David Y. Zhang MD, PhD, MPHProfessor

Director, Molecular PathologyDepartments of Pathology

Icahn School of Medicine at Mount Sinai , New York

Molecular diagnosis and monitoring of HPV infection

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%

Oncogenity HPV types

High risk HPV types

16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 67, 68, 70

Low risk HPV types

6, 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

Stratum corneum

Stratum granulosum

Stratum spinosum

Basal cells

E1,

E2,

E4,

E5,

E6,

E7

E1, E2

20-100 HPV DNA/cell

E4,

L1,

L

2

>10

00 H

PV

DN

A/c

ell

Keratinocytes release

Productive infection- HPV replication

HPV associated cancers

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.

HPV associated Cervical Cancer

NEJM 348:518-527, 2003

80%

10-15 y

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

Biological and clinical characteristics of HNSCC

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

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

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

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

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

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)

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

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

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

Cervical Cancer Screening: Summary

Saslow et al. Am J Clin Pathol. 2012; 137(4):516-42.

PCR detection of HPV

Genotyping HPV in tissue samples

Methods for HPV detection in tissueso 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)

HPV generic primer

Generic HPV PCR

Real-time PCR: High resolution melting dye for HPV

Melting curveReal time amplification

LC480

Real Time PCR and melting curves to detect HPV

HPV16 HPV18 TmHPV16: 76.5C +/- 2.5°CHPV18: 79.5C +/- 2.5°CHPV33: 72°C +/- 2.5°C

Internal Control: beta-actin gene

Tm: 85.5°C +/- 2.5°C

HPV16 specific signal

HPV18 specific signal

Real-time HPV Genotyping

• LightCycler-Red 640 for HPV16 • LightCycler-Red 670 for HPV18

HPV genotyping (HPV16)

HPV screening by consensus PCR

+ -

HPV16 and 18 PCR assays

HPV16 or 18

+ -

Sanger sequencing

HPV other genotype

HPV negative

HPV detection and genotyping

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

Proteomics and HNSCC

• 84 laryngeal SCC– HPV negative

• 225 antibodies– 61 detected– 16 differentially expressed

between T and NL– 13 upregulated and 3

downregulated

Signaling pathway altered in HNSCC

Prognosis markers and risk score