HPV Analysis of Head and Neck Squamous Cell Carcinomas based on Fine-Needle. Aspiration Specimens. William H. Westra M.D.

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1 HPV Analysis of Head and Neck Squamous Cell Carcinomas based on Fine-Needle Aspiration Specimens William H. Westra M.D. The Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore Maryland Key words: human papillomavirus, in-situ hybridization, p16 immunohistochemistry, fine needle aspiration (FNA), cell block, hybrid capture 2, branchial cleft cyst, cystic squamous cell carcinoma Correspondence to: William H. Westra, M.D. The Weinberg Bldg, Room N. Broadway Baltimore, Maryland Phone: Fax:

2 Epidemiologic trends of head and neck squamous cell carcinoma (HNSCC) HNSCC is the eighth most common cancer worldwide with approximately 650,000 new cases reported annually. In the United States, 45,660 cases of HNSCC were newly diagnosed in 2007, accounting for 3.2% of all incident malignancies. Tracking the epidemiology of head and neck cancer over extended periods of time has shown that these tumors are in flux both with respect to incidence and associated mortality. The declining overall incidence of head and neck cancer in the United States and elsewhere is partially offset by a disturbing rise in the incidence of oropharynx carcinoma (1). While the age-adjusted incidence of laryngeal, oral cavity, and hypopharyngeal cancers have been in decline, the incidence of oropharyngeal cancer has been on the rise, particularly among individuals under 45 years of age. This anomalous rise of oropharyngeal carcinoma bucks a prevailing notion that HNSCC incident rates can be controlled through smoking prevention and cessation. Indeed, the escalating incidence of oropharyngeal carcinoma in the absence of a parallel rise in smoking and alcohol consumption in the United States and Western Europe points to the participation of non-traditional behavioral and environmental factors driving these epidemiologic trends. Carcinogen exposure, diet, oral hygiene, infectious agents, family history and preexisting medical conditions all play a role, individually or in combination, in the development of HNSCC. While tobacco exposure and alcohol consumption are responsible for the vast majority of HNSCCs that occur in the oral cavity, larynx and hypopharynx; their role in tumorigenesis of the oropharynx is much less consequential. Instead, oncogenic human papillomavirus (HPV), particularly type HPV-16, has been established as a causative agent in up to 80% of oropharyngeal cancers (2, 3). With declining cigarette smoking in many areas of the world, HPV-16 infection is stepping in as a preeminent risk factor and is shifting the demographics of

3 HNSCC toward younger patients who do not smoke or drink. HPV-associated HNSCC is strongly associated with oral HPV infection and certain sexual practices that facilitate repeated viral exposure including early age of sexual debut, a high number of lifetime vaginal and oral sexual partners, frequent oral-genital and oral-anal contact, and infrequent use of barriers during sexual activity (4, 5). HPV-related squamous cell carcinoma is a distinct variant of HNSCC HPV-related HSCCs are distinct from their tobacco related HNSCCs in a number of important respects, and their growing dominance is changing the landscape of head and neck oncology and impacting on pathology practices. Patients with HPV-related cancer look very different than the prototypic patient with tobacco-related SCC of the oral cavity. They tend to be white males that are younger, of higher socioeconomic status and more highly educated. Importantly, they are often non-smokers who do not abuse alcohol. HPV-related SCC has a strong predilection for the oropharyngeal compartment of the oral cavity, particularly the lingual and palatine tonsils. In the United States, 60-80% of those SCCs arising in the oropharynx are caused by oncogenic HPV (particularly type 16), but they are much less frequently encountered in other anatomic sites of the head and neck including the oral cavity proper (6). The unique spectrum of molecular genetic alterations associated with HPV infection is of both biologic and diagnostic relevance. A key step in HPV-related carcinogenesis is the transcription of the viral oncoprotein E7. E7 is known to functionally inactive the Retinoblastoma (Rb) gene product, causing a perturbation of other key components of the critical Rb pathway. As one example, functional inactivation of Rb by E7 is known to induce up regulation of the p16 tumor suppressor gene product, reaching levels that can be readily detected by routine immunohistochemistry.

4 Indeed, p16 immunohistochemistry is now commonly used as a diagnostic assay to differentiate those oral SCCs that are HPV-related from those that are not (3). The presence of HPV is of considerable clinical relevance as these HPV-positive SCCs are associated with improved clinical outcomes relative to their HPV-negative counterparts. HPV-positivity correlates with a lower risk of tumor progression and death (7, 8). The mechanisms underlying this favorable prognosis may involve the combined effects of immune surveillance to viral-specific tumor antigens, an intact apoptotic response to radiation, and the absence of widespread genetic alterations associated with smoking. The development of therapeutic vaccine protocols to augment the host immune response to viral antigens holds promise as a strategy to further enhance tumor clearance. The histomorphology of HPV-related HNSCC HPV-SCC has a characteristic histologic appearance that sets them apart from non-hpvrelated oral SCCs. They typically take origin from the tonsillar crypts of the lingual and palatine tonsils. The surface epithelium does not show progression through the histologic stages of dysplasia that characterizes most conventional oral SCCs. When the surface epithelium is involved, it is usually due to secondary extension from a subjacent crypt. The tumor infiltrates as expansive lobules. The tumor lobules are often permeated by infiltrating lymphocytes. Overt keratinization is usually absent or only focally present. The cells have a high nuclear to cytoplasmic ratio that imparts a basaloid appearance a feature that is commonly taken as evidence to support a poorly differentiated tumor grade. In point of fact, these HPV-related SCCs closely resemble the specialized reticulated from which they are derived and might be more appropriately regarded as highly differentiated SCCs.

5 HPV as a biomarker HPV detection is recognized as a valid method to discern the presence and progress of disease encompassing all aspects of patient care including early cancer detection, prediction of therapeutic response, selection of therapeutic strategies, tumor surveillance, and determination of tumor origin. For those patients who present with metastatic spread to cervical lymph nodes in the absence of a primary tumor, a biomarker strategy takes advantage of the fact that some HNSCCs are caused by certain oncogenic viruses that target specific regions of the upper aerodigestive tract. In effect, detection of a specific virus in the metastasis implicates site of tumor origin. Most notably, detection of Epstein-Barr virus in a neck metastasis reliably points to tumor origin from the nasopharynx. The application of this approach has been expanded with the recognition that HPV-16 is an important causative factor for HNSCCs arising from the oropharynx but not for HNSCCs arising from non-oropharyngeal sites. In-situ hybridization detection of HPV-16 in a metastatic implant reliably points to the oropharynx as the site of tumor origin (9). HPV analysis can also be useful as a purely diagnostic tool when it comes to classification of pathologic processes - as when dealing with cystic squamous lesions of the lateral neck. Metastatic HPV-related OPSCCs involving cervical lymph nodes frequently undergo cystic degeneration causing clinical and even pathologic confusion with benign lymphoepithelial cysts. As branchial cleft cysts are consistently negative for HPV by ISH, detection of HPV in a squamous-lined cyst provides compelling evidence of its malignant nature and oropharyngeal origin (10).

6 Methods of HPV detection in clinical samples There is not yet a standard assay for HPV detection. Current methods included consensus and type-specific PCR techniques, real-time PCR assays to quantify viral load, DNA ISH, and immunohistochemical detection of surrogate biomarkers (e.g. p16 protein). Compared to PCR-based methods, in situ hybridization is a more practical tool for the diagnostic pathologist: 1) the development of nonfluorescent chromogens now allows visualization of DNA hybridization using conventional light microscope allowing for histologic evaluation of viral distribution in tissue samples; 2) the introduction of various signal amplification steps has significantly improved the sensitivity of this technique, even to the point of viral detection down to one viral copy per cell; 3) adaptation of ISH to formalin-fixed and paraffin-embedded tissues has made this technique compatible with standard tissue-processing procedures. P16 immunohistochemistry is often advocated as a surrogate marker of HPV based on the findings that HPV integration with transcription of viral oncoproteins induces overexpression of p16. As a surrogate marker, p16 overexpression is highly sensitive for the presence of HPV but not entirely specific. HPV detection in cytologic specimens The ability to adapt a tissue-targeted approach to aspirated cells represents a critical transition in the clinical implementation of a highly relevant biomarker. Use of aspirated cells as a substrate for HPV16 assessment could facilitate the diagnosis of a HNSCC, direct the search for its site of origin, predict clinical outcome, and select patients most likely to benefit from immunology-based therapy; all while abrogating the need for tissue acquisition via a more aggressive surgical procedure. The feasibility of an in situ hybridization approach for human

7 papillomavirus (HPV) detection in cervical lymph node aspirates has been confirmed in a limited number of studies (11, 12, 13, 14). Using an in-situ hybridization approach on aspirated cells processed as cell blocks, we detected HPV16 in 53% of metastatic oropharyngeal carcinomas but in none of those metastatic carcinomas arising from non-oropharyngeal sites (12). Detection of high risk HPV in fine needle aspirates of neck masses emphatically points to the oropharynx as the site of tumor origin. HPV analysis of fine needle aspirates also may be useful as a diagnostic tool, particularly for cystic squamous lesions of the lateral neck. Metastatic oropharyngeal carcinomas involving cervical lymph nodes frequently undergo cystic degeneration causing clinical confusion with benign cystic squamous lesions. Even fine needle aspiration cytopathology is not always helpful in separating benign lymphoepithelial cysts with reactive squamous atypia from cystic squamous cell carcinomas. Although the absence of high risk HPV may not be helpful in confirming the benign nature of a branchial cleft cyst, its presence provides compelling evidence for metastatic oropharyngeal carcinoma (10). The finding that p16 overexpression is strongly associated with the presence of HPV16 supports the role of p16 expression as a surrogate marker of HPV infection. P16 immunohistochemistry and HPV16 in situ hybridization are complementary, not redundant, in HPV analysis of fine needle aspirates. The intensity of p16 staining in fine needle aspirates appears to be inversely correlated with the integrity of the HPV positive tumor cells: the more degraded the specimen, the weaker the staining. In contrast, the HPV16 hybridization signal persists even in markedly degraded samples (12). Of further note, p16 staining in aspirates from cystic squamous lesions of the lateral neck is not specific for metastatic HPV-related HNSCC. P16 staining is noted in almost half of all branchial cleft cysts evaluated by FNA (10).

8 Future directions in HPV testing of FNA samples Application of an expanding armament of potentially useful biomarkers will undoubtedly strain access to this valuable resource and limit implementation unless current practices are modified to accommodate new applications. The clinical need to establish the status of HPV, p16 and other promising biomarkers in fine needle aspirates of neck masses places a broader expectation on the fine needle aspirate and argues for the routine preparation and retention of materials (e.g. cytospins, cell blocks) for these studies. One very promising approach involves the transfer of a liquid phase assay that is now widely used for assessment of cervical cancer risk. The Hybrid Capture 2 HPV DNA Test is an in vitro nucleic acid hybridization assay with signalamplification using microplate chemiluminescence for the detection of 18 types of human papillomavirus (HPV) DNA in cervical specimens. The Hybrid Capture 2 (HC2) system is an attractive assay in that it 1) is already widely commercially available for general clinical use, 2) uses a liquid transport media that can be used for short and long-term storage, 3) is able to distinguish low from high risk HPV, 4) covers all the high risk HPV types (including type 16) that are known to cause HNSCC, and 5) requires minimal specimen preparation and eliminates the need to process limited specimens as cell blocks. In a very limited validation study (12 study samples to date), we have found the assay to be highly reliable in discerning HPV status based on 100% correlation of HC2 analysis of cytologic specimens (brushings and FNAs) with in-situ hybridization analysis of paired surgical resection specimens (primary tumor resections and lymph node dissections). Of course application in the clinical application requires validation across a much larger number of study samples.

9 Conclusions In summary, the incidence of HPV-related HNSCC is on the rise. Its dominance is changing the head and neck oncologic landscape and re-shaping clinical and diagnostic practices. HPV is now established as a powerful biomarker for all aspects of patient care including prognosis, therapeutic selection, and tumor localization. HPV testing of all oropharyngeal carcinomas will soon be mandatory. But while the demands for HPV testing are increasing, opportunities for testing of tissue samples are diminishing. The sensitivity of HPV-related HNSCC has limited the role of surgical resection, and tissue biopsies of the oropharyngeal primary may not even be available in a substantial portion of patients with occult primaries. Accordingly, the clinical need to establish HPV status in cytologic samples places a more expansive mandate on the fine needle aspirate that goes beyond mere diagnosis (e.g. positive or negative for carcinoma). While HPV analysis of FNAs is feasible, reliable and practical assays must be further developed to enhance broad and routine application in the clinical arena.

10 References 1. Pai SI, Westra WH. Molecular pathology of head and neck cancer: Implications for diagnosis, prognosis, and treatment. Annu Rev Pathol. 2009; 4: Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl. Cancer Inst. 2000; 92: Singhi AD, Westra WH. Comparison of human papillomavirus in situ hybridization and p16 immunohistochemistry in the detection of human papillomavirus-associated head and neck cancer based on a prospective clinical experience. Cancer 2010; 116: D'Souza G, Kreimer AR, Viscidi R, Pawlita M, Fakhry C, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N. Engl. J Med. 2007; 356: Smith EM, Ritchie JM, Summersgill KF, Klussmann JP, Lee JH, et al. Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers. Int. J Cancer 2004; 108: Begum S, Cao D, Gillison M, Zahurak M, Westra WH. Tissue distribution of human papillomavirus 16 DNA integration in patients with tonsillar carcinoma. Clin Cancer Res. 2005; 11: Ang KK, Harris J, Wheeler R, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med. 2010; 363: Fakhry C, Westra WH, Li S, et al. Improved survival of patients with human papillomaviruspositive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst. 2008; 100:261-9.

11 9. Begum S, Gillison ML, Ansari-Lari MA, Shah K, Westra WH. Detection of human papillomavirus in cervical lymph nodes: a highly effective strategy for localizing site of tumor origin. Clin Cancer Res. 2003; 9: Cao D, Begum S, Ali SZ, Westra WH. Expression of p16 in benign and malignant cystic squamous lesions of the head and neck. Hum Pathol. 2009; 41: Umudum H, Rezanko T, Dag F, Dogruluk T. Human papillomavirus genome detection by in situ hybridization in fine-needle aspirates of metastatic lesions from head and neck squamous cell carcinomas. Cancer 2005;105: Begum S, Gillison ML, Nicol TL, Westra WH. Detection of human papillomavirus-16 in fine-needle aspirates to determine tumor origin in patients with metastatic squamous cell carcinoma of the head and neck. Clin Cancer Res 2007; 13: Zhang MQ, El-Mofty SK, Dávila RM. Detection of human papillomavirus-related squamous cell carcinoma cytologically and by in situ hybridization in fine-needle aspiration biopsies of cervical metastasis: a tool for identifying the site of an occult head and neck primary. Cancer 2008; 114: Jannapureddy S, Cohen C, Lau S, Beitler JJ, Siddiqui MT. Assessing for primary oropharyngeal or nasopharyngeal squamous cell carcinoma from fine needle aspiration of cervical lymph node metastases. Diagn Cytopathol. 2010; 38: