Jesper Bonde

Molecular Pathology Laboratory Dept. Pathology

Copenhagen University Hospital, Hvidovre

The HORIZON Study Different HPV tests, Different Horizons

DISCLOSURE STATEMENT

AxLab

BD Diagnostics Genomica

Hologic/Gen-Probe Qiagen

Roche Diagnostics Roche Pharma

Jesper Bonde, PhD, Dipl.Med.Sci Senior Researcher Laboratory Manager Molecular Pathology Laboratory Dept. Pathology Copenhagen University Hospital, Hvidovre, Denmark

The company partners accepted and acknowledged the protocol

prior to the study

All instrumentation and software were manufacturer issued, and

maintained for the duration of the study in both studies

The HORIZON study is a independent investigator driven study

Molecular HPV testing allows for improvements in cervical cancer

prevention and screening, either as triage modality or as stand

alone screening test

The introduction of DNA and RNA based methods opens up a wide

new horizon of methodologies to make the screening individualized

and highly specific for disease detection

Today

Tomorrow

The Vision

5

Key numbers, Denmark

• Country population 5.627.235 (2014) • Screening recommendations defined by the

National Board of Health • Regionally organized in the 5 regions, 12

screening laboratories • Only pathology departments perform screening

in DK (no microbiology!)

• All women 23-49 years: Every 3rd year • All women ≥50-65: Every 5th year

• Total turnover of samples/annually: 450.000 • Only LBC: SurePath 82% , ThinPrep 18% • Prescreening by automation, and • Computer assisted microscopy widely

implemented • HPV tests used in triage and test of cure; annual

turnover approx. 35.000

14.5% 21.4%

22.7%

10.3%

31.1%

42.096 (2012)

100.912 (2012)

87.259 (2012)

158.189 (2012)

55.946 (2012)

% of total population Number of screening tests/year

A doubble challenge – a window of opportunity

[Lynge et al., forthcoming]

Current indications for HPV tests by the Danish National Board of Health, 2012

Triage (ASCUS ≥30 years)

1st control after conisation (LEEP)

Primary screening ≥60 years (Check out test, DNA only, starting from 2013)

Annual turnover of HPV tests:

75,000 National/25,000 at Hvidovre Hospital from 2013

Equals: 1 of 6 LBC samples in our lab will have an HPV test by full

implementation of these indications

The Premise – clinical perspective

All cervical cancers caused by HR-HPV infection, active or latent over a period

of years

High sensitivity CIN2+ in the range of >90% favours HPV assays over cytology

(cytology variable from 55%-80%)

20% of newly diagnosed cervical cancers had a recent normal cytology

Of current cervical screening technologies, molecular HPV testing offers by far

best negative predictive value for detection of high grade CIN

A negative test result provides long term confidence that disease is not

imminent

Molecular HPV technology offers a new approach

The Premise – Laboratory perspective

Molecular HPV testing is more reproducible than cytology

Molecular HPV testing can be automated and QA/QC controlled

Not prone to intra-observer variability as cytology

Molecular HPV technology allows self-sampling as a new screening option

National and international QA/QC programs will eventually allow for performance evaluation in order to secure uniform services

Negative predictive value of HPV tests means longer, safe intervals reducing the cost of screening allowing a re-focusing on those women who really need follow up and/or treatment

Molecular HPV technology offers a new approach

What do we need from Molecular HPV screening tests?

“In the fight to reduce a rare disease, cervical cancer, let´s not introduce a common disease, hr-HPV.”

PRIMARY SCREENING • High Clinical Specificity • Good sensitivity EXIT TEST

• High sensitivity • Sample sufficiency control

TRIAGE • High Clinical Specificity • Good sensitivity TEST of CURE

• High sensitivity • Genotyping?

The Assays on the HORIZON

•Cobas®HPV test Real-time PCR assay, with co-detection of HPV HR and 16 and 18

•HC2® Hybridization assay with HR HPV detection

•APTIMA® RNA assay with HR HPV detection

•CLART® PCR-Microarray assay with simultaneous detection of 35 genotypes

Four HPV assays – four different technologies

QiaSymphony RCS HC2® work flow

Roche cobas®4800

workflow

Gen-Probe PANTHER®

APTIMA workflow

Genomica CLART®HPV2

The HORIZON Study

Comparing APTIMA, CLART, cobas & HC2 HPV assays in PRIMARY cervical cancer screening

The HORIZON Study

Clinical and analytical assay performance in a true screening study sample

5064 unselected samples taken straight from our routine

Tested on cytology plus the four HPV tests in a split sample fashion

All cytology positive followed up according to national guidelines

All HPV+ (on any test) cytology normal invited for an additional screening round at 18 months after baseline

= The Horizon Study

The HORIZON Study Main results from baseline and the 18 month follow up invitation

HPV prevalence on all 5,064 samples

Preisler et al., PLOS ONE, 2013 Rebolj et al., J.Mol.Med 2013 Goldman et al., Vaccine 2013 Rebolj et al, Plos One 2014 Bonde et al., forthcoming

Baseline data from the HORIZON study

16

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Genotype distribution in all 5064 unselected samples

Baseline data from the HORIZON study

Goldman et al., Vaccine 2013 Bonde et al., forthcoming

Horizon: The surprising variability in assay outcomes

Mo

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amp

les

Assay B C D A

Concordance of the 3 HPV DNA assays

All 5,064 samples

N 5,064

N (%) ≥1 test pos

1,636 (100%)

1 test pos 28%

2 test pos 20%

3 test pos 52%

4 test pos

(41%)

All 5,064 samples

23-29 years

30-65 years

N 5,064 1,534 3,256

N (%) ≥1 test pos

1,636 (100%)

731 (100%)

771 (100%)

1 test pos 28% 18% 38%

2 test pos 20% 21% 20%

3 test pos 52% 61% 41%

4 test pos

(41%)

(49%)

(30%)

All 5,064 samples

23-29 years

30-65 years

30-65 years

Screening samples

Follow-up samples

N 5,064 1,534 3,256 2,881 375

N (%) ≥1 test pos

1,636 (100%)

731 (100%)

771 (100%)

630 (100%)

141 (100%)

1 test pos 28% 18% 38% 40% 30%

2 test pos 20% 21% 20% 21% 19%

3 test pos 52% 61% 41% 39% 50%

4 test pos

(41%)

(49%)

(30%)

(29%)

(38%)

All 5,064 samples

23-29 years

30-65 years

30-65 years 30-65 years, primary screening samples

Screening samples

Follow-up samples

Normal cytology

Abnormal cytology

N 5,064 1,534 3,256 2,881 375 2,741 127

N (%) ≥1 test pos

1,636 (100%)

731 (100%)

771 (100%)

630 (100%)

141 (100%)

537 (100%)

93 (100%)

1 test pos 28% 18% 38% 40% 30% 45% 14%

2 test pos 20% 21% 20% 21% 19% 22% 13%

3 test pos 52% 61% 41% 39% 50% 34% 73%

4 test pos

41%

49%

30%

29%

38%

22%

68%

[Rebolj et al., Plos One 2014

Agreement of the four HPV assays

[Rebolj et al., Plos One 2014]

Sum of all proportions: 100% (all women testing positive on at least one HPV assay) Only 29% of all HPV+ samples tested positive on all 4 assays All combinations of positive tests for the four assays were observed in the data

Agreement of the four HPV assays: Primary screening, 30-65 years

Is this an odd finding, from an odd country, with odd data?

[Rebolj et al., Plos One 2014]

52% HC2 cobas 50% HC2 CLART 58% HC2 APTIMA

58% CLART cobas 42% CLART APTIMA 45% APTIMA cobas

Primary screening, 30-65 years

Pairwise agreement of the four HPV assays:

Heideman et al.: 60% Cuzick et al.: 65%

Lloveras et al.: 65%

Getman et al.: 48% Wu et al.: 50%

Cuzick et al.: 57%

Concordance for Linear Array, Cervista, Abbott & GP5+/6+

Gage et al., HC2 vs LA: 50%; Poljak et al., HC2 vs. Abbott: 67%; Carrozzi et al., HC2 vs. Abbott: 60%; Kurian et al., HC2 vs. Cervista: 49%; Quigley et al., HC2

vs. Cervista: 54%; Meijer et al., HC2 vs. GP5+/6+: 56%

[Nielsen et al., in preparation]

And this means:

Concordance in HPV assay positivity is Age, sceening history, cytology dependent

Poorer in women ≥30 years Poorer in screening samples

Poorer in women with normal cytology

What are the clinical consequences of these findings?

The HORIZON Study Different HPV tests, Different Horizons

Colposcopy referral: Triage by cytology

Sum of all proportions: 100% (all women testing positive on at least one HPV assay having abnormal cytology)

Only 68% of all HPV+/cyt+ women would be referred to colposcopy regardless of which of the four assays was used

Screening test Result

Cytology 9% became cytology abnormal colposcopy

Hybrid Capture 2 47% remained positive on HC2 colposcopy

cobas 49% remained positive on cobas colposcopy

CLART 37% remained positive on CLART colposcopy

APTIMA 37% remained positive on APTIMA colposcopy

Women aged 30-65 years with HPV+/cyt- samples in primary screening:

Results of repeated testing in 18 months

Assays find more or less the same CIN lesions…

(= a prerequisite for high sensitivity)

…but whether or not a given woman will be referred to follow-up:

• Repeat testing • Colposcopy, biopsies

• Treatment

depends on the HPV assay used for testing

We need a molecular triage for HPV screening

to fully deliver the promise of a superior screening

Summary

• All four HPV-assays have a relatively high sensitivity for CIN2+ and CIN3+

• But the specificity is low.

• A woman’s fate of becoming false positive depends on choice of HPV-assay

• New triage methods needed (methylation markers, genotyping, p16/ki67)

The HORIZON dilemma

• Triage with cytology does not solve the problem with the many HPV-test positive women

• Women with HPV+/cyt- tests have a higher risk of CIN2+ than HPV- women; therefore this group cannot be sent back to long-term interval, routine screening

– But three years intervals could be an option

• Scheduled retesting in 6-12 months is a burden for this relatively low risk group,

• Half of them would likely have tested negative had they been tested with another HPV-assay!

A short term challenge is to find a molecular triage with high specificity to

reduce the number of false positives

The HORIZON Study

But this should not impede, delay or cause concern for the implementation of primary

HPV screening

Its still ”better, faster, cheaper” than cytology

And finally –not to forget- molecular HPV screening allows us to reach out to non-responders in order to

improve the participation rate

Centre for Epidemiology & Screening, University of Copenhagen

Elsebeth Lynge, Professor Matejka Rebolj, PhD

The HORIZON Group

Sponsor Danish Strategic Research Council

Coorporate Partners Genomica Hologic/Gen-Probe Qiagen Roche

Dept. Pathology & Clinical Research Centre, Copenhagen University Hospital, Hvidovre

Carsten Rygaard, MD

Sarah Preisler, PhD Ditte Ejegod, PhD

Jette Junge, MD Jesper Bonde, PhD

a multi-diciplinary team