Comprehensive analyses of genetic and clinical factors in patients affected by Testicular Germ Cell Tumor

Introduction: Testicular Germ Cell Tumor (TGCT) is a multifactorial, polygenic, and complex disease. It is the most common malignancy of men in their reproductive ages. This neoplasm has one of the highest heritability (37–48,9%) based mainly on epidemiological and Genome-Wide Association Study data. Epidemiological studies support that there is an increased familial cancer risk among TGCT patients’ family members. However, the studies are heterogeneous and sometimes controversial. Despite of the growing body of evidence regarding the involvement of genetic factors in TGCT susceptibility, our knowledge about its genetic basis remains scarce. In the latest study, aiming at the evaluation of 48 established DNA Repair (DR) genes in the etiopathogenesis of TGCT, CHEK2 has been identified as a new susceptibility gene with moderate penetrance. In addition, in a recent case report, TGCT has been linked to Lynch syndrome. Objectives: In order to identify new clinical and genetic risk factors of TGCT, and to explore whether TGCT may be part of a more generalized cancer predisposition, we performed two projects: i) an epidemiological study, where we aimed to estimate the familial cancer risk among TGCT patients’ relatives (first-degree and grandparents), and ii) Whole Exome Sequencing (WES) in TGCT patients with positive family history of cancers, to identify monogenic causes of the malignancy and to determine the role of DR genes in the etiopathogenesis of TGCT. Materials and Methods: For both projects included in this thesis, the patients were recruited at the Andrology Unit, Department of Experimental and Clinical Biomedical Sciences "Mario Serio", Centre of Excellence DeNothe, University of Florence, Florence, Italy and at the Andrology Department, Fundació Puigvert, Universitat Autònoma de Barcelona, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Barcelona, Spain. In the epidemiological study, which was a multicentric, retrospective, case-control study, a total of 1407 subjects were enrolled. Among them, 592 were affected by TGCT, 352 had oncohematological (OH) malignancy, and 463 were fertile cancer-free controls. Statistical analyses were performed using SPSS Software. In the second project, WES was carried out for 32 TGCT and one Leydig tumor patients, who had two or more family members affected by any type of malignant tumors. DNA was extracted form peripheral blood lymphocytes and WES was performed by NovaSeq 6000 System (Illumina) using the SureSelect Human All Exon V6 (Agilent Technologies) kit. After filtering for rare (Minor Allele Frequency < 0,01), presumed as deleterious, non-synonymous and splicing variants, we performed a first cross with 653 DR genes, and a second cross with 1731 Mendelian autosomal dominant genes from OMIM and COSMIC databases. For further classification of the variants’ pathogenicity, we used the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) criteria. Finally, a series of bioinformatic analyses were carried out. Results: In the first, epidemiological study we found that TGCT patients’ relatives have significantly more cancers (p value = 0,0001) than controls’ relatives. As comparing the TGCT cohort with another type of malignancy, such as OH, which affects young males, we did not observe significant differences. This implies that OH patients also have an increased familial cancer risk (p value = 0,0045). Furthermore, we report some site-specific associations, other than TGCT aggregation in case of familial TGCT, and OH malignancy aggregation in case of familial OH. The novelty of our study is that we defined the semen phenotype for all subjects of the different cohorts, thus we were able to assess not only the impact of tumors versus non-tumors on familial incidence of neoplasms but also to compare whether non-normozoospermic subjects have more tumors among family members. We report a 1,57-fold higher risk (p value = 0,0048) for tumor development among family members if the patient had severe spermatogenic disturbances (azoospermia and severe oligozoospermia with TSC < 5 million). Another interesting finding of our study was that we observed significantly less siblings among TGCT (mean number of siblings: 1,16) and OH (1,09) cases in respect to controls (2,07). In the WES study, we identified rare, predicted as damaging, germline variants of DR genes in 5 out of 32 (15,6 %) TGCT patients. We report seven variants in seven genes in the five subjects. Five out of 7 variants were loss-of-function, whereas two were missense. Three variants were classified as pathogenic, two as likely pathogenic, and two as “hot” VUS, according to ACMG criteria classification. Our main findings are related to Lynch syndrome (LS). We found mutations in three established LS genes (MSH6, MLH3, MLH1) in three patients with typical LS-associated tumor types among their relatives. The other mutated genes are involved in homologous recombination (FANCD2, XRCC3), nucleotide excision repair (ERCC3), and oxidative DNA damage repair (MUTYH). Further important finding of our study was the identification of two patients, both carrying two variants in different DR genes and presenting typical tumor types for both genes among their family members. Conclusions and wider implications: Our results show association between TGCT, increased familial cancer incidence, and sub/infertility. Therefore, there might be a common link between spermatogenic defects and a systemic problem leading to higher morbidity including cancer development. The biological explanation for such a relationship could be an overall genomic instability/DNA repair defects in the family, which is reflected in the occurrence of multiple cancers and subfertility. WES is a powerful tool for searching monogenic causes of TGCT in highly selected patients as it has been in our study on selected TGCT patients with family aggregation of cancers. DR genes might have a role in the etiopathogenesis of TGCT, which could explain the increased frequency of cancers among TGCT patients’ relatives. We therefore suggest to perform sequencing of DR genes in selected TGCT cases with clear signs of familial predisposition to cancer. We also propose that TGCT may be part of the Lynch syndrome associated urological malignancies. Therefore, an onco-andrological screening is suggested for the male members of Lynch syndrome families. On the other hand, in case of pathogenic variants in DR genes, other types of malignancies could occur later in life, hence a careful follow-up of these patients is strongly advised.