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XIS IST pr promoter meth thylatio ion status as as pu putativ ive mole

• lecular bi

biomarker for

• r

tes estic icula lar ger erm cel cell l tum tumors

João

• ão Lob

Lobo Sand ndra ra Nu Nunes, nes, Vera ra Gon Gonçal alves, es, Dani niel ela a Barro rros-Silva, a, Annet nette e va van der r Berg, rg, Ad Gi Gillis, Le Leend endert rt HJ Looi Looijen jenga ga, Carm rmen en Jeró róni nimo, Rui Henr enriqu que

No conflicts of interest 11th September 2019

(T)GCTs: challenges

Prognosis Early diagnosis Targeted treatment Fertility preservation Resistance Response to therapy BIOMARKERS Epigenetics

• Dev. Biology

Lobo et al. Hum Pathol 2018

(T)GCTs are developmental cancers

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"

Development & (T)GCT biomarkers

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-chromosome inactivation

Lobo et al. (submitted)

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

X-chromosome inactivation

Lobo et al. (submitted)

METHYLATED DEMETHYLATED DEMETHYLATED EXPRESSED EXPRESSED NOT EXPRESSED

Rationale

XIST promoter (++region IV) consistently

demethylated in TGCTs,

(++Seminomas) No demethylated fragments in somatic male cancers XISTexpression in TGCTs (++Seminomas)

XIST and TGCTs

Looijenga et al. Am J Pathol 1997 Kawakami et al. Lancet 2004

Cancer survivors and fertility

Young-adults Overall good prognosis Long-term treatment side effects

Costa and Lobo et al. Epigenomics 2017

Spermatogenesis assessment

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

XIST and spermatogenesis

Richler et al. Nat Genet 1992

Rationale

Aims and Methodology

To explore the role of (de)methylated XIST promoter as a candidate biomarker For TGCTs For spermatogenesis status

Samples (2005- 2017) DNA extraction (Norgen kit/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

Validation of the series

Case 59 TE Case 59 SE Case 271 SE Case 271 EC

Mixed TGCTs

XIST methylated fragment – TGCTs

XIST promoter (de)methylation

XIST demethylated fragment – TGCTs

XIST promoter (de)methylation

XIST demethylated fragment – TGCTs

XIST promoter (de)methylation

XIST demethylated fragment – Cell lines

XIST promoter (de)methylation

High Resolution Melting (HRM) analyses

XIST promoter (de)methylation

XIST demethylated fragment - spermatogenesis

XIST promoter (de)methylation

XIST demethylated fragment - spermatogenesis

Context Sensitivity (%) Specificity (%) JS≥4 vs JS<4 75.7 100

Conclusions and perspectives

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?

XIST promoter: ongoing work

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 Jerónimo Leendert Looijenga

Department of Pathology Ângelo Rodrigues Paula Lopes Mariana Cantante Rita Guimarães Cancer Biology & Epigenetics Group Vera Gonçalves Daniela Barros Silva Sandra Nunes

Ethics approval: CES IPO 1/2018

IPO Porto The Netherlands

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

Urology Clinic Jorge Oliveira Joaquina Maurício Isaac Braga Department of Epidemiology Luís Antunes PMC Utrecht (Group Looijenga) Ad Gillis Annette van den Berg Rachita Lahri Dennis Timmerman Erasmus MC Rotterdam & LEPO Wolter Oosterhuis Lambert Dorssers Hans Stoop Willem Boellaard

Thank you for your attention!