xist promoter methylation status as putative molecular...
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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
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(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
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(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"
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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"
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XIST = X-inactive specific transcript (lncRNA, Xq13.2)X
-ch
rom
oso
me
ina
ctiv
ati
on
Lobo et al. (submitted)
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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
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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
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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
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Sper
ma
tog
enes
isa
sses
smen
t
JOHNSEN’S SCORE
Laborious
Subjective(low reproducibility)
Invasive (biopsy)
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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
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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
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Va
lida
tio
no
fth
ese
ries
Case 59TE
Case 59SE
Case 271SE
Case 271EC
Mixed TGCTs
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XIST methylated fragment – TGCTsX
IST
pro
mo
ter
(de)
met
hyl
ati
on
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XIST demethylated fragment – TGCTsX
IST
pro
mo
ter
(de)
met
hyl
ati
on
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XIST demethylated fragment – TGCTsX
IST
pro
mo
ter
(de)
met
hyl
ati
on
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XIST demethylated fragment – Cell linesX
IST
pro
mo
ter
(de)
met
hyl
ati
on
High Resolution Melting (HRM) analyses
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XIS
T p
rom
ote
r(d
e)m
eth
yla
tio
nXIST demethylated fragment - spermatogenesis
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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
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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?
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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
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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
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Thank you for your attention!