Editorial
Human Papillomavirus (HPV) Assays for Testing Fine-Needle Aspiration Specimens in Patients With Head and Neck Squamous Cell Carcinoma William C. Faquin, MD, PhD1,2
Human papillomavirus (HPV)-related oropharyngeal carcinoma is a distinct form of head and neck squamous cell carcinoma (HNSCC) at the morphologic, epidemiologic, and molecular levels.1-3 In contrast to nonHPV–related HNSCC, HPV-related oropharyngeal carcinoma has had a dramatic, increasing incidence in the United States over the past 3 decades.2 In many institutions, including ours, greater than 80% to 90% of oropharyngeal squamous cell carcinomas (SCCs), which occur primarily in the palatine tonsil and base of tongue, are associated with high risk-HPV (HR-HPV) types, predominantly HPV type 16 (HPV16). Cancer in these patients is driven by the production of the HPV E6 and E7 oncoproteins, which interfere with p53 and pRb tumor-suppressor pathways.1,4-6 A consequence of this includes the overexpression of p16, which, in selected clinical contexts, can be used as an immunohistochemical surrogate marker of HR-HPV infection.7 HPVrelated HNSCCs often are associated with an improved response to therapy; therefore, the diagnosis of HPVrelated HNSCC has potentially important therapeutic and prognostic implications.1,5 Because of the tendency of HPV-related HNSCC to metastasize to cervical lymph nodes, fine-needle aspiration (FNA) is frequently used for its initial evaluation and diagnosis. In this context, accurate and efficient testing methods for HR-HPV are needed for the evaluation of HNSCCs in cytologic preparations. The article by Guo et al in this issue of Cancer Cytopathology addresses the validity of using the Cervista HPV16/18 and Cervista HPV HR assays [Hologic, Inc., Marlborough, Mass], 2 of several US Food and Drug Administrationapproved methods for testing HPV in cervical cytology specimens, for the detection of HR-HPV in FNA specimens of HNSCC.8 The availability of valid methods, such as that described by Guo et al, for detecting HRHPV in FNA specimens of HNSCC is important for multiple reasons. First, as alluded to previously, the demonstration of HR-HPV in HNSCC has significant implications for patient prognosis, because many of these patients will have improved outcomes in terms of overall survival, progression-free survival, and disease-specific survival compared with patients who have non-HPV–related HNSCC.9-11 The typical patient with HPVrelated HNSCC is a middle-aged man, nonsmoker, and nondrinker with sexual risk factors for oral HPV infection, and low T and high N in the TNM staging system.3,5 That being said, a group of HR-HPV-related HNSCCs remains in which the prognosis appears to be further modified by other features, such as TP53 mutations and expression of Bcl-2.12,13 Biomarkers like these are currently the subject of much research, because they have the potential to influence the therapeutic approach in patients with HPV-related HNSCC. Although changes in the management of patients with HPV-related HNSCC, such as de-escalation therapies, are currently limited to clinical trials, in the very near future, the demonstration of HPV positivity is likely to Corresponding author: William C. Faquin, MD, PhD, Department of Pathology, Warren 219, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114; Fax: (617) 573-3389; [email protected] 1
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts; 2Harvard Medical School, Boston, Massachusetts
See referenced original article on pages 96-103, this issue. Received: October 30, 2013; Accepted: November 6, 2013 Published online December 11, 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21374, wileyonlinelibrary.com
92
Cancer Cytopathology
February 2014
HPV Assays for Testing Fine-Needle Aspiration Specimens in Patients With Oropharyngeal Carcinoma/Faquin
translate into modified treatment regimens and the use of novel targeted therapies for this group of patients.1,5,14 Because an FNA specimen, in many cases, may be the only diagnostic tissue sample obtained from the patient with oropharyngeal carcinoma, the need for developing valid testing methods for HR-HPV in cytologic preparations is critical.15 Testing for HR-HPV in FNA specimens of HNSCC can also serve to address certain diagnostic issues. It is noteworthy that, in a patient who had metastatic SCC without a known primary site, a demonstration of HR-HPV positivity in an FNA specimen from a cervical lymph node can localize the primary site with a high degree of specificity to the oropharynx. Since HPVrelated oropharyngeal SCCs typically arise from the specialized lymphoepithelium located deep within the crypts of the tonsillar-type mucosa, this subset of HNSCCs may go undetected during routine otolaryngologic examination. Because of this, HR-HPV testing on an FNA specimen of the metastatic lesion can provide key information for patient management. The determination of the primary tumor site for patients with metastatic HPV-related HNSCC offers the possibility of providing a more localized treatment, such as limiting radiotherapy to the oropharynx and neck as opposed to irradiating a much wider anatomic field, thereby leading to a reduction in both morbidity and treatment-related complications.5,14 Another diagnostic dilemma includes the FNA of a cystic squamous mass in the lateral neck of a middle-age or older patient in which the differential diagnosis includes a branchial cleft cyst versus metastatic HNSCC. The demonstration of HR-HPV associated with the squamous lesion supports that the lesion in question is metastatic HNSCC and implicates its origin from the oropharynx. It is worth noting that p16 immunochemistry on cell block material is not useful for solving this diagnostic problem, because branchial cleft cysts are sometimes positive for p16.16 Similarly, testing for HR-HPV in an FNA specimen of SCC at other head and neck sites or at a site distant from the head and neck can help to define the disease. This raises the question of when patients with HNSCC should be tested for HR-HPV. Much of the discussion surrounding this question has focused on surgical pathology specimens; however, as demonstrated by the examples mentioned above, testing for HR-HPV in FNA specimens is quickly moving to the forefront. At many institutions within the United States, routine testing for Cancer Cytopathology
February 2014
HR-HPV of any oropharyngeal carcinoma is done along with routine testing of any metastatic SCC of unknown primary origin in the head and neck, and the latter commonly involves testing of an FNA specimen. Both the College of American Pathologists (CAP) and the American Joint Committee on Cancer have recommended routine HPV testing as part of the pathologic workup of oropharyngeal SCCs. Most recently, Cancer Care Ontario issued evidence-based recommendations to perform routine testing for HPV on all oropharyngeal SCCs (available at: http://www.cancercare.on.ca/; accessed November 18, 2013) from adult patients.17 Cancer Care Ontario also recommends that neck lymph node tissue from patients who have metastatic SCC with an unknown primary be tested routinely for HPV status. The CAP is currently working on developing evidence-based guidelines for HPV testing in HNSCCs; and it is noteworthy that, with regard to the application of testing methods like the Cervista HPV assays, the CAP is including testing for HRHPV in cytologic specimens as part of the scope of its project. Questions that remain to be addressed include which test or combination of tests for HR-HPV is sufficient for a reliable diagnosis of HPV-related HNSCC and how those tests should be interpreted. A standard approach in the United States for the testing of HR-HPV status in HNSCCs has yet to be defined for surgical pathology specimens, let alone for cytologic specimens. A range of different detection methods with different targets (eg viral oncoproteins E6 and E7, HPV DNA, and HPV RNA) and differing sensitivity and specificity are available, but DNA in situ hybridization and polymerase chain reaction (PCR)-based amplification are the 2 methods most commonly in use.5 DNA in situ hybridization allows for direct visualization of the host cell containing integrated HPV DNA and, thus, is highly specific, but it is less sensitive than PCR-based amplification. In addition, many laboratories are using immunohistochemistry (IHC) for p16 as a surrogate marker of HR-HPV infection either alone or in combination with 1 of the other available molecular tests. IHC for p16 has the advantage of being inexpensive and having a sensitivity greater than 96% when properly interpreted (ie, both cytoplasmic and nuclear staining in greater than 50%-70% of tumor cells); however, p16 IHC alone has a specificity of only 79% to 82%.5,7,13 Although p16 IHC has a high degree of specificity for evaluating oropharyngeal SCC, it loses specificity when applied to 93
Editorial
nonsquamous lesions and to lesions outside of the oropharynx. Therefore, caution is advised when considering the use of p16 alone for samples from patients with HNSCC in which high specificity is needed. For this reason, many laboratories are using a combination of p16 IHC paired with DNA in situ hybridization for confirmation. Standard HPV testing methods used for surgical pathology specimens could be applied to cytology specimens using formalin-fixed, paraffin-embedded cell block material; however, very often, an FNA sample of HNSCC will consist only of a liquid-based specimen and/or glass slide smears. For this reason, the study by Guo et al helps to advance our ability to use cytologic samples to guide the management of HPV-related HNSCC.8 In addition to the Cervista HPV assays evaluated in the study by Guo and colleagues, 3 other assays are currently approved by the US Food and Drug Administration for the detection of HR-HPV in cervical cytology (Hybrid Capture II [Qiagen, Gaithersburg, Md], Cervista HPV HR [Hologic, Inc.], the Roche cobas HPV Test [Roche Molecular Systems, Pleasanton, Calif], and the APTIMA HPV Assay [Gen-Probe, Inc., San Diego, Calif]).18 With proper laboratory validation, in theory, each of these assays could be used for the detection of HR-HPV in FNAs of HNSCCs. The advantages of applying any of these standard liquidbased assays for cervical HPV determination to cytologic specimens of HNSCC include that these tests are already widely available to cytology laboratories, samples can be stored for modest periods of time, and they provide a quantitative result with clear-cut scoring. Although the various liquid-based assays are generally comparable for HR-HPV detection, there are some differences, with advantages and limitations between the systems. The Cervista assay described by Guo et al is not PCR-based but uses a proprietary Invader chemistry (Hologic, Inc.) to amplify and detect specific nucleic acid sequences.8 Using a series of 64 FNA specimens of HNSCC, Guo et al were able to demonstrate 96% agreement between the Cervista HPV assays and corresponding PCR-based HPV assays. In their study, Guo and colleagues did observe an elevated false-negative rate for the Cervista HPV HR assay; therefore, they recommend using the Cervista HPV16/18 assay as the first-line test in FNA specimens of HNSCC.8 This approach seems logical, because greater than 92% of HPV-related HNSCCs are 94
caused by HPV16. Solomides et al have also studied the Cervista HPV assays and demonstrated that they can be used not only for FNA specimens in PreservcCyt solution (Hologic, Inc.) but also for scraped cells from Papanicolaou-stained and Diff-Quik–stained slides.19 The Qiagen Hybrid Capture II system uses in vitro nucleic acid hybridization with signal amplification along with microplate chemiluminescence for the detection of 13 HR-HPV types. In recent study by Bishop et al using a series of 24 HNSCC specimens, 100% correlation was demonstrated between the Hybrid Capture II assay and DNA in situ hybridization of corresponding resection specimens.20 Two issues that have been identified with the Hybrid Capture II test include the lack of an internal control to verify the presence of adequate specimen DNA and potential cross-reactivity of its probe cocktail with certain untargeted HPV types. Our group has recently studied the performance of the Roche cobas HPV test for detection of HR-HPV in cytologic preparations of HNSCC.21 The Roche cobas test is a PCRbased assay that identifies 14 HR-HPV types in a single analysis. Although there is currently no clinical role for distinguishing between the different HR-HPV types in HNSCC, the Roche cobas test does specifically differentiate between HPV16 and HPV18 while concurrently detecting the other HR-HPV types as a single group. bGlobin amplification and detection are included in the Roche cobas test to help eliminate false-negative results. In our study of this test when applied to HNSCC, the sensitivity relative to in situ hybridization was 100%, but the specificity was lower at only 86%.21 The APTIMA HPV Assay is an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger oncoprotein RNA from 14 HR types of HPV. The APTIMA HPV Assay appears to be as sensitive as the other liquid-based methods; and, because it measures E6 and E7 expression, it may have increased specificity over other methods for HR-HPV infection.22 Efforts by multiple groups to apply the available assays for HR-HPV used in cervical cytology are helping to address the critical need for accurate, efficient, and affordable methods with which to determine HR-HPV status in HNSCCs. More and more, it is clear that the use in cytology of these tests, like that presented in this issue by Guo et al, will play a role in determining clinical trial eligibility as well as having potential diagnostic, prognostic, and general treatment implications. Cancer Cytopathology
February 2014
HPV Assays for Testing Fine-Needle Aspiration Specimens in Patients With Oropharyngeal Carcinoma/Faquin
FUNDING SUPPORT No specific funding was disclosed.
12.
CONFLICT OF INTEREST DISCLOSURES The author made no disclosures.
13.
REFERENCES 1.
Pai SI, Westra WH. Molecular pathology of head and neck cancer: implications for diagnosis, prognosis, and treatment. Annu Rev Pathol. 2009;4:49-70. 2. Chaturvedi AK, Engels EA, Anderson WF, Gillison ML. Incidence trends for human papillomavirus-related and -unrelated oral squamous cell carcinomas in the United States. J Clin Oncol. 2008;26: 612-619. 3. Gillison ML, D’Souza G, Westra W, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. J Natl Cancer Inst. 2008;100:407-420. 4. Strati K, Lambert PF. Role of Rb-dependent and Rb-independent functions of papillomavirus E7 oncogene in head and neck cancer. Cancer Res. 2007;67:11585-11593. 5. Bonilla-Velez J, Mroz EA, Hammon RJ, Rocco JW. Impact of human papillomavirus on oropharyngeal cancer biology and response to therapy: implications for treatment. Otolaryngol Clin North Am. 2013;46:521-543. 6. McLaughlin-Drubin ME, Munger K. Oncogenic activities of human papillomaviruses. Virus Res. 2009;143:195-208. 7. El-Naggar AK, Westra WH. p16 Expression as a surrogate marker for HPV-related oropharyngeal carcinoma: a guide for interpretative relevance and consistency. Head Neck. 2012;34:459-461. 8. Guo M, Dhillon J, Feng J, et al. Cervista HPV assays for fineneedle aspiration specimens are a valid option for human papillomavirus testing in patients with oropharyngeal carcinoma. Cancer Cytopathol. 2014;122:96-103. 9. O’Rorke MA, Ellison MV, Murray LJ, et al. Human papillomavirus related head and neck cancer survival: a systematic review and meta-analysis. Oral Oncol. 2012;48:1191-1201. 10. 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. 11. Fakhry C, Westra WH, Li S, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell
Cancer Cytopathology
February 2014
14.
15.
16.
17.
18. 19.
20.
21.
22.
carcinoma in a prospective clinical trial. J Natl Cancer Inst. 2008; 100:261-269. Nichols AC, Finkelstein DM, Faquin WC, et al. Bcl2 and human papilloma virus 16 as predictors of outcome following concurrent chemoradiation for advanced oropharyngeal cancer. Clin Cancer Res. 2010;16:2138-2146. Kumar B, Cordell KG, Lee JS, et al. EGFR, p16, HPV titer, bcl-xL and p53, sex, and smoking as indicators of response to therapy and survival in oropharyngeal cancer. J Clin Oncol. 2008;26:3128-3137. Chung CH, Schwartz DL. Impact of HPV-related head and neck cancer in clinical trials: opportunity to translate scientific insight into personalized care. Otolaryngol Clin North Am. 2012;45:795-806. Krane JF. Role of cytology in the diagnosis and management of HPV-associated head and neck carcinoma. Acta Cytol. 2013;57: 117-126. Cao D, Begum S, Ali SZ, et al. Expression of p16 in benign and malignant cystic squamous lesions of the neck. Hum Pathol. 2010; 41:535-539. Lacchetti C, Waldron J, Perez-Ordonez B, Kamel-Reid S, Cripps C, Gilbert R; and the Head and Neck Cancer Disease Site Group. Routine HPV Testing in Head and Neck Squamous Cell Carcinoma. A Quality Initiative of the Program in Evidence-Based Care (PEBC), Cancer Care Ontario (CCO). Evidence-Based Series 5-9. Toronto, Ontario, Canada: Cancer Care Ontario; 2013. Available at: http://www.cancercare.on.ca/. Accessed November 18, 2013 Lenhoff A. Five FDA-approved HPV assays. MLO Med Lab Obs. 2012;44:14, 16, 18. Solomides CC, Bibbo M, Wang ZX. Assessment of fine needle aspiration specimen adequacy for high-risk HPV detection and genotyping in oropharyngeal squamous cell carcinoma. Acta Cytol. 2012;56:196-198. Bishop JA, Maleki Z, Valsamakis A, et al. Application of the hybrid capture 2 assay to squamous cell carcinomas of the head and neck: a convenient liquid-phase approach for the reliable determination of human papillomavirus status. Cancer Cytopathol. 2012;120:18-25. Kerr DA, Pitman MB, Sweeney B, Arpin RN, Wilbur DC, Faquin WC. Performance of the Roche cobas 4800 high-risk human papilloma virus test in cytologic preparations of squamous cell carcinoma of the head and neck. Cancer Cytopathol. 2013; doi:10.1002/ cncy.21372. Nishino HT, Tambouret RH, Wilbur DC. Testing for human papillomavirus in cervical cancer screening: a review of indications and methodology. Cancer Cytopathol. 2011;119:219-227.
95
Human Papillomavirus (HPV) Assays for Testing Fine-Needle Aspiration Specimens in Patients With Head and Neck Squamous Cell Carcinoma William C. Faquin, MD, PhD1,2
Human papillomavirus (HPV)-related oropharyngeal carcinoma is a distinct form of head and neck squamous cell carcinoma (HNSCC) at the morphologic, epidemiologic, and molecular levels.1-3 In contrast to nonHPV–related HNSCC, HPV-related oropharyngeal carcinoma has had a dramatic, increasing incidence in the United States over the past 3 decades.2 In many institutions, including ours, greater than 80% to 90% of oropharyngeal squamous cell carcinomas (SCCs), which occur primarily in the palatine tonsil and base of tongue, are associated with high risk-HPV (HR-HPV) types, predominantly HPV type 16 (HPV16). Cancer in these patients is driven by the production of the HPV E6 and E7 oncoproteins, which interfere with p53 and pRb tumor-suppressor pathways.1,4-6 A consequence of this includes the overexpression of p16, which, in selected clinical contexts, can be used as an immunohistochemical surrogate marker of HR-HPV infection.7 HPVrelated HNSCCs often are associated with an improved response to therapy; therefore, the diagnosis of HPVrelated HNSCC has potentially important therapeutic and prognostic implications.1,5 Because of the tendency of HPV-related HNSCC to metastasize to cervical lymph nodes, fine-needle aspiration (FNA) is frequently used for its initial evaluation and diagnosis. In this context, accurate and efficient testing methods for HR-HPV are needed for the evaluation of HNSCCs in cytologic preparations. The article by Guo et al in this issue of Cancer Cytopathology addresses the validity of using the Cervista HPV16/18 and Cervista HPV HR assays [Hologic, Inc., Marlborough, Mass], 2 of several US Food and Drug Administrationapproved methods for testing HPV in cervical cytology specimens, for the detection of HR-HPV in FNA specimens of HNSCC.8 The availability of valid methods, such as that described by Guo et al, for detecting HRHPV in FNA specimens of HNSCC is important for multiple reasons. First, as alluded to previously, the demonstration of HR-HPV in HNSCC has significant implications for patient prognosis, because many of these patients will have improved outcomes in terms of overall survival, progression-free survival, and disease-specific survival compared with patients who have non-HPV–related HNSCC.9-11 The typical patient with HPVrelated HNSCC is a middle-aged man, nonsmoker, and nondrinker with sexual risk factors for oral HPV infection, and low T and high N in the TNM staging system.3,5 That being said, a group of HR-HPV-related HNSCCs remains in which the prognosis appears to be further modified by other features, such as TP53 mutations and expression of Bcl-2.12,13 Biomarkers like these are currently the subject of much research, because they have the potential to influence the therapeutic approach in patients with HPV-related HNSCC. Although changes in the management of patients with HPV-related HNSCC, such as de-escalation therapies, are currently limited to clinical trials, in the very near future, the demonstration of HPV positivity is likely to Corresponding author: William C. Faquin, MD, PhD, Department of Pathology, Warren 219, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114; Fax: (617) 573-3389; [email protected] 1
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts; 2Harvard Medical School, Boston, Massachusetts
See referenced original article on pages 96-103, this issue. Received: October 30, 2013; Accepted: November 6, 2013 Published online December 11, 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21374, wileyonlinelibrary.com
92
Cancer Cytopathology
February 2014
HPV Assays for Testing Fine-Needle Aspiration Specimens in Patients With Oropharyngeal Carcinoma/Faquin
translate into modified treatment regimens and the use of novel targeted therapies for this group of patients.1,5,14 Because an FNA specimen, in many cases, may be the only diagnostic tissue sample obtained from the patient with oropharyngeal carcinoma, the need for developing valid testing methods for HR-HPV in cytologic preparations is critical.15 Testing for HR-HPV in FNA specimens of HNSCC can also serve to address certain diagnostic issues. It is noteworthy that, in a patient who had metastatic SCC without a known primary site, a demonstration of HR-HPV positivity in an FNA specimen from a cervical lymph node can localize the primary site with a high degree of specificity to the oropharynx. Since HPVrelated oropharyngeal SCCs typically arise from the specialized lymphoepithelium located deep within the crypts of the tonsillar-type mucosa, this subset of HNSCCs may go undetected during routine otolaryngologic examination. Because of this, HR-HPV testing on an FNA specimen of the metastatic lesion can provide key information for patient management. The determination of the primary tumor site for patients with metastatic HPV-related HNSCC offers the possibility of providing a more localized treatment, such as limiting radiotherapy to the oropharynx and neck as opposed to irradiating a much wider anatomic field, thereby leading to a reduction in both morbidity and treatment-related complications.5,14 Another diagnostic dilemma includes the FNA of a cystic squamous mass in the lateral neck of a middle-age or older patient in which the differential diagnosis includes a branchial cleft cyst versus metastatic HNSCC. The demonstration of HR-HPV associated with the squamous lesion supports that the lesion in question is metastatic HNSCC and implicates its origin from the oropharynx. It is worth noting that p16 immunochemistry on cell block material is not useful for solving this diagnostic problem, because branchial cleft cysts are sometimes positive for p16.16 Similarly, testing for HR-HPV in an FNA specimen of SCC at other head and neck sites or at a site distant from the head and neck can help to define the disease. This raises the question of when patients with HNSCC should be tested for HR-HPV. Much of the discussion surrounding this question has focused on surgical pathology specimens; however, as demonstrated by the examples mentioned above, testing for HR-HPV in FNA specimens is quickly moving to the forefront. At many institutions within the United States, routine testing for Cancer Cytopathology
February 2014
HR-HPV of any oropharyngeal carcinoma is done along with routine testing of any metastatic SCC of unknown primary origin in the head and neck, and the latter commonly involves testing of an FNA specimen. Both the College of American Pathologists (CAP) and the American Joint Committee on Cancer have recommended routine HPV testing as part of the pathologic workup of oropharyngeal SCCs. Most recently, Cancer Care Ontario issued evidence-based recommendations to perform routine testing for HPV on all oropharyngeal SCCs (available at: http://www.cancercare.on.ca/; accessed November 18, 2013) from adult patients.17 Cancer Care Ontario also recommends that neck lymph node tissue from patients who have metastatic SCC with an unknown primary be tested routinely for HPV status. The CAP is currently working on developing evidence-based guidelines for HPV testing in HNSCCs; and it is noteworthy that, with regard to the application of testing methods like the Cervista HPV assays, the CAP is including testing for HRHPV in cytologic specimens as part of the scope of its project. Questions that remain to be addressed include which test or combination of tests for HR-HPV is sufficient for a reliable diagnosis of HPV-related HNSCC and how those tests should be interpreted. A standard approach in the United States for the testing of HR-HPV status in HNSCCs has yet to be defined for surgical pathology specimens, let alone for cytologic specimens. A range of different detection methods with different targets (eg viral oncoproteins E6 and E7, HPV DNA, and HPV RNA) and differing sensitivity and specificity are available, but DNA in situ hybridization and polymerase chain reaction (PCR)-based amplification are the 2 methods most commonly in use.5 DNA in situ hybridization allows for direct visualization of the host cell containing integrated HPV DNA and, thus, is highly specific, but it is less sensitive than PCR-based amplification. In addition, many laboratories are using immunohistochemistry (IHC) for p16 as a surrogate marker of HR-HPV infection either alone or in combination with 1 of the other available molecular tests. IHC for p16 has the advantage of being inexpensive and having a sensitivity greater than 96% when properly interpreted (ie, both cytoplasmic and nuclear staining in greater than 50%-70% of tumor cells); however, p16 IHC alone has a specificity of only 79% to 82%.5,7,13 Although p16 IHC has a high degree of specificity for evaluating oropharyngeal SCC, it loses specificity when applied to 93
Editorial
nonsquamous lesions and to lesions outside of the oropharynx. Therefore, caution is advised when considering the use of p16 alone for samples from patients with HNSCC in which high specificity is needed. For this reason, many laboratories are using a combination of p16 IHC paired with DNA in situ hybridization for confirmation. Standard HPV testing methods used for surgical pathology specimens could be applied to cytology specimens using formalin-fixed, paraffin-embedded cell block material; however, very often, an FNA sample of HNSCC will consist only of a liquid-based specimen and/or glass slide smears. For this reason, the study by Guo et al helps to advance our ability to use cytologic samples to guide the management of HPV-related HNSCC.8 In addition to the Cervista HPV assays evaluated in the study by Guo and colleagues, 3 other assays are currently approved by the US Food and Drug Administration for the detection of HR-HPV in cervical cytology (Hybrid Capture II [Qiagen, Gaithersburg, Md], Cervista HPV HR [Hologic, Inc.], the Roche cobas HPV Test [Roche Molecular Systems, Pleasanton, Calif], and the APTIMA HPV Assay [Gen-Probe, Inc., San Diego, Calif]).18 With proper laboratory validation, in theory, each of these assays could be used for the detection of HR-HPV in FNAs of HNSCCs. The advantages of applying any of these standard liquidbased assays for cervical HPV determination to cytologic specimens of HNSCC include that these tests are already widely available to cytology laboratories, samples can be stored for modest periods of time, and they provide a quantitative result with clear-cut scoring. Although the various liquid-based assays are generally comparable for HR-HPV detection, there are some differences, with advantages and limitations between the systems. The Cervista assay described by Guo et al is not PCR-based but uses a proprietary Invader chemistry (Hologic, Inc.) to amplify and detect specific nucleic acid sequences.8 Using a series of 64 FNA specimens of HNSCC, Guo et al were able to demonstrate 96% agreement between the Cervista HPV assays and corresponding PCR-based HPV assays. In their study, Guo and colleagues did observe an elevated false-negative rate for the Cervista HPV HR assay; therefore, they recommend using the Cervista HPV16/18 assay as the first-line test in FNA specimens of HNSCC.8 This approach seems logical, because greater than 92% of HPV-related HNSCCs are 94
caused by HPV16. Solomides et al have also studied the Cervista HPV assays and demonstrated that they can be used not only for FNA specimens in PreservcCyt solution (Hologic, Inc.) but also for scraped cells from Papanicolaou-stained and Diff-Quik–stained slides.19 The Qiagen Hybrid Capture II system uses in vitro nucleic acid hybridization with signal amplification along with microplate chemiluminescence for the detection of 13 HR-HPV types. In recent study by Bishop et al using a series of 24 HNSCC specimens, 100% correlation was demonstrated between the Hybrid Capture II assay and DNA in situ hybridization of corresponding resection specimens.20 Two issues that have been identified with the Hybrid Capture II test include the lack of an internal control to verify the presence of adequate specimen DNA and potential cross-reactivity of its probe cocktail with certain untargeted HPV types. Our group has recently studied the performance of the Roche cobas HPV test for detection of HR-HPV in cytologic preparations of HNSCC.21 The Roche cobas test is a PCRbased assay that identifies 14 HR-HPV types in a single analysis. Although there is currently no clinical role for distinguishing between the different HR-HPV types in HNSCC, the Roche cobas test does specifically differentiate between HPV16 and HPV18 while concurrently detecting the other HR-HPV types as a single group. bGlobin amplification and detection are included in the Roche cobas test to help eliminate false-negative results. In our study of this test when applied to HNSCC, the sensitivity relative to in situ hybridization was 100%, but the specificity was lower at only 86%.21 The APTIMA HPV Assay is an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger oncoprotein RNA from 14 HR types of HPV. The APTIMA HPV Assay appears to be as sensitive as the other liquid-based methods; and, because it measures E6 and E7 expression, it may have increased specificity over other methods for HR-HPV infection.22 Efforts by multiple groups to apply the available assays for HR-HPV used in cervical cytology are helping to address the critical need for accurate, efficient, and affordable methods with which to determine HR-HPV status in HNSCCs. More and more, it is clear that the use in cytology of these tests, like that presented in this issue by Guo et al, will play a role in determining clinical trial eligibility as well as having potential diagnostic, prognostic, and general treatment implications. Cancer Cytopathology
February 2014
HPV Assays for Testing Fine-Needle Aspiration Specimens in Patients With Oropharyngeal Carcinoma/Faquin
FUNDING SUPPORT No specific funding was disclosed.
12.
CONFLICT OF INTEREST DISCLOSURES The author made no disclosures.
13.
REFERENCES 1.
Pai SI, Westra WH. Molecular pathology of head and neck cancer: implications for diagnosis, prognosis, and treatment. Annu Rev Pathol. 2009;4:49-70. 2. Chaturvedi AK, Engels EA, Anderson WF, Gillison ML. Incidence trends for human papillomavirus-related and -unrelated oral squamous cell carcinomas in the United States. J Clin Oncol. 2008;26: 612-619. 3. Gillison ML, D’Souza G, Westra W, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. J Natl Cancer Inst. 2008;100:407-420. 4. Strati K, Lambert PF. Role of Rb-dependent and Rb-independent functions of papillomavirus E7 oncogene in head and neck cancer. Cancer Res. 2007;67:11585-11593. 5. Bonilla-Velez J, Mroz EA, Hammon RJ, Rocco JW. Impact of human papillomavirus on oropharyngeal cancer biology and response to therapy: implications for treatment. Otolaryngol Clin North Am. 2013;46:521-543. 6. McLaughlin-Drubin ME, Munger K. Oncogenic activities of human papillomaviruses. Virus Res. 2009;143:195-208. 7. El-Naggar AK, Westra WH. p16 Expression as a surrogate marker for HPV-related oropharyngeal carcinoma: a guide for interpretative relevance and consistency. Head Neck. 2012;34:459-461. 8. Guo M, Dhillon J, Feng J, et al. Cervista HPV assays for fineneedle aspiration specimens are a valid option for human papillomavirus testing in patients with oropharyngeal carcinoma. Cancer Cytopathol. 2014;122:96-103. 9. O’Rorke MA, Ellison MV, Murray LJ, et al. Human papillomavirus related head and neck cancer survival: a systematic review and meta-analysis. Oral Oncol. 2012;48:1191-1201. 10. 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. 11. Fakhry C, Westra WH, Li S, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell
Cancer Cytopathology
February 2014
14.
15.
16.
17.
18. 19.
20.
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22.
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