ORIGINAL ARTICLES
Hybrid Capture 2 Human Papillomavirus Testing of Fine Needle Aspiration Cytology of Head and Neck Squamous Cell Carcinomas Laleh Hakima, D.O.,1* Esther Adler, M.D.,1** Michael Prystowsky, M.D., Ph.D.,1 Bradley Schiff, M.D.,2 Nicolas Schlecht, Ph.D.,3 Amy Fox, M.D.,1 Mark Suhrland, M.D.,1 and Samer N. Khader, M.D.1
Background: Human papillomavirus (HPV) positive head and
neck squamous cell carcinoma (HNSCC) accounts for 25% of HNSCCs and frequently presents with neck lymph node metastases. We investigated utilizing cytology needle rinse material for HPV DNA testing by Hybrid Capture 2 molecular testing (HC2) as an alternative to p16 immunohistochemistry. Methods: Twenty-two cases of HNSCC presenting with neck lymph node metastasis were prospectively identified by assessment of Diff Quik stained cytology smears. An aliquot of the needle rinse material from the lymph node was analyzed for HPV status using standard HC2 protocol. P16 status was determined with immunohistochemistry on the cell block and/or surgically obtained tumor. Results: The mean age of patients with p16 negative HNSCC was 7 years older than p16 positive disease (Table I). Primary tumor subsites were as follows: 17 oropharynx, 1 hypophayrnx, 3 larynx, and 1 oral cavity (Table II). All ten p16 negative patients had a history of smoking compared with 33% of p16 positive. Only 3 (25%) of p16 positive tumors demonstrated keratinization, whereas 90% of the p16 negative tumors keratinized
1 Department of Pathology, Montefiore Medical Center, Bronx, New York 2 Department of Otolaryngology, Head & Neck Surgery, Montefiore Medical Center, Bronx, New York 3 Department of Epidemiology and Population Health, Montefiore Medical Center, Bronx, New York *Correspondence to: Laleh Hakima, DO, Department of Cytopathology, Montefiore Medical Center, 111 E 210th Street, Hofheimer Bldg, Room 415, Bronx, NY 10467-2401, USA. E-mail: hakima.laleh@gmail. com Received 10 July 2014; Revised 6 January 2015; Accepted 30 March 2015 DOI: 10.1002/dc.23278 Published online 12 May 2015 in Wiley Online Library (wileyonlinelibrary.com).
C 2015 WILEY PERIODICALS, INC. V
(Fig. 1). Twelve of 22 HNSCC cases (55%) were p16 positive, of which 7 (58%) tested positive for HPV by HC2. Ten cases (45%) were negative for p16, all of which were negative for HPV by HC2 (Table III). Conclusion: Molecular testing for HPV using HC2 on needle rinse material of FNA of HNSCC is a useful method of determining HPV status in HNSCC. Diagn. Cytopathol. 2015;43:683– C 2015 Wiley Periodicals, Inc. 687. V Key Words: Hybrid Capture 2; HPV; head and neck squamous cell carcinoma
Human papillomavirus (HPV) is a group of double stranded DNA viruses in the family Papillomaviridae that can infect a large range of animal hosts. Many of the viruses are species specific and tissue specific with predilections toward various epithelial and mucosal sites. In humans, HPV strains are classified as either low risk or high risk with low risk strains causing warts in skin or anogenital region and high risk strains causing dysplasia or carcinoma in various sites including anogenital and oropharynx. Regardless of the strain, the mechanism for viral integration into host cells and replication is the same. The HPV genome is divided into a set of genes expressed early, E1 through E6 and late genes, L1 and L2. The HPV virus infects the basal epithelial layer where the viral genome is maintained with support of the E genes. As the epithelium matures, the L genes are expressed which support amplification of the viral genome and viral assembly. The E6 and E7 genes have been shown to play the most significant role in the Diagnostic Cytopathology, Vol. 43, No 9
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Diagnostic Cytopathology DOI 10.1002/dc
HAKIMA ET AL.
progression to carcinoma. E6 binds to P53 and inhibits apoptosis and E7 binds to the retinoblastoma tumor suppression protein (pRB) resulting in unchecked epithelial proliferation.1,2 HPV infection is associated with 25% of head and neck squamous cell carcinomas (HNSCC) and up to 80% of oropharyngeal cancers.3–8 HPV16 is the most common strain associated with HNSCC, accounting for up to 90% of cases; however, other high-risk strains have been identified as well.2,9 Similar to cervix, the locations in the oropharynx that are most predisposed to HPV infection are those with a squamocolumnar junction which are the tonsillar crypts and glottides.10–12 Additionally, the lateral borders of the tongue and the floor of the mouth are the most common sites involved in the oral cavity. The gold standard for identification of HPV in formalin-fixed paraffin-embedded tissue is HPV PCR. In situ hybridization (ISH) for HPV and immunohistochemistry (IHC) for p16, a surrogate marker for HPV, can also both be performed on paraffin-embedded tissue.9 HPV ISH assays are more technically difficult to perform and interpret and require special equipment. P16 IHC is readily available and is performed in most hospital immunohistochemistry laboratories. For this reason, evaluation of p16 status has become the current standard of care.13 Several studies have shown p16 to be an imperfect test for HPV in the head and neck, although in all studies it was shown to be superior to HPV ISH. Although there are studies that show between 88 and 94% sensitivity and 82–88% specificity of p16 IHC,14,15 in a more recent study, Schlecht et al. demonstrated a sensitivity of 52% and a specificity of 93%.13 The findings by Schlecht et al. indicate that p16 IHC may not be as sensitive as previous literature indicates. This raises an important issue regarding the clinical management of patients with HNSCC where there are differences in prognosis and therapeutic decisions in patients with HPV positive and HPV negative cancer. Molecular testing for HPV on liquid-based cytology is in widespread use for gynecologic specimens. The Hybrid Capture 2 test (HC2) uses in vitro nucleic acid hybridization with chemiluminescent signal amplification to detect 13 high risk (HR) strains of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Utilizing needle rinse material for Hybrid Capture 2 testing would not only decrease turnaround time but would obviate the need for formalin-fixed tissue for determination of HPV status while implementing a more specific test.
Einstein College of Medicine. Fine-needle aspiration of cervical lymph nodes, clinically suspicious for metastatic squamous cell carcinoma of primary head and neck origin, were performed between January 2012 and May 2014. Diff-Quik stained smears were examined and a total of 22 cases positive for squamous cell carcinoma with excess residual material in the needle rinse were selected for the study. Informed consent was not indicated since cases were selected only after positive diagnoses had been made. No additional aspirations were performed for the study. Patient demographics, tobacco smoking history, and tumor histology were collected from review of charts, pathology slides, and pathology reports.
Hybrid Capture 2 Assay Fine-needle aspiration material was rinsed in Hank’s Balanced Salt Solution at the time of the procedure. Approximately 0.5–1.0 mL of residual needle rinse was aliquoted into SurePath media for HPV testing. Specimens were centrifuged and 230 lL of a mixture of specimen transport media:denaturing reagent was added to the precipitant. Denatured specimens were then loaded onto the Digene hybridization plates. Samples containing target high-risk HPV types hybridized with specific RNA probes and were captured onto a microplate well coated with antibodies. The RNA:DNA hybrids were then reacted with a secondary alkaline phosphatase conjugated antibody with multiple antibodies binding to each hybrid resulting in signal amplification. A detection reagent substrate was cleaved by the enzyme resulting in light emission. The relative light units (RLUs) were measured on a luminometer and a value greater than the cut off (CO) indicated the presence of high-risk HPV DNA in the specimen.
Immunohistochemistry
Methods
Immunohistochemical staining for p16 was performed on paraffin-embedded tissue from corresponding cytology cell blocks and/or surgical biopsies or resections for comparison. Deparaffinized tissue sections were incubated with a Pharmingen monoclonal mouse antibody (BD Biosciences, CA) at 1:50 dilution for 30 min at room temperature. A secondary Dako EnVisionTM biotinylated horseradish peroxidase-conjugated mouse antibody (K4001) was then added without dilution and incubated for 30 min at room temperature. Slides were stained with ultra-marque iaminobenzidine reagent for 2–4 min (Dako, CA; K3468) and counterstained with hematoxylin (Harris formula; Surgipath, IL). Positive and negative controls were included with each run and consisted of uterine cervix with severe dysplasia, with and without primary antibody, respectively.
Case Collection
Statistics
This was a prospective study approved by Institutional Review Boards at Montefiore Medical Center and Albert
Overall p16 status was determined by qualitative assessment of nuclear staining in tissue sections from cytology
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Diagnostic Cytopathology DOI 10.1002/dc
HYBRID CAPTURE 2 HPV TESTING OF FNA HEAD AND NECK SQUAMOUS CELL CARCINOMAS
cell blocks and/or surgical biopsies and resections. The concordance rate between HC2 assay test results and overall p16 status was determined by Kappa statistics. Tumor keratinization and smoking association was assessed using v2 tests.
Results Twenty-two fine needle aspiration cytology cases of cervical lymph nodes with metastatic squamous cell Table I. Clinical Pathologic Characteristics of p161 and p162 HNSCC
N (%) Male, N (%) Female, N (%) Age mean (range) Smoking history, N (%) Keratinization, N (%)
Total
p161
p162
22 19 (86%) 3 (14%) 67 14 (64%) 12 (55%)
12 (55%) 11 (50%) 1 (4%) 63 (36-88) 4 (18%) 3 (14%)
10 (45%) 8 (36%) 2 (9%) 70 (50-91) 10 (45%) 9 (41%)
Table II. Primary Tumor Site of Origin
Oropharynx, N (%) Larynx, N (%) Hypopharynx, N (%) Oral cavity, N (%)
Total
p161
p162
17 (77%) 3 (14%) 1 (5%) 1 (5%)
11 (92%) 0 0 1 (8%)
6 (60%) 3 (30%) 1(10%) 0
carcinoma, primary head and neck, were included in the study. The majority of patients were male (n 5 19, 86%) with a median age of 66 years old at diagnosis (Table I). All cases were tested for HPV by HC2 technology (n 5 22). Immunohistochemical staining for p16 was performed on surgically obtained tissue (n 5 20) and cytology cell blocks (n 5 2). Primary head and neck subsites of squamous cell carcinoma were oropharynx (n 5 17) followed by larynx (n 5 3), hypopharynx (n 5 1), and oral cavity (n 5 1) (Table II). Immunohistochemical staining for p16 was positive in 12 patients (55%), of which 7 (58%) tested positive by HC2 assay. Ten patients (45%) were p16 negative, all of whom were also negative by HC2 assay. Comparison of HC2 assay and p16 IHC results showed good agreement between the two methods (77%, Kappa 5 0.56, 95% CI: 0.54–0.91). Five patients (23%) were p16 positive but tested negative with HC2 (Table III). There was a strong correlation between p16 negative tumors and positive smoking history in the patients. All (100%) of p16 negative patients had a smoking history compared with only 4 (33%) of p16 positive patients (P 5 0.002) (Table I). P16 expression also inversely correlated with histologic tumor keratinization. Nine (75%) p16 positive tumors showed non-keratinizing SCC compared with1 (10%) p16 negative tumor (P 5 0.004) (Fig. 1).
Table III. HPV Hybrid Capture 2 Results Compared to p16 IHC
HPV positive, N (%) HPV negative, N (%)
Total
p161
p162
7 (32%) 15 (68%)
7 (58%) 5 (42%)
0 10 (100%)
Discussion HPV infection, primarily subtype 16, is frequently present and associated with good prognosis in primary head and
Fig. 1. Top row: (A) Keratinizing squamous cell carcinoma (Papanicolaou stain, 403); (B) Keratinizing squamous cell carcinoma (H&E, 403); (C) p16 negative. Bottom row: (D) Non-keratinizing squamous cell carcinoma (Papanicolaou stain, 403); (E) Non-keratinizing squamous cell carcinoma (H&E, 403); (F) p16 positive. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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neck squamous cell carcinomas.2,9 The exact prevalence of HPV virus varies depending on tumor primary subsite in the head and neck region and the detection method used. Immunohistochemical staining for p16 is one of the most commonly used methods as it is simple, cost effective, and available in most laboratories.9 P16 is a tumor suppressor gene which is inactivated in conventional, smoking associated, HNSCCs but upregulated in the presence of high risk HPV infection in non-keratinizing primary oropharyngeal tumors. The sensitivity and specificity of p16 expression as a surrogate maker for HPV detection varies in the literature. False positive results may occur due to technical errors and stain interpretation with respect to intensity and localization of the p16 protein expression.13–15 Insufficient cellularity on cell blocks obtained from FNA procedures further represents an obstacle in performing IHC. We evaluated HC2 technology as an alternative method for HPV detection in comparison to p16 IHC. There was 100% correlation between p16 IHC and HC2 in detecting HPV negative tumors. There were, however, five cases which were positive by p16 IHC and negative on HC2 (Table III). There is currently no standardized method for obtaining an aliquot of cytologyobtained material for HPV testing in HNSCC. The method we used in this study consisted of taking a small aliquot (0.5–1.0 mL) of the needle rinse fluid and placing into the SurePath medium. This method has the effect of diluting the specimen and could be an important underlying cause of false-negative results. On the other hand, without performing sequencing for HPV, it is possible that discordant p16 expression maybe falsely positive. Several other studies have investigated the use of HC2 molecular testing for HPV in HNSCC. Smith et al. studied the cost benefit of testing HPV status using HC2 on aspirate fluid obtained from cervical lymph node metastases of HNSCC versus p16 on paraffin-embedded tissue. They found that the HC2 assay correctly classified 21 out of 25 cases (84%) with a cost savings of between $113.74 and $364.63 per patient.16 The HPV status on cytology specimens (fine-needle aspirates and brushes) was also correctly classified by HC2 compared with p16 IHC and HPV ISH in Bishop et al. study. A single discordant case (P161/HPV2) was reported which tested positive by HC2 assay and was subsequently confirmed by real-time PCR.17 In both of the above studies, FNA material was initially evaluated for adequacy after which dedicated passes were performed and placed directly in SurePath medium for HC2. Jarboe et al. reported a series of 16 specimens from brushings of oral and pharyngeal squamous cell carcinomas. Tissue was available in 11 (69%) cases for HR-HPV ISH testing. All 16 cases were tested by HC2 for the presence of HPV. There was a high concordance rate 686
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between HR-HPV ISH and HC2 (82%). The results suggested HC2 maybe an effective method for determining HPV status in brushing/scraping specimens from oropharyngeal lesions.18,19 Another recent study evaluated 50 consecutive primary oropharyngeal squamous cell carcinomas for the presence of HPV. P16 IHC was performed on formalin-fixed paraffin-embedded tissue and 22/50 cases with available fresh tumor were also tested with Digene HC2 DNA and NucliSENS EasyQ HPVv1 RNA assays. The agreement rate between DNA and RNA detection in samples was 100%. The agreement rate between p16 IHC and HC2 detection was fair but not excellent (k 5 0.618). This study reports that HPV nucleic acid detection is more reliable and objective than p16 IHC and better correlates with survival.20 In conclusion, HC2 is a valid method of determining HPV status in HNSCC, especially in cases where the most accessible lesion is metastatic disease in a palpable lymph node. More studies are needed to optimize the technique and improve sensitivity.
References 1. Ragin CCR, Modugno F, Gollin SM. The epidemiology and risk factors of head and neck cancer: A focus on human papillomavirus. J Dent Res 2007;86:104–114. 2. Marur S, D’Souza G, Westra WH, Forastiere AA. HPV-associated head and neck cancer: A virus-related cancer epidemic. Lancet Oncol 2010;11:781–789. 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. Herrero R, Castellsague X, Pawlita M, et al. Human papillomavirus and oral cancer: The International Agency for Research on Cancer multicenter study. J Natl Cancer Inst 2003;95:1772–1783. 5. Applebaum KM, Furniss CS, Zeka A, et al. Lack of association of alcohol and tobacco with HPV16-associated head and neck cancer. J Natl Cancer Inst 2007;99:1801–1810. 6. Smith EM, Ritchie JM, Summersgill KF, et al. Human papillomavirus in oral exfoliated cells and risk of head and neck cancer. J Natl Cancer Inst 2004;96:449–455. 7. Schwartz SM, Daling JR, Doody DR, et al. Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 1998;90:1626–1636. 8. Gillison ML, Koch WM, Capone RB, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000;92:709–720. 9. Agoston ES, Robinson SJ, Mehra KK, et al. Polymerase chain reaction detection of HPV in squamous carcinoma of the oropharynx. Am J Clin Pathol 2010;134:36–34. 10. Boy S, Van Rensburg EJ, Engelbrecht S, Dreyer L, van Heerden M, van Heerden W. HPV detection in primary intraoral squamous cell carcinomas—Commensal, aetiological agent or contamination? J Oral Pathol Med 2006;35:86–90. 11. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus types in head and neck squamous cell carcinomas worldwide: A systematic review. Cancer Epidemiol Biomarkers Prev 2005;14:467–475. 12. Scheurer ME, Tortolero-Lunay G, Adler-Storthzz K. Human papillomavirus infection: Biology, epidemiology, and prevention. Int J Gynecol Cancer 2005;15:727–746.
Diagnostic Cytopathology DOI 10.1002/dc
HYBRID CAPTURE 2 HPV TESTING OF FNA HEAD AND NECK SQUAMOUS CELL CARCINOMAS 13. Schlecht NF, Brandwein-Gensler M, Nuovo GJ, et al. A comparison of clinically utilized human papilloma virus detection methods in head and neck cancer. Mod Pathol 2011;24:1295–1305.
and neck: A convenient liquid phase approach for the reliable determination of human papillomavirus status. Cancer Cytopathol 2012; 120:18–25.
14. Smeets SJ, Hesselink AT, Speel EJ, et al. A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen. Int J Cancer 2007;121:2465–2472.
18. Jarboe EA, Hunt JP, Layfield LJ. Cytomorphologic diagnosis and HPV testing of metastatic and primary oropharyngeal squamous cell carcinomas: A review and summary of the literature. Diagn Cytopathol 2012;40:491–497.
15. Schache AG, Liloglou T, Risk JM, et al. Evaluation of human papilloma virus diagnostic testing in oropharyngeal squamous cell carcinoma: Sensitivity, specificity, and prognostic discrimination. Clin Cancer Res 2011;17:6262–6271. 16. Smith DF, Maleki Z, Coughlan D, et al. Human papillomavirus status of head and neck cancer as determined in cytologic specimens using the hybrid-capture 2 assay. Oral Oncol 2014;50:600–604. 17. Bishop JA, Maleki Z, Valsamakis V, et al. Application of the hybrid capture 2 assay to squamous cell carcinomas of the head
19. Jarboe EA, Willis M, Bentz B, et al. Detection of human papillomavirus using hybrid capture 2 in oral brushings from patients with oropharyngeal squamous cell carcinoma. Am J Clin Pathol 2011;135:766–769. 20. Bussu F, Sali M, Gallus R, et al. Human papillomavirus (HPV) infection in squamous cell carcinomas arising from the oropharynx: Detection of HPV DNA and p16 Immunohistochemistry as diagnostic and prognostic indicators—A pilot study. Int J Radiat Oncol Biol Phys 2014;89:1115–1120.
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Hybrid Capture 2 Human Papillomavirus Testing of Fine Needle Aspiration Cytology of Head and Neck Squamous Cell Carcinomas Laleh Hakima, D.O.,1* Esther Adler, M.D.,1** Michael Prystowsky, M.D., Ph.D.,1 Bradley Schiff, M.D.,2 Nicolas Schlecht, Ph.D.,3 Amy Fox, M.D.,1 Mark Suhrland, M.D.,1 and Samer N. Khader, M.D.1
Background: Human papillomavirus (HPV) positive head and
neck squamous cell carcinoma (HNSCC) accounts for 25% of HNSCCs and frequently presents with neck lymph node metastases. We investigated utilizing cytology needle rinse material for HPV DNA testing by Hybrid Capture 2 molecular testing (HC2) as an alternative to p16 immunohistochemistry. Methods: Twenty-two cases of HNSCC presenting with neck lymph node metastasis were prospectively identified by assessment of Diff Quik stained cytology smears. An aliquot of the needle rinse material from the lymph node was analyzed for HPV status using standard HC2 protocol. P16 status was determined with immunohistochemistry on the cell block and/or surgically obtained tumor. Results: The mean age of patients with p16 negative HNSCC was 7 years older than p16 positive disease (Table I). Primary tumor subsites were as follows: 17 oropharynx, 1 hypophayrnx, 3 larynx, and 1 oral cavity (Table II). All ten p16 negative patients had a history of smoking compared with 33% of p16 positive. Only 3 (25%) of p16 positive tumors demonstrated keratinization, whereas 90% of the p16 negative tumors keratinized
1 Department of Pathology, Montefiore Medical Center, Bronx, New York 2 Department of Otolaryngology, Head & Neck Surgery, Montefiore Medical Center, Bronx, New York 3 Department of Epidemiology and Population Health, Montefiore Medical Center, Bronx, New York *Correspondence to: Laleh Hakima, DO, Department of Cytopathology, Montefiore Medical Center, 111 E 210th Street, Hofheimer Bldg, Room 415, Bronx, NY 10467-2401, USA. E-mail: hakima.laleh@gmail. com Received 10 July 2014; Revised 6 January 2015; Accepted 30 March 2015 DOI: 10.1002/dc.23278 Published online 12 May 2015 in Wiley Online Library (wileyonlinelibrary.com).
C 2015 WILEY PERIODICALS, INC. V
(Fig. 1). Twelve of 22 HNSCC cases (55%) were p16 positive, of which 7 (58%) tested positive for HPV by HC2. Ten cases (45%) were negative for p16, all of which were negative for HPV by HC2 (Table III). Conclusion: Molecular testing for HPV using HC2 on needle rinse material of FNA of HNSCC is a useful method of determining HPV status in HNSCC. Diagn. Cytopathol. 2015;43:683– C 2015 Wiley Periodicals, Inc. 687. V Key Words: Hybrid Capture 2; HPV; head and neck squamous cell carcinoma
Human papillomavirus (HPV) is a group of double stranded DNA viruses in the family Papillomaviridae that can infect a large range of animal hosts. Many of the viruses are species specific and tissue specific with predilections toward various epithelial and mucosal sites. In humans, HPV strains are classified as either low risk or high risk with low risk strains causing warts in skin or anogenital region and high risk strains causing dysplasia or carcinoma in various sites including anogenital and oropharynx. Regardless of the strain, the mechanism for viral integration into host cells and replication is the same. The HPV genome is divided into a set of genes expressed early, E1 through E6 and late genes, L1 and L2. The HPV virus infects the basal epithelial layer where the viral genome is maintained with support of the E genes. As the epithelium matures, the L genes are expressed which support amplification of the viral genome and viral assembly. The E6 and E7 genes have been shown to play the most significant role in the Diagnostic Cytopathology, Vol. 43, No 9
683
Diagnostic Cytopathology DOI 10.1002/dc
HAKIMA ET AL.
progression to carcinoma. E6 binds to P53 and inhibits apoptosis and E7 binds to the retinoblastoma tumor suppression protein (pRB) resulting in unchecked epithelial proliferation.1,2 HPV infection is associated with 25% of head and neck squamous cell carcinomas (HNSCC) and up to 80% of oropharyngeal cancers.3–8 HPV16 is the most common strain associated with HNSCC, accounting for up to 90% of cases; however, other high-risk strains have been identified as well.2,9 Similar to cervix, the locations in the oropharynx that are most predisposed to HPV infection are those with a squamocolumnar junction which are the tonsillar crypts and glottides.10–12 Additionally, the lateral borders of the tongue and the floor of the mouth are the most common sites involved in the oral cavity. The gold standard for identification of HPV in formalin-fixed paraffin-embedded tissue is HPV PCR. In situ hybridization (ISH) for HPV and immunohistochemistry (IHC) for p16, a surrogate marker for HPV, can also both be performed on paraffin-embedded tissue.9 HPV ISH assays are more technically difficult to perform and interpret and require special equipment. P16 IHC is readily available and is performed in most hospital immunohistochemistry laboratories. For this reason, evaluation of p16 status has become the current standard of care.13 Several studies have shown p16 to be an imperfect test for HPV in the head and neck, although in all studies it was shown to be superior to HPV ISH. Although there are studies that show between 88 and 94% sensitivity and 82–88% specificity of p16 IHC,14,15 in a more recent study, Schlecht et al. demonstrated a sensitivity of 52% and a specificity of 93%.13 The findings by Schlecht et al. indicate that p16 IHC may not be as sensitive as previous literature indicates. This raises an important issue regarding the clinical management of patients with HNSCC where there are differences in prognosis and therapeutic decisions in patients with HPV positive and HPV negative cancer. Molecular testing for HPV on liquid-based cytology is in widespread use for gynecologic specimens. The Hybrid Capture 2 test (HC2) uses in vitro nucleic acid hybridization with chemiluminescent signal amplification to detect 13 high risk (HR) strains of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Utilizing needle rinse material for Hybrid Capture 2 testing would not only decrease turnaround time but would obviate the need for formalin-fixed tissue for determination of HPV status while implementing a more specific test.
Einstein College of Medicine. Fine-needle aspiration of cervical lymph nodes, clinically suspicious for metastatic squamous cell carcinoma of primary head and neck origin, were performed between January 2012 and May 2014. Diff-Quik stained smears were examined and a total of 22 cases positive for squamous cell carcinoma with excess residual material in the needle rinse were selected for the study. Informed consent was not indicated since cases were selected only after positive diagnoses had been made. No additional aspirations were performed for the study. Patient demographics, tobacco smoking history, and tumor histology were collected from review of charts, pathology slides, and pathology reports.
Hybrid Capture 2 Assay Fine-needle aspiration material was rinsed in Hank’s Balanced Salt Solution at the time of the procedure. Approximately 0.5–1.0 mL of residual needle rinse was aliquoted into SurePath media for HPV testing. Specimens were centrifuged and 230 lL of a mixture of specimen transport media:denaturing reagent was added to the precipitant. Denatured specimens were then loaded onto the Digene hybridization plates. Samples containing target high-risk HPV types hybridized with specific RNA probes and were captured onto a microplate well coated with antibodies. The RNA:DNA hybrids were then reacted with a secondary alkaline phosphatase conjugated antibody with multiple antibodies binding to each hybrid resulting in signal amplification. A detection reagent substrate was cleaved by the enzyme resulting in light emission. The relative light units (RLUs) were measured on a luminometer and a value greater than the cut off (CO) indicated the presence of high-risk HPV DNA in the specimen.
Immunohistochemistry
Methods
Immunohistochemical staining for p16 was performed on paraffin-embedded tissue from corresponding cytology cell blocks and/or surgical biopsies or resections for comparison. Deparaffinized tissue sections were incubated with a Pharmingen monoclonal mouse antibody (BD Biosciences, CA) at 1:50 dilution for 30 min at room temperature. A secondary Dako EnVisionTM biotinylated horseradish peroxidase-conjugated mouse antibody (K4001) was then added without dilution and incubated for 30 min at room temperature. Slides were stained with ultra-marque iaminobenzidine reagent for 2–4 min (Dako, CA; K3468) and counterstained with hematoxylin (Harris formula; Surgipath, IL). Positive and negative controls were included with each run and consisted of uterine cervix with severe dysplasia, with and without primary antibody, respectively.
Case Collection
Statistics
This was a prospective study approved by Institutional Review Boards at Montefiore Medical Center and Albert
Overall p16 status was determined by qualitative assessment of nuclear staining in tissue sections from cytology
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Diagnostic Cytopathology, Vol. 43, No 9
Diagnostic Cytopathology DOI 10.1002/dc
HYBRID CAPTURE 2 HPV TESTING OF FNA HEAD AND NECK SQUAMOUS CELL CARCINOMAS
cell blocks and/or surgical biopsies and resections. The concordance rate between HC2 assay test results and overall p16 status was determined by Kappa statistics. Tumor keratinization and smoking association was assessed using v2 tests.
Results Twenty-two fine needle aspiration cytology cases of cervical lymph nodes with metastatic squamous cell Table I. Clinical Pathologic Characteristics of p161 and p162 HNSCC
N (%) Male, N (%) Female, N (%) Age mean (range) Smoking history, N (%) Keratinization, N (%)
Total
p161
p162
22 19 (86%) 3 (14%) 67 14 (64%) 12 (55%)
12 (55%) 11 (50%) 1 (4%) 63 (36-88) 4 (18%) 3 (14%)
10 (45%) 8 (36%) 2 (9%) 70 (50-91) 10 (45%) 9 (41%)
Table II. Primary Tumor Site of Origin
Oropharynx, N (%) Larynx, N (%) Hypopharynx, N (%) Oral cavity, N (%)
Total
p161
p162
17 (77%) 3 (14%) 1 (5%) 1 (5%)
11 (92%) 0 0 1 (8%)
6 (60%) 3 (30%) 1(10%) 0
carcinoma, primary head and neck, were included in the study. The majority of patients were male (n 5 19, 86%) with a median age of 66 years old at diagnosis (Table I). All cases were tested for HPV by HC2 technology (n 5 22). Immunohistochemical staining for p16 was performed on surgically obtained tissue (n 5 20) and cytology cell blocks (n 5 2). Primary head and neck subsites of squamous cell carcinoma were oropharynx (n 5 17) followed by larynx (n 5 3), hypopharynx (n 5 1), and oral cavity (n 5 1) (Table II). Immunohistochemical staining for p16 was positive in 12 patients (55%), of which 7 (58%) tested positive by HC2 assay. Ten patients (45%) were p16 negative, all of whom were also negative by HC2 assay. Comparison of HC2 assay and p16 IHC results showed good agreement between the two methods (77%, Kappa 5 0.56, 95% CI: 0.54–0.91). Five patients (23%) were p16 positive but tested negative with HC2 (Table III). There was a strong correlation between p16 negative tumors and positive smoking history in the patients. All (100%) of p16 negative patients had a smoking history compared with only 4 (33%) of p16 positive patients (P 5 0.002) (Table I). P16 expression also inversely correlated with histologic tumor keratinization. Nine (75%) p16 positive tumors showed non-keratinizing SCC compared with1 (10%) p16 negative tumor (P 5 0.004) (Fig. 1).
Table III. HPV Hybrid Capture 2 Results Compared to p16 IHC
HPV positive, N (%) HPV negative, N (%)
Total
p161
p162
7 (32%) 15 (68%)
7 (58%) 5 (42%)
0 10 (100%)
Discussion HPV infection, primarily subtype 16, is frequently present and associated with good prognosis in primary head and
Fig. 1. Top row: (A) Keratinizing squamous cell carcinoma (Papanicolaou stain, 403); (B) Keratinizing squamous cell carcinoma (H&E, 403); (C) p16 negative. Bottom row: (D) Non-keratinizing squamous cell carcinoma (Papanicolaou stain, 403); (E) Non-keratinizing squamous cell carcinoma (H&E, 403); (F) p16 positive. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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HAKIMA ET AL.
neck squamous cell carcinomas.2,9 The exact prevalence of HPV virus varies depending on tumor primary subsite in the head and neck region and the detection method used. Immunohistochemical staining for p16 is one of the most commonly used methods as it is simple, cost effective, and available in most laboratories.9 P16 is a tumor suppressor gene which is inactivated in conventional, smoking associated, HNSCCs but upregulated in the presence of high risk HPV infection in non-keratinizing primary oropharyngeal tumors. The sensitivity and specificity of p16 expression as a surrogate maker for HPV detection varies in the literature. False positive results may occur due to technical errors and stain interpretation with respect to intensity and localization of the p16 protein expression.13–15 Insufficient cellularity on cell blocks obtained from FNA procedures further represents an obstacle in performing IHC. We evaluated HC2 technology as an alternative method for HPV detection in comparison to p16 IHC. There was 100% correlation between p16 IHC and HC2 in detecting HPV negative tumors. There were, however, five cases which were positive by p16 IHC and negative on HC2 (Table III). There is currently no standardized method for obtaining an aliquot of cytologyobtained material for HPV testing in HNSCC. The method we used in this study consisted of taking a small aliquot (0.5–1.0 mL) of the needle rinse fluid and placing into the SurePath medium. This method has the effect of diluting the specimen and could be an important underlying cause of false-negative results. On the other hand, without performing sequencing for HPV, it is possible that discordant p16 expression maybe falsely positive. Several other studies have investigated the use of HC2 molecular testing for HPV in HNSCC. Smith et al. studied the cost benefit of testing HPV status using HC2 on aspirate fluid obtained from cervical lymph node metastases of HNSCC versus p16 on paraffin-embedded tissue. They found that the HC2 assay correctly classified 21 out of 25 cases (84%) with a cost savings of between $113.74 and $364.63 per patient.16 The HPV status on cytology specimens (fine-needle aspirates and brushes) was also correctly classified by HC2 compared with p16 IHC and HPV ISH in Bishop et al. study. A single discordant case (P161/HPV2) was reported which tested positive by HC2 assay and was subsequently confirmed by real-time PCR.17 In both of the above studies, FNA material was initially evaluated for adequacy after which dedicated passes were performed and placed directly in SurePath medium for HC2. Jarboe et al. reported a series of 16 specimens from brushings of oral and pharyngeal squamous cell carcinomas. Tissue was available in 11 (69%) cases for HR-HPV ISH testing. All 16 cases were tested by HC2 for the presence of HPV. There was a high concordance rate 686
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between HR-HPV ISH and HC2 (82%). The results suggested HC2 maybe an effective method for determining HPV status in brushing/scraping specimens from oropharyngeal lesions.18,19 Another recent study evaluated 50 consecutive primary oropharyngeal squamous cell carcinomas for the presence of HPV. P16 IHC was performed on formalin-fixed paraffin-embedded tissue and 22/50 cases with available fresh tumor were also tested with Digene HC2 DNA and NucliSENS EasyQ HPVv1 RNA assays. The agreement rate between DNA and RNA detection in samples was 100%. The agreement rate between p16 IHC and HC2 detection was fair but not excellent (k 5 0.618). This study reports that HPV nucleic acid detection is more reliable and objective than p16 IHC and better correlates with survival.20 In conclusion, HC2 is a valid method of determining HPV status in HNSCC, especially in cases where the most accessible lesion is metastatic disease in a palpable lymph node. More studies are needed to optimize the technique and improve sensitivity.
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