Methyleringsprocesser i forbindelse med triagering af HPV forandringer

Molekylærbiolog, ph.d. Dorthe Ørnskov

Klinisk patologi, Vejle Sygehus, Sygehus Lillebælt

Agenda

o Screening og Triage o Indførelse af HPV som screenings-test i Vejle.

o Mulige triage metoder

o Epigenetik

o HPV – opbygning, cyklus

o HPV methylering o Hvad viser litteraturen

o Hvilke metoder anvendes ofte

o Afrunding

Baggrund for studiet:

- Nye anbefalinger for cervix screening.

- Daværende HPV DNA metode var ikke velegnet til screening.

- Prøveantal ville stige.

Data fra Hvidovre studie (Horizon):

• 5000 screeningsprøver undersøgt med 4 forskellige HPV testmetoder

(Hybrid Capture 2, Cobas HPV, Aptima, Genomica).

• Stor diskrepans mellem HPV test resultaterne:

26,8% positive i Cobas HPV testen mod 20,4% Hybrid Capture 2.

• Dårlig reproducerbarhed af Cobas HPV testen: 90,7%.

• Vejle er det eneste sted i Danmark der bruger ThinPrep.

Formål:

• Undersøge hyppigheden af høj-risiko HPV i SLB’s optageområde i en screeningpopulation af kvinder 30-65 år.

• Undersøge og sammenligne to HPV DNA test: Cobas HPV test og HC2 .

– Sammenholde med cytologiske diagnoser

– Sammenholde med histologiske follow-up (CIN2+/3+)

• Undersøge reproducerbarheden af Cobas 4800 HPV testen.

• Evaluere workflow af de to test.

• Valg af HPV test til primærscreening test af 60-64 årige ( “check ud test”)

Metode og design

• 3000 konsekutive screeningsprøver fra kvinder 30-65 år.

• Alle kvinderne undersøges som vanligt med cytologi og følges op efter gældende nationale retningslinjer.

• På restmaterialet testes for HPV med Cobas og HC2 efter producentens instruktion.

• Ved uoverensstemmelse mellem de to metoder blev der genotypet med Linear Array og/eller Inno-Lipa testen.

• Alle Cobas positive og tilsvarende antal negative gen-testes med Cobas (reproducerbarhed).

• Alle Cobas HPV positive dobbeltfarvning med CINtec Plus.

Studie populationen

• I alt 4681 cervix-cytologiske prøver i perioden.

• 961 ekskluderet pga. alder ( <30 eller >65 år).

• 657 ekskluderet, da der ikke var nok materiale til alle test.

• 73 ikke medtaget pga. fejl.

• 2990 kvinder indgik i studiet (kvinder 30-65 år).

• 17 prøver var uegnet til cytologi undersøgelse.

• 8 prøver udgik pga. for lidt materiale til gentestning.

• Tilbage er der i alt 2965 prøver.

Tal fra screenings-studiet

HC2 positive HC2 negative

Cobas positive 298 56

Cobas negative 47 2553

11 ”failed” prøver er fratrukket, dvs. total antal er 2965-11 = 2954

Positiv procent Cobas: 12,0 %

Positiv procent HC2: 11,7 %

Enighed: 298+2553/2954 = 96,5 %

(Kappa værdi = 0,833)

Tal fra Screeningsstudiet

Cobas positive

Alder HPV16 HPV18 Other Dobbelt infektion

Cobas positive

Cobas negative Cobas failed HC2 positive HC2 negative

HC2 unclear

Total antal

30-39 46 (4.8%) 14 (1.5%) 139 (14.4%) 19 (2.0%) 179 (18.6%) 779 (81.0%) 4 (0.4%) 170 (17.7%) 791 (82.2%) 1 (0.1%) 962

40-49 22 (2.1%) 11 (1.0%) 87 (8.2%) 8 (0.8%) 112 (10.6%) 943 (89.0%) 5 (0.5%) 111 (10.5%) 949 (89.5%) 0 1060

50-59 4 (0.6%) 1 (0.2%) 44 (6.9%) 1 (0.2%) 48 (7.5%) 587 (92.3%) 1 (0.2%) 50 (7.9%) 586 (92.1%) 0 636

60-64 4 (1.3%) 0 (0.0 %) 12 (3.9%) 1 (0.3%) 15 (4.9) 292 (95.1%) 0 17 (5.5%) 290 (94.5%) 0 307

2965

Gennemsnit alder (år) 40,1 40,3 42,4 38,3 42,1 46,4 41,8 42,3 46,4 35,2

Cytologi

Normal 27 (1.0%) 13 (0.5%) 172 (6.3%) 0 204 (7.5%) 2498 (92.1%) 10 (0.4%) 192 (7.1%) 2519 (92.3%) 1 (0.04%) 2712

AGC 2 (18.2%) 2 (18.2%) 2 (18.2%) 1 (9.1%) 5 (45.5%) 6 (55.5%) 0 5 (45.5%) 6 (55.5%) 0 11

AIS 0 1 (100%) 0 0 1 (100%) 0 0 1 (100%) 0 0 1

ASCUS 11 (9.0%) 5 (4.1%) 38 (31.1%) 4 (3.3%) 50 (41.0%) 72 (59.0%) 0 52 (42.6%) 70 (57.4%) 0 122

LSIL 10 (18.9%) 1 (1.9%) 30 (56.6) 4 (7.5%) 37 (69.8%) 16 (30.2%) 0 40 (75.5%) 13 (24.5%) 0 53

ASCH 2 (12.5%) 0 11 (68.8%) 1 (6.3%) 12 (75.0%) 4 (25.0%) 0 12 (75.0%) 4 (25.0%) 0 16

HSIL 24 (48.0%) 4 (8.0%) 29 (58.0%) 11 (22.0%) 45 (90 %) 5 (10.0%) 0 46 (92.0%) 4 (8.0%) 0 50

total 76 26 282 21 354 2601 10 348 2616 1 2965

Cobas HPV positive og cytologi normale prøver

Antal Cobas

HPV positive

prøver i studiet

HPV positive og

cytologi normale

prøver i studiet

Estimeret antal

pr år. i SLB

optageområde

Prøver i alt 354 204 3407

60+ prøver 15 10 167

Kontrol

prøver 101 32 533

Hvordan skal de følges?

Krav til triage:

An ideal test should identify the small group of women at highest risk for treatable cervical precancers needing colposcopy referral, while reassuring the majority of women that their risk is low. In other words, an optimal strategy should identify as much disease as possible, while leading to as little colposcopy referral as possible.

Nicolas Wentzensen N. Lancet Oncol 2013;14(2):107-9.

Triage af HPV positive kvinder

o Cytologi (gør vi idag)

o HPV genotypning

o HPV mRNA

o p16/Ki-67 dual stain (CINtec Plus, Roche)

o Methylering (humane og virale gener)

o …

Se bla. Wentzensen et al., 2015 (J Clin Virol.)

Triage vhj HPV genotypning

Long-term HPV type-specific risks of

high-grade cervical intraepithelial

lesions: A 14-year follow-up of a

randomized primary HPV screening trial.

(Smelov et al., 2015, Int J Cancer)

Long-term risk of cervical intraepithelial neoplasia

grade 3 or worse according to high-risk human

papillomavirus genotype and semi-quantitative viral

load among 33,288 women with normal cervical

cytology.(Thomsen et al., 2015 , Int J Cancer)

Risiko for CIN3+ Risk

Women aged ≥30

years 8 years

follow-up 95% CI

HPV16 21.8 (18.0–25.6)

HPV18 (no 16) 12.8 (7.6–18.0)

HPV31 (no 16) 11.3 (7.7–14.9)

HPV33 (no 16) 12.9 (7.0–18.8)

Other hrHPV 3.9 (2.7–5.2)

hrHPV neg 0.6 (0.4–0.7)

CIN3+ Risk

HPV16/18/31/33 >28%

HPV35/45/52/58 14-18%

HPV39/51/56/59/66/68 <10%

Triage vhj. HPV mRNA

Comparing triage algorithms using HPV DNA genotyping, HPV E7 mRNA

detection and cytology in high-risk HPV DNA-positive women

Roosmarijn Luttmera, Johannes Berkhofb, Maaike G. Dijkstraa, Folkert J. van Kemenadea,

Peter J.F. Snijdersa, Daniëlle A.M. Heidemana, Chris J.L.M. Meijer. (J Clin Virol. 2015)

Conclusion: For detection of ≥CIN2 in hrHPV DNA-positive women, an

algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58

DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology.

CIN3+ Sen Spec. PPV NPV

1) mRNA+genotypning 77,5 52,2 32,6 88,6

2) HPV16/18 geno 72,5 52,2 31,2 86,4

3) cytologi 87,5 38,8 29,9 91,2

p16/Ki-67 som triage

The value of adding CINtec PLUS dual staining for p16 (INK4A)/KI-67 on COBAS HPV positive

women for detection of high grade CIN

Marianne Waldstrøm1,2, Janina Augustenas1, Rikke Kølby Christensen1 & Dorthe Ørnskov1, Department of Pathology , Vejle

Hospital, Denmark1 and Institute of Regional Health Services Research, University of Southern Denmark2.

Table 2

HPV results Total number

Cobas 16 positive (+/- 18, others) 60(80%) 15(20%) 75

Cobas 18 (+/- others) 14(56%) 11(44%) 25

Cobas other HPV types than 16,18 131(52%) 120(48%) 251

Total 205(58%) 146(42%) 351

HC2 positive 186 110 296

HC2 negative 19 36 55

CINtec PLUS

positive

CINtec PLUS

negative

Table 1CINtec PLUS

positive

CINtec PLUS

negative

Age (years)

30-39 108(61%) 68(39%)

40-49 50(45%) 62(55%)

50-64 47(75%) 16(25%)

Cytology

Normal 87 115

AGC 5 0

AIS 1 0

ASCUS 34 16

LSIL 25 11

ASCH 9 3

HSIL 44 1

Total 205 (58%) 146 (42%)

p16-Ki67 som triage

Table 3

Follow-up diagnoses

Cytology

Normal 4 3

ASCUS 3 1

LSIL 3 1

ASCH 1 0

Histology

Normal 26 16

CIN 1 12 10

CIN 2 15 3

CIN 3 49 3

AIS 1 0

Carcinoma 5 0

No follow-up available 86 109

Total 205 146

CINtec PLUS

positive

CINtec PLUS

negative

CIN3+ niveau (follow-up efter 6 mdr.) Sensitivitet Specificitet PPV NPV

Alle HPV positive prøver 94,8% 48,8% 26,8% 98,0%

HPV-other positive prøver 95,7% 52,2% 16,8% 99,2%

Metylering af humane gener som triage

A four-gene methylation marker panel as triage test in high-risk human papillomavirus positive

patients. (J.J.H. Eijsink et al., 2012, Int J of Cancer)

Combined Promoter Methylation Analysis of CADM1 and MAL: An Objective Triage Tool for High-Risk

Human Papillomavirus DNA–Positive Women. (Hesselink et al., 2011, Clinical Cancer Researche)

Combined CADM1/MAL methylation and cytology testing for colposcopy triage of

high-risk HPV-positive women. (De Strooper et al., 2014, Cancer Epi Biom Prev)

Follow-up of high-risk HPV positive women by combined cytology and bi-marker

CADM1/MAL methylation analysis on cervical scrapes. (Verhoef et al., 2015, Gynecol

Oncol).

Utility of methylation markers in cervical cancer early detection: appraisal of the state-

of-the-science. (Wentzensen et al., 2009, gynecol Oncol)

DAPK1, CADM1, RARB var hypermetylerede i alle studier

CIN3+: sen 84%, spec. 69%

CIN3+: sen 87,8%, spec. 53,6%

Epigenetik

Genetik handler om arvelighed og

hvordan vores gener videreføres fra

generation til generation

”Epi” fra græsk betyder ”ved siden af”

Epigenetikken, handler om de

arvelige forandringer, der ikke direkte

involverer ændringer i selve arvelige

materialet, DNA’et.

Epigenetik – genetisk identiske individer

By Kara Rogers January 16, 2012, Scientific American, 2013-04-02 Author：Pamela Prindle Fierro Source

Det centrale dogme

DNA

RNA

Protein

Regulering af gen-ekspression

Epigenetik

Epigenetik

Kilde: nihroadmap.nih.gov

Epigenetik

• Histon-modifikationer

• Metylering af DNA

• miRNA

• Imprinting

http://www.abcam.com/epigenetics/histone-modifications-a-guide

www.spandidos-publications.com

Metylering

CpG islands

DNMT = DNA methyltransferases

SAM-CH3 = S-adenosylmethionine

S-adenosyl homocysteine (SAH)

Human Papilloma Virus (HPV)

• HPV er ”non-enveloped, circular,

supercoiled, double-stranded

DNA”.

• HPV genomet er ca. 8000

nucleotides lang, og har 7-9 gener

(HSV 150.000 bp, E.coli 4,6x106

bp, Humane genome 3,3x109 bp).

HPV genomet

HPV optagelse i cellen

Livscyklus

Metylering: CpG sites i HPV16 – i alt 113

Analysis of CpG methylation sites and CGI among human papillomavirus

DNA genomes. (Galvan et al., 2011, BMC Genomics)

HPV Metylering

Target Antal artikler

LCR 12

L1-LCR 8

Whole genome 4

Review: Epigenetics of Human Papillomaviruses

Johannsen and Lambert, 2013, Virologi.

Methylated host cell gene promoter and human

papillomavirus type 16 and 18 predicting cervical lesions

and cancer. (Gasperov et al., 2015, PLOS ONE.)

L1 5’LCR Enhancer promoter

CpG sites i HPV HPV16

HPV metylering Human Papillomavirus 16, 18, 31 and 45 viral load, integration and

methylation status stratified by cervical disease stage. (Marongiu et al.,

2014, BMC Cancer)

Stor forskel i graden af

metylering blandt genotyperne

Forskel i evnen til at vise

forskelle ml low-grade og high-

grade dysplasie

HPV Metylering som triage

Validation of a DNA methylation HPV triage classifier in a screening

sample. (Lorincz et al., 2016 (Int J Cancer)).

• S5: metylering af L1 og L2 i HPV16, HPV18, HPV31, HPV33 samt promoteren i det

human gen EPB41L3.

• Screening cohorte i London, målt på hrHPV positive kvinder findes:

CIN3+ HPV16/18

genotypning

S5 classifier

Sensitivitet 0,58 (0,36-0,77) 0,84 (0,62-0,94)

Specificitet 0,69 (0,64-0,74) 0,63 (0,58-0,68)

Hyppigt anvendte metoder til at undersøge metylering:

Hyppigt anvendte metoder til at undersøge metylering:

Afrunding

• Skal vi interessere os for metylering som triage af

HPV DNA positive kvinder?

• Er cytologi på vej ud?