HPVProofer, a new molecular assay for the detection and typing of HPV with active E6E7 oncogenes

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Evaluation of HCII and HPV-Proofer
HPV testing related to histology (n351)
HCII and HPV-Proofer was done on the same smear
samples at the same time

• Conclusions
• The HPV-Proofer test revealed a higher
  prognostic value and higher specificity than the
  DNA test
• The HPV-Proofer test gives no age
  discrimination (not only gt30 years)

(Lie, 2005)
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A new molecular assay using mRNA as a target for
multiplex routine diagnostics of HPV induces cell
abnormalities
Frank Karlsen, PhD Head of RD
This product is designed, developed and produced
in compliance with Directive 98/79/EC of the
European Parliament and of the Council of 27
October 1998 on in vitro diagnostic medical
devices
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HPV 16, 18, 31, 33, 45
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Routine based extraction of mRNA
mRNA extraction is now a routine standard It is
reliable, easy to perform and cost effective
mRNA
DNA
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PAP SMEAR NORMAL
Latent Subclinical HPV
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Who are at risk of developing CC?

• Female
• HPV infection
• E6/E7 production
• PersistentE6/E7 production

E6/E7, the most oncogenic protein factor ever
discovered in relation to Cervical Cancer
1-2
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Expression of Full-length E7 protein
Short window of FL E7P and mRNA expression
Irreversible Highly expressed FL E6/E7P and mRNA
E7 level
Low/no detectable expression of FL E7 P or mRNA
Initiation of viral replication
Failure of control mechanisms
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Transient native infection
Model for relationship between
Viral events and Clinical
manifestation
No clinical symptoms
Presence of HPV
Condyloma/Dyskariosis
Persistent infection
Transforming infectionintegrated HPV
Dysplasia or Histological CIN II
Persistent Transforming infection
Persistent CIN III or Cancer in situ
Karlsen 2005
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Definition of Persistent Transforming Infection

• The normal viral life cycle is lost
• No virions are produced
• The replication of HPV is lost
• An irreversible high expression of E7 mRNA is
  established
• No full-length E6/E7 expression No cancer
• The E6 or E7 are more oncogenic than mutated p53

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Risk factors and cause of cervical cancer

• Risk factors
• Smoking
• Early sexual debut
• Multiple sexual partners
• Persistent HPV infection
• The one and only cause
• Cervical cancer develops due to presence of HPV
  E6/E7 oncoproteins
• Harald zur Hausen, Nature May 2002, Vol 2, 342-350

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Scientific Background HPV-types
( Prevalence)
HPV 16, 18, 31, 33, 45 98 (Muños et.al. 2004
IJC) 99 (Clifford et.al. 2003 BJC)
Other HPV subtypes found in cervical cancers
appear to be passengers, and not responsible for
the development of cervical cancer Dont nail
the passengers, nail the driver
HPV 35
HPV 6, 39, 51, 53, 56, 59, 66, 67, 68
(Time)
Cancer
Normal
ASCUS
CIN I
CIN II
CIN III
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Routine assay - Total quality control

• Providing three levels of information
• Identification of samples positive for individual
  HPV types 16, 18, 31, 33 and 45
• Identification of full length E6/E7 transcripts
  from five specific different HPV-types (16, 18,
  31, 33 45)
• Presence of full length E6/E7 mRNA indicating
  dysregulation
• HPV-Proofer does not detect HPV virions!
• No age discrimination (not only gt30 years)
• Each sample undergoes three duplex NASBA
  reactions
• Six results are always reported for each sample
• Negative controls are included each time to
  monitor contamination
• Positive controls are included for all HPV types
  to monitor reagent performance
• Intrinsic cellular control U1A mRNA (cellular
  housekeeping gene) monitors entire test procedure
  to eliminate possible false negatives
• It is not possible for the HPV-Proofer to detect
  DNA
• PreTect Analysis Software (PAS) for automated
  routine data analysis, interpretation and
  reporting

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Evidence based documentation of PreTect
HPV-Proofer as a diagnostic method

• Head to head comparison studies
• Prospective and retrospective studies
• Cross sectional studies
• Follow-up studies
• Population studies
• Verification and validation studies
• Studies of biopsies and cervical smears from
  women with cancer
• Studies of biopsies and cervical smears from
  women with CIN III

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Now documented in more than 15 clinical studies

• E6 and E7 mRNA expression in 4136 clinical
  samples (Molden, 2004, CEBP, 2005)
• CIN III study on 190 biopsies (Kraus, 2004
  British J Cancer 901407-1413)
• Validation study in Oslo (Lie, 2004, Gyn
  Oncology)
• Follow up study in Edinburgh
  (Cuscheri, 2004 J Med Virol.
  May73(1)65-70)
• Follow up of 77 ASCUS/CIN cases (Molden, 2005,
  Jan 11, IJC )
• Study of mRNA expr. in 204 sq. carcinoma samples
  (Kraus Molden, submitted 2005)
• Development and verification of HPV-Proofer
  (Molden, 2005 Poster )
• High risk infections without oncogene expression
• in cervical smears (Molden ,2004, Poster
  150 )
• Out-patience population study in DR Congo
  (Hovland, in prep. 2004, oral at Eurogin 2004)
• Karlstad follow-up study in Sweden (Bjerre,
  2004, Poster 134)
• High risk oncogene expression in progressive
  lesions (Nygaard, 2004, Poster 282 and oral
  pres.)
• Human papillomavirus E6/E7 mRNA expression in
  (Molden, 2005, in press, Gyn Oncology)
• women younger than 30 years of age
• Evaluation of the main transcripts in clinical
  samples (Molden, in prep 2005)
• Evaluation of the stability of mRNA (Kraus, in
  prep 2005)
• Predicting CIN2 when detecting HPV mRNA or DNA
• in women with a normal Pap smear.. (Molden et
  al., in prep 2005)

Accepted only after revision
21th international papillomavirus conference in
Mexico, March 2004
22th international papillomavirus conference in
Vancouver, April 2005
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Evaluation of HCII and HPV-Proofer
HPV testing related to histology (n351)
HCII and HPV-Proofer was done on the same smear
samples at the same time

• Conclusions
• The HPV-Proofer test revealed a higher
  prognostic value and higher specificity than the
  DNA test
• The HPV-Proofer test gives no age
  discrimination (not only gt30 years)

(Lie, KA2005)
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Clincal Studies HPV-Proofer vs. hc2
100
DNA test
80
60
40
DNA results not available
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0
LSIL n24
Cancer n19
Benign n105
ASCUS/AGUS n49
ASC-H/HSIL/ACIS n163
Unsuitable cytology23
Based on
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The importance of representativity

• Biopsies in the Norwegian study
• LEEP biopsies in the Swedish study

Cervical smears or cervical biopsies from women
with invasive cervical carcinoma
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Comparison between two studies

• Head to head comparison of two different
  commercial methods in Norway
• Independent performed blindly study by the
  Norwegian Radium Hospital, Ullevål hospital, ST
  Olavs Hospital and the Norwegian Cancer Registry
• 339 cytological normal and 269 cytological
  positive women was collected from an around 8000
  outpatient women in Norway. Nineteen cervical
  smears from women with cervical carcinoma was
  added to the study from the Norwegian Radium
  Hospital
• Biopsies were taken if the HPV test was positive
  or cytology revealed HSIL. 383 histological
  diagnosis was performed
• 107 high-grade lesions (out of 271 39) was not
  discovered with cytology
• Head to head comparison of two different
  commercial methods in Sweden
• Independent blindly study by the Central Hospital
  in Karlstad and the Karolinska University
  Hospital in Stockholm
• 260 CIN I and ASCUS was included from
  approximately 15000 outpatient women in Sweden
• LEEP biopsies were taken in the HCII test was
  positive or cytology was positive. 118 LEEP
  biopsies were collected
• 29 high-grade lesions (out of 40 72) were not
  discovered with cytology three month before LEEP
  conisation

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Results from this comparison

• The study in Norway
• 51 of the histological normal biopsies (n61)
  were positive with HCII
• 12 of the histological normal biopsies (n61)
  were positive with HPV-Proofer
• 90 of the smears collected from cervical
  carcinoma samples (n20) were positive with HCII
• 100 of the smears collected from cervical
  carcinoma samples (n20) were positive with
  HPV-Proofer
• The study in Sweden
• 73 of the histological normal LEEP biopsies
  (n18) were positive with HCII
• 73 of the histological normal LEEP biopsies
  (n18) were positive with cytology
• 0 of the histological normal LEEP biopsies
  (n18) were positive with HPV-Proofer

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Study in Oslo on 4136 cervical smears
(Molden et al., 2005)
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HPV-Proofer positive among true histological
biopsy lesions
(Kraus, 2004 British J Cancer 901407-1413)
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Rate of oncogene expression and DNA ploidy
aberrations in biopsies
Comparison of separate studies
100
100
100
100
86
90
90
84
80
80
70
70
60
60
53
55
50
50
HPV-Proofer
Aneuploid
40
40
30
30
20
20
10
10
0
0
0
0
CIN I
CIN II
CIN III
Cancer
CIN I
CIN II
CIN III
Cancer
(Skyldberg et al., 1999)
(Molden, 2004, Poster 283, Kraus, BJC, in
press 2004)
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Follow up study, Edinburgh

• 3445 women with reconfirmed normal cytology
• 54 were positive with PCR and followed up over
  two years
• All samples with persistent HPV were positive
  with PreTect HPV-Proofer
• All cytological moderate/severe samples after two
  years were positive with PreTect HPV-Proofer
• PreTect HPV-Proofer has twice as high specificity
  compared to PCR (76 versus 39) for HSIL
• Women positive with PreTect HPV-Proofer have
  significantly higher risk for having malignant
  cell changes

(Cuschieri, JMV, 2004)
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Comparison between HCII and HPV-Proofer among
customers of TDL (Sept-Nov 2004)
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Description of the test cases

• Severe dyskaryosis 9/760 1.2
• Moderate dyskaryosis 6/760 0.8
• Cytological HSIL 2
• Norwegian average 1, UK-Hart study 0.8
• Borderline 55/760 7.2
• Norwegian average 2.5, UK-Hart study 2.8
• Mild 52/760 6.8
• Norwegian average 1.9, UK-Hart study 1.2
• HCII negative/HPV-Proofer positive 0.8

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When to use PreTect HPV-Proofer

• In combination with cytology (ref. IARC
  2004)
• ASCUS/LSIL (identifies the dangerous cases)
• HSIL (control of treatment)
• Normal cytology (based on medical knowledge of
  the patient)
• Other areas
• Can be used in research on all kinds of samples
• Anal cancer (the same pathogenesis.)
• Penis cancer
• Head and neck cancer

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Experiences from using PreTect HPV-Proofer, Europe
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Conclusions from clinical studies

• 97 coverage rate in cervical cancer Kraus and
  Molden in press 2005
• 97 detection rate in CINIII Kraus Br J Cancer
  2004
• HPV-Proofer test revealed a higher prognostic
  value and higher specificity than HCII Lie, Gyn
  Oncol 2005
• Indications that HPV-Proofer gives prospective
  values for persistent CINII Nygård, in prep
  2005
• mRNA stable for routine diagnostics Hovland in
  prep 2005
• HPV-Proofer has the same sensitivity and
  significantly higher specificity than HPV-DNA
  tests 1Molden CEBP 2005, 2Hovland in prep 2005,
  3Cushieri J Med Virol 2004, 4Molden Int J
  Cancer 2005

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The does and doesn't of PreTect HPV-Proofer

• DOES
• Identifies production of E6/E7 oncoproteins
• Finds the primary factor of carcinogenesis
• Using HPV Proofer has totally different clinical
  implications compared to DNA-detection
• DOESN'T
• PreTect HPV-Proofer does NOT find HPV-viriones
• PreTect HPV-Proofer is NOT a HPV-test
• Cannot be replaced by a HPV-DNA test

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What Now ?

• This is not the end
• This is not the beginning of the end
• But it is the end of the beginning
• Sir Winston Churchill
• North Egypt Dec 1942

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