Differential methylation EPIC analysis discloses cisplatin-resistance related hypermethylation and tumor-specific heterogeneity within matched primary and metastatic testicular germ cell tumor patient tissue samples

João Lobo,Vera Constâncio,Rui Henrique,Joaquina Maurício,Rita Guimarães,Leendert H J Looijenga,Isaac Braga,Pedro Leite-Silva,Mariana Cantante,Carmen Jerónimo

doi:10.1186/s13148-021-01048-y

Abstract

Testicular germ cell tumors (TGCTs) are among the most common solid malignancies in young-adult men, and currently most mortality is due to metastatic disease and emergence of resistance to cisplatin. There is some evidence that increased methylation is one mechanism behind this resistance, stemming from individual studies, but approaches based on matched primary and metastatic patient samples are lacking. Herein, we provide an EPIC array-based study of matched primary and metastatic TGCT samples. Histology was the major determinant of overall methylation pattern, but some clustering of samples related to response to cisplatin was observed. Further differential analysis of patients with the same histological subtype (embryonal carcinoma) disclosed a remarkable increase in net methylation levels (at both promoter and CpG site level) in the patient with cisplatin-resistant disease and poor outcome compared to the patient with complete response to chemotherapy. This further confirms the recent results of another study performed on isogenic clones of sensitive and resistant TGCT cell lines. Differentially methylated promoters among groups of samples were mostly not shared, disclosing heterogeneity in patient tissue samples. Finally, gene ontology analysis of cisplatin-resistant samples indicated enrichment of differentially hypermethylated promoters on pathways related to regulation of immune microenvironment, and enrichment of differentially hypomethylated promoters on pathways related to DNA/chromatin binding and regulation. This data supports not only the use of hypomethylating agents for targeting cisplatin-resistant disease, but also their use in combination with immunotherapies and chromatin remodelers.

Highlights

Germ cell tumors (GCTs) comprise a heterogeneous group of neoplasms that arise in both genders—in the gonads and in extragonadal sites—and within a wide age range, from pediatric age to adolescence/adulthood and older age [1]
Clinical samples A total of twelve type II Testicular germ cell tumors (TGCTs) individual samples, belonging to four patients, were prepared for EPIC methylation array and included in the study: patient #1 with a primary testicular mixed tumor and a yolk sac tumor bone metastasis; patient #2 with a primary testicular seminoma and a seminoma lymph-node metastasis; patient #3 with a primary testicular embryonal carcinoma and four chemo-exposed metastases with viable embryonal carcinoma, two in the lung and two in lymph-nodes; and patient #4 with a primary testicular embryonal carcinoma and an embryonal carcinoma lung metastasis, who showed a complete response to cisplatin-based chemotherapy
Differential methylation related to clinical response to cisplatin we investigated the differential methylation between embryonal carcinoma samples of patient #3 and embryonal carcinoma samples of patient #4

Summary

Introduction

Germ cell tumors (GCTs) comprise a heterogeneous group of neoplasms that arise in both genders—in the gonads (testis and ovary) and in extragonadal sites (related to migration of primordial germ cells along the midline of the body)—and within a wide age range, from pediatric age (type I) to adolescence/adulthood (type II) and older age (type III) [1]. Of all seven distinct classes of GCTs, the type II tumors of the testis (TGCTs) are by far the most common and present most clinical challenges, including those related to early diagnosis, appropriate treatment strategies, adequate follow-up and emergence of metastatic disease and resistance to platin-based chemotherapy [3]). Global hypomethylation has been suggested to be in part responsible for the outstanding sensitivity to cisplatin This lack of studies in this niche is in part because tissue samples from metastatic locations with remaining viable tumor are rarely available, with most studies focusing on the investigation of chemo-naïve primary tumor samples [18], which has limitations. Recent studies have provided big data analyses on copy number variations and mutations [19,20,21,22], and an interesting recent study has provided strong and complete data on differential mRNA expression among sensitive and resistant cell lines [23], genome-wide methylation data in paired clinical samples is lacking

Objectives

Methods

Results

Conclusion