Methyleringsprocesser i forbindelse med triagering af HPV forandringer

Methyleringsprocesser i forbindelse med triagering af HPV forandringer Molekylærbiolog, ph.d. Dorthe Ørnskov Klinisk patologi, Vejle Sygehus, Sygehus Lillebælt

Agenda o o o o o Screening og Triage o Indførelse af HPV som screenings-test i Vejle. o Mulige triage metoder Epigenetik HPV opbygning, cyklus HPV methylering o Hvad viser litteraturen o Hvilke metoder anvendes ofte 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 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 Total antal 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) CIN3+ Risk HPV16/18/31/33 >28% HPV35/45/52/58 14-18% HPV39/51/56/59/66/68 <10% 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+ Women aged 30 years Risk 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)

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) 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 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.

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 Waldstrøm 1,2, Janina Augustenas 1, Rikke Kølby Christensen 1 & Dorthe Ørnskov 1, Department of Pathology, Vejle Hospital, Denmark 1 and Institute of Regional Health Services Research, University of Southern Denmark 2. Table 1 CINtec 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%) Table 2 HPV results CINtec PLUS positive CINtec PLUS negative 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

p16-ki67 som triage Table 3 Follow-up diagnoses Cytology CINtec PLUS positive CINtec PLUS negative 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 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) CIN3+: sen 84%, spec. 69% 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). CIN3+: sen 87,8%, spec. 53,6% Utility of methylation markers in cervical cancer early detection: appraisal of the stateof-the-science. (Wentzensen et al., 2009, gynecol Oncol) DAPK1, CADM1, RARB var hypermetylerede i alle studier

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

Metylering www.spandidos-publications.com

CpG islands DNMT = DNA methyltransferases SAM-CH 3 = 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 4,6x10 6 bp, Humane genome 3,3x10 9 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.

CpG sites i HPV Methylated host cell gene promoter and human papillomavirus type 16 and 18 predicting cervical lesions and cancer. (Gasperov et al., 2015, PLOS ONE.) HPV16 L1 5 LCR Enhancer promoter

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 highgrade 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?