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Baarmoederhalskanker : het cervicaaluitstrijkje en HPV. Moleculaire diagnostiek voor HPV

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(1)

Baarmoederhalskanker :

het cervicaaluitstrijkje en HPV

Moleculaire diagnostiek voor HPV

16-11-2004

Reinoud Cartuyvels Klinisch laboratorium

(2)

Moleculaire diagnostiek van HPV

Microbiologie

Diagnose van HPV

– cytologie – serologie – moleculaire diagnostiek • hybridisatie • PCR • RT-PCR
(3)

Humaan Papilloma Virus (HPV)

familie van de Papovaviridae

kleine (52 - 55 nm), naakte

virussen

icosahedrische capside

(72 capsomeren)

(4)

HPV: genoom

• structuur gelijkaardig voor alle papillomavirussen

• “early region” (E1-E7): met genen noodzakelijk voor DNA-replicatie en cellulaire transformatie

• “late region” (L1-L2): codeert voor virale capsiden

• “regulatory region”: informatie

ivm. de controle op transcriptie en replicatie.

(5)
(6)

HPV: types

> 100 types

typering

op basis van nucleïnezuursequentie

> 10% variatie = nieuw type 2 – 10% varaiatie = subtype < 2% variatie = variant

sequentiële nummering op basis van volgorde van

ontdekken

(7)

HPV: types

2 majeure phylogenetische takken met verschillende

voorkeur voor infectieplaats

cutaan:

gekeratiniseerd squameus epitheel

(8)

HPV: epidemiology

“Warts: an intriguing and ancient disease”

(Diversity and molecular evolution of papillomaviruses. Van Ranst M. Thesis KUL, 1994, 6.)

– Celsus (100 A.D.): thymus

“At thymion nominatur, quod super corpus quasi verrucula eminet, ad cutem tenue, supra latius, subdurum et in summo perasperum. Idque summum colorem floris thymi repraesentet, undi ei nomen est, ibque facile finditur et cruentatur.”

widespread in higher vertebrates

– highly species specific

(9)

HPV: epidemiology

very common disease

– cutane wratten

• common wart (61%):

4 – 20% school-aged children

• plantait wart (34%): less common

• juvenil or flat wart (4%)

– condyloma acuminatum

(10)

HPV: epidemiology

> 50% sexually active women at least once infected

prevalence 15% (18-28 yr)

rapidly increasing

incidence of condylomata

STD clinic, UK: 1971

1994

k

: rate of attendance grew by

394%

l

: 594%

(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)

HPV: pathogenicity

HPV infection mostly

transient

:

– rest prevalence < 35 yr: 1-2%

long

interval

(primary infection – carcinoma)

role of

cofactors

(genicity, smoling): oncogenesis

probably multiple step process

(19)

HPV: laboratory diagnosis

cervical cytology

Pap smear

liquid based cytology

serology

(20)

HPV serology

ELISA

based detection of antibodies

– L1-VLPs

– recombinant E6-7

serum or mucosal, IgA or IgG

type specific (low titers)

– good specificity (> 90%)

reaction indicates past or current infection

– < 70% develops detectable antibodies

(21)

HPV serology

formats vary (direct vs. indirect)

VLP production not standardised

– different expression systems, preparative methods, QC approach

no gold standard for setting treshold for + results

few inter-laboratory comparisons

(22)

HPV: moleculaire diagnostiek

complex because of different HPV types

hybridisatie

– hybrid capture

– blot methods

– in situ hybridisation

PCR:

type specifiek vs. consensus

– PCR

(23)

HPV

HPV HYBRID

CAPTURE

CAPTURE

Liquid hybridisation

techniek

– 1995 tube format, 1999 microtiter format

Chemoluminiscent

detection of 18 types

– semi quantitative signal

– RNA probes react with DNA targets

– RNA-DNA hybrids captured and detected with monoclonal antibody technique

(24)

HPV

HPV HYBRID

CAPTURE

CAPTURE

MVR, 2000

(25)

HPV

HPV Hybrid

Capture

Capture

II

II

Assay

Assay

low risk

probe mix

HPV types 6, 11, 42, 43, 44

high risk

probe mix

HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,

58, 59, 68

good inter-laboratory precision!!

(26)

HPV

HPV Hybrid

Capture

Capture

II

II

Assay

Assay

+

easy

suitable for automation

no special facilities necessary to avoid cross-contamination (no target amplification)

-cross reaction

other high risk types not detected (66, 73, MM4 (related to 82) )

geographically different HPV type prevalence: screening for most prevalent types?

(27)

HPV

HPV Hybrid

Capture

Capture

II

II

Assay

Assay

FDA recommend

cut-off of 1 RLU

(equivalent to 1 pg

HPV DNA (10

5

copies) per 1 ml sampling buffer)

cross-reaction

with HPV-types, non-represented in

the probe mix

cut-off 1 pg/ml:

type 53, 66, 67, 73

cut-off 10 pg/ml:

type 53, 67

cross-reaction with high risk types: beneficial?

(28)
(29)

HPV

HPV Hybrid

Capture

Capture

III

III

Assay

Assay

(

(

Digene

Digene

)

)

• RNA probes binds target sequence

• biotinylated capture oligonucleotides: captures target sequence into streptavidine coated well of microtiter plate

• signal by RNA probe that hybridize to other regions

• blocker oligonucleotides to reduce aspecific hybridisation

Rapid Capture System: robotic handling of 96 well

microtiter plate for high volume testing

(30)

INFORM® HPV cocktails utilize proprietary probe formulations.

The Family 16 probe cocktail has an affinity to HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59.

The Family 6 Probe cocktail has an affinity to HPV genotypes 6, 11, 42, 43, and 44.

(31)
(32)

HPV PCR assays

small portion of genome targeted

allows testing of samples with poor quality DNA

small changes in virus (variants or integration) may

give false negative results

type specific assay:

– generally target E6/E7 region

consensus assays

– generally target L1 region

– type(s) determined by type specific hybridisation, restriction digestion or sequencing

(33)

HPV PCR assays

• consensus primers to highly conserved region of L1 gene (potentially capable of detecting all mucosal HPV types)

GP5/6 +/- 150 bp GP5+/6+ (extended version)

MY09/11 +/- 450 bp PGMY09/11 (modified version)

type distinction by gel electrophoresis, dot blot, (reverse) line strip hybridisation

SPF PCR +/- 65 bp

potentially increased sensitivity

(34)
(35)

HPV PCR assays

PCR: good agreement for MY06/11 and GP5+/6+ protocols

1. several conditions can affect test performance

(sampling, transport, storage, primers, reaction conditions, DNA polymerase, ability to detect multiple types, …)

Need for validated protocols, reagents and reference samples!

(36)

HPV PCR assays

2. samples with multiple HPV types

HC2: no discrimination between different types in multiple type infection

PCR: GP5+/6+ detects only 47% of multiple HPV type sample, MY09/11 detects 90%

Difference in sensitivity for distinctive HPV types:

multiple primer systems are more sensitive for multiple type infection than consensus primer systems

(37)

HPV PCR assays

PGMY09/11 systems

– 2 pools of primers (5, 13)

– amplification of MY09/11 450 bp target

– detects all known genital HPV types

– very good sensitivity (10 HPV genomes per PCR reaction)

– minimizing risk of potential mismatches

– increased detection of multiple infections

SPF-PCR (Innogenetics)

(38)

HPV PCR assays

Amplicor MWP (Roche Molecular Diagnostics)

– pool of primers : amplification of 170 bp fragment: (short fragment: increased sensitivity)

– 13 high risk types

– amplification with Taq Gold DNA polymerase

– amplicon immobilized by pool of capture molecules

– bound to wells of microtiter well plate (MWP)

(39)
(40)

Viral load determination

High viral load infections are probably

clinically more relevant!

Yltatalo: high viral load: risk for Ca x30

Joseffson: 20% highest viral load risk for Ca in situ x60 Woodman: abnormal smears less likely associated with

low-viral load types

Ylitatalo N, Cancer Res 2000;60: 6027-32 Joseffson A, J Clin Microbiol 1999; 37: 490-6 Woodman CB, Lancet 2001; 357: 1831-6

(41)
(42)

HPV Real-Time PCR assays

multiplex RT-PCR

– more HPV types

– cellular gene amplification: control for amount of input DNA

(43)

HPV Real-Time PCR assays

Caution! “Viral load” is difficult to estimate because of

– requires measure of number of cells: adjustment for cell load is absolute requirement (not fulfilled in HC2 and many PCR protocols)

– uneven tissue distribution: control for lesion size and proportion of infected to normal cells is extremely difficult

– variation in sampling

clinical applications

of viral load determination

remains to be demonstrated

(44)

HPV Genotyping Chips

(Bio Medlab Company)

oligonucleotide micro-array

based detection system of

22 HPV types

detection of multiple type

infection is possible

(45)
(46)

HPV screening strategies

Liquid based cytology

Hybrid Capture II

(RT)-PCR

(47)
(48)

TRIAGE

TRIAGE ASCUS

WITH

WITH

REPEAT CYTOLOGY

REPEAT CYTOLOGY

Cochrane meta-analysis, Marc Arbyn, 2002

Sensitivity: 60%

Specificity: 80%

(49)

TRIAGE ASCUS WITH HPV DNA TESTING

Cochrane meta-analysis, Marc Arbyn, 2002

AUC:

85%

Sensitivity: 85%

Specificity: 71%

(50)

TRIAGE ASCUS WITH DIGENE HCII TESTING

Cochrane meta-analysis, Marc Arbyn, 2002

AUC:

86%

Sensitivity: 93%

Specificity: 67%

(51)

CONCLUSIE

Digene Hybrid Capture II

HPV DNA diagnostiek is

beter dan repeat pap smear

(52)

TRIAGE LSIL WITH REPEAT CYTOLOGY

Cochrane meta-analysis, Marc Arbyn, 2002

Sensitivity: 77%

Specificity: 46%

(53)

TRIAGE LSIL WITH HPV DNA TESTING

Cochrane meta-analysis, Marc Arbyn, 2002

AUC:

85%

Sensitivity: 87%

Specificity: 47%

(54)

TRIAGE LSIL WITH DIGENE HCII TESTING

Cochrane meta-analysis, Marc Arbyn, 2002

AUC:

86%

Sensitivity: 95%

Specificity: 28%

(55)

CONCLUSIE

Digene Hybrid Capture II

HPV DNA diagnostiek

is onvoldoende specifiek

(56)

HPV screening strategies

Cytology: ASCUS

HPV DNA testing

HPV DNA testing

HPV DNA testing

Cytology if HR+

Cytology and HPV DNA testing

(57)

HPV screening strategies

HPV DNA testing as alternative

+

superior sensitivity of HPV DNA testing

-

transient infection

majority never develops premalignant lesions

(58)

HPV screening strategies

HPV DNA testing

Cytology if HR+

Colposcopy if abnormal cytology

+

combines

high sensitivity of HPV DNA testing (high NPV)

high specificity of cytology (high PPV)

possibly cost effective if

cost of HPV DNA testing comes down screening interval can be lengthened

(59)

HPV screening strategies

Combined cytology and HPV DNA testing

+

combines

high sensitivity of HPV DNA testing (high NPV) high specificity of cytology (high PPV)

⌫ increase screening interval if negative HPV DNA and cytology result

• FDA approved combined testing (04-2004) for

l

> 30 yr (age limit avoids unnecessary colposcopy referral for transient infection)

• American Cancer Society and American College of

(60)
(61)

HPV Vaccination

Phase III trials very successful

Glaxo SK type 16, 18

References

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