Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
TITLE Advanced diagnostic methods in diagnosis of oral cancers – A Review AUTHORS (1) Dr. Vishnudas Dinesh Prabhu, M.D.S , Professor, Department of Oral Pathology & Microbiology, Yenepoya Dental College & Hospital, Yenepoya University, Mangalore – Karnataka. (2) Dr. Shakil M. (M.D.S), Post Graduate student, Dept of Department of Oral Pathology & Microbiology, Yenepoya Dental College & Hospital, Yenepoya University, Mangalore – Karnataka. (3) Dr. Soniya Adyanthaya. M.D.S, Assistant Professor, Dept of Department of Oral Pathology & Microbiology, Yenepoya Dental College & Hospital, Yenepoya University, Mangalore – Karnataka. (4) Dr. Maji Jose. M.D.S, Professor and Head, Dept of Department of Oral Pathology & Microbiology, Yenepoya Dental College & Hospital, Yenepoya University, Mangalore – Karnataka. Corresponding Author: Dr. Vishnudas Dinesh Prabhu Professor Department of Oral Pathology & Microbiology Yenepoya Dental College & Hospital, Yenepoya University, Mangalore – Karnataka. E-mail : [email protected] ABSTRACT: Advances in the analysis of DNA, RNA, and proteins in tissues have permitted improved biologic insights into neoplastic diseases including oral cancers and also many developmental, inflammatory, metabolic, and infectious diseases. Molecular diagnostic methods as an investigative tool in the field of histopathology have evolved into the most widespread, diagnostically useful techniques in the assessment of disease states. It is likely that, in the future, these methods will increasingly enter into the day-to-day diagnosis and management of patients. The pathologist will continue to play a fundamental role in diagnosis and will likely be in a pivotal position to guide the implementation and interpretation of these tests as they move from the research laboratory into diagnostic pathology. Advanced molecular diagnostic methods and their application to diagnostic pathology of oral cancers are reviewed in this article. KEY WORDS; CANCER, DIAGNOSIS, IMMUNOHISTOCHEMISTRY, TUMOR MARKERS, MOLECULAR ONCOLOGY INTRODUCTION The diagnosis of cancer relies primarily on invasive tissue biopsy, as noninvasive diagnostic tests are generally insufficient to define a disease process of cancer. The conventional histopathology based on light microscopy, however, has recently been complemented with ultrastructure, immunohistochemistry and molecular diagnostics. Even though the biopsy study is fundamental, it is a diagnostic method with limited sensitivity where one of the most important features is the subjective interpretation of the examining pathologist. Changes occur at the molecular level before they are seen under the microscope and before clinical changes occur. These issues underline the importance of discovering and developing new diagnostic methods, improving the existing ones and discovering new therapeutics targets for oral neoplastic diseases. 1,2,3,4 In recent decades, we have seen a dramatic switch from histopathological to molecular methods of disease diagnosis. Strategies have been developed whereby, from single tissue sample, different techniques can be performed in order to fully characterize molecular phenotype and genotype of tumors. Immunohistochemistry, cytogenetics, analysis of DNA content and molecular genetic assays which have been added as valuable adjuncts to light microscopy in oral cancer diagnosis are discussed. IMMUNOHISTOCHEMISTRY Immunocytochemistry is a technique for identifying cellular or tissue constituents (antigens) by means of antigen-antibody interactions, the site of antibody binding being identified either by direct labeling of the antibody, or by use of a secondary labeling method. The selection of antibodies for immunohistochemical testing is made on the basis of their tumor specificity and the likelihood that they will react with the tumor under evaluation. IHC has greatly reduced the number of unclassified tumors and has been of major assistance in defining metastatic tumors of unknown primary site. It is of great value in the diagnosis of undifferentiated tumors where light microscopy is unable to discern diagnostic features. Few interpretations are: Expression of cytokeratins strongly suggests an epithelial origin; leukocyte common antigen (LCA) is evidence of lymphoid origin while expression of S-100 protein and HMB 45 is characteristic of malignant melanoma.5 Immunohistochemistry can help in the diagnosis of undifferentiated tumors which include poorly differentiated carcinoma, , amelanotic melanoma or, less commonly sarcoma. Presence of neural markers like neuron specific enolase and synaptophysin are suggestive of neuroectodermal tumors, and the markers of skeletal muscle differentiation, desmin and myoglobin, are indicative of rhabdomyosarcoma. 5 The interpretation in IHC should be taken care with strict adherence to laboratory practice. A panel of antibodies is generally recommended to characterize a diagnostic problem. TUMOR MARKERS Tumor markers are biologic or biochemical substances produced by tumors and secreted into blood, urine, other body fluids or body tissues of some patients with certain types of cancer in higher than normal amounts. A tumor marker may be produced by tumor itself, or by the body in response to the presence of cancer or certain non-cancerous conditions. Tumor markers can be detected by various methods including antigen-antibody based techniques.6 Tumor marker levels are not elevated in every person with cancer- especially in the early stage of disease. The field of tumor markers is ever expanding with many new candidate markers either in clinical use or under active evaluation. Usually multiple tumor markers are associated with individual malignancies; vice versa individual tumor markers may be associated with various malignancies. Thus, the use of multiple markers based on the combination pattern for the selected malignancy will improve sensitivity and specificity of the detection.7 MOLECULAR ONCOLOGY At a molecular level, a cancer cell may be distinguished from its normal counterpart by abnormalities in structure or expression of certain genes. These abnormalities, directly or indirectly, result in disturbance of cell cycle regulation and induce dysregulated growth in cancer cells. Molecular oncology studies the alterations in genetic and biochemical processes at the molecular level. It helps in establishing a definitive diagnosis and classification of tumors based on the recognition of complex profiles ('finger-prints') or unique molecular alteration that occur in specific tumor types. The changes can be studied on chromosomes, DNA or RNA. CHROMOSOME ANALYSIS Chromosome abnormalities are frequently found in malignant cells. Many abnormalities may be specific to tumor types. In hematological malignancies, individual abnormalities can be easily analyzed on bone marrow aspirate samples. Conventional cytogenetic analysis is a fundamental method in the study of small round cell tumours. It reveals translocations and other structural abnormalities as well as numerical aberrations.8 Detection of chromosome abnormalities has traditionally been performed through banding analysis of metaphase chromosome for solid tissue biopsies. Fluorescence in situ hybridization (FISH) technique is applicable to interphase cells and is more sensitive compared to conventional cytogenetics. It involves hybridization of conjugated probes to chromosomes, and visualization of the probe by fluorescent microscopy. FISH seems to be emerging as a major focus for development of molecular tests for subclassification and prognostication in lymphomas.9 It can readily identify gains or losses of whole chromosomes or chromosome arms. Several genetic translocations associated with common subtypes of lymphoma can be conveniently detected by FISH.10 Comparative genomic hybridization (CGH) is a newly described method that globally assays for chromosomal gains and losses in genomic complement. Comparative genomic hybridization (CGH) enables the screening of the whole genome and reveals DNA sequence copy number changes (losses, gains and high-level amplications).11 CGH has shown to be especially useful in obtaining cytogenetic data of solid tumours which often fail to produce mitotic cells in cell culture or in which the cytogenetic complexity prevents any real interpretation of the karyotype. Recurrent alterations in a specific tumour type have led to the identification of target oncogenes12 or tumour suppressor genes.13 Fresh, fresh frozen or paraffin-embedded tumour tissue samples can be used. Normal cell contamination should be avoided and the proportion of tumour cells should be high in order to ensure the highest possible sensitivity of CGH.14 The molecular cytogenetic techniques like FISH and CGH are increasingly been used in addition to conventional cytogenetics to properly discern various chromosomal abnormalities in tumor samples. Genetic analysis in oral cancer/ Molecular genetic assays: Malignancy is considered as a process caused by the accumulation of multiple genetic alterations, which affect the cell cycle as well as normal cell differentiation. These alterations are mainly acquired (somatic) although some of them may be inherited and when they activate protooncogenes, inactivate tumour suppressor genes or affect enzymes, which repair DNA, they could lead to a malignant transformation. Most of the oral cavity carcinogens are chemical (tobacco), physical (radiation) and infectious (Human papilloma virus, Candida) mutagenic agents that may cause changes in gene and chromosome structure by point mutations, deletions, insertions and rearrangements. However, some of these changes may occur spontaneously. These genetic alterations, which occur during carcinogenesis, can be used as targets for detecting tumour cells in clinical samples.2,4,15 Not all mutations in cancer genes are apparent at cytogenetic level, so it has become increasingly important to identify genes themselves, and relevant changes within their structure. Genes and their encoded proteins, may serve as markers of lesions with high risk of progression to malignant disease—or they may be predictive of patient response to treatment and survival. 16-20 The gene alterations can be broadly classified into following: Oncogenes: normal cell proteins that become abnormally activated. Tumor suppressor genes: normal antiproliferative genes or their products that becomes inactivated. DNA repair genes: become inactivated, causing accumulation of potentially damaging mutations. Regulators of apoptosis: inactivation of pro-apoptotic genes or activation of anti-apoptotic genes promotes cell survival.5 Overexpression of the cell cycle–associated oncoproteins cyclin D1 and MDM2, as well as underexpression of the tumor suppressor proteins p53, p16, and p27, may be important tumor markers.16-20 In some oral cancers the antiapoptotic proteins Bcl-X and Bcl-2 are overexpressed.21,22 Moreover, expression of the proapoptotic protein Bax has been positively correlated with increased sensitivity to chemotherapeutic agents in head and neck carcinomas.23 Growth factor receptors like EGFR and HER-2/neu are oncogenes that encode transmembrane receptors that are believed to increase cell cycle, cell motility, and angiogenesis and are overexpressed in many types of malignancy, including head and neck carcinomas.24-28 Squamous cell carcinomas that overexpress EGFR are associated with a poorer outcome than those that do not overexpress this receptor.29,30 Proteins involved in signal transduction (ie, the process whereby interactions between a receptor on the cell surface and its ligand are transmitted to the nucleus of the cell), such as ras (GTP-binding proteins), and nuclear regulatory proteins, such as myc (transcriptional activator proteins), are also abnormally expressed in oral cancers. Mutations in the tumour suppressor gene p53 are the most frequent genetic alterations in human cancer and show a variable frequency in oral cancer. 26 2. Epigenetic alterations, Loss of hetrozygosity and Microsatellite instability The applicability of other molecular markers such as epigenetic alterations (hypermethylation of promoter regions) and genomic instability such as loss of hetrozygosity (LOH) and microsatellite instability (MSI) has also been studied.31,32 The main epigenetic modification in tumours is methylation and it seems that the changes in the methylation patterns can play an important role in tumorigenesis. These epigenetic alterations are often associated with the loss of genetic expression and their occurrence seems to be essential for the multiple necessary genetic events. So malignant progression takes place because these alterations can inactivate DNA repairing genes. Rosas et al. detected abnormal hypermethylation patterns in p16, MGMT and DAP-K genes in smears of patients suffering from head and neck cancer by methylation specific Polymerase Chain Reaction (PCR).31 Huang et al. used PCR techniques to amplify DNA from exfoliated cytology samples and found that 66% of the tumours studied showed LOH at one position in the p53 sequence, while 55% showed LOH at some other location.33 PCR analyses have also been used for the detection of microsatellite markers, i.e. short repetitive DNA sequences. Microsatellite regions are distributed along the genome and have been widely and satisfactorily used as molecular markers for carcinogenesis. Alterations in these regions have been used as clonal markers and for detecting tumoral cells among normal cells.34,35 Several studies have demonstrated these by using microsatellite markers that alterations in certain regions of chromosomes 3p, 9p, 17p and 18q are associated with the development of head and neck squamous cell carcinomas. 36,37 Analysis of DNA content: Molecular detection methods involve the analysis of nucleotide sequences within nucleic acid to detect the presence of malignant cells in fluids and tissues. DNA is, in general, stable in tissues and cells after removal. RNA, on the other hand, is unstable and highly degradable by endogenous RNAses released by lysosomes or dead cells. The analysis is usually done on total cellular DNA using southern blot (SB) procedure or polymerase chain reaction (PCR) in which regions of DNA are amplified and more easily identified. PCR-based methods can be used to detect gene rearrangements and translocation-derived fusion genes associated with Ewing tumours and lymphomas.8 Messenger RNA (mRNA) detection of genes and their products is done by the techniques like northern blot, reverse transcription-PCR (RT-PCR) and in situ hybridization. A relatively newer technique like micro-array methods allows measurement of differential expression of all the genes including those of low abundance. Microarrays exploit the unique feature of single-stranded DNA to hybridize to complementary DNA sequences, thereby permitting sequence specific identification of DNA and allow us to simultaneously monitor the transcription of thousands of genes in parallel.38,39 In oral cancer it has been used to classify & predict the biologic process underlying this malignancy. Premalignant & malignant oral lesions contain genetic changes that are present prior to phenotypic & morphologic changes. Identification of gene expression profiles will allow clinicians to differentiate harmless white lesions from pre-cancerous lesions or from very early cancer.37,38 Conclusion: A good understanding of molecular biology of head & neck cancer along with an improved understanding of the various advanced diagnostic techniques will definitely contribute to early detection & future therapeutic improvements. Molecular genetic tests may shape the new insights into the pathogenesis of oral cancers, diagnostic approach, and may lead to new therapeutic options. We are still at an early stage of the integration of molecular genetic tests into diagnosis of oral cancer. The future for implementation of molecular genetic testing to benefit patients with head and neck cancer is bright, with methods either established already or on the brink of readiness for application in diagnostic laboratories. Professionals in the field of histopathology must now work hard to ensure that clinical needs and technical effectiveness are the major factors in determining which tests are brought into standard use over the next few years. References: 1. Epstein JB, Zhang L, Rosin M. Advances in the diagnosis of oral premalignant and malignant lesions. J Can Dent. 2002;68:617–621. 2. Ogden GR, Cowpe JG, Green MW. Detection of field change in oral cancer using oral exfoliative cytologic study. Cancer. 1991;68:1611–1615. 3. El-Naggar AK, Mao L, Staerkel G, Coombes MM, Tucker SL, Luna MA, et al. Genetic heterogeneity in saliva from patients with oral squamous carcinomas: implications in molecular diagnosis and screening. J Mol Diagn. 2001;3:164–170. 4. Spafford MF, Koch WM, Reed AL, Califano JA, Xu LH, Eisenberger CF, et al. Detection of head and neck squamous cell carcinoma among exfoliated oral mucosal cells by microsatellite analysis. Clin Cancer Res. 2001;7:607. 5. Jordan R.C.K, Daniels T.E, Greenspan J.S, Regezi J.A. Advanced diagnostic methods in oral and maxillofacial pathology. Part II: Immunohistochemical and immunofluorescent Methods. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:56-74. 6.Tumor markers in clinical practice: general rinciples and guidelines. Sharma S. Indian J Med Pediatr Oncol 2009;30 (1):1-8. 7. Sanjay R, Reddy M, NDVN Shyam. Tumor markers in oral neoplasia. Indian journal of dental advancements 2010;2(1):103-114. 8.Tarkkanen M and Knuutila S. The diagnostic use of cytogenetic and molecular genetic techniques in the assessment of small round cell tumours. Current Diagnostic Pathology. 2002; 8: 338-348. 9. Martin-Subero JI, Chudoba I, Harder L, et al. Expanding the possibilities of combined morphologic, immunophenotypic and genetic single cell analyses. Am J Pathol 2002;161:413–420. 10. Wilkins B.S. Molecular genetic analysis in the assessment of lymphomas. Current Diagnostic Pathology 2004;10: 351–359. 8. Kallioniemi A, Kallioniemi O-P, Sudar D et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 1992; 258: 818–821. 9. Visakorpi T, Hyytinen E, Koivisto P et al. Invivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat Genet 1995; 9: 401–406. 10. Hemminki A, Tomlinson I, Markie D et al. Localization of a susceptibility locus for Peutz–Jeghers syndrome to 19p using comparative genomic hybridization and targeted linkage analysis. Nat Genet 1997; 15: 87–90. 11. Kallioniemi O-P, Kallioniemi A, Piper J et al. Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chromosomes Cancer 1994; 10: 231–243. 12. 45. Boyle JO, Mao L, Brennan JA, Koch WM, Eisele DW, Saunders JR, Sidransky D. Gene mutations in saliva as molecular markers for head and neck squamous cell carcinomas. Am J Surg. 1994;168:429–32. 13. Bartkova J, Lukas J, Muller H, Strauss M, Gusterson B, Bartek J. Abnormal patterns of D-type cyclin expression and G1 regulation in human head and neck cancer. Cancer Res 1995;55:949-956. 14. Jordan RC, Bradley G, Slingerland J. Reduced levels of the cellcycle inhibitor p27kip1 in epithelial dysplasia and carcinoma of the oral cavity. Am J Pathol 1998;152:585-590. 15. Michalides R, van Veelen N, Hart A, Loftus B, Wientjens E, Balm A. Overexpression of cyclin D1 correlates with recurrence in a group of forty-seven operable squamous cell carcinomas of the head and neck. Cancer Res 1995;55:975-978. 16. Regezi JA, Dekker NP, McMillan A, Ramirez-Amador V, Meneses-Garcia A, RuizGodoy Rivera LM, et al. p53, p21, Rb, and MDM2 proteins in tongue carcinoma from patients <35 versus >75 years. Oral Oncol 1999;35:379-383. 17. Schoelch ML, Regezi JA, Dekker NP, Ng IO, McMillan A, Ziober BL, et al. Cell cycle proteins and the development of oral squamous cell carcinoma. Oral Oncol 1999;35:333-342. 18. Pena JC, Thompson CB, Recant W, Vokes EE, Rudin CM. BclxL and Bcl-2 expression in squamous cell carcinoma of the head and neck. Cancer 1999;85:164-170. 19. Schoelch ML, Le QT, Silverman S Jr, McMillan A, Dekker NP, Fu KK, et al. Apoptosisassociated proteins and the development of oral squamous cell carcinoma. Oral Oncol 1999;35:77-85. 20. Guo B, Cao S, Toth K, Azrak RG, Rustum YM. Overexpression of Bax enhances antitumor activity of chemotherapeutic agents in human head and neck squamous cell carcinoma. Clin Cancer Res 2000;6:718-724. 21. Grandis JR, Melhem MF, Barnes EL, Tweardy DJ. Quantitative immunohistochemical analysis of transforming growth factoralpha and epidermal growth factor receptor in patients with squamous cell carcinoma of the head and neck. Cancer 1996;78:1284-1292. 22. Grandis JR, Melhem MF, Gooding WE, Day R, Holst VA, Wagener MM, et al. Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Natl Cancer Inst 1998;90:824-832. 23. Ke LD, Adler-Storthz K, Clayman GL, Yung AW, Chen Z. Differential expression of epidermal growth factor receptor in human head and neck cancers. Head Neck 1998;20:320327. 24. Press MF, Pike MC, Hung G, Zhou JY, Ma Y, George J, et al. Amplification and overexpression of HER-2/neu in carcinomas of the salivary gland: correlation with poor prognosis. Cancer Res 1994;54:5675-5682. 25. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707-712. 26. Wong DT. TGF-alpha and oral carcinogenesis. Eur J Cancer B Oral Oncol 1993;29B:3-7. 27. Maiorano E, Favia G, Maisonneuve P, Viale G. Prognostic implications of epidermal growth factor receptor immunoreactivity in squamous cell carcinoma of the oral mucosa. J Pathol 1998;185:167-174. 28. Rosas SL, Koch W, da Costa Carvalho MG, Wu L, Califano J, Westra W, et al. Promoter hypermethylation patterns of p16, O6-methylguanine-DNA methyltransferase, and deathassociated protein kinase in tumors and saliva of head and neck cancer patients. Cancer Res. 2001;61:939–942. 29. López M, Aguirre JM, Cuevas N, Anzola M, Videgain J, Aguirregaviria J, et al. Gene promoter hypermethylation in oral rinses of leukoplakia patients – a diagnostic and/or prognostic tool? Eur J Cancer. 2003;39:2306–2309. 30. Huang MF, Chang YC, Liao PS, Huang TH, Tsay CH, Chou MY. Loss of heterozygosity of p53 gene of oral cancer detected by exfoliative cytology. Oral Oncol. 1999;35:296–301. 31. Mao L, Lee DJ, Tockman MS. Microsatellite alterations as clonal markers in the detection of human cancer. Proc Natl Acad Sci USA. 1994;91:9871–9875. 32. Sidransky D. Molecular markers in cancer diagnosis. J Natl Cancer Inst Monogr. 1995;17:27–29. 33. El-Naggar AK, Hurr K, Batsakis JG, Luna MA, Goepfert H, Huff V. Sequential loss of heterozygosity at microsatellite motifs in preinvasive and invasive head and neck squamous carcinoma. Cancer Res. 1995;55:2656–2659. 34. Califano J, Riet VDP, Westra W, Nawroz H, Clayman G, Piantadosi S, et al. Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res. 1996;56:2488–2492. 35. King HC, Sinha AA. Gene expression profile analysis by DNA microarrays: promise and pitfalls. J Am Med Assoc 2001;286:2280-2288. 36. Lockhart D J, Winzeler E A. Genomics, gene expression and DNA arrays. Nature 2000; 405: 827--836. 37. Kuo WP, Whipple ME. Gene expression profiling by DNA microarrays and its application to dental research. Oral Oncol 2002;38:650–656. 38. Winston patrick, Whipple M.E. Deciphering gene expression profiles generated from DNA microarrays & their applications in oral medicine. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:584-591. 39. Mohr S, Leikauf GD, Keith G, Rihn BH. Microarrays as cancer keys: an array of possibilities. J Clin Oncol 2002;20:3165-3175.