© AMK

Thessaloniki

15.9.2012

Detection and Typing of HPV DNA:

validated and non-validated

methods

Andreas M. Kaufmann

Clinic for Gynecology

Charité Campus Benjamin Franklin

and Campus Mitte

Berlin, Germany

andreas.kaufmann@charite.de GTI@CBF

Why HPV-based Screening and why Genotyping?

• Primary cervical cancer screening

• Triage of suspicious findings

• Longitudinal follow up

• Risk assessment

• Epidemiology

Introduction of Primary HPV Screening

• The Netherlands, Mexico, England…

• Combination of high sensitivity of HPV tests with

high specificity of cytology

• Algorithm: organized program

– 2-3 PAP, 20-30 years

– HPV screening test every 3-5 years

– HR-HPV positive => cytology triage

– PAP positive => colposcopy

Risk assessment:

Development of CIN3 in HPV Positives

© AMK© AMKKhan et al., JNCI 97:1072-9 (2005)

20514 women

>30 years

initial normal cytology

10 years follow-up

Incidence CIN3

HPV16+ 21%

HPV18+ 18%

HR-HPV+ 1.5%

LR-HPV/- 0.5%

Same risk

<30 vs >30

16

18

HRLR

HPV

Risk of Persistence and Progression

Depends on HPV Type

Infection

LSIL HSIL CxCa

CIN 1 CIN 2 CIN 3/CIS

high HR-HPV: 16, 18 (31, 45)10-25% 70-85%

HR-HPV: 33, 35, 39, 51, 52, 56, 58, 59, 68, 73, 8270-85% 20-35%

PAPIII PAPIIID PAPIV

© AMK

Epidemiology: HPV-Prevalence and Age 11.851 women in Denmark

0

10

20

30

40

50

60

15-1

8 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

60-6

2

66-6

875

+

HR

LR

womentested(%)

Krüger-Kjaer, et al. 2007Baseman and Koutsky, J Virol:32S, 2005

age

© AMK© AMK

Cumulative Risk of new HPV Infections (all types)

in Women by Age

Cohort study in Bogotá

Bosch et al., Vaccine 26 suppl.10 (2008) K1-K16

Persistent Infections with HPV Increase with Age

Castle et al., JID 191 (2005)

<25 25-34 35-44 45-54 55-64 65 Alter

Pe

rsis

ten

ce

Oncogenic

HPV16

Non-Oncogenic

HPV61

0%

10%

20%

30%

40%

50%

60%

<25 25-34 35-44 45-54 55-64 65

%

Oncogenic

HPV16

Non-Oncogenic

HPV61

0%

10%

20%

30%

40%

50%

60%

0%

10%

20%

30%

40%

50%

60%

50%

60%

Mean persistence: 5.6 years

Example from follow-up

Patient 29 years, HPV16 positive dysplasia

29.09.2010 pre-therapy:

Smear taken in Dysplasia clinic, PAP IVa/HSIL, CIN 3

Genotyping: HPV16 strong (1347 MFI), HPV 59 borderline positive (9 MFI)

02.11.2010 at conisation surgery:

Smear targeted from lesion

Genotyping: HPV16 strong positive (1131 MFI), HPV 59 not detectable

04.02.2011 at follow-up visit:

PAP IIID/LSIL

Genotyping: HPV 59 strong positive (560 MFI), HPV 16 not detectable !

colposcopic aspect atyp. Condylomata

Generic HPV-test „HR-HPV still positive“ => failure of conisation=>

reconisation

Genotyping test „other HPV type“ means other risk => successful

surgery=> no immediate reconisation, because of lower risk for recurrence.

Taken 4.2.2011

Courtsey Dr. G. Cichon

Aceto white

Schillers iodine

© AMK

Potential Sampling During Progression

Deposit from partner?

High virus load

High cell load

Progression,

Ansammlung

von Mutationen

E6/E7

p16/Ki67

DNA

mRNA

Protein E6/E7

HPV General Primer-PCR

E6-E7: Oncogenes

E1-E5: regulatory

genes

L1-L2: Capsid

L1

L2

E4

E6E7URR

E5

E1

E2

HPV 167905 bp

L1 GP-PCR

Systems:

MY09/11

GP5+/6+

SPF10

450 bp

150 bp

65 bp

General primer amplify all HPV types

Targets L1, E1, E2, E6/E7

• mRNA

• Proteins

© AMK

Which test is performed in your laboratory?

What is the cut-off for clinical specificity?

Categories of Assay Systems

• Detection of generic HR-HPV presence/infection

• Detection of generic HR-HPV plus genotypes HPV16/18

• Detection of individual HPV genotypes

Commercially Available MethodsTest HPV Spectrum geno- Company

Probe-based typing

Hybrid Capture 2 (HC2) 13 HR/5 LR Qiagen

Cervista HPV 14 HR Hologic

PCR-based

Abbott RealTime High-Risk HPV 14 HR-HPV G+ Abbott

GenoID Real Time PCR HPV Test 14 HR/6 LR G+ Gene ID

AMPLICOR® HPV Test 13 HR Roche

Cobas 4800 HPV Test 12 HR +16/18 G+ Roche

Line Array® 19 HR/18LR G+ Roche

Papillo Check 18 HR/6 LR G+ GreinerBioOne

F-HPV-typingTM Multiplex (E6/7) 13 HR/2LR G+ Genomed

CLART®HPV2 21 HR/13 LR G+ Genomica

Innolipa HPV Genotyping Extra 18 HR/7 LR G+ Innogenetics

Multiplexed Genotyping MPG (DKFZ) 18 HR/7 LR G+ Multimetrix

mRNA-based

NucliSensEasy®HPV 5 HR G+ Biomerieux

APTIMA® (mRNA) 14 HR (G+) Genprobe/

Hologic

E6/E7 Protein-based (in devel.)

AVCE6 Rapid HPV 14 HR Arbor Vita

Commercially available MethodsTest HPV Spectrum geno- Company

Probe-based typing

Hybrid Capture 2 (HC2) 13 HR/5 LR Qiagen

Cervista HPV 14 HR HOLOGIC

PCR-based

Abbott RealTime High-Risk HPV 14 HR-HPV G+ Abbott

GenoID Real Time PCR HPV Test 14 HR/6 LR G+ Gene ID

AMPLICOR® HPV Test 13 HR Roche

Cobas 4800 HPV Test 12 HR +16/18 G+ Roche

Line Array® 19 HR/18LR G+ Roche

Papillo Check 18 HR/6 LR G+ GreinerBioOne

F-HPV-typingTM Multiplex (E6/7) 13 HR/2LR G+ Genomed

CLART®HPV2 21 HR/13 LR G+ Genomica

Innolipa HPV Genotyping Extra 18 HR/7 LR G+ Innogenetics

Multiplexed Genotyping MPG (DKFZ) 18 HR/7 LR G+ Multimetrix

mRNA-based

NucliSensEasy®HPV 5 HR G+ Biomerieux

APTIMA® (mRNA) 14 HR (G+) Genprobe/

Hologic

E6/E7 Protein-based (in devel.)

AVCE6 Rapid HPV 14 HR Arbor Vita

Analytical sensitivity: How many HPV genomes necessary for detection?

specificity: is there cross-reaction between HPV types?

Refers to all HPV infections, important in epidemiology

Clarification for Test Performance/Quality

Clinical sensitivity: How many CIN2+ detected by HPV test?

specificity: How many false positives HPV+/CIN- are detected?

Refers to clinically relevant infections only,

important in (primary) screening

Therefore use tests with sufficient CLINICAL VALIDATION

e.g. FDA approval

good balance necessary:

high sensitivity for CIN2+ to reliably detect disease AND

minimal detection of HR-HPV infections w/o disease

Hybrid Capture II0.7

50.8

0.8

50.9

0.9

51

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

GenomicaHPV-Proofer

p16INK4a

APTIMALinear Array

Amplicor

Specificity

Sen

sit

ivit

y

0.8

Detection of CIN3+

17Szarewski et al., Epidemiol Biomarkers Prev 2008; 17(11): 3033–42)

@

© AMK

14 HR-HPV types included in a HPV test

Most commonly used assay Hybrid Capture 2

and GP5+/GP6+ PCR-EIA

HC2

Validated in large prospective clinical trials (women >30 years)

Has clinical sensitivity for CIN2+ of 95%

Has clinical specificity for CIN2+ of 90.7 to 94.1%

New test formats should be compared to HC2 and reach at least

90% of HC2 sensitivity

98% of HC2 specificity

Requirements of HPV test formats

Meijer et al., J Clin Virol. 2009

© AMK

Quality control

• Smear quality (taken under colposcopic view?)

• Lab infrastructure (contamination risk, PCR problem)

• Validated test

• Internal control for cellular DNA!

• Monitoring of procedures

• Intra/inter Lab reproducibility

• Proficiency testing of standards from reference labs

© AMK

Assay examples: Hybrid Capture 2

Probe-based

13 HR-HPV (a probe)

5 LR-HPV (b probe)

Semi quantitative

No internal control

Clinically defined cut-off

(5000 copies HPV16)

Problem: cross reactivity

with 12 other HPV types

2003 FDA approved

„gold standard assay“

Iftner et al., www.zervita.de

© AMK

Automated sample

preparation and PCR

Real time PCR

Evaluated by

Fluorescent probes

Assay examples: cobas 4800 system (Roche)

PCR-based

12 HR-HPV (a probe)

HPV 16&18 (b probe)

Quantitative result

Higher analytical sensitivity

Cut-off adapted to HC2

No cross reaction

No contamination risk

2011 FDA approved

Athena Studie: „Addressing the need for advanced screening“

47.000 women, US multi-center study, double-blind

Cobas HPV test (Roche) 12 hrHPV and HPV16/18 genotyping

93,5% Sensitivity, 69,3% Specificity for CIN3, 0,3% false negative

Identification of women with highest absolute/relative risk for CIN3+

10% of HPV16 and/or 18 positive with negative cytology had CIN3+

16% of HPV16 and/or 18 positive with ASC-US cytology had CIN3+

Wright TC et al., Am J Clin Pathol. 2011 Oct;136(4):578-586

CIN 3 Risk with HPV16/18+ Test

Genotyping => risk assessment

Assay examples: APTIMA® Technologies

E6/E7 mRNA-based

Sample Processing

mRNA

Target Capture

Isothermal transcription-mediated

Amplification of Target

Hybridization of DNA Probes (AE) Detection of Hybridized

Probes DKA/HPA24

109 fold amplification!

APTIMA HPV Assay

• 14 high risk HPV subtypes (pool)

• qualitatively detects HPV E6/E7 mRNA

• High analytical sensitivity

• clinical sensitivity comparable to HC2

• clinical specificity higher due to mRNA detection

• Includes internal control

• CE marked in Europe for diagnostic screening of liquid based

cytology (LBC) and APTIMA Cervical Specimen Collection

and Transport Kit samples

• APTIMA HPV Genotyping assay for types 16 and 18/45 will

be available CE marked end of 2012

• 2011 FDA approved25

© AMK

Assay examples: InnoLipa (full genotyping)

PCR-based Generic PCR (SPF10)

Amplification

28 HPV types

18 HR-HPV

10 LR-HPV

Full genotyping,

Multiple infections

No clinical cut-off,

for epidemiology

Not for primary

HPV screening!

Triage (genotype)

Not FDA approved

Iftner et al., www.zervita.de

Assay examples: AVCE6, rapid onsite test (in eval.)

Protein-based

© AMK

Summary and Conclusion

HPV tests used in clinical practice have to be

• validated for clinical sensitivity and specificity

• quality controlled and

• approved

This is fulfilled by:

• HC2 (Qiagen), gold standard

• Cervista (Hologic), (not described)

• cobas-4800 (Roche), genotyping 16&18

• Aptima (Gen-probe/Hologic), higher specificity (?)

PCR-based genotyping is too sensitive but information for

triage or follow-up may be important.

Rather for epiemiology.

New tests on the horizon for onsite (pre)-testing

or meaningful biomarkers for progressing disease.

© AMK

Thank you!

And please ….

screen the mothers

AND VACCINATE THE DAUGHTERS

27th INTERNATIONAL PAPILLOMAVIRUS CONFERENCE AND CLINICAL WORKSHOP

September 17-22, 2011Clinical Workshop and Main Conference, ICC

September 16, 2011Preclinical Workshop „Gynecologic Oncology“, Charité

Berlin, Germany

© AMK

Anteil der einzelnen HPV-Typen an allen

identifizierten HPV-Typen (n=407) bei 235 Personen

0%

5%

10%

15%

20%

25%

30%

16 53 42 66 18 56 59 51 39 52 82 43 70 73 58 6 33 11 45 35 68

HPV Genotypisierungstest:

Gynäkologie, Labor Dr. Kaufmann, Charité/CBF

Zur Veröffentlichung eingereicht!

Aber: HPV16 ca. 50% in CxCa

HPV18 ca. 20% in CxCa

© AMK

HPV-Typen bei Zervixkarzinom in EuropaHPV16/18-Impfstoff mit Kreuzprotektion

0 20 40 60 80 100

+ 52

+ 58

+ 45

+ 31

+ 33

+ 18

1656.7%

75.5%

79.9%

84.1%

88.0%

89.0%

89.8%

HPV Typ

Clifford, G.M. et al.: Br. J Cancer 2003; 88: 63-73

85.4%

Das Krebsrisiko ist für HPV16 vermutlich 1 log-Stufe höher als für

die restlichen high-risk HPV-Typen.

Karzinogenitäts-Gruppen

1 karzinogen für Menschen

2A vermutliche karzinogen für Menschen

2B möglicherweise karzinogen für Menschen

3 nicht klassifizierbar bezügliche der Karzinogenität für Menschen Lancet Oncology 10, 2009

A review of human carcinogensInternational Agency for Research on Cancer

Das Dilemma: HPV Nachweis vs Zytologie

Cuzick et al., Int J Cancer 2008

HPV Test: hohe Sensitivität

Aber Zytologie: hohe Spezifität

Alternative:

HPV im primären

Screening?

(hohe Sensitivität)

Zytologie als Triage?

(hohe Spezifität)

NPV als Vorteil?

Zur Zeit:

Zytolog. Screening

(hohe Spezifität)

HPV als Triage

nach Therapie

Real life data: unsere HPV Genotypisierung

27.9.2011, niedergelassene Gynäkologen, Berlin

46 Proben

31x HPV negativ

15x HPV positiv

5x multiple Infektionen

16, 33

31,35,45,59

16, 39

51

16

56

54

16, 52

18

70

39

53,68

16grzw.

51

16

LR

LR

! hHR

! hHR

! hHR

! hHR

! hHR

! hHR

26 J

18 J

32 J

30 J

39 J

46 J