Petra Pateisky Dietmar Pils, Lorenz Kuessel, Ladislaus Szabo,

Christine Staudigl, Arndt Schmitz, René Wenzl, Iveta Yotova

Contact: petra.pateisky@meduniwien.ac.at

Circulating miRNAs

biomarkers for Endometriosis?

Why biomarkers for endometriosis?

!  prevalence: 10% of women in their reproductive age !  heterogenous symptoms - delay of diagnosis !  diagnosis – management – surgery?

!  clinical examination, palpation, Imaging techniques – TIE

!  Subgroup of patients

Giudice LC, Kao LC. Endometriosis. Lancet2004;364(9447):1789-99

Function of miRNAs

Ohlsson Teague et al. Human Reproduction Update, Vol.16, No.2 pp. 142–165, 2010

microRNAs - circulating - tissue-bound

miRNAs are involved in several processes within endometriosis

Study Aims

search for differences in the plasma of secreted miRNAs of patients with versus without endometriosis

evaluate the correlation of differentially secreted miRNAs with the disease Can we predict endometriosis with a model taking into account miRNA-expression

signatures?

miRNAs in the plasma

microRNA – based arrays Search in the blood of patients - qPCR

!  Evaluation of found differences in bigger patients cohort

!  Change of expression differences within course of disease?

Fold change in comparison to one control patient

Pre-Screen: 3 endometriosis-patients 3 control-patients

Fold Change (comparing to control group)Group 1

Position Mature ID Fold Change CommentsA01 hsa-let-7a 1,7777 OKAYA02 hsa-miR-1 0,2717 AA03 hsa-miR-100 0,9138 OKAYA04 hsa-miR-106b 3,9449 OKAYA05 hsa-miR-10b 2,8284 OKAYA06 hsa-miR-122 1,4044 OKAYA07 hsa-miR-124 0,4763 AA08 hsa-miR-125b 0,3711 OKAYA09 hsa-miR-126 1,257 OKAYA10 hsa-miR-133a 0,2932 BA11 hsa-miR-133b 1,257 BA12 hsa-miR-134 0,2449 AB01 hsa-miR-141 0,0718 AB02 hsa-miR-143 0,4538 OKAYB03 hsa-miR-146a 1,1487 OKAYB04 hsa-miR-150 1,1567 OKAYB05 hsa-miR-155 0,79 AB06 hsa-miR-17 2,9282 OKAYB07 hsa-miR-17* 2,6574 OKAY

Phase I: Prescreening samples: 4 pools of plasma samples composed of n=20 individual plasma Pool1: controls–proliferative phase Pool2: controls-secretory phase Pool3: endometriosis-proliferative phase Pool4:endometriosis- secretory phase

Phase II: Discovery Samples: Individual plasma samples used in Phase I n=20

Phase III: Validation New control/patient samples; n=74

Method:. Q-PCR miRNA screening miRCURY LNA™ Universal RT microRNA PCR, ready to use human Panel IV3 (Exiqon)

Data analysis R-package software and GenEx (Exicon), Candidate identification and diagnostic model building

Data analysis; model revision training and validation

Project Workflow

Project Workflow - details

1.Sample quality control Hemolysis check: optical density (OD415nm) and Q-PCR for miRNA23a and miRNA 451

2.miRNA-Isolation&Pool building Equal amount of individual samples miRNAs, followed by cDNA synthesis (Exiqon)

4. Q-PCR miRNA screening miRCURY LNA™ Universal RT microRNA PCR, human Panel I, V3 (Exiqon) - 384 including controls

5. Data analysis/ Candidates total expressed genes: 154 candidates: n=75

1. Q-PCR miRNA screening N=75 candidates (including 6 controls)

2. Data analysis, Candidate identification and diagnostic model building (Lasso/penalized support vector machines (SVM) Total candidate genes: 36

Phase I: Prescreening samples: 4 pools of plasma samples composed of n=20 individual plasma

Phase II: Discovery samples: Individual plasma samples used in Phase I n=20

1.Q-PCR miRNA screening Total candidate miRNAs: 36 (plus 6 controls)

2. Data analysis; model revision training and validation

Final list of biomarker candidates for negative selection validation

Phase III: Validation New control/patient samples; n=74

Results – Phase I, Pool Analysis

!  Panel of 372 spotted miRNAs !  4 Plasma Pools !  154 miRNAs differently expressed

!  Heat map – expression profile

Phase I - Results

27 miRNAs- disease associated changes ; 12 miRNA – phase specific changes

Unique Expression Pattern

miRNA con_prol con_sec endo_prol endo_sec hsa-miR-a NA NA 35.69 33.78 hsa-miR-b NA NA 34.07 34.48 hsa-miR-c NA NA 35.33 34.93 hsa-miR-d NA NA 36.39 34.46 hsa-miR-e NA NA 36.95 35.83 hsa-miR-f NA NA 35.68 36.73 hsa-miR-g 35.22 34.64 NA NA hsa-miR-h 36.24 35.63 NA NA

Phase II - Discovery

2 independent analysis

75 miRNAs (incl. 6 house keeping) 20 same patiens - individually Normalization: Geometric mean of 6 house keepings 67 miRNAs + 6 house keeping for model

statistical models – prediction possible? 2 Models for target prediction

Phase II – Discovery

Penalized Support Vector Machine (SVM) model. „high dimension, low sample size“ analysis. x= -1 controls, X= 1 endometriosis

Model Building: 67 miRNAs 4 clinical parameters: age, BMI, VAS dysmen, phase

LASSO / elastic net: logistic regression with feature selection

Phase II - Discovery

Phase III – Validation: measure 36 miRNAs (+6 controls) in an independent patient set

Phase III

!  74 patients as a separate cohort EM – n=43 (58%), controls – n= 31 (42%) rAFS I/II – n=18 (42%), rAFS III/IV – n=25 (58%)

!  model of Phase II not applicable to larger cohort !  Why? - high biological variability of patient cohort?

miRNA P-value logFC

hsa.miR.-1 <0.01 14.525

hsa.miR.-2 <0.05 0.4409

hsa.miR.-3 <0.05 0.8123

hsa.miR.-4 <0.05 0.9983

hsa.miR.-5 <0.05 15.459

Single expression analysis:

Conclusion and Outlook

! miRNA-expression profiles differ ! Next steps: !  link miRNA-expression with different entities in

extra analysis (DIE, OMA, superficial endometriosis)

! screen tissue of patients – correlation changes of miRNA in the tissue and plasma??

! Biological background of disease – regulated genes?

Endometriosis Center Vienna: www.meduniwien.ac.at/endometriosezentrum

Function of miRNAs

!  non-coding, posttranscriptional regulation of gene expression

repression of translation of proteins post-transcriptional silencing or stimulating

transcript degradation ! Regulation of 1/3 from the cellular transcriptome !  eutopic endometrium vs. ectopic lesions

!  role in cancer, myocardial infarction, recurrent pregnancy loss