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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