qualitative study of provider perspectives of structural barriers to cervical cancer screening among first nations women. Womens Health Issues. 2013;23(5):e319-325. 33. Walsemann KM, Gee GC, Ro A. Educational attainment in the context of social inequality: new directions for research on education and health. Am Behav Sci. 2013;57(8):1082-1104. 34. Bowleg L. The problem with the phrase women and minorities: Intersectionality-an important theoretical framework for public health. Am J Public Health. 2012;102(7):1267-1273. 35. Gordon Nembhard J, Chiteji N, eds. Wealth Accumulation and Communities of Color in the United States: Current Issues. Ann Arbor, MI: University of Michigan Press; 2006. Cancer Control 161 36. Conley D. Being Black, Living in the Red: Race, Wealth, and Social Policy in America. Los Angeles: University of California Press; 1999. 37. West C. Race Matters. Boston, MA: Beacon Press; 1993. 38. Yin D, Morris CR, Bates JH, et al. Effect of misclassified underlying cause of death on survival estimates of colon and rectal cancer. J Natl Cancer Inst. 2011;103(14):1130-1133. 39. Hall S, Schulze K, Groome P, et al. Using cancer registry data for survival studies: the example of the Ontario Cancer Registry. J Clin Epidemiol. 2006;59(1):67-76. 40. Lenfant C, Freidman L, Thom T. Fifty years of death certificates: the Framingham Heart Study. Ann Intern Med. 1998;129(12):1066-1067. 41. Brown BW, Brauner C, Minnotte MC. Noncancer deaths in white adult cancer patients. J Natl Cancer Inst. 1993;85(12):979-987. 42. Maskarinec G, Sen C, Koga K, et al. Ethnic differences in breast cancer survival: status and determinants. Womens Health. 2011;7(6):677-687. 162 Cancer Control April 2016, Vol. 23, No. 2 Infectious Diseases Report Bordetella pertussis Infection in Patients With Cancer Abraham Yacoub, MD, Sowmya Nanjappa, MD, Tyler Janz, and John N. Greene, MD Summary: We illustrate 2 cases of pneumonia associated with Bordetella pertussis infection in 72-year-old and 61-year-old patients with cancer receiving myelosuppressive therapy after hematopoietic stem cell transplantation. Bacterial infections are a significant cause of morbidity and mortality in patients with cancer, and those receiving hematopoietic stem cell transplant, solid organ transplant, or myelosuppressive therapy are at increased risk. The infection was detected and the 2 patients had good outcomes following azithromycin treatment. Pertussis, also known as whooping cough, is a contagious respiratory illness that has become a public health challenge due to decreased immunity of the pertussis vaccine. Therefore, it is critical to recognize pertussis early in the course of the disease. Introduction The lung is one of the most frequently involved organs in a variety of complications in the immunocompromised host.1 Among the pulmonary complications that occur in persons who are immunocompromised, infection is the most common and is associated with high rates of morbidity and mortality.1 The most commonly encountered type of infection is bacterial in origin.2 Before the development of vaccines, Bordetella pertussis infection was a significant threat among immunocompetent hosts.3 Bordetella vaccination has significantly reduced the number of infections, but immunity appears to be short lived.4 In addition, a large portion of the population remains susceptible to infection from B pertussis.5 We present 2 cases of B pertussis infection that led to pneumonia in patients with cancer. The medical staff members at our institution were up to date on their vaccinations, and no secondary cases of pertussis were reported. Case Reports Case 1 A woman aged 72 years with metastatic ovarian cancer who was receiving chemotherapy with intrathecal methotrexate, gemcitabine, and carboplatin presented with a 12-day history of productive cough with yelFrom the Division of Infectious Diseases (AY, JNG) and International Medicine (AY), Departments of Internal Medicine (SN) and Oncologic Sciences (SN), H. Lee Moffitt Cancer Center & Research Institute, and the Morsani College of Medicine (AY, SM, JNG), Department of Biomedical Sciences (TJ), University of South Florida, Tampa, Florida. Submitted January 20, 2016; accepted March 5, 2016. Address correspondence to John N. Greene, MD, Department of Infectious Diseases, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612-9497. E-mail: [email protected] No significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. April 2016, Vol. 23, No. 2 low sputum and shortness of breath associated with the coughing spells. She had tried a cough suppressant with minimal relief. She denied fever, chills, sweats, hemoptysis, ill contacts, and recent travel. Her vital signs were within normal limits, and findings on physical examination were significant for crackles heard throughout the lung fields. Her white blood cell count was 4,910/µL with a normal differential. The patient denied receiving pneumocystis prophylaxis in the past and tested negative for Pneumocystis jiroveci. The result from a nasopharyngeal swab sent for a respiratory viral polymerase chain reaction (PCR) panel was positive for B pertussis. PCR testing was negative for adenovirus, Chlamydia pneumoniae, coronavirus, metapneumovirus, rhinovirus, enterovirus, influenza types A and B, Mycoplasma, parainfluenza virus types 1 to 4, and respiratory syncytial virus. Computed tomography (CT) of the chest showed dependent areas of subpleural consolidation and patchy, ground-glass opacities (Fig 1). She was discharged home with a 5-day course of oral azithromycin (250 mg daily). Repeat PCR testing 1 week later was negative for B pertussis. She reported improvement of her symptoms 3 weeks later. Case 2 A man aged 61 years admitted to our hospital with a 5-year history of chronic lymphocytic leukemia and a matched, unrelated donor stem cell transplantation 12 days prior complained of a 1-day history of nonproductive cough, sore throat, and fatigue. He denied fever, chills, shortness of breath, hemoptysis, nausea, vomiting, ill contacts, and recent travel. His vital signs were all within normal limits. Finding on physical examination was remarkable for coarse breath sounds heard across the left lung fields. His white blood cell count was 560 µL, absolute neutrophil Cancer Control 163 Fig 1. — Computed tomography of the chest showing dependent areas of subpleural consolidation and patchy, ground-glass opacities. count was 190 µL, and lymphocyte count was 200 µL. The patient was receiving pneumocystis prophylaxis and tested negative for P jiroveci. The result from a nasopharyngeal swab sent for a respiratory viral PCR panel was positive for B pertussis. PCR testing was negative for adenovirus, C pneumoniae, coronavirus, metapneumovirus, rhinovirus, enterovirus, influenza types A and B, Mycoplasma, parainfluenza virus types 1 to 4, and respiratory syncytial virus. Radiography of his chest showed an interval increase in opacification of the perihilar lungs. CT of his chest demonstrated patchy, bilateral airspace disease and ground-glass nodularity, which was greatest in the left lower lobe and bilateral small pleural effusions (Fig 2). Rapid strep testing was negative, and blood cultures did not show any growth after 3 days. He was treated with a 7-day course of oral azithromycin (250 mg daily) and was followed-up in the clinic. He reported complete resolution of his symptoms 2 weeks later. Repeat PCR testing for B pertussis performed on day 26 was negative. Discussion B pertussis, a gram-negative aerobic, pleomorphic coccobacilli, is known to cause whooping cough, which is a respiratory tract infection characterized by paroxysmal cough.6-9 B pertussis is transmitted by large droplets produced during coughing, sneezing, or talking. These droplets generally travel fewer than 3 feet and can be deposited on mucosal surfaces of susceptible individuals.10 B pertussis belongs to the Bordetella genus, which now includes 9 species, and B pertussis, B parapertussis, and B bronchiseptica are the 3 most studied.11 Infection is initiated by the attachment of B pertussis to the cilia of epithelial cells of the upper respi164 Cancer Control Fig 2. — Computed tomography of the chest demonstrating patchy, bilateral airspace disease and ground-glass nodularity (greatest in the left lower lobe). ratory tract. Evasion of host defenses is facilitated by adenylate cyclase toxin (CyaA) and pertussis toxin.12 CyaA enters the neutrophils and catalyzes the excessive production of cyclic adenosine monophosphate, which intoxicates the cells such that phagocytosis is compromised. Similar to CyaA, pertussis toxin adversely affects phagocytosis and inhibits the migration of lymphocytes and macrophages to areas of infection.12 Local tissue damage of the ciliated epithelial cells may be due to tracheal cytotoxin, dermonecrotic toxin, and perhaps CyaA.12 Although B pertussis mainly causes pulmonary issues, it has been documented to be associated with otitis media,13 encephalopathy,14 bacteremia,15 hemolytic uremic syndrome,16-18 infantile hypertrophic pyloric stenosis,19 multiple sclerosis, amyotrophic April 2016, Vol. 23, No. 2 lateral sclerosis,01 cerebral ataxia, 21 and Wegener granulomatosis.22 Cough is one of the most common complaints among patients with B pertussis infection.23 Diagnosis can be made by PCR assay because it is a rapid, efficient, and specific technique with superior sensitivity compared with that of culture.24-26 Five other cases have been cited in the literature of pertussis in patients with cancer and a mix of hematological malignancies and solid tumors (Table).15,27-30 It is worthy of note that these cases were published prior the advent of PCR assay and several were based on culture of the organism from extrapulmonary sources (eg, blood, empyema fluid).15,27-30 The recommended antimicrobial agents for the treatment or chemoprophylaxis of pertussis in patients who are immunocompromised or immunocompetent are azithromycin, clarithromycin, and erythromycin.31 Trimethoprim/sulfamethoxazole can also be used.32 Erythromycin, clarithromycin, and azithromycin are preferred for the treatment of pertussis in persons who are at least 1 month of age.31 For infants younger than 1 month of age, azithromycin is preferred for postexposure prophylaxis and treatment because azithromycin has not been associated with infantile hypertrophic pyloric stenosis, unlike erythromycin.33,34 Azithromycin should be used with caution because it is associ- ated with prolongation of the QT interval.35 For patients allergic or intolerant to these agents, doxycycline or levofloxacin is an alternative option (rather than no treatment).35 If symptoms do not improve after a trial of macrolides, then resistance should be suspected.36,37 Use of vaccination against B pertussis has been well studied.38 The diphtheria, tetanus, and acellular pertussis (DTaP) vaccine is indicated only for children younger than 7 years of age.38 However, the adult tetanus, diphtheria, and acellular pertussis (Tdap) vaccine is a 3-dose series with high tetanus and pertussis content that may be more immunogenic in recipients of hematopoietic stem cell transplantation, so it should be considered as the initial vaccination for this patient population, regardless of age.38 Furthermore, Suzuki et al30 suggested that acellular pertussis vaccine can be considered for patients after receiving hematopoietic stem cell transplantation, even if they are adults or older than 7 years of age. Conclusions We presented 2 patients who had good outcomes following azithromycin treatment. Due to the advent of and availability of polymerase chain reaction–based diagnostic testing, the rate of Bordetella pertussis infection among patients who are immunocompromised and have cancer is expected to increase.39 Such test- Table. — Studies of Bordetella pertussis Infection in Patients With Cancer Study Age, y Sex Malignancy Symptoms Source of Culture/ PCR Treatment Outcome Case 1 72 F Ovarian cancer Productive cough Shortness of breath Pharynx PCR assay Azithromycin Improved Case 2 61 M Chronic lymphocytic leukemia Nonproductive cough Sore throat Fatigue Pharynx PCR assay Azithromycin Improved Florax 27 16 M ALL Nonproductive cough Pharynx PCR Roxithromycin Symptoms resolved after 2 mo of treatment MacLean29 — — Bronchogenic lung cancer — — — — Senturk 28 64 F Non–small-cell lung cancer Dyspnea Chest pain Empyema PCR assay Erythromycin 2 mg/d Improved Suzuki30 10 M ALL Dry cough Sleep disturbances Plasma serum PCR assay Cephalosporin Clarithromycin Poor Troseid15 63 M Multiple myeloma Cough Hoarseness Fever Blood culture Clarithromycin Poor ALL = acute lymphoblastic leukemia, F = female, M = male, PCR = polymerase chain reaction. April 2016, Vol. 23, No. 2 Cancer Control 165 ing can be easily done via a nasopharyngeal swab. The literature is scant regarding pertussis in adults — and this is particularly true among those with cancer — due to the historical difficulty of accurate diagnostic testing.39 References 1. Oh YW, Effmann EL, Godwin JD. Pulmonary infections in immunocompromised hosts: the importance of correlating the conventional radiologic appearance with the clinical setting. Radiology. 2000;217(3):647-656. 2. Conces DJ Jr. Bacterial pneumonia in immunocompromised patients. J Thorac Imaging. 1998;13(4):261-270. 3. Hewitt M, Canning BJ. Coughing precipitated by Bordetella pertussis infection. Lung. 2010;188(suppl 1):S73-S79. 4. Ward JI, Cherry JD, Chang SJ, et al; APERT Study Group. Bordetella pertussis infections in vaccinated and unvaccinated adolescents and adults, as assessed in a national prospective randomized Acellular Pertussis Vaccine Trial (APERT). Clin Infect Dis. 2006;43(2):151-157. 5. Bloom DE. The value of vaccination. Adv Exp Med Biol. 2011;697:1-8. 6. Shumilla JA, Lacaille V, Hornell TM, et al. Bordetella pertussis infection of primary human monocytes alters HLA-DR expression. Infect Immun. 2004;72(3):1450-1462. 7. Skerry CM, Mahon BP. A live, attenuated Bordetella pertussis vaccine provides long-term protection against virulent challenge in a murine model. Clin Vaccine Immunol. 2011;18(2):187-193. 8. Greenberg D, Bamberger E, Ben-Shimol S, et al. Pertussis is under diagnosed in infants hospitalized with lower respiratory tract infection in the pediatric intensive care unit. Med Sci Monit. 2007;13(11):CR475-CR480. 9. Bettiol S, Thompson MJ, Roberts NW, et al. Symptomatic treatment of the cough in whooping cough. Cochrane Database Syst Rev. 2010;(1):CD003257. 10. Sandora TJ, Gidengil CA, Lee GM. Pertussis vaccination for health care workers. Clin Microbiol Rev. 2008;21(3):426-434. 11. Guiso N. Bordetella pertussis and pertussis vaccines. Clin Infect Dis. 2009;49(10):1565-1569. 12. Mattoo S, Cherry JD. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev. 2005;18(2):326-382. 13. Decherd ME, Deskin RW, Rowen JL, et al. Bordetella pertussis causing otitis media: a case report. Laryngoscope. 2003;113(2):226-227. 14. Halperin SA, Marrie TJ. Pertussis encephalopathy in an adult: case report and review. Rev Infect Dis. 1991;13(6):1043-1047. 15.Trøseid M, Jonassen TØ, Steinbakk M. Isolation of Bordetella pertussis in blood culture from a patient with multiple myeloma. J Infect. 2006;52(1):e11-e13. 16. Cohen-Ganelin E, Davidovits M, Amir J, et al. Severe Bordetella pertussis infection associated with hemolytic uremic syndrome. Isr Med Assoc J. 2012l;14(7):456-458. 17. Chaturvedi S, Licht C, Langlois V. Hemolytic uremic syndrome caused by Bordetella pertussis infection. Pediatr Nephrol. 2010;25(7):1361-1364. 18. Pela I, Seracini D, Caprioli A, et al. Hemolytic uremic syndrome in an infant following Bordetella pertussis infection. Eur J Clin Microbiol Infect Dis. 2006;25(8):515-517. 19. Morrison W. Infantile hypertrophic pyloric stenosis in infants treated with azithromycin. Pediatr Infect Dis J. 2007;26(2):186-188. 20. Flore D. Diagnosis and treatment of multiple sclerosis and amyotrophic lateral sclerosis: neuropathies from Bordetella pertussis. Am J Ther. 2003;10(5):377-379. 21. Setta F, Baecke M, Jacquy J, et al. Cerebellar ataxia following whooping cough. Clin Neurol Neurosurg. 1999;101(1):56-61. 22. Janda WM, Santos E, Stevens J, et al. Unexpected isolation of Bordetella pertussis from a blood culture. J Clin Microbiol. 1994;32(11):2851-2853. 23. Melo N, Dias AC, Isidoro L, et al. Bordetella pertussis, an agent not to forget: a case report. Cases J. 2009;2(1):128. 24. van der Zee A, Agterberg C, Peeters M, et al. A clinical validation of Bordetella pertussis and Bordetella parapertussis polymerase chain reaction: comparison with culture and serology using samples from patients with suspected whooping cough from a highly immunized population. J Infect Dis. 1996;174(1):89-96. 25. Birkebaek NH, Heron I, Skjødt K. Bordetella pertussis diagnosed by polymerase chain reaction. APMIS. 1994;102(4):291-294. 26. Gilberg S, Njamkepo E, Du Chatelet IP, et al. Evidence of Bordetella pertussis infection in adults presenting with persistent cough in a French area with very high whole-cell vaccine coverage. J Infect Dis. 2002;186(3):415-418. 27. Florax A, Ehlert K, Becker K, et al. Bordetella pertussis respiratory infection following hematopoietic stem cell transplantation: time for universal vaccination? Bone Marrow Transplant. 2006;38(9):639-640. 28. Senturk E, Sen S, Telli M. Empyema due to Bordetella pertussis in an adult patient with lung cancer [in English, Spanish]. Arch Bronconeumol. 166 Cancer Control 2012;48(7):263-264. 29. MacLean DW. Bordetella pertussis infection in patients with bronchogenic carcinoma. Lancet. 1981;1(8218):503-504. 30. Suzuki N, Mizue N, Hori T, et al. Pertussis in adolescence after unrelated cord blood transplantation. Bone Marrow Transplant. 2003;32(9):967. 31. Dierig A, Beckmann C, Heininger U. Antibiotic treatment of pertussis: are 7 days really sufficient? Pediatr Infect Dis J. 2015;34(4):444-445. 32. Altunaiji S, Kukuruzovic R, Curtis N, et al. Antibiotics for whooping cough (pertussis). Cochrane Database Syst Rev. 2007;(3):CD004404. 33. Cooper WO, Griffin MR, Arbogast P, et al. Very early exposure to erythromycin and infantile hypertrophic pyloric stenosis. Arch Pediatr Adolesc Med. 2002;156(7):647-650. 34. Santos N, Oliveira M, Galrinho A, et al. QT interval prolongation and extreme bradycardia after a single dose of azithromycin [in English, Portuguese]. Rev Port Cardiol. 2010;29(1):139-142. 35. Flanagan MP. How to manage a pertussis outbreak in your practice. Fam Pract Manag. 2005;12(7):31-34. 36. Guillot S, Descours G, Gillet Y, et al. Macrolide-resistant Bordetella pertussis infection in newborn girl, France. Emerg Infect Dis. 2012;18(6):966-968. 37. Lewis K, Saubolle MA, Tenover FC, et al. Pertussis caused by an erythromycin-resistant strain of Bordetella pertussis. Pediatr Infect Dis J. 1995;14(5):388-391. 38. Rubin LG, Levin MJ, Ljungman P, et al; Infectious Diseases Society of America. 2013 IDSA clinical practice guideline for vaccination of the immunocompromised host [Erratum appears in Clin Infect Dis. 2014;59(1):144]. Clin Infect Dis. 2014;58(3):e44-e100. 39. McGuiness CB, Hill J, Fonseca E, et al. The disease burden of pertussis in adults 50 years old and older in the United States: a retrospective study. BMC Infect Dis. 2013;13:32. April 2016, Vol. 23, No. 2 Pharmacy Report Megestrol Acetate–Induced Adrenal Insufficiency Sowmya Nanjappa, MD, Christy Thai, PharmD, Sweta Shah, MD, and Matthew Snyder, PharmD Summary: A man aged 65 years with metastatic renal cell carcinoma presented for evaluation after a recent fall. A thorough workup of the case was performed and secondary adrenal insufficiency induced by the administration of megestrol acetate was determined to be the cause. Adrenal insufficiency is a serious disorder that is a potential adverse event of megestrol acetate, a medication used to help patients with cancer cachexia increase their appetite and gain weight. This association is not well recognized in clinical practice, so this case highlights the importance of distinguishing possible endocrine complications induced by the long-term administration or sudden discontinuation of megestrol acetate. Introduction Adrenal insufficiency is a serious complication that is a potential adverse event of megestrol acetate, a medication used to help patients with cancer cachexia increase their appetite and gain weight.1,2 However, this association is not well recognized in clinical practice.3 Case Report A man aged 65 years with hypertension and stage 4 clear cell renal cell carcinoma (RCC) with metastases to the brain, lungs, bones, and pancreas was admitted for evaluation after a recent fall. His past surgical history was significant for left nephrectomy and adrenalectomy nearly 20 years ago. After his RCC did not respond to sunitinib and pazopanib treatment, his chemotherapy regimen was changed about 1 month ago to twice daily oral axitinib (5 mg). During his admission, he was hypotensive and his mental status was altered. Vitals were blood pressure of 71/49 mm Hg, respiratory rate of 18 breaths/minute, heart rate of 101 beats/minute, temperature of 98.8 °F, and oxygen saturation of 96% on room air. Findings on physical examination were unremarkable, except for tenderness and swelling in his left lower extremities secondary to the recent fall. Findings on complete blood count with a differential and comprehensive metabolic panel were all within normal limits. Findings following a workup for sepsis and brain imaging were negative. After ruling out potential causes, including infecFrom the Departments of Internal Medicine (SN), Pharmacy (CT, MS), H. Lee Moffitt Cancer Center & Research Institute, and University of South Florida Morsani College of Medicine (SS), Tampa, Florida. Submitted February 26, 2016; accepted March 4, 2016. Address correspondence to Sowmya Nanjappa, MD, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612. E-mail: [email protected] No significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. April 2016, Vol. 23, No. 2 tion, brain metastases, dehydration, electrolyte imbalances, and hypothyroidism, a workup for adrenal insufficiency was performed because hypotension persisted while his mental status improved. A morning serum cortisol level was below 1.0 µg/dL. A cosyntropin stimulation test revealed serum cortisol levels of 5.9 and 8.9 µg/dL at 30 and 60 minutes, respectively, with a pretest plasma adrenocorticotropic hormone (ACTH) level below 5 pg/mL, indicating secondary adrenal insufficiency. A review of the patient’s medication list unveiled no agents commonly known to induce adrenal insufficiency with one exception. He had recently been treated with daily megestrol acetate (oral suspension 625 mg) for about 4 weeks prior to his current admission, and this dose had been continued for the initial portion of this hospitalization. We suspected that this agent might be the likely culprit, so it was discontinued. Oral hydrocortisone 20 mg every morning/10 mg every evening was initiated for adrenal insufficiency, and his blood pressure significantly improved. Steroids were discontinued prior to a follow-up appointment 3 months following discharge. Repeat serum cortisol level and ACTH value were 20 µg/dL and 68 pg/mL, respectively; these results confirmed resolution of the adrenal insufficiency secondary to megestrol acetate. Discussion Adrenal insufficiency is a serious, potentially life-threatening condition that stems from primary adrenal failure or secondary adrenal disease owing to an impaired hypothalamic–pituitary–adrenal (HPA) axis.1 The disorder can chronically cause fatigue, anorexia, weakness, abdominal pain, and hypotension, whereas the acute syndrome may induce hypotensive crisis, fever, clouded sensorium, and myopathy.1 Those affected typically require stress-dose steroids for surgical proCancer Control 167 cedures or severe illness and glucocorticoid replacement on a long-term basis. Although management of the disorder is relatively straightforward, making an accurate and timely diagnosis often proves to be challenging in the oncology setting. Patients with cancer frequently experience these symptoms, sometimes as a result of disease progression, surgery, radiation, chemotherapy, or a combination of all of these. Oftentimes, cachexia seen over time with adrenal insufficiency can mimic wasting characteristic of metastatic disease.4 Paraneoplastic syndromes and malnutrition may cause severe electrolyte abnormalities, clouding, or covering up of the evidence of the role of adrenal insufficiency in such irregularities.4 In addition to causing adrenal insufficiency–associated symptoms, such as fatigue, weakness, myopathy, and pain, chemotherapy and immunotherapy agents can directly give rise to the disorder.5-7 The tyrosine kinase inhibitors sunitinib and imatinib and, more frequently, the checkpoint-blocking immunotherapies ipilimumab, nivolumab, and pembrolizumab have been reported to cause adrenal insufficiency.5-7 Given the limited data available, it will be interesting to see whether the effects of these agents on adrenal function extend to other members of their respective classes as their use continues to increase. Medications commonly used as supportive care can also induce adrenal insufficiency.1,7-10 The detrimental impacts of the long-term administration and abrupt discontinuation of steroids on adrenal function are well known.1 Use of glucocorticoids is also common in patients with cancer, and these agents play a critical role in the treatment of various hematological malignancies, immune-related adverse events,7,8 the relief of symptoms secondary to brain metastases,9 and pain management.10 Similarly, megestrol acetate, which is a synthetic progestin with antiestrogenic properties, is often used to help patients with cancer cachexia increase their appetite and gain weight.2 Secondary to its inherent glucocorticoid-like effect,2 the agent is thought to cause suppression of the HPA axis at the hypothalamus through negative feedback, thus resulting in low levels of serum cortisol and plasma ACTH.11,12 Although stimulating the appetite through the use of megestrol acetate was useful in a patient like ours, megestrol acetate has been associated with thrombosis, hyperglycemia, hypertension, osteoporosis, hypogonadism, and adrenal insufficiency.3 Its clinical benefit is often outweighed by these potential adverse events, many of which are already at a heightened risk of occurring in this patient population (eg, thrombosis). Several questions regarding the length of time needed for megestrol acetate–induced adrenal suppression and subsequent restora168 Cancer Control tion of HPA axis function after discontinuation of the drug are still unknown. Leinung et al13 conducted a trial that initiated 80 mg megestrol acetate 3 times a day in study patients with normal cortisol and ACTH levels prior to therapy. After 1 month, both values significantly decreased, and study participants also displayed a significantly decreased response to cosyntropin stimulation.13 An interventional trial found that 6 of 7 study patients with adrenal suppression recovered normal function within 2 weeks, with the remaining patient doing so in 6 weeks (NCT00575029). However, general data are lacking, so definitive timeframes are uncertain. Nevertheless, our case describes a patient who developed treatment-related adrenal insufficiency after 1 month of therapy, but successfully recovered in 3 months after the medication was discontinued. Although adrenal insufficiency is a reported potential adverse event of megestrol acetate, it is not well recognized in clinical practice.3 Adrenal suppression can stem from abrupt discontinuation of the agent. However, as in our case, megestrol acetate has been found to cause this effect in patients actively taking the agent, thus complicating the diagnosis.2,14,15 Postulated mechanisms for the latter scenario include a dual agonist–antagonist action (binding to the glucocorticoid receptor while simultaneously preventing endogenous glucocorticoids from doing so), a greater tendency to suppress the HPA axis rather than induce glucocorticoid-like effects, and concurrent acute stress or illness in a patient whose HPA axis is already suppressed.11,15 Retrospectively, the patient was not taking any other medication likely to cause such an effect. The origin of our patient’s adrenal insufficiency was also clouded by numerous factors, including prior left adrenalectomy, tyrosine kinase inhibitor use, and metastatic RCC. This case of megestrol-induced adrenal insufficiency was only discovered after extensive workup and elimination of other possible causes. Conclusions Although obtaining and analyzing a patient’s medication history may be challenging, thorough and accurate assessment of previous and current medication use is a necessity that proved to be pivotal in this case. Given the potentially severe complications of adrenal insufficiency, this report underscores the importance of recognizing possible endocrine complications induced by the long-term administration or sudden discontinuation of megestrol acetate. However, further study is still needed to draw definitive conclusions on the effects of megestrol acetate on adrenal function. Acknowledgment: We thank the patient for his cooperation throughout the duration of the case. April 2016, Vol. 23, No. 2 References 1. Bornstein SR. Predisposing factors for adrenal insufficiency. N Engl J Med. 2009;360(22):2328-2339. 2. Delitala AP, Fanciulli G, Maioli M, et al. Primary symptomatic adrenal insufficiency induced by megestrol acetate. Neth J Med. 2013;71:17-21. 3. Dev R, Del Fabbro E, Bruera E. Association between megestrol acetate treatment and symptomatic adrenal insufficiency with hypogonadism in male patients with cancer. Cancer. 2007;110(6):1173-1177. 4. Yeung SC, Escalante CP, Gagel RF. Medical Care of Cancer Patients. Shelton, CT: BC Decker; 2009. 5. Charmandari E, Nicolaides NC, Chrousos GP. Adrenal insufficiency. Lancet. 2014;383(9935):2152-2167. 6. Bilgir O, Kebapcilar L, Bilgir F, et al. Is there any relationship between imatinib mesylate medication and hypothalamic-pituitary-adrenal axis dysfunction? Int J Clin Pract. 2010;64(1):45-50. 7. Villadolid J, Amin A. Immune checkpoint inhibitors in clinical practice: update on management of immune-related toxicities. Transl Lung Cancer Res. 2015;4(5):560-575. 8. Sionov RV, Spokoini R, Kfir-Erenfeld S, et al. Mechanisms regulating the susceptibility of hematopoietic malignancies to glucocorticoidinduced apoptosis. Adv Cancer Res. 2008;101:127-248. 9. Ryken TC, McDermott M, Robinson PD, et al. The role of steroids in the management of brain metastases: a systematic review and evidencebased clinical practice guideline. J Neurooncol. 2010;96(1):103-114. 10. Leppert W, Buss T. The role of corticosteroids in the treatment of pain in cancer patients. Curr Pain Headache Rep. 2012;16(4):307-313. 11. Mann M, Koller E, Murgo A, et al. Glucocorticoidlike activity of megestrol. A summary of Food and Drug Administration experience and a review of the literature. Arch Intern Med. 1997;157(15):1651-1656. 12. Naing KK, Dewar JA, Leese GP. Megestrol acetate therapy and secondary adrenal suppression. Cancer. 1999;86(6):1044-1049. 13.Leinung MC, Liporace R, Miller CH. Induction of adrenal suppression by megestrol acetate in patients with AIDS. Ann Intern Med. 1995;122(11):843-845. 14.González Villarroel P, Fernández Pérez I, Páramo C, et al. Megestrol acetate-induced adrenal insufficiency. Clin Transl Oncol. 2008;10(4):235-237. 15. Mehta K, Weiss I, Goldberg MD. Megace mystery: a case of central adrenal insufficiency. Case Rep Endocrinol. 2015;2015:147265. April 2016, Vol. 23, No. 2 Cancer Control 169 Pathology Report Presacral Noncommunicating Enteric Duplication Cyst Shabnam Seydafkan, MD, David Shibata, MD, Julian Sanchez, MD, Nam D. Tran, MD, Marino Leon, MD, and Domenico Coppola, MD Background: Gastrointestinal (GI) tract duplication cysts or enteric duplication cysts are rare congenital malformations sometimes found on the mesenteric aspect of segments of the alimentary tract. Enteric duplication cysts are lined by normal GI epithelium and may be classified as foregut, mid-gut, and hindgut cysts. Except in very rare cases of retroperitoneal enteric duplication cysts, these cysts communicate with the GI tract and share a common blood supply. Concurrent congenital malformations are not uncommon and malignant transformation within enteric duplication cysts has also been reported. Methods: We describe a case of a noncommunicating enteric duplication cyst in a 52-year-old woman. Results: The patient presented with a presacral cystic mass requiring frequent drainage procedures that was primarily believed to be of neural origin. Upon resection, the lesion contained heterotopic tissue, including ciliated bronchial epithelium, squamous and transitional epithelia, and pancreatic and gastric tissue. Focal, low-grade intestinal adenoma was present, but malignancy was not detected in this case. Conclusion: To our knowledge, this is the sixth reported case of a noncommunicating enteric duplication cyst in the English medical literature. Introduction An enteric duplication cyst is a cystic or tubular congenital malformation located on the mesenteric side of the gastrointestinal (GI) tract that firmly attaches to or shares the gut wall. Theories regarding the origin of GI duplications include diverticulization, canalization defects, intrauterine vascular accident, split notochord theory, and the traction hypothesis between the intestinal endoderm and the overlying structures.1-4 Enteric duplication cyst is rare, with a prevalence of 1 out of 4,500 individuals, and it may originate from anywhere in GI tract.5-7 Enteric duplication cysts are lined with epithelium similar to that of the adjacent normal GI mucosa and are classified by location as foregut, midgut, or hindgut.5,8 In order of decreasing frequency, the most common sites of enteric duplication cysts are the ileum, stomach, and appendix, with the rarest site being the gastroduodenal region.9,10 Criteria to establish the diagnosis of enteric duplication cyst include the presence of a double-walled cyst, simulating the normal anatomy From the Departments of Anatomic Pathology (SS, ML, DC), Gastrointestinal Oncology (DS, JS), Chemical Biology & Molecular Medicine Program (NDT, DC), Tumor Biology Program (DC), H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida. Address correspondence to Domenico Coppola, MD, Department of Anatomic Pathology, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612. E-mail: [email protected] Submitted March 11, 2015; accepted January 15, 2016. No significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. Dr Shibata is now with Department of Surgery at the University of Tennessee Health Science Center, Memphis, Tennessee. 170 Cancer Control of the intestine, lined with enteric-type epithelium, and sharing a common blood supply with its neighboring alimentary tract.11 Rare subtypes of retroperitoneal enteric duplication cysts do not communicate with the intestine and have their own exclusive blood supply.2,12 A patient’s presenting symptoms depend on the anatomical location, size, and the inner lining of the cyst and may include abdominal pain, rectal bleeding, and hematuria. Enteric duplication cyst may also be incidentally discovered in asymptomatic patients. However, enteric duplication cysts become symptomatic in a person’s early childhood and present in the newborn period as an abdominal mass. Few adulthood cases of enteric duplication cyst have been reported.8,11,13 Intussusception, volvulus, or intestinal obstruction and, in rare cases, malignant transformation may also occur.8,14 In more than 50% of cases, other associated malformations such as vertebral defects and other GI duplications may be present.10 Ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI) remain the main diagnostic tools.14 Herein we present a case of enteric duplication cyst initially believed to be of neural origin. In addition, we provide a review of the literature for similar cases and discuss the diagnostic challenges presented by enteric duplication cysts. Case Report A 52-year-old woman presented with a history of a cystic presacral mass that was partially and initially resected via an abdominal approach in 2006. She sustained multiple recurrences, which were all managed by percuApril 2016, Vol. 23, No. 2 taneous image-guided drainage. She was subsequently referred to our treatment team approximately 7 years after her initial diagnosis with worsening rectal pain. Findings on both CT and pelvic MRI revealed a posterior pelvic bilobed cystic mass extending from the puborectalis posteriorly to the sacrum and coccyx and displacing the rectum anteriorly (Fig 1). The lesion also involved part of the levator muscle and had 2 cystic components measuring 3.4 and 3.2 cm, respectively. The lesion showed high-signal intensity on T1-weighted sequences and somewhat low-signal intensity on T2-weighted sequences. The lesion did not show suspicious enhancement, and the vagina and bladder were not involved. The decision was made to proceed with surgical resection via a local, transcoccygeal Kraske approach. To allow for intraoperative identification of rectothecal communication (in the event of pseudomeningocele), preoperative lumbar puncture with intrathecal injection of fluorescein dye was performed. During surgery, a large cystic mass to the left of the midline crossing anterior to the coccyx in a dumbbell shape was identified. The mass was adherent to the posterior rectal wall and appeared to caudally extend to the level of the external sphincter. It was completely excised by the Kraske approach. The cystic mass had no communication with the rectum or the thecal sac. Gross examination revealed an irregular fragment of fibrous and adipose tissue measuring 7 × 6 × 3 cm and weighing 77 g. Upon sectioning the specimen, 3 cystic lesions were found, ranging from 3 to 3.5 cm in their greatest dimensions. Two of the cysts were previously opened and were connected to each other. The third cyst was independent and contained a large amount of tan-brown colored viscous material. The inner walls of the cysts were pink-tan to dusky red in color, focally erythematous and hemorrhagic, and averaged less than 0.1 cm in thickness. The fibroadipose tissue surrounding the cysts was grossly unremarkable. Routine hematoxylin and eosin stain was done, and immunohistochemical stains were obtained with a BenchMark automated platform (Ventana Medical Systems, Tucson, Arizona) using the ultra-view DAB Detection Kit (Ventana) and antibodies cytokeratin (CK) CK7, CK20, transcription termination factor 1 (TTF1), p63, smooth muscle myosin heavy chain, and GATA binding protein 3. A trichrome special stain was also performed. Immunostain for p16 and synaptophysin were not contributory. The immunostains for p53 and Ki67 were also performed. Fig 1. — Axial magnetic resonance imaging showing a presacral dumbbell shape lesion with high signal on T1-weighted images (T1 FL 2D FS). A B Results Histopathology examination showed the cysts to be lined by intestinal-type epithelium with focal adenomatous changes surrounded by a thick muscle coat (Fig 2). The lesions demonstrated heterotopic tissue, April 2016, Vol. 23, No. 2 Fig 2A–B. — (A) Histology of the entire cyst at low power (H & E, × 10). (B) Component of the cyst lined by adenomatous colonic-type epithelium (H & E, × 100). H & E = hematoxylin and eosin. Cancer Control 171 including ciliated bronchial epithelium (Fig 3A), squamous and transitional epithelia (Figs 3B and 3C), and pancreatic and gastric tissue (Fig 3D). The adenomatous epithelium and the adjacent intestinal mucosa were positive for CK7 and negative for CK20. The TTF1 immunostain was negative. The squamous and urothelial epithelia were positive for p63 (Fig 4). The inner and outer smooth-muscle layers surrounding 1 of the cysts was highlighted by smoothmuscle myosin and trichrome special stain. Ki-67 and p53 immunostains did not support high-grade dysplasia or invasive carcinoma. Findings after trichrome special stain, which was used to highlight the layers of muscularis propria, supported the final diagnosis (Fig 5). of the location and include proximity to the GI tract, a 2-layered coat of well-developed smooth muscle, and a GI-type epithelium.15,16 Discussion Enteric duplication cysts are rare congenital lesions that can originate from anywhere in the GI tract from the oropharynx to the anus, including the retrorectal and presacral space.1-4 The key diagnostic features for enteric duplication cysts remain the same regardless Fig 4. — p63 immunostain marking the squamous epithelium (immunohistochemistry, × 400). A B C D Fig 3A–D. — Heterotopic components within the cyst. (A) Ciliated epithelium (H & E, × 400). (B) Squamous epithelium (H & E, × 200). (C) Mucinous metaplasia (H & E, × 400). (D) Pancreatic ectopia (H & E, × 200). H & E = hematoxylin and eosin. 172 Cancer Control April 2016, Vol. 23, No. 2 Fig 5. — Trichrome special stain highlighting the layers of muscularis propria, supporting the final diagnosis (immunohistochemistry, × 400). Rectal duplication cysts belong to the family of developmental cysts and usually present as unilocular cystic masses located in the prerectal space.17 They are lined with intestinal or respiratory epithelium and are associated with well-defined muscle layers and myenteric plexus.17 Enteric duplication cysts may have various types of presenting features.18 For example, enteric duplication cysts found in the duodenum may have communication with the biliary tract or the pancreatic duct.6 Those of gastric origin may involve the pancreas and may contain heterotopic tissues such as gastric mucosa, pancreatic tissue, or bronchial ciliated epithelium, or all 3 types, as was observed in our case.19-21 Correlating the radiological findings with the histopathological features is useful for making the correct diagnosis of enteric duplication cyst. In our case, the enteric duplication cysts did not communicate with the GI tract. Such noncommunicating, isolated enteric duplication cysts have GI epithelium and a wall similar to that seen in regular enteric duplication cysts but without an anatomical association with the alimentary tract. This feature has previously been reported, but it is rare: Approximately 8 cases of this type have been reported in the English literature.20 Of the cases reported, 2 were infected cases of isolated duplication cysts. One was an isolated enteric duplication cyst with features of a classic enterogenous duplication cyst, and 1 case showed mucinous cystadenoma arising within an isolated duplication cyst of the ileum.20,21 Differential Diagnosis Clinically, the differential diagnosis of a retrorectal mass is broad and includes both benign and malignant entities ranging from inflammatory, congenital or developmental cysts, neurogenic and osseous lesions like chordoma, or anterior sacral meningocele.16,22 April 2016, Vol. 23, No. 2 Retrorectal developmental cysts include different types of lesions such as duplication cysts, tailgut cysts, epidermoid cysts, dermoid cysts, and teratomas. Retrorectal developmental cysts are rare and may be misdiagnosed as a supralevator abscess or a complex anal fistula.23 A retrorectal mass with a major cystic component is highly suggestive of a developmental disorder, and the epidermoid, dermoid, and rectal duplications cysts are more often unilocular.24 On CT, tailgut cysts appear as retrorectal masses with well-defined smooth borders and uneven attenuation. Calcifications and invasion of surrounding tissues are uncommon findings for tailgut cysts.25 Tailgut cysts have a female predominance, whereas enteric duplication cysts are equally present in both sexes.15,16 In general, duplication cysts are unilocular developmental cysts with GI- or respiratory-type epithelium. The GI epithelium reveals villi, crypts, and glands that replicate normal gut mucosa. By contrast, tailgut cysts are usually multicystic or multiloculated, lined with a variety of stratified squamous, transitional, stratified columnar, ciliated pseudostratified columnar, and gastric-type mucosa. However, when seen in conjunction with enteric duplication cysts, the mucosa usually is associated with a regular and continuous 2-layer muscle coat containing a nerve plexus. This feature is characteristic of duplication cysts and is not seen in tailgut cysts, where the smooth muscle is disorganized and focally well formed.17,25-30 Cuboidal, transitional, or columnar epithelium can be seen in enterogenous cysts and tailgut cystic hamartomas. Oftentimes, they are multilocular, and disorganized smooth-muscle fibers can be identified surrounding their thin walls.22 A developmental cyst with stratified squamous epithelium and skin appendages is suggestive of a dermoid cyst, whereas no skin appendages are seen in the epidermoid subtype. These cysts lack smooth muscle in their walls. Teratomas are also included in the differential diagnosis of developmental retrorectal cysts. Teratomas contain heterogeneous structures from all 3 embryonic layers. Mature teratomas manifest as complex, well-circumscribed cystic and solid masses with calcifications along with epithelial lining, skin adnexa, heterologous mesenchymal tissues, and neural tissues. Primitive endoderm, mesoderm, and ectoderm are characteristic of immature teratoma. Germ cell–derived neoplastic cells are seen in malignant teratomas. Intralesional calcifications, bony destruction of the coccyx and sacrum, and hypoattenuating fat within the lesions are features of teratomas and were not seen in our case.22,24,27,28,31-33 Developmental cysts are generally benign but do have the potential to undergo premalignant and malignant transformations. The presence of adenocarcinoma within a completely isolated duplication cyst Cancer Control 173 has been reported.34,35 These cases may manifest as bowel-like cysts with no anatomical and luminal connection with the adjacent GI tract and with a separate vascular pedicle. Development of adenocarcinomas and neuroendocrine tumors has also been reported in rectal tailgut cysts. One-half of all reported cases of presacral carcinoids are thought to have developed in a preexisting tailgut cyst or teratoma.29,32,34,36,37 Conclusions Enteric duplication cysts are rare congenital anomalies that should be considered in the differential diagnosis of a retrorectal mass. Careful review of the clinical presentation, radiological features, and gross examination of specimens and its surrounding tissue will help to narrow the diagnostic possibilities, while histopathology and immunohistochemistry are critical to establish the final diagnosis of enteric duplication cyst. Acknowledgment: We thank Jackie Hattle for technical assistance during the preparation and submission of the manuscript and the Pathology Histology Laboratory for the preparation of the stains. References 1.Mandhan P, Ehsan TM, Al-Sibai S, et al. Noncommunicating multiple intra-abdominal enteric duplication cysts. Afr J Paediatr Surg. 2014;11(3):276-278. 2.Gümüş M, Kapan M, Gümüş H, et al. Unusual noncommunicating isolated enteric duplication cyst in adults. Gastroenterol Res Pract. 2011;2011:323919. 3. Atalar MH, Cankorkmaz L, Ozer H, et al. A huge duplication cyst of the ileum. Pol J Radiol. 2013;78(3):70-73. 4. Sheikh MA, Latif T, Shah MA, et al. Ileal duplication cyst causing recurrent abdominal pain and melena. APSP J Case Rep. 2010;1(1):4. 5. Stern LE, Warner BW. Gastrointestinal duplications. Semin Pediatr Surg. 2000;9(3):135-140. 6. Lopez-Fernandez S, Hernandez-Martin S, Ramírez M, et al. Pyloroduodenal duplication cysts: Treatment of 11 cases. Eur J Pediatr Surg. 2013;23(4):312-316. 7. Gebesce A, Korkmaz M, Keles E, et al. Importance of the ultrasonography in diagnosis of ileal duplication cyst. Gastroenterol Res Pract. 2013;2013:248625. 8. Blank G, Königsrainer A, Sipos B, et al. Adenocarcinoma arising in a cystic duplication of the small bowel: case report and review of literature. World J Surg Oncol. 2012;10:55. 9. Rasool N, Safdar CA, Ahmad A, et al. Enteric duplication in children: Clinical presentation and outcome. Singapore Med J. 2013;54(6):343-346. 10. Bal HS, Kisku S, Sen S, et al. A retroperitoneal enteric duplication cyst communicating with the right upper ureter in an infant. BMJ Case Rep. 2014;9:2014. 11. Choi SO, Park WH, Kim SP. Enteric duplications in children--an analysis of 6 cases. J Korean Med Sci. 1993;8(6):482-487. 12.Ho YC. Total colorectal and terminal ileal duplication presenting as intussusception and intestinal obstruction. World J Gastroenterol. 2012;18(43):6338-6340. 13. Patel MP, Meisheri IV, Waingankar VS, et al. Duplication cyst of the pylorus--a rare cause of gastric outlet obstruction in the newborn. J Postgrad Med. 1997;43(2):43-45. 14. Deesomsak M, Aswakul P, Junyangdikul P, et al. Rare adult gastric duplication cyst mimicking a gastrointestinal stromal tumor. World J Gastroenterol. 2013;19(45):8445-8448. 15. Liaqat N, Latif T, Khan FA, et al. Enteric duplication in children: a case series. Afr J Paediatr Surg. 2014;11(3):211-214. 16. Joyce EA, Kavanagh DO, Winter DC. A rare cause of low back pain: Report of a tailgut cyst. Case Rep Med. 2012;2012:623142. 17. Raisolsadat SM, Zabolinejad N, Tabrizian-Namini F, et al. Tailgut cyst in an infant with imperforate anus: a case report. Iran J Pediatr. 2013;23(5):597-600. 18.Glasgow SC, Dietz DW. Retrorectal tumors. Clin Colon Rectal Surg. 2006;19(2):61-68. 19. Baek SW, Kang HJ, Yoon JY, et al. Clinical study and review of 174 Cancer Control articles (Korean) about retrorectal developmental cysts in adults. J Korean Soc Coloproctol. 2011;27(6):303-314. 20. Kim H, Kim JH, Lim JS, et al. MRI findings of rectal submucosal tumors. Korean J Radiol. 2011;12(4):487-498. 21. Kang JW, Kim SH, Kim KW, et al. Unusual perirenal location of a tailgut cyst. Korean J Radiol. 2002;3(4):267-270. 22. Jang SH, Jang KS, Song YS, et al. Unusual prerectal location of a tailgut cyst: a case report. World J Gastroenterol. 2006;12(31):5081-5083 23. Gunkova P, Martinek L, Dostalik J, et al. Laparoscopic approach to retrorectal cyst. World J Gastroenterol. 2008;14(42):6581-6583. 24.Bathla L, Singh L, Agarwal PN. Retrorectal cystic hamartoma (tailgut cyst): report of a case and review of literature. Indian J Surg. 2013;75(suppl 1):204-207. 25. Graadt van Roggen JF, Welvaart K, de Roos A, et al. Adenocarcinoma arising within a tailgut cyst: Clinicopathological description and follow up of an unusual case. J Clin Pathol. 1999;52(4):310-312. 26. Cho BC, Kim NK, Lim BJ, et al. A carcinoembryonic antigen-secreting adenocarcinoma arising in tailgut cyst: clinical implications of carcinoembryonic antigen. Yonsei Med J. 2005;46(4):555-561. 27. Alzaraa A, Mousa H, Dickens P, et al. Idiopathic benign retroperitoneal cyst: a case report. J Med Case Rep. 2008;2:43. 28. Chhabra S, Wise S, Maloney-Patel N, et al. Adenocarcinoma associated with tail gut cyst. J Gastrointest Oncol. 2013;4(1):97-100. 29. Govaerts K, Van Eyken P, Verswijvel G, et al. A bronchogenic cyst, presenting as a retroperitoneal cystic mass. Rare Tumors. 2012;4(1):e13. 30. Ramakrishna HK. Intestinal duplication. Indian J Surg. 2008;70(6):270-273. 31. Ekbote G, Pokharkar AB, Moon P. A rare case of perforated tubular ileal duplication in 72-year-old male. Indian J Surg. 2013;75(suppl 1):418-420. 32. De Roeck A, Vervloessem D, Mattelaer C, et al. Isolated enteric duplication cyst with respiratory epithelium: case report and review of the literature. Eur J Pediatr Surg. 2008;18(5):337-339. 33. Pins MR, Compton CC, Southern JF, et al. Ciliated enteric duplication cyst presenting as a pancreatic cystic neoplasm: report of a case with cyst fluid analysis. Clin Chem. 1992;38(8 pt 1):1501-1503. 34. Shin SY, Cho MY, Ryu H, et al. Adenocarcinoma originating from a completely isolated duplication cyst of the mesentery in an adult. Intest Res. 2014;12(4):328-332. 35. Okamoto T, Takamizawa S, Yokoi A, et al. Completely isolated alimentary tract duplication in a neonate. Pediatr Surg Int. 2008;24(10):1145-1147. 36. Spada F, Pelosi G, Squadroni M, et al. Neuroendocrine tumour arising inside a retro-rectal tailgut cyst: Report of two cases and a review of the literature. Ecancermedicalscience. 2011;5:201 37. Liang JJ, Alrawi S, Fuller GN, et al. Carcinoid tumors arising in tailgut cysts may be associated with estrogen receptor status: case report and review of the literature. Int J Clin Exp Pathol. 2008;1(6):539-543. April 2016, Vol. 23, No. 2 Symposium Report Second Annual Moffitt Anatomic Pathology Symposium Dr Magliocco, chair of the Department of Anatomic Pathology, welcomed a near-capacity audience made up of pathologists, histotechnologists, researchers, and pathology residents and medical students at the 2nd Annual Moffitt Anatomic Pathology Symposium: Practicing Pathology in a Changing World that took place in Clearwater Beach, Florida, on September 26, 2015. Th focus of the symposium was on the updates of contemporary and emerging information and technology that impact practicing pathologists in an ever-changing world. Invited speakers included Drs Odze and Schnitt, both from Harvard Medical School in Boston, who shared their expert knowledge in gastrointestinal (GI) and breast pathology, as well as Theresa B. Burchette, Lawrence Patton Jr, John J. Shelley, and Steven Westra. Experts from the H. Lee Moffitt Cancer Center & Research Institute included Drs Magliocco, Bui, Centeno, Coppola, Hussaini, Khalil, Khazia, and Rosa, as well as Mr Hicks and Ms Balasi, Molina, and Snyder. Drs Minton and Malafa also presented topics in molecular pathology, digital pathology, and GI and breast cancers. The Ask the Expert Digital Pathology session was an interactive discussion of real breast and GI pathology cases among the experts and the audience. A total of 22 scientific abstracts were proffered by students, residents, fellows, and staff. Dr Altiok, chair of the research section of the Department of Anatomic Pathology, presented awards to the 3 best submissions. Dr Ryzhova and colleagues earned the Best Poster award for their work titled “Mutation Profiling of High-Grade Serous Ovarian Adenocarcinomas Suggests STK11 and NOTCH1 as Candidate Biomarkers.” This paper reported identification of STK11 mutations as possible biomarkers for platinum-therapy response and detailed actionable mutations in several patients with ovarian cancer. The Best Research Project prize was awarded to Dr Dettloff and colleagues for their work “Utility of a microRNA-Based Assay for Diagnosing Tumor Origin in Soft Tissue Sarcomas.” The investigators examined the recent experience at Moffitt Cancer Center with noncoding microRNA tumor profiling of tumors of uncertain origin. They listed several situations in which the technique can be particularly useful, such as the identification of germ-cell tumors. They also recognized pitfalls of the technique, such as a bias toward sarcoma, possibly representing a biological shift in recurrent or previously treated tumors. The Best Clinicopathologic Correlation award was won by Dr Liu and colleagues for their poster titled “Spindle Cell/Sclerosing Rhabdomyosarcoma Is a New and Underdiagnosed Entity: A Clinical and Tissue Microarray Immunohistochemical Study With Literature Review.” This work emphasized the utility of using tissue microarray to screen previously encountered cases with antibodies directed against emerging biomarkers. Opening Remarks Anthony M. Magliocco, MD The esoteric and molecular laboratories in the Department of Pathology have implemented many new equipment platforms and assays to support the personalized medicine programs at Moffitt Cancer Center. These include the introduction of new next-generation sequencing assays for solid tumors and hematological malignancies, the arrival of the NanoString platform (NanoString Technologies, Seattle, Washington) to enable multiplex gene-expression profiling, and the performance of the Prosigna assay (NanoString Technologies) for the analysis of early breast cancer on site. Molecular experts continue to closely work with the personalized medicine services at Moffitt Cancer Center; a training fellowship program is also offered in moApril 2016, Vol. 23, No. 2 lecular pathology. Future directions will focus on the development of new, broader panels and technologies to monitor circulating tumor cells and free nucleic acid in patients with cancer (liquid biopsy). The laboratory also acknowledges the support from the Moffitt Foundation and the Morsani Family. Summaries of Select Platform Presentations Progression of Neoplasia in Barrett Esophagus Robert D. Odze, MD Barrett esophagus is a result of columnar metaplasia of the esophagus induced by gastroesophageal reflux disease that, through a progressive sequence of molecular changes, renders the mucosa at increased risk for cancer via a metaplasia–dysplasia–carcinoma sequence. The lecture focused on the diagnostic criteCancer Control 175 ria, controversies, and difficulties with regard to diagnosing and treating dysplasia in patients with Barrett esophagus. The lecture highlighted areas of new developments and future trends. The risk of malignancy and the treatment of patients with different types and grades of dysplasia were discussed along with new methods of endoscopic therapy and ablation. The roles of pathologists in evaluating biopsies and endoscopic mucosal resection specimens were also addressed. Progression of Neoplasia in Irritable Bowel Disease Robert D. Odze, MD Similar to Barrett esophagus, cancer in patients with irritable bowel disease (IBD) develops via an inflammation–dysplasia–carcinoma sequence. New developments have been made in regard to the gross (endoscopic) and microscopic classification of neoplasia in IBD. These new developments were discussed at length as well as various areas of controversy with regard to the diagnosis and management of dysplasia in patients with IBD. The criteria and limitations of dysplasia grading were discussed in detail, as were areas in need of future research and newly developed technologies for the treatment of patients with IBD and dysplastic lesions. The Continuing Dilemma of Ductal Carcinoma in Situ Stuart J. Schnitt, MD The term ductal carcinoma in situ (DCIS) encompasses a heterogeneous group of lesions that vary in their clinical presentation, distribution in the breast, pathological features, genetic and molecular alterations, and their potential to progress to invasive breast cancer. During the past 30 years, widespread use of screening mammography has resulted in a 9-fold increase in the incidence of DCIS. As a result, approximately 50,000 new cases of DCIS are diagnosed each year in the United States, and this lesion accounts for nearly 20% of breast “cancers” detected by mammography. Treatment of patients with DCIS is aimed at preventing the development of invasive breast cancer, and this is best thought of as prophylactic management. Current treatment options include mastectomy, excision, radiotherapy, and excision, any of which may be accompanied by endocrine therapy. Clinical follow-up studies have demonstrated that not all patients with DCIS will progress to invasive breast cancer. However, after decades of research, it is still not possible based on clinical and pathological features alone to reliably identify which patients with DCIS are likely to progress to invasive breast cancer and require aggressive treatment and, conversely, which patients are unlikely to progress and whose cancer may be adequately managed by excision alone or perhaps even no treatment beyond diagnostic biopsy. Although a variety of bio176 Cancer Control markers, as well as a newer molecular assay, is available to help stratify risk among patients with DCIS, the clinical value of biomarkers remains to be determined. Newer treatment strategies are assessing the role of neoadjuvant endocrine therapy and trastuzumab for estrogen receptor–positive and ERBB2 (formerlly HER2 or HER2/neu)–positive DCIS, respectively. In addition, clinical trials evaluating the role of “active surveillance” in patients with low-risk DCIS are underway. Furthermore, results from studies of the tumor microenvironment are providing new insights into the factors related to the progression of DCIS to invasive breast cancer. A better understanding of the molecular alterations associated with the progression of DCIS to invasive breast cancer may lead to new methods to distinguish those patients with DCIS likely to recur or progress from those who are not and to the identification of new targets for treatment and prevention. Clinical Decision-Making Based on Molecular Pathology Susan Minton, MD Genomic testing is now available and used in both the early- and late-stage settings of breast cancer to aid in the discussion of therapeutic options for select patients. With the development and approval of several targeted therapies for the treatment of metastatic breast cancer, the need is increasing to expand genomic testing to determine the most optimal therapy for patients. The availability of next-generation sequencing provides more data for the physician to utilize when making new treatment decisions. Several factors, such as the particular line of therapy, patient comorbidities, and previous biological behavior of the cancer, can help differentiate which test may be more beneficial in a particular circumstance. Prognostic genomic tests can enhance treatment decisions for early stage breast cancer. Several are now available in the early treatment setting and provide information about possible early and late risk of recurrence. Some of the advantages and limitations of the most commonly used tests were reviewed through case discussions. Evaluation of Postchemotherapy Breast Specimens Marilin Rosa, MD Neoadjuvant chemotherapy has become an established treatment modality for locally advanced and large breast carcinomas. In addition, its use provides an opportunity to evaluate patient response to chemotherapeutic drugs as well as to potentially change a patient’s treatment course if satisfactory results are not being achieved. The pathological evaluation of postchemotherapy breast specimens is challenging and requires careful correlation with previous biopsy results, radiographic imaging (especially pre- and posttreatment tumor sizes), and clinical information. PathApril 2016, Vol. 23, No. 2 ological complete response (pCR) is strongly associated with long-term outcome (disease-free and overall survival rates); therefore, histological evaluation is the gold standard for the evaluation of tumor response to therapy. The definition of pCR in the setting of breast carcinoma varies among institutions, but it is generally considered as the absence of residual invasive carcinoma within the breast and the lymph nodes. When examining a postchemotherapy specimen, the residual tumor, fibrotic tumor bed, or both should be grossly identified and mapped in its larger dimensions. The residual tumor should be generously sampled because estimations of residual tumor size and cellularity are necessary. To determine the rate of pCR is necessary in order to identify chemotherapy changes in the fibrotic tumor bed, at the site of biopsy, or both. Histological chemotherapy changes include hyalinized vascular stroma, stromal fibrosis, edema and elastosis, presence of macrophages, inflammation, and hemosiderin pigmentation, among others. In the absence of these, additional sampling is required and correlation with clinical and radiological findings is necessary. Multidisciplinary Gastrointestinal Case Presentation Kevin Neill, MD, et al We presented the case of a 33-year-old woman with a locally advanced, solid pseudopapillary neoplasm (SPN) of the pancreas that later metastasized to the liver. The presentation had a multidisciplinary format. Dr Neill began with a brief summary of the patient’s complete clinical course and the histopathology of solid pseudopapillary neoplasms. Dr Khazanchi presented a video on the endoscopic ultrasonographical imaging characteristics of solid pseudopapillary neoplasms. Dr Coppola then presented a comprehensive review of the current scientific literature on the pathology, molecular alterations, miRNA, and gene expression profiles of solid pseudopapillary neoplasms. Dr Malafa presented an overview on pancreaticoduodenectomy procedures, surgical management of solid pseudopapillary neoplasms, and expected surgical outcomes in relation to the patient case featured in this presentation. Dr Kis closed with a summary of the chosen interventional radiology treatment course, which included multiple chemoembolization procedures, chemoradiotherapy, and microwave ablation surgery. Incorporating Digital Pathology Into Pathology Practice Marilyn M. Bui, MD, PhD Digital pathology, including telepathology, whole-slide imaging, and image analysis is gaining more adoption in pathology practice. This talk discussed the current state of using digital pathology in surgical pathology practice in the United States based April 2016, Vol. 23, No. 2 on a literature review, communication with early adopters, and institutional and personal perspectives. Helpful resources, updates in technology, best-practice examples, practical issues using whole-slide imaging, such as validation, and the guidelines of telepathology were discussed. This talk also shed light on the bright future of digital pathology in surgical pathology and its opportunities in education and research. Leadership Challenges in Histology Malissa Snyder Histotechnologists in leadership roles face many challenges on a continual basis, and yet they must maintain a strong emphasis on patient care. The histology laboratory produces high volumes of work and must also keep quality control while maintaining appropriate turnaround times, which can be achieved by workload measurements and maintaining adequate staffing levels. This workshop discussed strategies for prioritizing workflow and managing fast turnaround times in a high-pressure environment. Tips for troubleshooting common problems, tools for effective communication between all team members in the laboratory, and tips for managing difficult personalities were discussed. Challenges in Immunohistochemistry Jodi Balasi With new antibodies emerging and the demand for these antibodies to be integrated into the pathologist’s diagnosis, histologists are challenged to test and validate new antibodies into their current immunohistochemistry menu. Whether a histotechnologist is new to the process or is a seasoned professional, the process always starts with the optimization and validation of the antibody. This workshop highlighted the immunohistochemistry protocol set up, testing, documentation, and validation of in vitro diagnostic, analyte specific reagent, and research-use only antibodies. Selected Abstracts Accepted for Poster Presentations 1. Further Characterization of Circulating Tumor Cells With User-Defined Antibodies John Puskas, Gisela Cáceres, Carolyn Loret de Mola, Mike Gruidl, and Anthony Magliocco H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Evaluation of circulating tumor cells (CTCs) is emerging as an important component of the liquid biopsy and provides important information for oncologists about their patients. CTCs provide a real time evaluation of current disease state in patients and provide information of tumor evolution in metastatic sites that cannot be biopsied. Also, monitoring CTCs may provide pharmacodynamic information regarding the effect of treatment to cause cell cycle arrest or Cancer Control 177 dampen signaling pathways. This could be used to more quickly discontinue futile treatments, enabling a switch to alternate options or maintain treatments that appear effective. The most recognizable CTC phenotype is EpCAM+, cytokeratin 8, 18 or 19+, DAPI+, and CD45- cells and CTCs are measured in the peripheral blood (PB) using the FDA-approved CellTracks System for metastatic breast, colorectal and prostate cancers. To use this technology for other applications the assay requires modification that includes capture antibody selection (CD146, N-cadherin and O-cadherin), unique cell surface markers (Her2/neu and AR) and nuclear markers (Ki67) and validation using cell lines, creating a LDT. The technology is limited by the number of available markers and the ability to measure the expression level of the protein in the captured cells as well as the viability which is 24–96 hours. Recently we have verified the use of HER2/neu, Ki67 and androgen receptor (AR) user-defined antibodies for further characterization of CTCs and are applying these new assays for use in clinical trials. 2. Comparing and Contrasting Decalcification Solutions: Cellular and Nuclear Preservation Malissa Snyder and Lindsey Martinez H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida The goal of this project is to preserve nuclear and cellular detail by finding the best decalcification solution. Preserving antigenicity is also a main goal in order to achieve the highest quality of immunohistochemical staining. Three decalcification solutions were used by testing surgical and bone marrow solutions and comparing the H&E stains and pertinent Immunohistochemistry stains. Three decalcification solutions were used: nitric acid (Nitrical), formic acid (Formical), and formic acid with formalin (Formical-4). Six surgical specimens were tested, and of the six, the Formical-4 yielded the best results. Four bone marrow specimens were also tested with the three different decalcification solutions, and again, the Formical-4 gave the best results. The Formical-4 preserved the nuclei and cytoplasmic components of the cells of both specimen types the best. All of the immunohistochemistry stains that were tested also yielded the best staining results. 3. Cancer Cell Line Doubling Time Describes Epithelial-Mesenchymal Transition Bernadette M. Boac,1 Yin Xiong,1,2 Douglas C. Marchion,1,2 Forough Abbasi,1,2 Beman R. Khulpateea,1 E. Clair McClung,1 Stephen H. Bush,1 Sharon E. Robertson,1 Johnathan M. Lancaster,1–3 and Anthony M. Magliocco1,3,4 1 Department of Women’s Oncology, 2 Chemical Biology and Molecular Medicine Program, 3 Department of Oncologic Sciences, 4 Department 178 Cancer Control of Anatomic Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Background: Patients with aggressively dividing cancers are generally diagnosed at an advanced stage of disease, have higher tumor burden, and carry a poorer prognosis. We sought to identify molecular signaling pathways associated with fast dividing (high doubling rate) cancer cells. Methods: Pathways were identified using SAM (significance analysis of microarrays) correlation of Affymetrix HG-U133_Plus_2 expression data and doubling time in 59 human cancer cell lines encompassing 9 cancer cell types. PCA (principal component analysis) was used to analyze the pathways association with overall survival using institutional as well as publically available clinicogenomic datasets containing a total of 2,205 patients with a broad range of human cancers, including ovarian, breast, lung, leukemia, brain, and colon. Results: Pearson’s correlation of doubling times and genomic expression data identified the differential expression of 3,811 probesets (FDR < 0.05: 1,110 upregulated; 2,701 downregulated) representing 47 molecular signaling pathways (FDR<0.05, fold change > 2) to be associated with fast dividing cancer cells. Twentytwo pathways (P < 0.05) were found to be associated with overall survival in 2 or more datasets. The integrins pathway was associated with survival in 5 datasets comprising of breast, colon, and ovarian cancers. Conclusion: Several pathways associated with cancer cell doubling time may influence overall survival and thus be good therapeutic targets for drug development. Surprisingly, the majority of these pathways are descriptive of epithelial-mesenchymal transition biology. 4. Validation of a Rapid, Sensitive, and Inexpensive QClamp BRAF Assay for the Detection of Codon 600 Specific Mutations Ravi Kothapalli,1 James Saller,1 Carolyn Loret de Mola,1 and Anthony Magliocco1,2 1Clinical Testing Development, 2 Department of Anatomic Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida BRAF mutations occur in approximately 8% of all human cancers and around 50% in melanoma and papillary thyroid carcinoma. Identification of these mutations provides potentially valuable theranostic and prognostic information in cancer patients. DiaCarta (Hayward, CA) has developed a QClamp BRAF Codon Specific Mutation Test in Codon 600 based on xenonucleic acid mediated PCR clamping technology. Validation of this assay is performed prior to implementing it for routine use in clinical laboratory. DNA patient samples that were selected have a BRAF mutation at the 600 codon whereas others are negatives. Allelic frequencies of between 50% to 0.156% BRAF, V600E were April 2016, Vol. 23, No. 2 made by mixing appropriate portions of BRAF mutant DNA and wild type DNA obtained from Horizon Diagnostic (Cambridge, UK) and used in the assay. DiaCarta BRAF assay can reliably detect up to 0.156 % allelic frequency. The results of the assay are 100% in agreement with Nextgen sequencing or pyro sequencing. The main advantage of this assay is to detect the BRAF mutation in ultra-low concentrations of DNA (10 ng of total DNA) as obtained from specimens including FFPE, liquid biopsies, cytology and tissues samples. The BRAF V600E mutation may serve as a very sensitive biomarker when the DiaCarta assay is used. Assays with high analytical sensitivity will likely be required for effective screening and minimal residual disease monitoring in liquid biopsy samples, as they can detect mutations in samples of DNA with high contaminating normal DNA background and identification of rare subclones within heterogeneous tumor samples. 5. Mutation Profiling of High-Grade Serous Ovarian Adenocarcinomas Suggests STK11 and NOTCH1 as Candidate Biomarkers Elena Ryzhova,1 Douglas C. Marchion,2,3 Forough Abbasi,2,3 Liang Nong,1 Alexandra Gartel, 4 Yin Xiong,2,3 and Anthony Magliocco1,5 1Clinical Testing Development Laboratory, 2Department of Women’s Oncology, 3Chemical Biology and Molecular Medicine, 4Molecular Laboratory, 5Department of Anatomic Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Introduction: The standard treatment of high grade serous ovarian cancer (HGS-OvCa) with platinum-taxane chemotherapy is followed by the development of chemoresistant disease in a significant proportion of patients. Molecular mechanisms underpinning chemoresistance remain obscure. In this study we performed mutation profiling of patients with varying responses to chemotherapy to investigate whether platinum-resistant phenotype can be linked to a particular molecular signature. Methods: DNA samples from 37 HGS-OvCa patients classified as either complete responders (CR, n = 18) or incomplete responders (IR, n = 19) were tested in Agena BioScience OncoPanel. The OncoPanel relies on the iPlex-MassArray methodology and permits parallel screening of 214 mutations in 26 oncogenes and tumor suppressor. Results: Frameshift or nonsense mutations in STK11 were found in 22% of the CR group suggesting a significant association with the disease phenotype (Fisher’s exact test, p=0.046). Further, mutations in NOTCH1 were identified in 16% samples regardless of platinum-taxane response status. NOTCH has been previously associated with certain hematologic and solid tumors; however it was not described in the context of HGS-OvCa. Lastly, well-established actionable April 2016, Vol. 23, No. 2 mutations in KRAS, EGFR, and BRAF were detected in patients demonstrating CR and IR. Conclusions: We have identified STK11 mutations as possible biomarkers for platinum-therapy response. Additionally, we have found that NOTCH1 could be a candidate for therapeutic targeting in HGSOvCa. Furthermore, we have identified several HGSOvCa specimens that harbor known actionable mutations. These data suggest that routine multiplexed mutation profiling of HGS-OvCa patients may facilitate the proper selection of available therapeutic options and/or clinical trials. 6. Development and Validation of a MassArray Custom GliomaPanel for Testing IDH1 and IDH2 Mutations in a Clinical Setting Elena Ryzhova,1 Rob Macaulay,2 Liang Nong,1 Ravi Kothapalli,1 Linda McIntosh,1 Gisela Caceres,1 Alexandra Gartel,3 Janella Lal,3 Shital Gandhi,3 Hema Liyanage,5 Robin Everts,5 Carolyn Loret de Mola,1 Dahui Qin,3 Peter Forsyth,4 and Anthony Magliocco1,2 1Clinical Testing Development, 2 Department of Anatomic Pathology, 3Laboratory of Molecular Pathology, 4Department of Neuro Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, 5 Applications and Technology, Agena Bioscience, San Diego, California Introduction: IDH1 and IDH2 mutations are associated with favorable outcome in low-grade gliomas. The common methods of IDH1, 2 testing are characterized by either limited spectrum of detected mutations (IHC) or low sensitivity (Sanger sequencing). To overcome these limitations we developed high throughput, sensitive and multiplexed GliomaPanel test using Agena Biosciences’ MassARRAY technology. Methods: GliomaPanel was designed to interrogate all the essential mutations in IDH1 and IDH2. Additionally, it includes 116 hot-spot targets in 17 genes with potential clinical or research utility. The detection method relies on a single nucleotide extension followed by MALDI-TOF mass spectrometry. Results: The panel was validated in accordance with the CAP guidelines and included the total of 78 FFPE diagnostic samples. For the majority of samples, the mutation status was pre-determined by IHC. The established GliomaPanel accuracy, specificity and sensitivity were 99%, 100%, and 97% respectively. Notably, the rare IDH1 R132C mutation was detected in several patients that were IDH1 negative in IHC tests. Additionally, R132C mutation at low frequency (20%) was identified in one patient, whose IDH1 status was negative in both IHC and Sanger sequencing. The discrepancies were resolved by Sanger sequencing confirming GliomaPanel results. Finally, we demonstrated that 70% of DNA samples, which failed NGS testing acceptance criteria, were accurately processed by GliomaPanel. Cancer Control 179 Conclusions: GliomaPanel offers cost-effective high-throughput solution for accurate of IDH1 and IDH2 profiling. The method detects a broad range of mutations, is highly sensitive, has relaxed DNA quality requirements, and represents a valuable alternative to conventional methods for targeted mutation profiling. 7. Higher Frequency of Isolated PMS2 Loss in Colorectal Tumors From Colombian Population Rania Shamekh,1 Mauro Cives,2 Jaime Mejia,3 and Domenico Coppola4 1Department of Pathology, University of South Florida, Tampa, Florida, 2Department of GastroIntestinal Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, 3Department of Pathology, Institutode Patologia Mejia Jimenez, Cali, Colombia, 4Department of Anatomic Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of death worldwide. It accounts for over 9% of all cancer. One of the pathogenic factors of CRC is germ line mutation, leading to alteration and inactivation of the MMR genes. The aim of the study is to compare the frequency of alterations in MMR protein expression in CRC of Caucasian as compared to Colombian patients. Forty-five Colombians and 48 Caucasians with CRC were studied. The tumors’ microsatellite instability (MSI) status was determined in primary CRC by immunohistochemistry using the automated Ventana Ultra. The combined loss of MLH1 and PMS2 was the most common alteration in both Colombian (11%; 5 out of 45) and Caucasian (12%; 6 out of 48) CRC patients. Interestingly, the loss of PMS2 expression in the presence of intact MLH1 was the second most common alteration in Colombians (8%; 4 out of 45) which was never seen in the Caucasians cohort (P = 0.05). The loss of MLH1 alone and the combined loss of MSH6 and PMS2 expression was only observed in one out of 45 (2%) in Colombians but not in Caucasians. The combined loss of MSH2 and MSH6 was not observed in any of the patients studied. The current findings highlight the significant difference in alterations of MMR protein expression in CRC patients from Colombia compared to Caucasians. These findings are novel and warrant further studies in larger cohorts. 8. Human Lung Cancer Derived Cancer Associated Fibroblasts Express a Variety of Immunosuppressive Mechanisms Arjun Rammohandas, David Noyes, Melanie MediavillaVarela, Liliana Lyviv, Dillan Villavisanis, and Scott Antonia H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 180 Cancer Control Immunotherapy has emerged as clinical efficacious in a variety of different cancer types. In particular this modality has been revolutionary in the treatment of lung cancer with anti-PD1/PD-L1 producing objective tumor responses in approximately 20% of patients and a dramatic impact on overall survival. As impactful as this has been, a large number of lung cancer patients do not benefit. This is due to the fact that there are numerous ways that tumors evade rejection by T cells, including a number of immunosuppressive mechanisms that can be operational within the tumor microenvironment including immune checkpoint ligands, immunosuppressive cytokines and enzymes, and immunosuppressive cells. One of these is the cancer associated fibroblast (CAF), though the precise way that these cells inhibit T-cells has been unknown. Freshly resected tumors not necessary for pathology were harvested, mechanically and enzymatically disaggregated and digested cells were plated in complete RPMI and allowed to adhere and proliferate CAFs. We have successfully propagated dozens of CAF and tumor infiltrating lymphocyte (TIL) lines and characterized a portion of them so far. Characterization was accomplished with immune-phenotypic characterization through flow cytometry. ELISA was used to determine immunosuppression with CAFs and TILs. Cytokine production was analyzed with multiple analyte profiling. Data were confirmed with qRT-PCR and Western blot analysis. Here we show that several human lung cancer derived CAFs constitutively express PD-L1 and CD73, and can be induced to express MHC class II, HVEM, IL-6, CXCL8, IDO, and NOS, all of which are immunosuppressive. These data suggest that a single manipulation targeting CAFs could influence CAF phenotype and cytokine secretion affecting the tumor microenvironment. 9. Decreased Stromal Expression of Caveolin-1 as a Potential Biomarker for Ovarian Serous Carcinomas Daryoush Saeed-Vafa,1 Douglas C. Marchion,2 Susan M. McCarthy,1 Lu Chen,3 Ardeshir Hakam,1 Emily C. McClung,2 Robert M. Wenham,2 Sachin M. Apte,2 Dung-Tsa Chen,3 Anthony M. Magliocco,1 and Jenny Permuth-Wey4,5 Departments of 1Anatomic Pathology, 2Women’s Oncology, 3Biostatistics and Bioinformatics, 4Cancer Epidemiology, 5Gastrointestinal Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Background: Previous studies have demonstrated that Caveolin-1 (Cav-1) can act as a tumor suppressor in various malignant neoplasms. It has recently been shown that there is reduced expression of Cav-1 in the stroma of malignant tumors as compared to the stroma of their benign counter parts and that this expression may be correlated with histological grade, specifically April 2016, Vol. 23, No. 2 in ovarian serous tumors. To our knowledge this difference has yet to be quantified. This study utilized the Automated Quantitative Software Analysis (AQUA) system and attempted to quantify the difference in the level of expression of Cav-1 in the reactive stroma of ovarian serous carcinomas as compared to stroma from benign ovaries. Material/Methods: Cav-1 expression was studied via a multiplex immunofluorescence assay in five tissue microarrays containing duplicate 1 mm cores from various grades of ovarian serous carcinomas and benign ovaries, and was quantified via an AQUA algorithm specifically designed to target stromal Cav-1 expression. Results: The study included cores representing reactive stroma from 117 unique ovarian serous carcinoma cases and 30 cases demonstrating stroma from benign ovaries. There was a statistically significant decrease in the expression of Cav-1 in the reactive stroma of ovarian serous carcinomas as compared to the stroma of benign ovaries, with mean AQUA scores, quantifying the amount of Cav-1, of 161.1 and 324.9 respectively (P < .0001). Conclusion: Our findings demonstrate that decreased stromal Cav-1 expression may be associated with malignant ovarian serous carcinomas. Studies are underway to evaluate stromal Cav-1 expression as a novel prognostic marker and rational therapeutic target for ovarian cancer. 10. Utility of a microRNA-Based Assay for Diagnosing Tumor Origin in Soft Tissue Sarcomas Jennifer Dettloff, Evita Henderson-Jackson, and Marilyn M. Bui H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Background: An accurate and definitive diagnosis plays a critical role in precision medicine. However, soft tissue sarcomas frequently present as diagnostic challenges due to their rarity, diversity and overlapping with other malignancies, both morphologically and immunohistochemically. MicroRNA (miRNA) is a master control of post-transcriptional regulation of proteincoding genes, some of which influence oncogenesis. MiRNA-based testing is an emerging tool in diagnosing tumor origin. This study is intended to investigate its utility in diagnosing soft tissue sarcomas. Materials and Methods: The MCC archives contained 44 cases that had been sent for a commercially available miRNA-based tumor-of-origin test. Of those 44 cases 33 had sarcoma in the differential diagnosis. The data included pre-testing working diagnosis, miRNA test result, and the post-testing multidisciplinary decision. Results: Of the 33 cases we used clinical, histopathologic and immunohistochemical findings to determine the most likely diagnosis as the gold standard. April 2016, Vol. 23, No. 2 There were 23/33 cases (69%) that yielded a result of sarcoma. Of those only 8/23 cases (35%) appeared to actually be sarcomas. The test appeared especially weak at differentiating sarcoma from carcinoma/ melanoma, which was the differential in 17 cases. Of those 17 cases the clinical and pathologic features favored: carcinoma/melanoma in 11/17, sarcoma in 3/17, and 3/17 were equivocal. The majority of the probable carcinoma/melanoma cases appeared clinically to be metastatic. Cytokeratin or EMA expression was seen in all probable carcinoma cases except one adrenal cortical carcinoma. Seven of 33 tested as mesothelioma, none of which clinically or pathologically appeared to be mesothelioma, including one suspected chordoma; these results were therefore considered uninformative. Three of the 33 cases included a differential diagnosis specifically between a germ cell tumor (GCT) and a sarcoma. One of these three cases, although the clinical impression was lymphoma or sarcoma, tested for seminomatous GCT, which eventually lead to the correct diagnosis of a seminoma. In the second of these three cases, a patient with a history of a testicular tumor presented with a new groin mass. The test confirmed the correct diagnosis of a new primary sarcoma. The third of these cases also carried the differential of GCT due increased HCG clinically; the miRNA test was helpful in helping rule out that differential. Four of the 33 cases yielded a test result of lymphoma (B- or T-cell origin). Two of these were of predicted dendritic cell origin and the miRNA test was helpful; one was a medullary carcinoma of the breast and one was a sarcoma. 11. Incidental Metastatic Medullary Carcinoma of the Colon Involving an Extraterritorial Adenoma: Diagnostic Pitfall and Impact on Patient Care Carolina Dominguez, Cory Porteus, Diana Braswell, Sherma Zibadi, and Kun Jiang H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Context: Microsatellite instability associated colorectal medullary carcinoma (MC) has been gaining attention recently due to its impact on clinical management and prognostication. Usually, MC is composed of sheets of tumor cells with pushing borders and lymphoplasmacytic banding. Rarely does MC present as a mucinous adenocarcinoma or a pure signet ring cell carcinoma. It is believed that MC possesses less aggressive properties compared with microsatellite-stable tumors; however, we recently encountered an opposite example, with lymphovascular invasion and distant metastasis in MC. Design: We present a unique case of metastatic signet ring cell MC, which was incidentally identified involving a noncontiguous distant adenoma following screening colonoscopy, so as to emphasize these Cancer Control 181 unique and essential histological features and to caution diagnostic pitfalls. Results: A 58-year-old man presents for a routine colonoscopy. A 2-cm adenoma was removed from hepatic flexure, which showed signet ring cell carcinoma, with loss of MLH1/PMS2 and retained MSH2/MSH6 immunohistochemically. Next generation sequencing showed no mutation in BRAF, EGFR, and Ras. The cauterized margin was benign. Significantly, biopsy of a noncontiguous 0.5 cm adenoma (35 cm distal) showed rare signet ring cells within lymphovascular spaces, with identical morphology and immunophenotype as seen in MC. Due to this unexpected finding, patient received chemotherapy, awaiting resection. Conclusions: MC could present as signet ring call carcinoma, with multifocal lymphovascular invasion and aggressive clinical behavior; understanding the molecular basis of MC and awareness of its histological features and clinical impact are pivotal for reaching the correct diagnosis and for improving patient management. 12. Metastatic Basal Cell Carcinoma: Recent Moffitt Cancer Center Experience T. Mendoza, Farah Khalil, and J. Messina H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Introduction: Basal cell carcinoma (BCC) of the skin has the lowest incidence of metastasis of any epithelial malignancy, even though it is the most common primary carcinoma. Methods: Cases of metastatic BCC encountered in our practice were included. The subtype of primary BCC was documented on review of the cases. The diagnosis of metastatic BCC was based on standard histologic criteria: histomorphologic confirmation with primary cutaneous BCC and supportive immunohistochemical studies were performed. Results: Six cases of metastatic basal cell carcinoma were included for study. The histologic subtypes included: four infiltrative BCCs, one infiltrative BCC with basosquamous features, and one basosquamous type BCC. The age of patients at diagnosis of the metastatic BCC ranged from 50 to 77. All were men. The sites of metastasis included: lung (3 patients), lymph nodes and lung (2 patients) and bone (1 patient). The time to metastasis was available in five cases and ranged from 4 years to 20 years; the median time was 7 years. Of the two patients with lymph node and lung metastases, one patient had a lymph node metastasis 9 years after diagnosis of the primary and a lung metastasis 16 years after diagnosis. The lymph node and lung metastases in the other patient were found 20 years after diagnosis. Significant therapy and survival information was available for four patients not recently diagnosed: four patients were treated in a hedgehog inhibitor trial 182 Cancer Control (vismodegib). Three of those four also underwent radiation therapy. One patient died of disease progression at one year, one patient had disease progression at two years, one patient had stable disease at two years and one patient was lost to follow-up shortly after therapy. Parenthetically, one of the six cases was identified as having a PTCH1 genetic alteration, which is a target of hedgehog inhibitor therapy. Conclusion: The metastatic BCC in our experience preferentially spread to the lung, followed infiltrative BCCs, and occurred a median of seven years after diagnosis. 13. Neuroendocrine Tumors (NETs) of the Thymus: Experience of a Single Cancer Center Sameer Al Diffalha1 and Farah Khalil1,2 1Department of Pathology and Surgical Oncology H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, 2University of South Florida Department of Pathology and Cell Biology, Tampa, Florida Background: Neuroendocrine tumors (NETs) of the mediastinum and thymus are rare. The term carcinoid was first presented in 1907 by Oberndorfer. Rosai and Higa were the first to acknowledge the existence of carcinoid tumors in the thymus. The median age for thymic NETs is 59 years and it is more common in males (M: F ratio 3:1). It has an annual incidence of 0.01/100,000 in the United States with ~ 400 cases reported in the literature to date. The prevalence of thymic NET is ∼ 3% of the total number of NETs at all sites and is estimated to account for approximately 2% to 4% of all anterior mediastinal neoplasms. It may be part of multiple endocrine neoplasia type 1 syndrome (MEN-1, 5%–15%). Material and Methods: We searched our database for all thymic neuroendocrine tumors that had been diagnosed between 1987 and 2015 (August). During this interval, there were 27 patients who had been diagnosed with thymic neuroendocrine tumor. Three patients had typical carcinoid tumor and 23 patients had atypical carcinoid tumor or high-grade neuroendocrine tumor. Tumorigenesis: The first hypothesis proposes that the NET tumor cells arise from ectopic tissues and the other hypothesis suggests that they arise from teratomatous components. Molecular Biology: NETs have a distinct gene expression from the normal thymus. The development of NETs may involve different genes, each of which may be associated with several different abnormalities that include point mutations, gene deletions, DNA methylation, chromosomal losses and chromosomal gains. The foregut, mid-gut and hindgut NETs have different molecular pathways. Foregut NETs have frequent deletions and mutations of MEN1, mid-gut NETs have losses of chromosome 18, 11q and 16q and hindgut NETs April 2016, Vol. 23, No. 2 express transforming growth factor alpha and the epidermal growth factor receptor. There are chromosomal imbalances that have been found by some investigators in thymic tumors, which include gains of chromosome Xp, 7p, 7q, 11q, 12q and 20q, and losses at 6q, 6p, 4q, 3p, 10q, 11q and 13q. Loss of heterozygosity (LOH) at chromosome 1p has been reported in two thymic neuroendocrine tumors. Loss of chromosomes 3, 9p21, Y and gain of chromosome 19p were discovered in one case. Bi Y.F. et al found evidence of Wnt pathway activation and consistently elevated level of cyclin D1 in AC tumors but, c-Myc expression changed only moderately. Prognosis: Thymic NETs have more aggressive behavior than their pulmonary equivalents. The most important prognostic factors with validated significance are: Tumor size, histological grade, paraneoplastic symptoms, Thymic Masaoka staging and surgical resection. Ki67 proliferation index is the key player in the stratification of patients with good and bad prognosis in other neuroendocrine tumors. Regional lymph nodes are often affected and distant metastases are common. Management: Surgical resection remains the typical management for NETs. Surgery followed by chemotherapy has become standard for metastatic bronchial and thymic NETs. Tyrosine kinase inhibitors (eg, sunitinib) and TOR inhibitor everolimus have been stated to show better prognosis in a small series. Cisplatinum-based regimens have also been of value in thymic NETs. SCLC is sensitive to chemotherapy and since the biological characteristics of LCNEC are similar to those of SCLC, both are treated with chemotherapy regimens. 14. Lymphoepithelioma-Like Hepatocellular Carcinoma Mimicking Hodgkin Lymphoma: Diagnostic Pitfalls and Impact on Management Ian Martin, Rania Shamekh, Julie Gibbs, Ardeshir Hakam, and Kun Jiang H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Introduction: Hepatocellular lymphoepitheliomalike carcinoma (LELC) is a unique, poorly differentiated carcinoma featuring dense lymphoid infiltrate surrounding neoplastic hepatocytes. To date, only a few cases have been reported. The etiological connection with immunosuppression or Epstein–Barr virus has not been determined. Morphologically, hepatocellular LELC mimics certain types of lymphoma, especially Hodgkin lymphoma, posing diagnostic challenges. As LELC patients generally have a better prognosis than those with conventional hepatocellular carcinoma, the correct diagnosis is crucial for patient care. Methods: We present a case of hepatocellular LELC that was misdiagnosed and managed as Hodgkin April 2016, Vol. 23, No. 2 lymphoma at an outside institution during a 6-month period. The aim of the study is to emphasize the key histological and immunophenotypical characteristics of LELC in an attempt to reinforce its diagnostic features and impact on clinical course. Results: A 71-year-old male was referred by his oncologist for a second opinion regarding his liver mass and hilar adenopathy, which was originally interpreted as Hodgkin’s lymphoma by an outside institution 6 months prior. The liver biopsy was reviewed at our institution. It showed a poorly differentiated carcinoma with dense lymphocytic infiltrate admixed with few large and prominent epithelial cells. Notably, no eosinophils or neutrophils were identified within the lymphoid infiltrate. The epithelial cells were immunoreactive for AE1/3, HepPar-1, and EMA, indicating hepatocellular origin. They were negative for CD15, CD30, and CD45. Importantly, the Ki-67 index was 80%, which is highly suspicious for malignancy. Furthermore, repeat bone marrow biopsy, flow cytometry, and lymph node biopsies were unremarkable. The diagnosis of hepatocellular LELC was established. The patient’s clinical management was subsequently adjusted following this diagnosis. Conclusions: This case demonstrates that hepatocellular LELC could be misinterpreted as a lymphoproliferative disease, potentially derailing clinical management. An astute histological and immunophenotypical examination plus awareness of this entity’s clinical impact are pivotal for appropriate patient management. 15. A Rare Case of Combined Small Cell Lung Cancer With Adenocarcinoma and Squamous Cell Carcinoma Sameer Al Diffalha1 and Farah Khalil1,2 1Department of Pathology and Surgical Oncology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, 2University of South Florida College of Medicine, Department of Pathology and Cell Biology, Tampa, Florida Introduction: Combined small cell lung cancer (c-SCLC) is relatively rare and occurs in approximately 2% to 14% of all cases of small cell lung cancer (SCLC). c-SCLC is defined by the World Health Organization (WHO) as small cell carcinoma combined with an additional component consisting of any non–small-cell histologic type, including adenocarcinoma, squamous cell carcinoma (SCC), and large cell neuroendocrine carcinoma (LCNEC). Most of the combined cases are SCLC with either adenocarcinoma or squamous component. Rare cases with up to 3 different morphologic tumors have been reported, and individual tumors containing up to 4 different morphologic constituents have been described. Case Presentation: We present the case of a 78-year-old male with a smoking history of 2 to 3 Cancer Control 183 packs of cigarettes per day for 35 years. The patient was referred for evaluation of right upper lobe (RUL) lung mass to Moffitt Cancer Center. The CT scan showed a 3.0 × 2.3 × 2.5 × cm RUL lung mass with mildly prominent mediastinal and hilar lymphadenopathy. The patient elected to proceed with right thoracotomy with right upper lobectomy, intercostal muscle flap, and lymphadenectomy. Microscopic examination showed combined well-differentiated adenosquamous carcinoma and small cell carcinoma. Next generation sequencing did not identify clinically relevant results. Tumor Genesis, Molecular: The mechanism of c-SCLC is believed to be the simultaneous appearance of both SCLC and other histological types, or the partial malignant transformation from non-SCLC. Tyrosine kinase inhibitors (TKIs) are active against different mutations in EGFR. While EGFR mutations are very rare in SCLC, they are quiet common (about 15%–20%) in c-SCLC, usually in nonsmoking females with adenocarcinoma component. Prognosis: In general, patients with c-SCLC have poor prognosis due to the small cell component. However, c-SCLC has worse outcome due to increased resistance to chemotherapy (CT) and/or radiation therapy (RT) than “pure” SCLC. Management: While there are available guidelines for treating SCLC, the optimal treatment for cSCLC, which will improve prognosis has not been adequately determined. Few studies suggest surgery for very early stage c-SCLC, which might improve the patient’s outcome. However others recommend starting CT and/or RT and then following with surgical intervention for the residual NSCLC components. Conclusion: Combined tumors in any organ site raise interesting biologic questions about the pathogenesis and relationship of the individual components. Moreover, an accurate understanding of c-SCLCs is of great practical importance because treatment strategies are significantly different for NSCLC and SCLC. 16. Diagnostic Dilemma: Incidentally Identified Disseminated Peritoneal Mantle Cell Lymphoma Mimicking Peritonitis Following Esophagogastrectomy and Splenectomy for an Invasive Esophageal Adenocarcinoma Cory Porteus, Carolina Dominguez, Diana Braswell, Elizabeth M. Sagatys, and Kun Jiang H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Introduction: Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell lymphoma that represents 7% of non-Hodgkin cases, composed of CD5-positive naive B-cell of mantle zone surrounding germinal center follicles. MCL is clinically challenging to manage, with an ominous prognosis. MCL involves lymph nodes, spleen, bone marrow, and gastrointestinal tract 184 Cancer Control organs. MCL involvement of peritoneal cavity and omentum is extremely rare, if not unheard of. Methods: Here we present a novel case of disseminated peritoneal and omental MCL mimicking postsurgery peritonitis after surgical resection of an esophageal tumor. Results: A 67-year-old gentleman was referred to our cancer center recently for an esophagogastrectomy and splenectomy due to esophageal adenocarcinoma and splenomegaly. Incidental MCL was diagnosed in the splenectomy. Following the surgeries he developed high fever and acute abdomen, tentatively managed as surgery-related peritonitis up to 3 months; he finally underwent debriding omentectomy. Microscopic examination illustrates numerous perivascular and nodular atypical lymphocytes aggregates with associated fat necrosis. No metastatic carcinoma was seen. Immunohistochemistry demonstrates that the lymphocytes are positive for CD20, PAX5, and BCL-1 (cyclin D1). Special stains and cultures for microorganisms were negative. These findings indicate diffuse peritoneal involvement by MCL, ruling out an infectious etiology. Conclusion: This is the first report of post-surgery peritoneal dissemination by MCL mimicking peritonitis and omentitis, implicating the surgery as the main culprit of peritoneal lymphomatosis, suggesting that peritoneal seeding and lymphomatosis could be potential complication of surgeries in MCL patients. Additionally, our case demonstrates that peritoneal MCL recurrence could be under recognized in patient(s) even after curative resection of an unrelated entity; the underlying mechanism is likely related to wound contamination, peritoneal trauma, postoperative immunosuppression and potentially various surgical routes. 17. Quantification of Focal Oligodendroglial Hyperplasia in a Patient With Epilepsy H.C. Rutherford,1 J.A.E. Radic,2 R.J.B. Macaulay,1 R.M. Sadler,3 T. Bardouille,4 A. Ross,5 and D.B. Clarke2 1 Department of Anatomic Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, Divisions of 2Neurosurgery and 3Neurology, University of South Florida, Tampa, Florida, 4Biomedical Translational Imaging Centre, IWK Health Centre, Halifax, Nova Scotia, Canada, 5Division of Nuclear Medicine, Department of Diagnostic Radiology, Dalhousie University, Halifax, Nova Scotia, Canada A 39-year-old female with medically refractory epilepsy underwent MRI, EEG as well as magneto-encephalography, and fluorodeoxyglucose positron emission tomography (PET). A lesion in the right inferomesial frontal lobe was identified, and subdural electrode recordings demonstrated seizure onset from this area. The patient underwent surgical resection of the lesion. Histopathology revealed focally increased cell density in the subcortical white matter. Overlying cortex was April 2016, Vol. 23, No. 2 unremarkable, with no dysmorphic or disorganized neurons, and no balloon cells to suggest focal cortical dysplasia (FCD). The supernumerary cells were morphologically similar to oligodendrocytes, and occupied normally myelinated white matter. Rare mildly reactive astrocytes were found. There was a gradual tapering of cell density towards deeper white matter regions. No perinuclear halo, microcalcification, microcystic change, nuclear pleomorphism or mitotic activity was evident, militating against oligodendroglioma (ODG). There was no pooling of mucinous material or ‘floating’ neuron to suggest dysembryoplastic neuroepithelial tumor (DNT). Immunohistochemistry (IHC) for IDH1 R13H and BRAF V600E were negative. Quantification of oligodendrocytes in high (3350.5 cells/mm2), compared to low (1683.6 cells/mm2) density areas in adjacent normal-appearing white matter was highly statistically significant (P < .001). A descriptive diagnosis of focal oligodendroglial hyperplasia (ODH) was eventually proposed. ODH is a poorly characterized subtype of microdysgenesis distinct from FCD, and a rare cause of medically refractory epilepsy. No carefully documented case is available in the medical literature for comparison. Histologically, it is important that ODH is differentiated from ODG and DNT. Because of the paucity of published cases, the clinical impact of surgical resection of ODH found within the seizure onset zone is uncertain. The patient continues to have 1-2 nocturnal partial (focal with dyscognitive features) seizures per month, but has had no convulsive seizure since her surgery (4.5-year follow-up). This report quantifies the density of oligodendrocytes within different neighboring portions of ODH and documents its morphologic and IHC features through analysis of a right inferomesial frontal lobe lesion. 18. Electronic Atlas of Epithelioid Soft Tissue Tumors: A Pilot for Pathology Atlas “App” Development Robert P. Seifert1 and Marilyn M. Bui1,2 1Department of Pathology and Cell Biology, University of South Florida Morsani College of Medicine, Tampa, Florida, 2Department of Anatomic Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Background: The “Atlas of Pancreatic Pathology and Cytopathology” apps developed by Johns Hopkins Pathology allow for accessible, rapid review of pancreatic histology. However, pathology, as a specialty, is lagging in integrating handheld devices into training and daily practice. An electronic atlas “app” for handheld devices can correlate clinical and histologic features quickly. The scarcity of pathology apps can be attributed to lack of time, funding and programming knowledge. To address this gap, we developed a proofof-concept atlas app encompassing soft tissue tumors with epithelioid morphology. April 2016, Vol. 23, No. 2 Methods: A free video game development platform (GameMaker Studio v1.4, YoYo Games, Dundee, Scotland) was selected because it is export platform agnostic and requires minimal programming knowledge. This platform has a relatively low entry cost: $299/export module. Static digital images were retrieved from our institution’s whole slide image database. A gallery user interface was designed within GameMaker including query and filter functions. Results: The app currently includes 11 diagnoses spanning 170 images; each with a brief description along with expected immunohistochemical staining patterns. Our app also acts as a template, allowing pathologists with no programming knowledge to create an atlas of their own. Following a brief tutorial, enduser pathologists may take our framework, add their own diagnoses and publish their atlas to the web. Conclusions: With minimal programming knowledge and expense, a multiplatform pathology app can be developed. This app will be tested in a pathology residency focus group to further evaluate its efficacy. This may provide a unique opportunity to enhance pathology education in the absence of resources. 19. Spindle Cell/Sclerosing Rhabdomyosarcoma Is a New and Underdiagnosed Entity: A Clinical and Tissue Microarray Immunohistochemical Study With Literature Review Yunguang Liu, Maryam Tahmasbi, and Marilyn M. Bui Department of Anatomic Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida Background: In adults rhabdomyosarcoma (RMS) accounts for less than 2% of all sarcomas. Spindle cell/sclerosing RMS is a newly recognized variant by 2013 WHO Classification of Tumors of Soft Tissue and Bone. It accounts for 5% to 10% of RMS with male to female ratio up to 6:1. One previous study suggested that spindle cell RMS may have not as rare as previously thought. Recently we encountered a case that was thought to be leiomyosarcoma of soft tissue. However, after further review and work up, the diagnosis was revised to spindle cell/sclerosing RMS. Therefore we intend to study how often spindle cell/sclerosing RMS is misdiagnosed for leiomyosarcoma and how to avoid this diagnostic pitfall. Materials and Methods: Literature review was conducted to identify the most sensitive and specific immunohistochemical (IHC) markers of RMS and the expression pattern of myogenin and myoD1 in leiomyosarcoma. Subsequent to the index case, 3 cases with previously diagnosed leiomyosarcomas were evaluated in real time and subjected to myogenin and myoD1 stains. A tissue microarray (TMA) consisting of 42 cases of previously diagnosed leiomyosarcoma was subjected to IHC of myogenin (MYF4, 1:20-1:40 dilution, Newcastle, UK) and myoD1 (EP212, 0.01–1.0 µg/ml, Rocklin, CA). Cancer Control 185 Results: According to the literature review myogenin and myoD1 are sensitive (97%) and specific (90% vs 91%) markers for RMS. Most authors agree that one of these markers is necessary for a diagnosis of RMS. Leiomyosarcoma rarely expresses myogenin or myoD1, but co-expression was not reported. In comparing the index case and the three subsequent cases, two of the three cases were reclassified as RMS (one spindle cell, one pleomorphic). The leiomyosarcoma TMA (age range 26-82, mean ± SE 55.8 ± 2.2; male 15, female 27; GYN origin 9, non-GYN origin 33) revealed one case expressing myoD1 not myogenin. Repeat staining on the corresponding whole slide section of the tumor rendered the same result. Conclusions: Spindle cell/sclerosing RMS can be sometimes misdiagnosed as leiomyosarcoma due to the overlapping histological features and immunohistochemical profile. However, judicious use of myogenin and myoD1 can be helpful to achieve an accurate and definitive diagnosis. Co-expression of myogenin and myoD1 is strong confirmation. save the date 3 RD ANNUAL MOFFITT CANCER CENTER PATHOLOGY SYMPOSIUM Transforming Discovery into Practice A 2.5 day program geared to pathologists, cancer clinicians, and laboratory professionals focused on improving practice by implementation of recent discoveries in biomarkers, molecular genomics, laboratory improvements, and personalized medicine. Friday, February 3 – Sunday, February 5, 2017 Sarasota, Florida 186 Cancer Control April 2016, Vol. 23, No. 2 Ten Best Readings Relating to Ocular Oncology Onken MD, Worley LA, Char DH, et al. Collaborative Ocular Oncology Group report number 1: prospective validation of a multi-gene prognostic assay in uveal melanoma. Ophthalmology. 2012;119(8):1596-1603. The prognostic performance of a gene expression profiling assay covering 15 genes was assessed in a prospective, multicenter study. Primary posterior uveal melanomas were assigned to classes 1 (low metastatic risk) and 2 (high metastatic risk) prognostic categories. The assay had a high technical success rate and provided a highly significant improvement in rates of prognostic accuracy over clinical tumor, node, and metastasis classification. Jakobiec FA, Mendoza PR. Eyelid sebaceous carcinoma: clinicopathologic and multiparametric immunohistochemical analysis that includes adipophilin. Am J Ophthalmol. 2014;157(1):186-208. This retrospective, clinicopathologic study sought to review the fine cytopathological features and immunohistochemistry of eyelid sebaceous carcinoma. The authors concluded that immunohistochemistry can make a significant contribution to the diagnosis of sebaceous carcinoma, and that p53 and vesicular granular adipophilin positivity could reliably supplement routine microscopic diagnosis of infiltrative tumors and could be used in paraffin sections, thereby obviating the oil red O staining of frozen sections. Corrêa ZM, Augsburger JJ. Independent prognostic significance of gene expression profile class and largest basal diameter of posterior uveal melanomas. Am J Ophthalmol. 2016;162:20-27. The authors of a prospective, interventional case series set out to determine whether select clinical prognostic factors for metastatic uveal melanoma were prognostically significant in multivariate models that incorporated a gene expression profile class of tumor cells. Gene expression profile class was the strongest prognostic factor for metastatic death, followed by tumor larger basal diameter, thickness, and intraocular tumor location. This study also showed that larger basal diameter and gene expression profile class of tumor were both independent prognostic factors for metastasis and metastatic-related death. Plaza JA, Mackinnon A, Carrillo L, et al. Role of immunohistochemistry in the diagnosis of sebaceous carcinoma: a clinicopathologic and immunohistochemical study. Am J Dermatopathol. 2015;37(11):809-821. The diagnosis of sebaceous carcinoma, which is an uncommon malignant epithelial neoplasm with a predilection for the periocular region, can be difficult to discern upon the initial presentation of a patient because the condition can clinically and histopathologically resemble other common benign and malignant epithelial lesions. A diagnosis of sebaceous carcinoma is made by confirmation of sebaceous differentiation of neoplastic cells; however, its recognition may be sometimes difficult and may require ancillary studies. The results of this study suggest that adipophilin represents a sensitive and reliable marker for diagnosing sebaceous carcinoma. Including various epithelial markers in the panel could also be of help if adequately used. Field MG, Decatur CL, Kurtenbach S, et al. PRAME as an independent biomarker for metastasis in uveal melanoma. Clin Cancer Res. 2016;22(5):1234-1242. Some class 1 uveal melanoma tumors are known to metastasize. Among class 1 tumors, the most significant predictor of metastasis was PRAME messenger RNA expression. The 5-year actuarial rate of metastasis was 0% for class 1 (PRAME–), 38% for class 1 (PRAME+), and 71% for class 2 tumors. The rate of median metastasis-free survival for patients with class 1 (PRAME+) tumors was 88 months compared with 32 months for patients with class 2 tumors. PRAME expression was associated with SF3B1 mutations and larger tumor diameter. The study findings suggested that PRAME is an independent prognostic biomarker in uveal melanoma; therefore, its presence is associated with an increased risk of metastasis in patients with class 1 or disomy 3 tumors. April 2016, Vol. 23, No. 2 Riemens A, Bromberg J, Touitou V, et al. Treatment strategies in primary vitreoretinal lymphoma: a 17-center European collaborative study. JAMA Ophthalmol. 2015;133(2):191-197. In this study, researchers evaluated the outcomes of treatment regimens used for primary vitreoretinal lymphoma for the prevention of subsequent central nervous system lymphoma (CNSL). Therapy to prevent CNSL included ocular radiotherapy, ocular chemotherapy, or both (group A); extensive systemic treatment (group B); and a combination of ocular and extensive treatment (group C). CNSL developed in 32% of group A, 43% of group B, and 39% of group C. The 5-year cumulative survival rate was lower in those with CNSL compared with those without CNSL (68%) Cancer Control 187 and was similar among all treatment groups. The most common adverse event was acute renal failure. The researchers of this study were unable to prove that use of systemic chemotherapy could prevent CNSL. Chan CC, Rubenstein JL, Coupland SE, et al. Primary vitreoretinal lymphoma: a report from an International Primary Central Nervous System Lymphoma Collaborative Group symposium. Oncologist. 2011;16(11):1589-1599. Primary vitreoretinal lymphoma (PVRL) commonly masquerades as posterior uveitis and has a unique tropism for the retina and central nervous system (CNS). More than 15% of patients with primary CNS lymphoma develop intraocular lymphoma, usually occurring in the retina, vitreous, or both areas. Conversely, 65% to 90% of patients with PVRL develop CNS lymphoma. PVRL is most often diagnosed using both histology to identify lymphoma cells in the vitreous or retina and immunohistochemistry to indicate monoclonality. Optimal therapy for PVRL has not yet been defined, but it is sensitive to radiotherapy and exhibits high responsiveness to intravitreal methotrexate or rituximab. Although systemic chemotherapy alone can result in high response rates in patients with PVRL, a high relapse rate is seen in this population. from follicular lymphoma. The researchers noted that an undescribed “multifocal RLH” syndrome must be distinguished from follicular lymphoma. Conjunctival RLH can usually be managed surgically without radiotherapy, but “multifocal RLH” required systemic treatment in 2 of 3 patients. Follicular lymphoma requires systemic chemotherapy if discovered beyond stage 1E. Al-Jamal RT, Cassoux N, Desjardins L, et al. The pediatric choroidal and ciliary body melanoma study: a survey by the European Ophthalmic Oncology Group. Ophthalmology. 2016;123(4):898-907. In this retrospective, multicenter, observational trial, European researchers collected comprehensive data on choroidal and ciliary body melanoma in children in order to determine whether children younger than 18 years of age, those of the male sex, and those without ciliary body involvement have a more favorable survival prognosis than young adults 18 to 24 years of age, those of the female sex, and those with ciliary body involvement. By multivariate analysis, being a young adult, having a higher tumor, node, and metastasis stage, and being of the female sex independently predicted less favorable rates of survival. Ciliary body involvement and cell type were not associated with rates of survival. Grossniklaus HE. Progression of ocular melanoma metastasis to the liver: the 2012 Zimmerman lecture. JAMA Ophthalmol. 2013;131(4):462-469. Grossniklaus evaluated the immunohistochemical and histological findings of uveal melanoma that metastasizes to the liver. Stage 1 metastases were identified in the sinusoidal spaces of 90% of study patients; these metastases were avascular and lacked mitotic activity. Stages 2 and 3 metastases were found in all study patients. Immunohistochemical stains were positive for S100 or HMB45 in all tumors. Overall, stage 1 metastases outnumbered stage 2 metastases (which outnumbered stage 3 metastases). The mean vascular density and mitotic index increased from stage 2 to stage 3 metastases. The architecture of stage 2 metastases mimicked the surrounding hepatic parenchyma, whereas stage 3 metastases exhibited either lobular or portal growth patterns. Stacy RC, Jakobeic FA, Schoenfield L, et al. Unifocal and multifocal lymphoid hyperplasia vs follicular lymphoma of the ocular adnexa. Am J Ophthalmol. 2010;150(3):412-426. This study was conducted so researchers could characterize the differentiating histopathological and immunophenotypic features of reactive lymphoid hyperplasia (RLH) and follicular lymphoma of the ocular adnexa. Microscopic analysis with immunohistochemical staining can be reliably used to distinguish RLH 188 Cancer Control April 2016, Vol. 23, No. 2
