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!
