FONDAZIONE PROSSIMO MIO [2025 Update]
To investigate the genetic components of testicular germ cell tumors (TGCC) and identify genetic factors that can distinguish unilateral TGCC from metachronous or synchronous bilateral TGCC, we enrolled a cohort of 106 patients [Group 1] with unilateral (N=89) or bilateral (N=17) TGCC. These patients were recruited from the Operative Unit 1 (UO1) of the Urological Research Institute (URI) and the Urology Unit of the IRCCS Ospedale San Raffaele (Milan, Italy), in collaboration with the Italian Testicular Cancer Association (AITT).
Our goal is to characterize germline variants and identify genetic profiles that can serve as markers of early health deterioration.
Furthermore, this project aims to provide a better understanding of TGCC predisposition and the risk of recurrence, ultimately enabling the development of potential preventive strategies.
All patients underwent a detailed medical and sexual history, including age at the time of surgery and any prevalent comorbidities, assessed using the Charlson Comorbidity Index (CCI). Venous blood samples were collected from each patient to measure hormone levels, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (tT), estradiol (E2), inhibin B (InhB), sex hormone-binding globulin (SHBG), and tumor markers, including alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (beta-HCG), and lactate dehydrogenase (LDH). When possible, seminal fluid parameters were also evaluated to further characterize the reproductive health of the patients. Additionally, we have also started collecting medical history from first-degree relatives of Group 1 patients [Group 2]. Similarly, biological samples from Group 2 were collected directly at OU1, during follow-up visits if present, or via home collection kits.
For the entire Group 1, saliva samples were obtained for DNA extraction. Whole exome sequencing (WES) using Illumina technology was performed by UO2. After sequencing, a variant calling file (VCF) was generated. Subsequently, the variants were filtered and classified according to the guidelines of the American College of Medical Genetics (ACMG).
For the specific purposes of this project, an ad hoc genetic library was created to prioritize genes already known to be associated with TGCC susceptibility, as well as those generally involved in the functional aspects of the testis, germ cell biology, germ cell cancer, and infertility.
Preliminary analyses have suggested five genes with mutations of interest. Subsequent investigations, based on tissue-specific expression and functional characteristics, narrowed the selection to two genes.
Among these, MLH1 emerged as a priority candidate. It is a key component of the DNA mismatch repair (MMR) system, previously associated with cancer predisposition. Specifically, we focused on the K377E variant of MLH1, which involves the replacement of a basic amino acid with a positive charge with an acidic amino acid with a negative charge. However, according to ACMG criteria, this variant must be classified as a variant of uncertain significance (VUS), as it has not been previously reported as a pathogenic mutation in cancer. The clinical implications of MMR gene expression in TGCC have not been adequately studied. Interestingly, infertile males show a well-documented association with carcinoma in situ of the testis. Based on these results, we hypothesized a model in which MLH1 transcription, translation, and meiotic recombination processes may be influenced by the K377E variant. Reduced or impaired protein activity could stimulate positive feedback to overcome a functional deficiency.
Although TGCC is a rare neoplasm, representing only 5% of urological cancers, its incidence has increased in recent decades and is expected to grow further. Despite the identification of several risk factors, it is difficult to attribute the high relative risk of TGCC solely to shared environmental components. The decisive genetic factors for TGCC have yet to be identified, and the possible involvement of MLH1 in both tumorigenesis and germ cell function suggests a potential pathogenetic explanation for the epidemiological association between TGCC and male infertility. However, at present, the pathogenetic function of the identified gene cannot be defined with certainty.
This preliminary investigation requires further research to improve the understanding of the gene-tumor relationship. To proceed with this project, we intend to prioritize all genetic variants identified through WES analysis in patients from Group 1 and Group 2.
