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CASE REPORT Genomic alterations in human epidermal growth factor receptor 2 (HER2/ERBB2) in head and neck squamous cell carcinoma Christine H. Chung, MD,1,2,3* Alexis Germain, BS,4 Rathan M. Subramaniam, MD, PhD,5 Andreas M. Heilmann, PhD,4 Kyle Fedorchak, BS,4 Siraj M. Ali, MD, PhD,4 Vincent A. Miller, MD,4 Robert A. Palermo, MD,6 Carole Fakhry, MD, MPH2 1 Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 2Department of Otolaryngology–Head and Neck Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 3Department of Head and Neck–Endocrine Oncology, Moffitt Cancer Center & Research Institute, Tampa, Florida, 4Foundation Medicine, Cambridge, Massachusetts, 5Department of Radiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 6Department of Pathology, Greater Baltimore Medical Center, Baltimore, Maryland. Accepted 5 August 2016 Published online 21 September 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.24587 ABSTRACT: Background. Despite recent advances, survival outcomes for those with metastatic or recurrent head and neck squamous cell carcinoma (HNSCC) have remained poor. Novel approaches should be investigated to improve outcomes. Methods. A retrospective chart review was performed of a patient who presented with a TNM classification III HNSCC of the oropharynx, positive for human papillomavirus (HPV) who had a complete response to a human epidermal growth factor receptor 2 (HER2)-targeted therapy. Amplification rates of HER2 in the HNSCC Cancer Genome Atlas Network (TCGA) dataset and the FoundationOne genomic profiling dataset were evaluated. INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) arises from heterogeneous anatomic mucosal sites, including the oral cavity, pharynx, and larynx. HNSCC is the sixth most common malignancy globally, with an annual incidence of over half a million cases.1 Risk factors include tobacco, alcohol use, and oral human papillomavirus (HPV) infection, particularly in the oropharynx.2,3 Prognosis for HNSCC is dependent upon anatomic site and stage, but tends to be poor in the setting of recurrent/metastatic HNSCC.4 Despite recent advances, the 2-year survival rate for patients with recurrent or metastatic HNSCC has not improved significantly, remaining between 28% and 55%.4,5 The current standard of care *Corresponding author: C. H. Chung, Department of Head and Neck– Endocrine Oncology, Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive FOB-2, Suite #5.2106, Tampa, FL 33612. E-mail: christine. [email protected] Conflict of Interest: A.G., A.H., K.F., S.M.A., and V.A.M. are employees of Foundation Medicine. C.H.C. received research funding from Boehringer Ingelheim for preclinical research and honoraria from Bristol-Myers Squibb and Lilly Oncology for serving in scientific advisory boards. All other authors have no conflict of interest to declare. Results. Comprehensive genomic profiling of the tumor obtained from the dermal metastasis identified amplification of HER2. Data from TCGA and FoundationOne showed that the frequency of HER2 alteration was not observed to vary significantly with HPV tumor status. Conclusion. This case demonstrates the application of genomic profiling to guide treatments in a patient with HNSCC with advanced metastatic C 2016 Wiley Periodidisease refractory to standard of care therapies. V cals, Inc. Head Neck 39: E15–E19, 2017 KEY WORDS: head and neck squamous cell carcinoma, human epidermal growth factor receptor 2 (HER2), genomic profiling, trastuzumab, biomarker for patients with metastatic HNSCC is palliative chemotherapy and/or cetuximab with supportive care.5,6 The observation that epidermal growth factor receptor (EGFR) is overexpressed in 80% to 90% of patients with HNSCC prompted initial investigations into utilizing EGFR-targeted therapy in this disease.7,8 To date, the antiEGFR monoclonal antibody, cetuximab, is the only Food and Drug Administration-approved targeted agent in the management of recurrent HNSCC that has been shown to improve overall survival when given with platinum and 5fluorouracil.5 However, genomic alterations, such as gainof-function EGFR mutations are extremely rare, and EGFR amplification, although identified more frequently, has not been associated with increased response or survival in patients treated with cetuximab and other EGFR-targeted agents.9–11 To build upon the experience gained from successful development of cetuximab and continued attempts to identify predictive biomarkers of clinical benefit for EGFR inhibitors, we need to further investigate potential oncogenic drivers and utilize the genomic data to identify targeted agents in HNSCC. In this study, we report on the case of a patient with amplification of known oncogene, human epidermal growth factor receptor 2 (HER2/ERBB2), detected through comprehensive genomic profiling, that responded to an HER2-targeted therapy in combination with chemotherapy. HEAD & NECK—DOI 10.1002/HED JANUARY 2017 E15 CHUNG ET AL. CASE REPORT A 52-year-old man with an 8 pack-year smoking history presented with a left tonsillar squamous cell carcinoma, T classification 3, N classification 1, M classification 0, and positive for HPV type 16 by in situ hybridization. The patient was treated with cisplatin, 5-fluorouracil, and radiotherapy as standard of care.12 However, 2 years after the completion of the primary chemoradiation regimen, he experienced a local recurrence involving the ipsilateral tonsil, which was treated with a radical tonsillectomy. Negative margins were achieved. Six months later, the patient had a regional recurrence. He underwent a neck dissection of levels I to IV. A single node in level II was positive for squamous cell carcinoma with extracapsular extension. Four months later, he was found to have a solitary lung metastasis, which was treated with thoracotomy and wedge resection. Approximately 8 months later, he developed another neck recurrence in the left level IV. Subsequent neck dissection identified 5 malignant lymph nodes with extracapsular extension, perineural invasion, and lymphovascular invasion. He received postoperative carboplatin, paclitaxel, and radiation concurrently. However, within 8 months, he developed recurrence involving subcarinal and hilar lymph nodes. The patient participated in a phase I melanoma antigen E-A3 and HPV16-specific vaccine trial13 but the disease continued to progress. Five months after completion of this trial, he developed a dermal metastasis with extension into the parotid gland and underwent local resection, reconstruction, and brachytherapy. Within a month, he developed new dermal metastatic nodules along the surgical scar. The patient was experiencing pain and fatigue and assessed as having an Eastern Cooperative Oncology Group performance status of 1. At this point, a dermal metastasis was submitted for comprehensive genomic profiling. An excision of a dermal metastasis was submitted for histopathology as well as comprehensive genomic profiling using the FoundationOne assay (Foundation Medicine, Cambridge, MA). Hybridization capture of 3734 exons from 236 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer was applied to 50 ng of DNA extracted from the formalin-fixed paraffin-embedded tumor and sequenced to a median coverage depth of 3639.14 Comprehensive genomic profiling of the dermal metastasis identified HER2 amplification (13 copies), RICTOR amplification, MLL2 E766*, and FGF10 amplification. The HER2 amplification and corresponding overexpression were confirmed with immunohistochemistry (Her2-neu, clone 4B5 rabbit monoclonal antibody; Figure 1A) and fluorescent in situ hybridization (karyotype: nuc ish 17cen[D17Z1 3 1–4], 17q11.2[HER-2 3 2–17]20; Figure 1B) although concordance between FoundationOne and fluorescent in situ hybridization has been established.14 The patient was treated with trastuzumab and paclitaxel and achieved complete response confirmed by positron emission tomography (PET) and CT scans (Figure 1C and 1D). Approximately 8 months after initiation of the palliative regimen, the patient’s disease progressed (Figure 1E). DISCUSSION This case demonstrates the use of trastuzumab to achieve a complete response in a patient with advanced E16 HEAD & NECK—DOI 10.1002/HED JANUARY 2017 metastatic disease, demonstrating efficacy of anti-HER2 targeted therapy in HER2-amplified HPV-positive HNSCC. HPV positivity defines a subset of patients with HNSCC with a more favorable outcome than HPVnegative patients independent of clinical prognostic factors, with better overall survival and reduced likelihood of recurrence.15–17 This has been attributed to increased response to chemoradiation and the lack of field cancerization seen in generally tobacco-induced HPV-negative HNSCC.18 Recent genomic data supports the differentiation of HNSCC as 2 clinically distinct entities stratified by the HPV tumor status, which inherently has implications for use of targeted therapy.9,19,20 HER2 (ERBB2), a member of the ErbB/EGFR family, encodes the receptor tyrosine kinase, HER2. Overexpression of HER2 has been observed in 7% to 39% of patients with HNSCC, a wide range likely, in part, due to variations in immunohistochemical methodology.21 The variations in the quantification of protein expression levels depending on the sample fixation and processing, specificity and sensitivity of the antibodies, immunohistochemical staining methods, interpretative errors, etc., have been identified as major problems in biomarker development.22–25 Studies indicate that patients with HNSCC with HER2 alterations exhibit shorter overall survival and increased likelihood of recurrence.26 Despite frequent overexpression of HER2, genomic alterations of this gene are uncommon.9 In the HNSCC Cancer Genome Atlas Network dataset of 279 patients, putative high-level amplification of HER2 was reported in 2.2% of samples, and mutation in 1.8% (cBioPortal, August 2015).12 Data from FoundationOne genomic profiling of 619 HNSCC cases was also analyzed to compare the frequency of HER2 amplification and mutation between HPV-positive and HPV-negative cases (Table 1). The frequency of HER2 alteration was not observed to vary significantly with HPV tumor status (p 5 .91), and was not statistically different from the The Cancer Genome Atlas (TCGA) dataset in either amplifications (p 5 .45) or mutations (p 5 .28). The relationship between primary site and HPV status was additionally evaluated (Table 2). Consistent with other reports, HPV positivity was strongly associated with HNSCC tumors arising from oropharyngeal primary sites compared with those from other regions of the head and neck (p < .00001).15,27 Trastuzumab is a monoclonal antibody that targets the extracellular domain of HER2. It has most commonly been used to treat gastric and breast cancers, although successful cases of its use in other HER2-amplified cancers have been reported.28–31 Some have postulated that HER2 may be a potential target in HNSCC in general due to its frequent overexpression, but an early study that treated patients with a combination of trastuzumab, platinum, and paclitaxel showed no increase in response rate with the addition of trastuzumab to a platinum-based chemotherapy regimen. None of the patients involved in this trial had alteration or amplification of HER2, and so the lack of response is not surprising.32 HER2 amplification has been shown to be the most reliable predictor of response to trastuzumab.33 The impressive response to trastuzumab in this patient highlights the significance of genomic profiling in HER2 ALTERATIONS IN HNSCC FIGURE 1. (A) ERBB2 (human epidermal growth factor receptor 2 [HER2]) immunohistochemical 21, (B) ERBB2 (HER2) fluorescence in situ hybridization amplified, (C) positron emission tomography (PET) scan before trastuzumab and paclitaxel treatment, (D) PET scan after 9 weeks of trastuzumab and paclitaxel treatment, and (E) PET scan at 8 months after initiating trastuzumab and paclitaxel treatment. HEAD & NECK—DOI 10.1002/HED JANUARY 2017 E17 CHUNG ET AL. TABLE 1. Frequency of ERBB2 amplifications and mutations in 619 head and neck squamous cell carcinoma from FoundationOne genomic profiling sorted by primary sites and human papillomavirus status. Oropharynx; HPV-positive Oropharynx; HPV-negative Nonoropharynx; HPV-positive Nonoropharynx; HPV-negative Total oropharynx Total nonoropharynx Total HPV-positive Total HPV-negative Total no. of HNSCC cases No. of cases with ERBB2 amplification No. of cases with an activating ERBB2 mutation No. of total cases with ERBB2 alteration 170 88 35 326 258 361 205 414 3 (1.76%) 1 (1.14%) 0 (0%) 5 (1.53%) 4 (1.55%) 5 (1.38%) 3 (1.46%) 6 (1.45%) 6 (3.53%) 1 (1.14%) 0 (0%) 12 (3.68%) 7 (2.71%) 12 (3.32%) 6 (2.92%) 13 (3.14%) 9 (5.29%) 2 (2.27%) 0 (0%) 17 (5.20%) 11 (4.26%) 17 (4.71%) 9 (4.39%) 19 (4.59%) Abbreviations: HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus. advanced metastatic head and neck cancer to identify targetable alterations. Beyond the established agent, trastuzumab, a number of other anti-HER2 targeted therapies may also have potential for use in patients with HNSCC with HER2 amplification or activation, such as presented here. Pertuzumab and lapatinib have been used to target HER2 in breast cancer, and afatinib has been used in the lungs. Particularly, the ErbB family inhibitor, afatinib, has been explored in phase II and III trials in HNSCC and exhibited improved progression-free survival over chemotherapy in second-line and antitumor activity comparable to that of cetuximab.34,35 Thus, the identification of HER2 amplification may be valuable in providing multiple potentially effective therapeutic options. Furthermore, the approach shown here has potential for application in other forms of head and neck disease. In particular, HER2 amplification has been observed in as many as 38.5% of salivary duct carcinomas.36,37 Two separate case studies have shown successful treatment of this diagnosis using trastuzumab.38,39 However, the limitation of targeted therapies has been development of resistance even after achieving initial complete response. This patient developed progression of disease after approximately 8 months of the trastuzumabcontaining regimen. The exact resistance mechanism is unclear because the patient was deceased without a repeat biopsy at the time of progression. One can speculate an emergence of a resistant clone with oncogenic dependency in the phosphotidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway as the patient’s tumor exhibited RICTOR amplification. Some studies have shown alterations within the PI3K/mTOR pathway, TABLE 2. The number of cases of head and neck squamous cell carcinoma from FoundationOne genomic profiling sorted by primary sites and human papillomavirus status. Oropharynx Nonoropharynx No. of HNSCC cases No. of HPV-positive cases No. of HPV-negative cases 258 361 170 (65.9%) 35 (9.7%) 88 (34.1%) 326 (90.3%) Abbreviations: HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus. E18 HEAD & NECK—DOI 10.1002/HED JANUARY 2017 such as PIK3CA gain-of-function mutations or phosphatase and tensin homolog loss, to be characteristic of HPV-positive HNSCC and one of the resistance mechanisms of trastuzumab.19,40,41 Additional possible mechanism of the HER2 inhibitor resistance is shifting the growth signal to other ErbB/EGFR family members, such as EGFR, HER3, or HER4. Therefore, a therapeutic approach to combine the ErbB/EGFR family receptor inhibitors with PI3K/mTOR inhibitors or completely block the ErbB/EGFR pathway by combining EGFR and HER2 inhibitors may be explored. Although genomic testing is highly promising, clinical implementation of genomic alteration-based treatment selection is still in its developmental phase. The main challenges are conducting trials in patients with rare genomic alterations and relatively uncommon cancers, such as HER2 in HNSCC and salivary duct carcinoma. This challenge is potentially alleviated by testing the efficacy of therapeutic agents in multiple tumor types with a common alteration. Prospective evaluation of clinical trials enrolling patients based on the genomic alteration rather than organ sites of the disease is ongoing at Eastern Cooperative Oncology Group-ACRIN Cancer Research Group (MATCH, NCT02465060) and the National Cancer Institute (NCT01827384). Support and further development of such innovative trials must be a priority. REFERENCES 1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893–2917. 2. Hashibe M, Brennan P, Benhamou S, et al. Alcohol drinking in never users of tobacco, cigarette smoking in never drinkers, and the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium. J Natl Cancer Inst 2007;99:777–789. 3. D’Souza G, Kreimer AR, Viscidi R, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med 2007;356:1944– 1956. 4. Fakhry C, Zhang Q, Nguyen–Tan PF, et al. Human papillomavirus and overall survival after progression of oropharyngeal squamous cell carcinoma. J Clin Oncol 2014;32:3365–3373. 5. Vermorken JB, Mesia R, Rivera F, et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer. N Engl J Med 2008;359:1116– 1127. 6. Vermorken JB, Trigo J, Hitt R, et al. Open-label, uncontrolled, multicenter phase II study to evaluate the efficacy and toxicity of cetuximab as a single agent in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck who failed to respond to platinum-based therapy. J Clin Oncol 2007;25:2171–2177. 7. Rubin Grandis J, Melhem MF, Gooding WE, 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. HER2 8. Chung CH, Ely K, McGavran L, et al. Increased epidermal growth factor receptor gene copy number is associated with poor prognosis in head and neck squamous cell carcinomas. J Clin Oncol 2006;24:4170–4176. 9. Cancer Genome Atlas Network. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature 2015;517:576–582. 10. Licitra L, Mesia R, Rivera F, et al. Evaluation of EGFR gene copy number as a predictive biomarker for the efficacy of cetuximab in combination with chemotherapy in the first-line treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck: EXTREME study. Ann Oncol 2011;22:1078–1087. 11. Stewart JS, Cohen EE, Licitra L, et al. Phase III study of gefitinib compared with intravenous methotrexate for recurrent squamous cell carcinoma of the head and neck [corrected]. J Clin Oncol 2009;27:1864–1871. 12. Forastiere AA, Goepfert H, Maor M, et al. Concurrent chemotherapy and radiotherapy for organ preservation in advanced laryngeal cancer. N Engl J Med 2003;349:2091–2098. 13. Zandberg DP, Rollins S, Goloubeva O, et al. A phase I dose escalation trial of MAGE-A3- and HPV16-specific peptide immunomodulatory vaccines in patients with recurrent/metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN). Cancer Immunol Immunother 2015;64:367–379. 14. Frampton GM, Fichtenholtz A, Otto GA, et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol 2013;31:1023–1031. 15. Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 2010;363:24–35. 16. Rischin D, Young RJ, Fisher R, et al. Prognostic significance of p16INK4A and human papillomavirus in patients with oropharyngeal cancer treated on TROG 02.02 phase III trial. J Clin Oncol 2010;28:4142–4148. 17. Posner MR, Lorch JH, Goloubeva O, et al. Survival and human papillomavirus in oropharynx cancer in TAX 324: a subset analysis from an international phase III trial. Ann Oncol 2011;22:1071–1077. 18. Kang H, Kiess A, Chung CH. Emerging biomarkers in head and neck cancer in the era of genomics. Nat Rev Clin Oncol 2015;12:11–26. 19. Chung CH, Guthrie VB, Masica DL, et al. Genomic alterations in head and neck squamous cell carcinoma determined by cancer gene-targeted sequencing. Ann Oncol 2015;26:1216–1223. 20. Lechner M, Frampton GM, Fenton T, et al. Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV1 and HPV- tumors. Genome Med 2013;5:49. 21. Cavalot A, Martone T, Roggero N, Brondino G, Pagano M, Cortesina G. Prognostic impact of HER-2/neu expression on squamous head and neck carcinomas. Head Neck 2007;29:655–664. 22. Ooi A, Takehana T, Li X, et al. Protein overexpression and gene amplification of HER-2 and EGFR in colorectal cancers: an immunohistochemical and fluorescent in situ hybridization study. Mod Pathol 2004;17:895–904. 23. Chung CH, Zhang Q, Hammond EM, et al. Integrating epidermal growth factor receptor assay with clinical parameters improves risk classification for relapse and survival in head-and-neck squamous cell carcinoma. Int J Radiat Oncol Biol Phys 2011;81:331–338. 24. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 2007;25:118–145. 25. Grimm EE, Schmidt RA, Swanson PE, Dintzis SM, Allison KH. Achieving 95% cross-methodological concordance in HER2 testing: causes and implications of discordant cases. Am J Clin Pathol 2010;134:284–292. ALTERATIONS IN HNSCC 26. Brunner K, Fischer CA, Driemel O, Hartmann A, Brockhoff G, Schwarz S. EGFR (HER) family protein expression and cytogenetics in 219 squamous cell carcinomas of the upper respiratory tract: ERBB2 overexpression independent prediction of poor prognosis. Anal Quant Cytol Histol 2010;32:78– 89. 27. Chung CH, Zhang Q, Kong CS, et al. p16 protein expression and human papillomavirus status as prognostic biomarkers of nonoropharyngeal head and neck squamous cell carcinoma. J Clin Oncol 2014;32:3930–3938. 28. Slamon DJ, Leyland–Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783–792. 29. Disel U, Germain A, Yilmazel B, et al. Durable clinical benefit to trastuzumab and chemotherapy in a patient with metastatic colon adenocarcinoma harboring ERBB2 amplification. Oncoscience 2015;2:581–584. 30. Weiler D, Diebold J, Strobel K, Aebi S, Gautschi O. Rapid response to trastuzumab emtansine in a patient with HER2-driven lung cancer. J Thorac Oncol 2015;10:e16–e17. 31. Kelly RJ, Carter C, Giaccone G. Personalizing therapy in an epidermal growth factor receptor-tyrosine kinase inhibitor-resistant non-small-cell lung cancer using PF-00299804 and trastuzumab. J Clin Oncol 2010;28: e507–e510. 32. Gillison ML, Glisson BS, O’Leary E, et al. Phase II trial of trastuzumab (T), paclitaxel (P) and cisplatin (C) in metastatic (M) or recurrent (R) head and neck squamous cell carcinoma (HNSCC): response by tumor EGFR and HER2/neu status. J Clin Oncol 2006;24:5511. 33. Lassus H, Leminen A, Vayrynen A, et al. ERBB2 amplification is superior to protein expression status in predicting patient outcome in serous ovarian carcinoma. Gynecol Oncol 2004;92:31–39. 34. Machiels JP, Haddad RI, Fayette J, et al. Afatinib versus methotrexate as second-line treatment in patients with recurrent or metastatic squamouscell carcinoma of the head and neck progressing on or after platinum-based therapy (LUX-Head & Neck 1): an open-label, randomised phase 3 trial. Lancet Oncol 2015;16:583–594. 35. Seiwert TY, Fayette J, Cupissol D, et al. A randomized, phase II study of afatinib versus cetuximab in metastatic or recurrent squamous cell carcinoma of the head and neck. Ann Oncol 2014;25:1813–1820. 36. Kondo Y, Kikuchi T, Esteban JC, et al. Intratumoral heterogeneity of HER2 protein and amplification of HER2 gene in salivary duct carcinoma. Pathol Int 2014;64:453–459. 37. Chiosea SI, Williams L, Griffith CC, et al. Molecular characterization of apocrine salivary duct carcinoma. Am J Surg Pathol 2015;39:744–752. 38. Kadowaki S, Yatabe Y, Hirakawa H, et al. Complete response to trastuzumab-based chemotherapy in a patient with human epidermal growth factor receptor-2-positive metastatic salivary duct carcinoma ex pleomorphic adenoma. Case Rep Oncol 2013;6:450–455. 39. Nardi V, Sadow PM, Juric D, et al. Detection of novel actionable genetic changes in salivary duct carcinoma helps direct patient treatment. Clin Cancer Res 2013;19:480–490. 40. Nagata Y, Lan KH, Zhou X, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004;6:117–127. 41. Morrow PK, Wulf GM, Ensor J, et al. Phase I/II study of trastuzumab in combination with everolimus (RAD001) in patients with HER2overexpressing metastatic breast cancer who progressed on trastuzumabbased therapy. J Clin Oncol 2011;29:3126–3132. HEAD & NECK—DOI 10.1002/HED JANUARY 2017 E19