XIST promoter methylation status as putative molecular biomarker for

testicular germ cell tumorsJoão Lobo

Sandra Nunes, Vera Gonçalves, Daniela Barros-Silva, Annette van der Berg,Ad Gillis, Leendert HJ Looijenga, Carmen Jerónimo, Rui Henrique

No conflicts of interest11th September 2019

(T)G

CTs

: ch

alle

ng

es

Prognosis

Early diagnosis

Targeted treatment

Fertilitypreservation

Resistance

Response to therapy

BIOMARKERS

Epigenetics

Dev. BiologyLobo et al. Hum Pathol 2018

(T)G

CTs

are

dev

elo

pm

enta

lca

nce

rs

Lobo et al. Int J Mol Sci 2019; Oosterhuis & Looijenga, Nat Rev Cancers 2019;

“Germ cell tumors are at the crossroads between developmental biology and cancer"

Dev

elo

pm

ent

& (

T)G

CT

bio

ma

rker

s

Almstrup and Lobo et al. (submitted)

“Developmental biology as a driver for uncovering novel disease biomarkers"

XIST = X-inactive specific transcript (lncRNA, Xq13.2)X

-ch

rom

oso

me

ina

ctiv

ati

on

Lobo et al. (submitted)

XIST = X-inactive specific transcript (lncRNA, Xq13.2)X

-ch

rom

oso

me

ina

ctiv

ati

on

Lobo et al. (submitted)

METHYLATED DEMETHYLATED DEMETHYLATED

EXPRESSED EXPRESSEDNOT EXPRESSED

Rationale

XIST promoter (++region IV) consistently

demethylated in TGCTs,

(++Seminomas)

No demethylated fragments in somatic male cancers

XIST expression in TGCTs (++Seminomas)

XIS

T a

nd

TGC

TsLooijenga et al. Am J Pathol 1997

Kawakami et al. Lancet 2004

Ca

nce

r su

rviv

ors

an

d f

erti

lity

Young-adults

Overall good prognosis

Long-term treatmentside effects

Costa and Lobo et al. Epigenomics 2017

Sper

ma

tog

enes

isa

sses

smen

t

JOHNSEN’S SCORE

Laborious

Subjective(low reproducibility)

Invasive (biopsy)

XIST is expressed (=demethylated) in the testis (spermatogenesis) around

the time of entering meiosis

This process of XIST activation in spermatogenesis is regulated by

METHYLATION

XIS

T a

nd

sper

ma

tog

enes

is

Richler et al. Nat Genet 1992

Rationale

Aim

sa

nd

Met

ho

do

log

y

To explore the role of (de)methylated XIST promoter as a candidate biomarker

For TGCTs

For spermatogenesis status

Samples (2005-2017)

DNA extraction

(Norgenkit/PC8)

Bisulfite treatment (Zymo kit)

qMSP(ABI 7500

RT PCR System)

250 TGCTs (tumor components)

54 testicular parenchyma (JS)

4 (T)GCT cell lines

Same primer set reported by Kawakami et al

Va

lida

tio

no

fth

ese

ries

Case 59TE

Case 59SE

Case 271SE

Case 271EC

Mixed TGCTs

XIST methylated fragment – TGCTsX

IST

pro

mo

ter

(de)

met

hyl

ati

on

XIST demethylated fragment – TGCTsX

IST

pro

mo

ter

(de)

met

hyl

ati

on

XIST demethylated fragment – TGCTsX

IST

pro

mo

ter

(de)

met

hyl

ati

on

XIST demethylated fragment – Cell linesX

IST

pro

mo

ter

(de)

met

hyl

ati

on

High Resolution Melting (HRM) analyses

XIS

T p

rom

ote

r(d

e)m

eth

yla

tio

nXIST demethylated fragment - spermatogenesis

XIS

T p

rom

ote

r(d

e)m

eth

yla

tio

nXIST demethylated fragment - spermatogenesis

Context Sensitivity (%) Specificity (%)

JS≥4 vs JS<4 75.7 100

Co

ncl

usi

on

sa

nd

per

spec

tive

s

To explore the role of (de)methylated XIST promoter as a candidate biomarker

For TGCTs

For spermatogenesis status

Testicular germ cell tumors

Early (differential) diagnosis

Follow-up

Especially for Seminoma

Spermatogenesis status

Improve selection of patients for TESE or other

fertility preservation techniques

Non-invasively?

XIS

T p

rom

ote

r: o

ng

oin

gw

ork

HRM – High-resolution melting

Detection of TGCTs in liquid biopsies (serum/plasma)

Patient monitoring

Upfront assessment of male patients’ fertility (semen samples)

Liquid biopsies

Supervising team:Rui Henrique

Carmen JerónimoLeendert Looijenga

Department of PathologyÂngelo Rodrigues

Paula LopesMariana Cantante

Rita Guimarães

Cancer Biology & Epigenetics GroupVera Gonçalves

Daniela Barros SilvaSandra Nunes

Ethics approval: CES IPO 1/2018

IPO Porto

The Netherlands

Project Funding: POCI-01-0145-FEDER-29043Doctoral Grant: SFRH/BD/132751/2017

Urology ClinicJorge Oliveira

Joaquina MaurícioIsaac Braga

Department of EpidemiologyLuís Antunes

PMC Utrecht (Group Looijenga)Ad Gillis

Annette van den BergRachita Lahri

Dennis Timmerman

Erasmus MC Rotterdam & LEPOWolter OosterhuisLambert Dorssers

Hans StoopWillem Boellaard

Thank you for your attention!