molecular aspects of hpv in relation to cervical cancer

Molecular aspects

of HPV in relation to

cervical cancer,

epidemiology and

vaccines Roland Sahli, Institute of Microbiology, CHUV, University of Lausanne

WHO HPV Reference Laboratory for Europe

Human Papillomaviruses

Biology: structure

HPV

7900 pb

E1

E2 E4

L2

L1

E5

E6 E7

Buck, C. B., N. Cheng, et al. (2008).

J Virol 82(11): 5190-5197.

Baker, T. S., W. W. Newcomb, et al. (1991).

Biophys J 60(6): 1445-1456.

Epitheliotropic viruses

• High genetic diversity (< 90% nucleotide sequence homology: distinct genotype)

– Genotype ≡ Serotype (important for prophylactic vaccine design)

• Highly specific

– Toward the host (slow co-evolution over millions of years)

• Reptiles, birds, mammals

– Toward the infected tissue (cutaneous, mucosal)

• Cutaneous: HPV-1, -2, -4, …

• Mucosal: HPV-6, -11, -16, -18, ….

• Induce cell proliferation

– Benign tumors (warts, laryngeal papillomas, condyloma): productive cycle

of all HPV

– Cancerous tumors (cervical cancer, oro-pharyngeal cancers): non

productive virus-host cell interaction associated more frequently with

infection by High Risk (HR) HPV

• The site and mode of production hide the virus from the host defense

– Viral persistence

Biology: HPV main characteristics

More than 150 HPV genotypes

Source: IARC

Biology: HPV genetic diversity

A9: HPV-16 group

A7: HPV-18 group

70% of cervical cancer cases are due to HPV-16 and -18

Certain genotypes are oncogenic

HPV genotypes according to their prevalence in cancer vs. normal cases

Biology: HPV genotype and oncogenic risk

Combes, J. D., P. Guan, et al. (2015). Int J Cancer. 136(3): 740-742

Productive infection : benign tumor

Micro wound is thought to serve as entry site for the virus. Mitotic activity of

healing basal cells may favor entry of the viral genome in their nucleus (Pyeon, D., S. M. Pearce, et al. 2009. PLoS Pathog 5(2): e1000318.)

Week 6-12

Week 0

Biology: HPV productive infection

Virion production takes place in infected keratinocytes during their terminal differentiation

Division of infected cells is necessary for viral DNA replication. It is induced by the viral E6/E7 oncogenes, to overcome growth arrest associated with terminal differentiation

Doorbar, J. Clinical Science (2006) 110, 525-541

The viral DNA is integrated into the chromosomes

of malignant tumor cells: dead end for the virus

Maintenance and continuous expression of E6/E7 is mandatory for tumor cell growth:

natural selection of cells with integrated viral DNA

Carcinogenesis: non productive interaction between HPV and its host cell

Principles of virology, 2nd edition

Cancer cell survival depends on the continuous

expression of the viral oncogenes E6/E7

J. Doorbar, Clinical Science (2006) 110, 525-541

H e L a c e lls (H P V 1 8 E 6 /E 7 )

0 .5 1 .0 1 .5 2 .0 2 .5 3 .0

-2 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

M O I (lo g 1 0 ) : A d E 2 e x p re s s in g H P V 1 6 E 2 (in h ib ito r o f E 6 /E 7 ) o r A d e G F P c o n tro l

Ce

ll s

urv

iva

l

R. Sahli, unpublished

Carcinogenesis: histologic progression and viral oncogene expression

Mechanism of cellular

transformation

Carcinogenesis: regulatory pathways in response to viral oncogene expression

Doorbar, J., N. Egawa, et al. (2015). Rev Med Virol 25 Suppl 1: 2-23.

•HR HPV differ from LR HPV by their ability to efficiently promote cell immortalization and telomerase activation, as well as by promoting the degradation of pRb and p53. •Many interactions between the E6 and E7 proteins with their respective cellular partners can differentiate LR HPV and HR HPV with regard to cell transformation.

The HPV life cycle is virtually hidden

from the immune system

• Cycle within the

epithelium

• Virions are released

from dead cells during

desquamation

• Neither cytolysis nor

necrosis

– Weak to no inflammation

• Favors viral persistence

– Risk of genetic alterations

Stanley, M. A. (2012). Clin Microbiol Rev 25(2): 215-222. Stanley, M. (2008). Gynecol Oncol 109(2 Suppl): S15-21.

Biology: the immune response against HPV is in a defavourable context

Cervical cancer is a rare outcome of a persistent infection

(>15 ans) by a high risk genotype

Adapted from Syrjänen and Syrjänen: Papillomavirus infections in human pathology. Wiley & Sons, Chichester; 2000.p. 143-165

CIN = cervical intraepithelial neoplasia; HPV = Human PapillomaVirus; HSIL = high grade squamous intraepithelial neoplasia

LSIL = low grade squamous intraepithelial neoplasia.

>99 % high

risk HPV

1-2 y-----------------------------------10 y and + (cancer 15-20 y)

Initial

HPV

infection

CIN 1, LSIL CIN 2/3, HSIL Cancer

~20% high

risk HPV

< 0.5-1% of the

initial infections Persistent Infections

90% HPV infections

are cleared within 2 y

Cervical cancer screening: natural history of HPV infection

Virological aspects of cervical

cancer prevention Prior to infection

Neutralisation of infection through prophylactic vaccination

Bivalent (Cervarix: 16, 18) or quadrivalent (Gardasil: 6,11,16, 18)

vaccines are available (potential: 70% of cases)

Nonavalent vaccine (Gardasil 9: 6,11,16,18, 31, 33, 45, 52, 58) will soon

be available (potential: 80-90% of cases)

Post infection

Identification of "at risk" patients

High risk HPV detection as an adjunct to cytology

High risk HPV detection can be used for primary screening under

specific conditions

Treatment of tumors via therapeutic vaccines

Prevention of cervical cancer: role of virology

PRIOR TO INFECTION

PROPHYLACTIC VACCINES

Exposed loops at the surface of L1

pentamers display genotype- and

conformation-specific neutralizing epitopes

Bishop, B., J. Dasgupta, et al. (2007). J Biol Chem 282(43): 31803-31811.

HPV16

HPV11

HPV18

HPV35

Immunodominant neutralizing Ab generated by L1 VLPs are:

type-specific not cross-neutralizing

Highly homologous L1 VLPs share cross-neutralisation epitopes which are less immunogenic than the type-specific ones Stanley et al. 2006. Vaccine 24S3 106-113

Prophylactic vaccine: neutralisation epitopes

Only limited cross-protection is observed

from L1 VLPs

Chen, X. S., R. L. Garcea, et al. (2000). Mol Cell 5(3): 557-567.

Highly variable positions are seen on the surface of the VLPs in extended loops

Neutralization epitopes are positioned in genotype-specific areas, and HPV variant selection may be driven by escape from neutralization by the host Limited cross-protection is favourable for HPVs in terms of evolution/selection

Prophylactic vaccine: cross neutralisation

Improved vaccine formulation Gardasil-9: HPV vaccine formulation (6, 11, 16, 18, 31, 33, 45, 52, 58)

http://clinicaltrials.gov/show/NCT00543543 Joura, E. A., A. R. Giuliano, et al. (2015). N Engl J Med 372(8): 711-723.

Prophylactic vaccine: Gardasil-9

http://clinicaltrials.gov/show/NCT00543543

POST INFECTION

The effect of vaccination on cervical cancer burden will not be seen

for decades

The majority of HPV infections are clinically irrelevant, and

their rate vary with the age of the patients

Cervical cancer screening using HPV tests: context

Schiffman MH. Epidemiology of cervical human papillomaviruses. In: zur Hausen H,

ed. Human pathogenic papillomaviruses. Heidelberg: Springer-Verlag, 1994

•HPV can be detected in a cytologically normal context (transient infections) •HPV positive results without clinical relevance is particularly high in patients aged less than 30 •HPV testing is highly sensitive but poorly specific •Positive predictive value can be improved by taking persistence in consideration

Characteristics and use of HPV tests

HPV tests

•HPV molecular tests •High analytical sensitivity

•High negative predictive value

•Low positive predictive value

•(Semi-) Quantitative HPV tests may be

easier to adapt for primary screening

(threshold of detection) •Against clinical endpoint (≥CIN2)

•Against HCII

•Patients > 30 years old Meijer, C.J. et al. (2009). Int J Cancer 124: 516-520

•HPV genotyping assays

•Treatment follow up, triage of ASC-US

•Epidemiology •Sensitivity: LOD 50 (HPV16/18)-500 ge

•Specificity: > 97%

Snijders et al., J Pathol 2003; 201: 1–6.

A. The viral load is associated with the grade of the disease

B. Persistence is associated with a

higher relative risk of HSIL Schlecht et al., JAMA 2001; 286: 3106

C. As un adjunct to pathology

D. Vaccine evaluation requires

genotyping

HPV testing for primary screening

• DNA assays

– Hybrid capture (HCII): gold std, targets the whole genome

– Cobas taqman HPV (ROCHE), RealTime High-risk HPV (ABBOTT)

• HPV16/HPV18 + (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, [66], [68])

• Validated against HCII: performances non inferior to HCII

• Distinguish HPV16 and 18 from the other HR genotypes

• Cannot evaluate persistence of other HR HPV

– Seegene Anyplex II HR assay

• HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, [66], [68]

• Can evaluate persistence

• Under clinical validation (Prof. Meijer's laboratory)

• RNA assays

– Aptima (Hologic)

• HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, [66], [68]

Biased selection of HPV assays

Hybrid capture assay (HC2)

HPV screening: signal amplification

Commercial assay (Qiagen/Digene), standardized, approved for

clinical use

Group-specific (13 HR HPV)

Sensitivity and specificity depend on the threshold of detection

(pg/ml): sensitivity (1 pg/ml) > 97%

Cross reactions with several LR types: clinical specificity may be

suboptimal

Cannot assess persistence of a specific genotype

Reference test against which new assays can be validated

Hybridization in solution

PCR

Measurement of the amplicons after the last cycle (classical PCR): qualitative Measurement of the amplicons at each synthesis step with fluorescent probes (Real time PCR): quantitative

RealTime High Risk HPV test (Abbott )

16/18, HR

Cobas 4800 HPV test (Roche)

16/18, HR

HPV screening: target amplification

In vitro transcription

Target:

E6/E7 mRNA

APTIMA

14 HR genotypes

(16, 18, 31, 33, 35,

39, 45, 51, 52, 56,

58, 59, 66 et 68)

(> 95% CC)

Commercial assay (Hologic), standardized, approved for clinical use

Sondes

Fluorescence

upond probe

binding

HPV screening: target amplification

No test is perfect

° CIN2+ prevalence: ~28% (27.4% in ||) + Szarewski, A., L. Ambroisine, et al. (2008). Cancer Epidemiol Biomarkers Prev 17(11): 3033-3042. | Cuzick, J., L. Ambroisine, et al. (2010). J Med Virol 82(7): 1186-1191. || Denton, K. J., C. Bergeron, et al. (2010). Am J Clin Pathol 134(1): 12-21.

Test Method Target PPV°

HCII + HC 13 HR HPV, DNA 36.1%

Amplicor + PCR 13 HR HPV, DNA 33.5%

Abbott hr

HPV | Real time

PCR

14 HR HPV, DNA

(HPV16 and18,

and 12 other types)

38.9 (all),

57.6 (16),

34.2 (18)

Nuclisens

EasyQ +

TMA 5 HR HPV, E6/E7

RNA

52.0%

APTIMA + TMA 14 HR HPV, E7

RNA

39.9%

Linear Array +

PCR-

RBH

37 (HR + LR) HPV,

DNA

37.7%

p16INK4A Imm. Cell. proliferation

marker

52.3%+ ,

36-50% ||

Median age: 29, IQR 26-36

HPV screening: value of various tests on abnormal smears

Genotyping assays

• Can be used as an adjunct to validated assays

• Persistence evaluation

• Treatment follow up

• Research tool

• INNO-LiPA, Linear Array, PGMY-CHUV and other in-

house assays

• Seegene Anyplex HPV28

HPV assays

L1 target

1 50 100 150 200 250 300 350 400 450

Similarity

SPF: Inno Lipa

GP5+/6+ : Multimetrix

PGMY: Linear Array (LA), PGMY-CHUV

HPV

P97

E1

E2 E4 L2

L1

E5

E6 E7

PGMY11 PGMY09

The sequence of the MY amplicon predicts the genotype determined by whole genome analysis

PGMY-CHUV: preferably with DNA from cervical smears and unfixed biopsy samples Se: LOD95 10 gen-eq (>98% vs LA) Sp: 100% (vs sequencing) SPF and GP5+/6+: valid for FFPE samples Se similar to PGMY-CHUV Sp SPF < PGMY-CHUV (proficency panels)

HPV screening: genotyping

PGMY-CHUV

HPV

P97

E1

E2 E4 L2

L1

E5

E6 E7

PGMY11

PGMY09

+

HLA PCR/gel

hyb

ridizatio

n

Genotypes

Acc

ess

D

atab

ase

Parallel array of type-specific probes

Detection

Used as an adjunct to cytology; Genotype-specific: persistence

LOD: 5-50 IU (HPV16/18), 50-500 ge (others); Probe specificity = 100%;

Low cost method transferred through WHO to ref. lab. worldwide Estrade, C., P. A. Menoud, et al. (2011). J Clin Microbiol 49(10): 3474-3481.

Estrade, C. and R. Sahli (2014). J Clin Microbiol 52(11): 4033-4035.

HPV genotyping: in house test

5 PGMY11 13 PGMY09 1RSMY09 2 HLA primers AmpliTaq Gold 1.5 or 3 mM MgCl2

Seegene Anyplex HPV28 assay

HPV genotyping: Real time PCR

28 genotypes can be identified in only 2 reactions!

Used as an adjunct to cytology; Genotype-specific: persistence

LOD: 5-50 IU (HR HPV); Probe specificity = 100%

HPV genotyping: assay validation

Comparison of the Seegene H28 assay

to the PGMY-CHUV assay


Analysis of discordant samples

Multiple infections status is associated with discordance for HPV42 Lower viral load is associated with discordance except for HPV51 and HPV68

Comparison of Seegene HPV28 assay to PGMY-CHUV



HPV51 discordants

DNA sequencing performed by Seegene on our HPV51 discordants revealed mismatches with the Seegene HPV28 assay primers and probe A new version was thus implemented (RUO-HPV51 kit) and compared to HPV28 kit using PGMY-CHUV as a reference

Significant improvement of HPV51 detection by RUO-HPV51 compared with HPV28 kit (p<0.004 by 2 tailed McNemar's test)

HPV68 discordants


The PGMY primers are known to miss a subtype of HPV68 (HPV68a) Inefficient priming thus occurs. However HPV68a at high viral load can be detected by the PGMY-primers DNA sequencing of a rare HPV68a amplicon identified the PGMY09_L primer as the closest HPV68a compatible primer RSPGMY09_L was thus designed and added to the PGMY primer cocktail

HPV genotyping: validation of PGMY-CHUV version 2

Correction of the HPV68a defect in

PGMY-CHUV version 2


Future of cervical cancer screening

• Combine high specificity of cytology with high sensitivity of HPV testing

– Screening interval

– Improvement of cytology in the vaccine era • Cytology will be more difficult to interpret because the

disease prevalence will diminish (low PPV)

• Take advantage of identifying persistent vs. transient infections

• How to target the 50% cancer cases from women who do not consult?

Way to improve cervical cancer screening?

Acknowledgements

• Christine Estrade

• Joakim Dillner

• Elizabeth Unger

Low cross protection confered by the first

generation vaccines against non-vaccine

HPV types in HPV-naive women

Van de Velde, N., M. C. Boily, et al. (2012). J Natl Cancer Inst. 104(22): 1712-1723.

In addition, the time-dependent lower cross-protection observed in studies Patricia (4 years), HPV-007 (6 years) and HPV-023 (9 years) suggests waning of cross-protection.

Prophylactic vaccine: cross protection

La VPN des test HPV est très élevée –

Probabilité très faible de lésions à 5 ans

Apport des tests HPV: augmentation de l’intervalle de temps entre visites

P[CIN3+ à 5 ans] si les deux types de tests sont positifs: 33%!

P[CIN3+ à 5 ans] si le test HPV est positif: 8%!

P[CIN3+ à 5 ans] si le test cytologique est positif: 3%

P[CIN3+ à 5 ans] si les deux types de tests sont négatifs: < 0.2%

P[CIN3+ à 5 ans] si le test HPV est négatif: < 0.3%

P[CIN3+ à 5 ans] si le test cytologique est négatif: 0.8%

Test HPV: capture d’hybride (HCII) ou PCR (GP5+/6+)

Dillner, J., M. Rebolj, et al. (2008). "Long term predictive values of

cytology and human papillomavirus testing in cervical cancer

screening: joint European cohort study. BMJ 337: a1754.

Cytologie et génotypage pourraient être combinés

sans augmentation exagérée du nombre de tests

Naucler, P., W. Ryd, et al. (2009). Efficacy of HPV DNA testing with cytology triage and/or repeat HPV DNA testing in

primary cervical cancer screening. J Natl Cancer Inst 101(2): 88-99.

Triage organisé (Suède) pour femmes de 34-38 ans; Test HPV validé pour tri

primaire et capable de génotypage; Persistance à > 1 an; Thin Prep facilite la

gestion des échantillons

Apport des tests HPV: amélioration de l’efficience par le génotypage

Cytology vs. HPV

Primary screening

Mayrand, M. H. et al. (2007). N Engl J Med 357(16): 1579-1588. Patientes de plus de 30 ans

•Superior sensitivity of HPV test

•Superior specificity of PAP

•Neither method is perfect (see low PPV)

HPV triage improves protection

against cervical cancer

Ronco, G., J. Dillner, et al. (2014). Lancet 383(9916): 524-532.

This reflects the vey high sensitivity of HPV testing

“HPV-based screening provides 60-70% greater protection against invasive cervical carcinomas

compared with cytology. Data of large-scale randomised trials support initiation of HPV-based

screening from age 30 years and extension of screening intervals to at least 5 years.”

HPV triage vs. cytology

Long-term prediction of impact of bivalent,

quadrivalent and nonavalent HPV vaccines

Van de Velde, N., M. C. Boily, et al. (2012). "Population-Level Impact of the Bivalent, Quadrivalent, and Nonavalent Human Papillomavirus Vaccines: A Model-Based Analysis." J Natl Cancer Inst. Published online 27.10.2012

Tests et méthodes de détection HPV

Category Target Assay principle

Method Genotyping

Target amplification

DNA

PCR: end-point hybridization read-out

Amplicor / PGMY-Linear array (Roche) Y

SPF10-LiPa (DDL) Y

GP5+/6+-PCR-EIA N

MGP PCR-Luminex Y

BSGP5+/6+-PCR-MPG Y

Digene HPV genotyping RH/LQ tests (Qiagen) Y

PapilloCheck (Greiner-Bio-one) Y

CLART HPV2 (Genomica) Y

Real-time PCR-based

Abbott RealTime High Risk HPV test 16/18, HR

Cobas 4800 HPV test (Roche) 16/18, HR

mRNA

RT-PCR HPV 16 E6*I RT-PCR-EIA 16?

TMA NucliSENS EasyQ HPV (Norchip/Biomerieux) Y, 5 HR

TMA Aptima HPV assay (Gen-Probe) N, 14 HR

Signal amplification

DNA

Liquid phase Hybrid Capture 2 (Qiagen-Digene) N

Cervista HR HPV (Hologic) N

In situ Ventana Inform HPV (Ventana) NA

Dako Gen point (Dako) NA Adapté de Snijders, P. J., D. A. Heideman, and C. J. Meijer. 2010. Methods for HPV detection in exfoliated cell and tissue specimens. APMIS 118:520-8.

Caractéristiques cliniques de tests

HPV bien étudiés (CIN2+)

Test Sensibilité Spécificité VPP

HCII 99.6% 28.4% 36.1%

Amplicor 98.9% 21.5% 33.5%

PreTect 73.6% 73.1% 52.0%

APTIMA 95.2% 42.2% 39.9%

Linear Array 98.2% 32.8% 37.7%

Population: patientes avec cytologie anormale, prévalence de CIN2+ par

conséquent supérieure à celle d’une population en tri primaire

Szarewski, A., L. Ambroisine, et al. (2008). "Comparison of predictors for high-grade cervical

intraepithelial neoplasia in women with abnormal smears." Cancer Epidemiol Biomarkers Prev 17(11):

3033-3042.

Meta-analyse des données comparatives de

test mRNA vs. DNA (CIN2+)

Burger, E. A., H. Kornor, et al. (2011). "HPV mRNA tests for the detection of cervical intraepithelial neoplasia: a systematic review." Gynecol Oncol 120(3): 430-438.

The review suggests that mRNA tests have diagnostic relevance, but additional studies and economic evaluations must be conducted in order to make a solid conclusion regarding the clinical applicability of HPV mRNA testing. APTIMA vs. EasyQ vs. HCII/PCR

molecular aspects of hpv in relation to cervical cancer

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