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TRANSCRIPT
Deirdre Pierry MCLS, MT(ASCP), SM, MBCM
Chromosome Crawl
Fifty Ways to Leave Your Lover: HPV Diagnostics
• Review the biology of the Human Papillomaviruses (HPV) & discuss the role of HPV infecGon in carcinogenesis.
• Discuss viral targets for vaccines & diagnosGc tests. • Discuss current commercial assay opGons for detecGng HPV infecGon – both IVD and LDT opGons.
• Explore the current understanding of the role of HPV in cancers beyond the cervix.
Objectives
Conflict of Interest Disclaimer: I have been a Speaker for IncellDx and have received discounted reagents as a part of Field Studies for Arbor Vita, Inc.
Human Papovaviridiae
• Non-‐enveloped, circular dsDNA, ~7,900 bp • Icosahedral capsid (72 capsomers each
capsomer = 5 copies of L1 protein) • > 120 types
Human Papovaviridiae
HPV vaccine targets: L1 capsid proteins
HPV Test targets: - L1 gene - E6, E7 genes - E6, E7 mRNA - p16 - E6, E7 proteins - E2 DNA length
HPV Genome Organization
Early Genes: E1 = Replication E2 = Replication & Transcription E4 = Viral release E5 = Transcription factors, Immune evasion E6 = Oncogene; binds p53 E7 = Oncogene, binds pRb
LCR = Promoter elements Viral ORI - replication
Late Genes: L1 = Major capsid protein L2 = Minor capsid protein
• Squamous Cell Carcinomas – account for 80-‐90% of all cervical cancers.
• Adenocarcinomas • `
Cervical Cancers: Types
- Arise within the ectocervical squamous cells. - HPV 16 is the primary type found in these lesions. - The incidence of integrated HPV form increases with increased lesion severity.
- Arise within glands located in endocervical cells. - HPV 18 strongly associated with these lesions. - The broad use of Pap smears has not decreased the incidence of adenocarcinoma as this cell population not consistently sampled.
Squamous Cell Carcinomas: Terms CYTOLOGY (Bethesda Terms) NILM = Negative for Intracellular Lesion or Malignancy ASCUS = Atypical Squamous Cells of Uncertain Significance ASC-H = Atypical Cells can’t r/o HSIL LSIL = Low Grade Intraepithelial Lesion HSIL = High Grade Intraepithelial Lesion
HISTOLOGY WNL = Within Normal Limits CIN 1 = Cervical Intraepithelial Neoplasia CIN 2 = Cervical Intraepithelial Neoplasia CIN 3 = Cervical Intraepithelial Neoplasia
Natural History of HPV Infection 75%
50%
10%
1.3% 0.4% 0%
10%
20%
30%
40%
50%
60%
70%
80%
PopulaGon Wide HPV Exposure
Exposure to HR HPV Persistent High Grade CIN
Invasive Disease Death
HPV Genome Targets
E. Burd Clin Micro Rev. 2003 (16) J. Doorbar Disease Markers 2007 (23) 297-313
HPV Type Specific Primers - HPV Detection +/- - HPV typing - Quantitation
L1 Consensus Primers - HPV Detection +/- - HPV typing methods
HPV Integration – results in loss of E2 – useful for measuring integration?
Full Array of Commercial Assays - hr HPV DNA-based screening assays - hr HPV DNA-based screening assays with concurrent or reflex HPV 16, HPV 18 genotyping - HPV DNA-based genotyping assays
- HPV E6, E7 mRNA-based screening assays
- In situ hybridization (NA based)
- Protein IHC (p16, ki67, E6)
Characteristics of the Ideal HPV Test • High clinical sensi%vity à test will detect high risk HPV infecGons that will lead to cervical disease. [high NegaGve PredicGve Value (NPV) for disease] • High clinical specificity à test will NOT detect transient high risk HPV infecGons that will not lead to cervical disease.
[high PosiGve PredicGve Value (PPV) for disease]
How Does HPV Cause Cancer?
- Integration of HPV genome into Host chromosomes - Disruption of E2 gene leads to loss of negative feedback control over oncogenes. - Transcription of E6 & E7 oncogenes upregulated.
- E6 & E7 proteins deregulate the Host cell growth cycle by binding & inactivating P53 tumor supressor proteins, cell cyclins and cyclin-dependent kinases
Syrjanen, S. Ann. Med 1999 31:175-187
Assay Method Specimen Types
HPV Types Deteected
Internal Control?
Genotyping
Digene Hybrid Capture 2
HC Rapid Capture System
ThinPrep® 16,18,31,33,35,39,4551,52,56,58,59,68
NO Reflex
cobas® 4800 HPV
Real Time PCR cobas 4800
Cobas collecGon ThinPrep ® (SurePath –
LDT)
16,18,31, 33, 35, 39, 45, 51, 52, 56, 58,
59, 66, 68
ß-‐globin Yes HPV 16,18
Roche Amplicor HPV
PCR ThinPrep® SurePath®
16,18,31, 33, 35, 39, 45, 51, 52, 56, 58,
59, 68
ß-‐globin
NO
AbboI RealTime HPV
Real Time PCR m2000sp m2000rt
ThinPrep® 16,18,31, 33, 35, 39, 45, 51, 52, 56, 58,
59, 66, 68
ß-‐globin
Yes HPV 16,18
Hologic Cervista HPV
HR
Invader / Cleavase chemistry
ThinPrep® (SurePath –
LDT)
16,18,31, 33, 35, 39, 45, 51, 52, 56, 58,
59, 66, 68
Human Histone 2 gene
Reflex to HPV 16, 18
HPV DNA Assays: Features
HPV DNA Detection: Digene Hybrid Capture 2 (HC 2)
The Digene HC2 HPV DNA Test is a Capture Hybridization assay with signal- amplification. - detects (HPV) DNA in cervical specimens. - Results differentiate 2 HPV DNA groups: low-risk and high-risk types. - NOT designed for genotyping, no Internal Control, cross reactivity with LR-HPVs & false negative issues w/SurePath LBC samples.
HPV DNA Detection: cobas® 4800 HPV
• DNA Amplification based assay detects 14 hrHPV types • Automated sample prep & processing. • Real Time PCR, Internal Control for sample adequacy. • Clinically validated for ASCUS triage.
HPV DNA Detection: Hologic Cervista
§ Cleavase® Invader (signal amplification) based assay for hr HPV DNA Screening. Internal Control for sample adequacy. § Detects all 14 HPV types designated as high-risk.
§ Semi-automated processing.
HPV DNA Detection: Abbott RealTime HPV
• DNA Amplification based assay detects 14 hrHPV types. • Automated sample prep & processing. • Real Time PCR based, Internal Control for sample adequacy. • Concurrent individual genotyping for HPV 16/18 and a pool of 12 other hr types: (31,33,35,39,45,51,52,56,58,59,66,68).
Assay Method Specimen Types
HPV Types Deteected
Internal Control?
Linear Array (Roche)
PCR Reverse Line Blot
ThinPrep®
16,18,31,33,35,39,45,51,52,56,58,59,
68
ß-‐globin
CE Marked
Inno-‐LIPA (Innogene%cs)
PCR Line Probe Assay
ThinPrep®
16,18,31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
HLA-‐DPβ1 CE Marked
PapilloCheck (Greiner Bio-‐
One)
PCR DNA Array Hyb.
ThinPrep® SurePath®
16,18,31, 33, 35, 39, 45, 51, 52, 56,
58, 59, 68
Yes CE Marked
HPV CLART® 2 (Genomica)
PCR DNA Array Hyb
Swab ThinPrep®
SurePath® -‐ LDT
16,18, 26,31, 33, 35, 39, 45, 51, 52, 53,56, 58, 59, 66,
67,68,69,70,73,82,85
CFTR – genomic DNA Plasmid –
amplificaGon
CE Marked HR & LR types in 1 reacGon
Additional DNA Assays
HPV DNA Detection: CE Marked Roche
LINEAR ARRAY HPV Genotyping Test (CE-IVD) – not available in US § Detects 37 high- and low-risk HPV genotypes. § HPV genotypes include 6, 11, 16, 18, 26, 31, , 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9), 81, 82, 83, 84, IS39, and CP6108
COBAS Amplicor HPV (CE) – not available in US § PCR-based test for detecting 13 high-risk HPV genotypes – HPV DNA detection § This system amplifies target DNA using PCR followed by nucleic acid hybridization. § A 96-microwell plate is used and requires only a small sample of 250 µL for testing. § Concurrent isolation and amplification of the ß-globin gene assesses cellular adequacy and inhibition
HPV DNA Detection: CE Marked Innogenetics Inno-‐LIPA
Genotyping Test (CE-IVD) – not available in US; widely used X-US § Amplification of HPV L1 target followed by reverse line blot detection. § HPV genotypes include 16,18,31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. § Assay useful to troubleshoot discordant results of other assays.
HPV DNA Detection: Genomica HPV 2 CLART
• DNA Amplification based assay detects BOTH LR & hrHPV types. • Detects individual genotypes in single reaction – no reflexing. • PCR based, Internal Control for sample adequacy. • Detection performed on a microarray; automated Reader. • Can detect concurrent infections.
Assay Method Specimen Types
HPV Types Deteected
Internal Control?
Comments
Ap%ma HPV Assay (Hologic)
TMA ThinPrep ® (SurePath ™–
LDT)
16,18,31,33,35,39,4551,52,56,58,59,66,68
Yes Reflex HPV 16, HPV 18/45 GT
HPV OncoTect (IncellDx)
ISH, Flow Cytometry
ThinPrep ® SurePath ™
Detects overexpression of
E6,E7 mRNA
Yes No GT necessary
PreTect Proofer v.2 (Norchip)
NASBA PreTect™ ThinPrep ® SurePath ™
16,18,31, 33,45 Yes ISC
CE Marked (X-‐US)
NucliSENS EasyQ HPV (bioMerieux)
NASBA ThinPrep ®
16,18,31, 33,45
Yes U1A
CE Marked IVD
HPV E6,E7 mRNA Assays: Features
HPV E6,E7 mRNA Assays: Aptima HPV & HPV 16, 18/45
- Transcription Mediated Amplification based assay targets E6 & E7 mRNA for amplification. - Internal Control to assess sample adequacy. - Automated sample processing and detection on Tigris DTS or Panther platforms. - HPV 16, HPV 18/45 genotyping available as reflex assay.
HPV E6,E7 mRNA Assays: IncellDX HPV OncoTect
- HPV OncoTect is a flow cytometry based in situ hybridization test for the detection of HPV E6/E7 mRNA in intact ectocervical cells.
- Raw data provides information on the quantity of E6, E7 mRNA in each cell & the percentage of cells in the sample that overexpress E6, E7 mRNA.
HPV E6,E7 mRNA Assays: PreTect Proofer
- NASBA – based assay that detects E6, E7 mRNA from the five most frequently identified HR HPVs - Internal Control (U1A) in addition to 5 synthetic HPV Positive Assay Controls. - Several studies show Proofer has higher specificity, lower sensitivity than HPV DNA methods (CIN 2+ lesions) – this may be do to limited # HPVs targeted.
-‐ Immunohistochemistry Stains for HPV-‐associated proteins (E6 oncoprotein, p16 INK4, ki67) -‐ Luminex – based “liquid microarray” genotyping assays. -‐ HPV IntegraGon assays (based on detecGng “short” or integrated forms of HPV E2)
-‐ RUO assays invesGgaGng hot spots where HPV DNA has been integrated into Host chromosome.
Additional HPV Assays
• CLINICAL sensiGvity & CLINICAL specificty are important & potenGally beqer predictors of paGent safety than ANALYTICAL sensi / speci.
• All the ANALYTICAL sensiGvity in the world does not increase the clinical sensiGvity of HPV assays.
-‐ it MAY, however result in overcalling true posiGves & result in decreased clinical specificity.
• Excessive analyGcal sensiGvity can result in unnecessary procedures and treatment of healthy women.
Stoler, MH. Am J Clin Path 2010: 134(2): 193-‐9
Patient Safety & Molecular HPV Testing