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

 Cervical  Cancers:  The  Setting  

Cox, JT OBG Management 2012 ;24(3)

 Cervical  Cancers:  The  Setting  

•  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

Normal  Epithelium  

Nature Reviews Immunology 2004; 4:46-55

Squamous  Cell  Carcinomas:  Cytology  

Natural  History  of  HPV  Infection  

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  Infection:  View  1  

Woodman, CB Nature Reviews Cancer 2007 (7): 11-22

HPV  Infection:  View  2  

Banks, L. Nature Reviews Cancer 2012;12:877-886

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

Other  Biomarkers?  

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:    Digene  Hybrid  Capture  2  (HC  2)  

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.

•  Lateral  Flow  Assays  for  E6  Protein  –  Arbor  Vita  Onco  E6  

Additional  HPV  Assays  

-­‐  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  

What  About  HPV  in  Other  Sites?  

What  About  HPV  in  Other  Sites?  

Stanley, M. Nature 488; S10 (30 Aug 2012)

What  About  HPV  in  Other  Sites?