1. David Y. Zhang MD, PhD, MPH Professor Director, Molecular Pathology Departments of Pathology Icahn School of Medicine at Mount Sinai, New York Molecular diagnosis and monitoring of HPV infection 2

2. HPV Papillomaviridae family Non-enveloped 50-55 nm, icosahedral capsid Circular genome, dsDNA virus >100 HPV types – Based on L1 gene sequence – HPV subtypes 2-10% – HPV variants <2%

3. OncogenityHPV types High risk HPV types16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 67, 68, 70 Low risk HPV types6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 74, 81, 83, 84 Probable high risk types 26, 51, 53, 56, 66, 69, 82 Mucosal HPV types

4. Stratum corneum Stratum granulosum Stratum spinosum Basal cells E1, E2, E4, E5,E6,E7 E1, E2 20-100 HPV DNA/cell E4, L 1, L 2 >1000 HPV DNA /cell Keratinocytes release Productive infection- HPV replication

5. HPV associated cancers

6. Prevalence of HPV cervical infection In women in the world: – HPV infection prevalence 2-44% – HR HPV prevalence 15.1% – ≥ 30 age HR HPV prevalence 5-10% HPV 16 is most common type in women with normal cervical cytology But cervical cancer is a rare complication of HPV infection Baseman JG, et al. J Clin Virol 2005, 32S;16-24.

7. HPV associated Cervical Cancer NEJM 348:518-527, 2003 80% 10-15 y

8. Head and neck cancers Epidemiology of HNSCC – 50,000 new cases/y – 13,000 deaths/y Subdivision by location – Oral Cancer – Laryngeal Cancer – Nasopharyngeal Cancer Histology types – SCC Keratinized Non-keratinized – NPC

9. Biological and clinical characteristics of HNSCC

10. Anal cancer ~0.16% of men and women born today will have cancer of the anus, anal canal, or anorectum sometime during their life Approx 5260 new cases annually in US – 2000 in men and 3260 in women Anal canal lesions may have more aggressive biology

11. SCC of the Anal Canal Histology: keratinizing, nonkeratinizing (transitional) and basaloid Anal canal is 5 times more common than anal margin Incidence is 1/10 that of rectal cancer Transformation Zone: dentate line (Transitional urothelium-like) The most common presenting symptom is bleeding >50% of patients with anal pain A small number of patients will be asymptomatic Most patients are diagnosed late stage

12. Molecular diagnosis of HPV Hybrid Capture II—Qiagen (Digene) – HR and LR Cervista—Hologic (Thirdwave Technology) – HR and reflex to 16/18 Roche Amplicor PCR—Roche – HR and 16/18 PCR/linear probe array—Roche – 37 HPV types In situ hybridization

13. Roche cobas 4800 System 23 inches cobas x 480 instrument Testing from primary specimen tube Specimen barcodes automatically read by system for positive specimen ID Batches of 24 or 96 cobas z 480 analyzer Real-Time PCR Based on LC technology 96 well plate format 4 channel detection 66 inches

14. Roche cobas ® 4800 HPV Test Qualitative multiplex assay – 14 high-risk genotypes 200 nucleotides within the polymorphic L1 region – 3 categories: HPV type 16 & 18, and others –  -globin as an endogenous internal control It will assess not only extraction and amplification procedures but also serves as a collection control Ensures that sufficient sample is collected and prepared

15. Limit of Detection (LOD) LOD was chosen at the clinical cutoff (to detect high-grade disease of  CIN2) to achieve pre-defined sensitivity estimates (93%) in the intended use populations (ASC-US). – Assessed by use of plasmids (HPV31, HPV16, and HPV18) or cell lines SiHa (contains 1- 2 copies of HPV16 per cell) and HeLa (20-40 copies of HPV18 per cell)

16. Indications To screen patients >21 y.o. with ASC-US cervical cytology test results to determine the need for referral to colposcopy. To be used in patients >21 y.o. with ASC-US cervical cytology results, to assess the presence or absence of high-risk HPV genotypes 16 and 18. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management. The results of this test are NOT intended to prevent women from proceeding to colposcopy. 21 years and older FDA approved claims

17. Indications In women >30 y.o., the cobas® HPV Test can be used with cervical cytology to adjunctively screen to assess the presence or absence of high risk HPV types. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management. In women >30 y.o., the cobas® HPV Test can be used to assess the presence or absence of HPV genotypes 16 and 18. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management. 30 years and older

18. Additional Indications HPV DNA test for women 25 and older that can be used alone to help a health care professional assess the need for a woman to undergo additional diagnostic testing for cervical cancer – positive for HPV 16 or HPV 18 should have a colposcopy – positive for one or more of the 12 other high-risk HPV types should have a Pap test to determine the need for a colposcopy Patient’s risk for developing cervical cancer in the future FDA approved on 4/24/14

20. Cervical Cancer Screening: Summary Saslow et al. Am J Clin Pathol. 2012; 137(4):516-42.

21. PCR detection of HPV Genotyping HPV in tissue samples

22. Methods for HPV detection in tissues o Specimens Blank slides (15) and H&E (one) Tissue block Fresh tissue o DNA extraction Maxwell 16 o PCR method: SYBR green real time PCR followed by melting curve analysis: o HPV detection using GP5+/GP6+ primers (L1 region) Hybridization Probe based real time PCR: o HPV genotyping: HPV16 and 18 (E6 region)

23. HPV generic primer

24. Generic HPV PCR

25. Real-time PCR: High resolution melting dye for HPV Melting curve Real time amplification LC480

26. Real Time PCR and melting curves to detect HPV HPV16HPV18 Tm HPV16: 76.5C +/- 2.5°C HPV18: 79.5C +/- 2.5°C HPV33: 72°C +/- 2.5°C

27. Internal Control: beta-actin gene Tm: 85.5°C +/- 2.5°C

28. HPV16 specific signal HPV18 specific signal Real-time HPV Genotyping LightCycler-Red 640 for HPV16 LightCycler-Red 670 for HPV18

29. HPV genotyping (HPV16)

30. HPV screening by consensus PCR + - HPV16 and 18 PCR assays HPV16 or 18 + - Sanger sequencing HPV other genotype HPV negative HPV detection and genotyping

31. Protein Pathway Array for protein analysis Protein extraction Gel electrophoresis Nylon membrane/plate Immunoblot Image analysis Data acquisition Signaling network Beads Binding of antibodies Samples (cells, tissues, FFPE) Genomic array information

32. Proteomics and HNSCC 84 laryngeal SCC – HPV negative 225 antibodies – 61 detected – 16 differentially expressed between T and NL – 13 upregulated and 3 downregulated

33. Signaling pathway altered in HNSCC

34. Prognosis markers and risk score