HPV Testing in Head and Neck Think before you order p16 Cancer - PowerPoint PPT Presentation

5/27/2017 HPV Testing in Head and Neck Think before you order p16 Cancer Annemieke van Zante MD/PhD Head & Neck Pathology and Cytopathology The WHO Head and Neck 2017 Disclosures I have nothing to disclose Oropharygeal squamous cell carcinoma: � HPV-positive � HPV-negative 1

5/27/2017 Why? SEER DATA Despite reductions in smoking… Cervical Cancer Oral Cavity and Pharynx the incidence of cancer of the oropharynx has increased Histomorphology Incidence of HPV-attributed Oropharyngeal cancer now Morphological spectrum of HPV+ exceeds Cervical cancer ororpharyngeal SCC: - Basaloid - Papillary Attributable to any HPV type No. - Lymphoepithelioma-like Cancer Average annual no. Cervical 11,771 10,700 (90.6%) - Sarcomatoid All oropharyngeal 15,738 11,000 (70.1%) cancers Viens LJ, Henley SJ, Watson M, et al. Human Papillomavirus–Associated Cancers — United States, 2008– 2012. MMWR Morb Mortal Wkly Rep 2016;65:661–666. 2

5/27/2017 Histomorphology Histomorphology Why test? Why test? � Many HPV+ patients may be cured by single � As a group, patients with HPV+ modality therapy (surgery or radiation alone). oropharynx SCC have improved clinical outcomes compared to patients with � NCCN Treatment Guidelines are currently the conventional, HPV-negative SCC. same for both HPV+ and HPV- tumors, but many clinicians will take a less aggressive � Given significant implications on patient approach in HPV+ cases. prognosis, HPV status will be integrated into the American Joint Committee on � While patients with HPV+ disease have a good Cancer (AJCC) staging in new 8 th Ed. prognosis, the side effects from multi-modality therapy are significant. 3

5/27/2017 Clinical utility Where is the oropharynx? If metastatic SCC of unknown primary involves and groin node and is HPV+ � Look in the anogenital tract If metastatic SCC of unknown primary involves an upper cervical lymph node and is HPV+ � Look in the oropharynx How should we test? What HPV test is the ”best”? Assay Sensitivity Specificity PPV NPV � Need a technically practical, reproducible, Method and easily interpreted marker for high P16 IHC 97% 82% 80% 97% risk HPV within tumor cells P16: High sensitivity, lower specificity � Widely available, inexpensive, standardized � Utilization and interpretation should follow best practice guidelines and consensus statements for consistent reporting * Br J Cancer. 2013 Apr 2; 108(6): 1332–1339. Validation of a novel diagnostic standard in HPV-positive oropharyngeal squamous cell carcinoma. A G Schache, T Liloglou, J M Risk, T M Jones, X-J Ma, H Wang, S Bui, Y Luo, P Sloan, R J Shaw and M Robinson. 4

5/27/2017 What HPV test is the ”best”? What HPV test is the ”best”? Assay Sensitivity Specificity PPV NPV Assay Sensitivity Specificity PPV NPV Method Method P16 IHC 97% 82% 80% 97% P16 IHC 97% 82% 80% 97% hrHPV DNA 94% 91% 89% 95% hrHPV DNA 94% 91% 89% 95% ISH ISH DNA qPCR 91% 87% 83% 93% DNA ISH: Lower sensitivity, better specificity, technically challenging DNA PCR: Lower sensitivity/specificity Cannot distinguish passenger virus from driver * Br J Cancer. 2013 Apr 2; 108(6): 1332–1339. Validation of a novel diagnostic standard in HPV-positive * Br J Cancer. 2013 Apr 2; 108(6): 1332–1339. Validation of a novel diagnostic standard in HPV-positive oropharyngeal squamous cell carcinoma. A G Schache, T Liloglou, J M Risk, T M Jones, X-J Ma, H Wang, oropharyngeal squamous cell carcinoma. A G Schache, T Liloglou, J M Risk, T M Jones, X-J Ma, H Wang, S Bui, Y Luo, P Sloan, R J Shaw and M Robinson. S Bui, Y Luo, P Sloan, R J Shaw and M Robinson. What HPV test is the ”best”? High risk HPV RNA in situ Assay Sensitivity Specificity PPV NPV Method P16 IHC 97% 82% 80% 97% hrHPV DNA 94% 91% 89% 95% ISH DNA qPCR 91% 87% 83% 93% hrHPV RNA 97% 93% 91% 98% ISH* mRNA ISH detects transcriptionally active HPV oncogenes E6/E7 with high sensitivity and specificity…. But technically challenging * Br J Cancer. 2013 Apr 2; 108(6): 1332–1339. Validation of a novel diagnostic standard in HPV-positive oropharyngeal squamous cell carcinoma. A G Schache, T Liloglou, J M Risk, T M Jones, X-J Ma, H Wang, S Bui, Y Luo, P Sloan, R J Shaw and M Robinson. 5

5/27/2017 hrHPV RNA in situ Reconsidering P16 � The correlation between hrHPV RNA ISH and � High sensitivity, high specificity p16 IHC is very high.* � New protocols and reagents for � An endogenous cell cycle protein overexpressed in tumor cells with automated platforms transcriptionally-active high risk HPV. � Standardization will be challenging � E6H4, MTM Lab, ER 1 20’ (BOND) Predilute � Widely available, technically practical, reproducible, and easily interpreted * Mirghani H, Casiraghi O, Amen F, et al. Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice. Mod Pathol. 2015;28(12):1518-1527. P16 Immunohistochemistry What is positive? � P16 IHC is positive in tissue specimens (non-cytology) when there is at least 70% nuclear and cytoplasmic expression with at least moderate to strong intensity . � Staining must be both nuclear and cytoplasmic to be considered positive. 6

5/27/2017 P16 interpretation What is positive? � With these criteria, sensitivity of p16 approaches 100%. � The specificity of p16 in the oropharynx is ~85-95%. � Excellent inter-rater agreement (κ = .97) Keratinizing HPV+ SCC CAP guidelines coming soon… 1. Pathologists should use p16 IHC as a surrogate for hrHPV on all new oropharynx cancers. 2. Additional HPV-specific testing is at the discretion of the pathologist. 7

5/27/2017 What about other head and CAP guidelines coming soon… neck sites? 3. Pathologists should not routinely � The PPV of p16 IHC for HPV in non- perform low-risk HPV testing on patients oropharyngeal SCC is low (25-50%). with head and neck carcinomas. � There is no proven prognostic difference 4. HPV status is not a reliable marker for based on HPV status outside of the aggressive behavior in non-SCC. Don’t oropharynx. test other tumor types. � DO NOT routinely test non-oropharyngeal SCC. What about other head and What about recurrences? neck sites? But I’ve heard that HPV DNA is present in 25% of laryngeal and sinonasal Recurrence can occur outside of the carcinomas? boundaries of the oropharynx. � Using RNA-based HPV detection methods � P16 status can be helpful to distinguish HPV may be an etiologic agent in 2% of between a new primary tumor and SCC outside the oropharynx. recurrence. � The prognostic significance is unknown. � P16 status is misleading. 8

5/27/2017 What about metastases? 1. New diagnosis: HPV testing is indicated in patients with Two scenarios: metastatic SCC of unknown primary in a cervical upper or mid jugular chain lymph 1. New diagnosis of cancer. Primary site node (levels 2 and 3). has not been established 2. Diagnosis has been established and � Note that p16 status is suggestive of patient has a new metastatic lesion. oropharyngeal origin. 20 to 30% of aggressive head and neck cutaneous SCCs overexpress p16 unrelated to high risk HPV. 2. New metastasis Testing FNAs High risk HPV testing is indicated for patients 1. FFPE cell block from FNA with oropharyngeal SCC and a new metastatic � P16 IHC lesion: � hrHPV in situ � When the original tumor was not tested. 2. Liquid based specimens � When there is diagnostic uncertainty � Published sensitivities and specificities >90% regarding recurrence vs. new primary. � Roche cobas (Roche Molecular Systems) � Cervista HR and HPV16/18 (Hologic) P16 is positive in a ≈25% lung SCC. HPV specific testing is indicated if p16 is positive. � Hybrid-Capture 2 (Qiagen) 9

5/27/2017 P16 on FNA cell block � How should p16 be interpreted on cell block specimens? � Recent studies suggest that thresholds as low as 10-15% positive cells may be valid for cell blocks. � Xu B, Ghossein R, Lane J, Lin O, Katabi N. The utility of p16 immunostaining in fine needle aspiration in p16-positive head and neck squamous cell carcinoma. Hum Pathol . 2016;54193-200. Holmes BJ, Maleki Z, Westra WH. The Fidelity of p16 Staining as a Surrogate Marker of Human � Papillomavirus Status in Fine-Needle Aspirates and Core Biopsies of Neck Node Metastases: Implications for HPV Testing Protocols. Acta Cytol . 2015;59(1):97-103. Jalaly JB, Lewis JS, Jr., Collins BT, et al. Correlation of p16 immunohistochemistry in FNA biopsies with � corresponding tissue specimens in HPV-related squamous cell carcinomas of the oropharynx. Cancer Oropharyngeal Carcinoma: A Unique Human Papillomavirus-Associated Tumor of the Head and Neck. Jordan, Richard; Gillison, Maura; van Zante, Annemieke. Pathology Case Reviews: July/August 2011 - Cytopathol . 2015;123(12):723-731. Volume 16 - Issue 4 - pp 173-175 P16 on FNA cell block Conclusions 1. Obtain viable tumor cells in intact fragments. 1. Perform hrHPV testing on all new oropharyngeal SCC by p16 IHC. 2. Do not interpret anucleate squamous • Reporting p16 status for non-oropharyngeal debris or necrotic specimens. SCC may be misleading and is not recommended. 3. Equivocal specimens should be sent for 2. The cutoff for positive p16 on tissue is 70%. HPV specific testing (ISH or PCR). 10

5/27/2017 P16+ Oropharyngeal SCC Let’s eat! � Histological grading or subtyping is not currently advocated. � The diagnosis should include “HPV positive” or “P16 positive.” 11