The use of massive parallel sequencing techniques for genetic testing of hereditary cancer syndromes provides the possibility of analyzing multiple genes at the same time with limited costs. However, novel genes are being found in melanoma-prone families, but in many cases a causal relation has not been yet established. Moreover, the same gene is often found associated with multiple tumors, suggesting that spectrum of non-melanoma cancers in melanoma tumor syndromes could be broader than that currently known. This thesis describes the effort to tackle these issues, with a particular focus on two genes involved in DNA damage repair: the Ataxia-Telangiectasia Mutated (ATM) and BRCA1-associated protein 1 (BAP1) genes.
During the last year of my PhD I completed the first and carried out the second of the following two studies: \u201cGermline ATM variants predispose to cutaneous melanoma: a joint analysis across the genomel and melanostrum consortia\u201d, and \u201cBAP1-tumor predisposition syndrome and susceptibility to breast cancer\u201d.GERMLINE ATM VARIANTS PREDISPOSE TO CUTANEOUS MELANOMA: A JOINT ANALYSIS ACROSS THE GENOMEL AND MELANOSTRUM CONSORTIA.
ATM germline pathogenic variants predispose to several types of cancers, such as breast cancer and pancreatic cancer, and we recently found rare loss-of-function variants (LOF) in melanoma-prone families. However, considering that current literature on melanoma is non univocal, and that ATM is a large gene with a high number of variants of unknown significance (VUS), it is unclear whether ATM is a melanoma predisposition gene.
To assess if rare germline ATM variants are enriched in melanoma, we conducted and coordinated a multicentric study in collaboration with the GenoMel and MelaNostrum consortia. From 10 countries, we collected 261 multiple primary melanoma (MPM) cases, 941 apparently sporadic cases, and 734 melanoma-prone families probands who underwent ATM genotyping via panel, exome or whole genome sequencing. Nonsense, frameshift and canonical splice variants were considered LOF. Missense variants with an allele frequency (AF) above that expected of ataxia-telangiectasia heterozygous carriers (0.005), or found homozygous in more than two GnomAD individuals, were excluded, whereas the remaining missense variants were included as VUS. We then compared the (AF) of selected ATM variants in our cohort and in GnomAD non-Finnish Europeans (NFE).
We found 19 ATM LOF (18 unique) in 10 familial, 3 MPM and 6 sporadic cases, a frequency higher than that of the GnomAD NFE cohort (AF 0.005 vs 0.002, OR=2.68, p=0.0002), especially when restricting the analysis to familial+MPM cases (AF 0.006 vs 0.002, OR=3.66, p=0.0004). VUSs had a similar, albeit weaker association, both when assessing all (AF 0.05 vs 0.033, OR=1.53, p<0.001) and familial+MPM cases (AF 0.07 vs 0.033, OR=2.22, p<0.001). In 2 families with >1 genotyped cases, LOF co-segregated with melanoma, and one showed partial co-segregation. These results show that ATM variants are enriched in melanoma cases, suggesting that ATM is a melanoma-predisposition gene.BAP1-TUMOR PREDISPOSITION SYNDROME AND SUSCEPTIBILITY TO BREAST CANCER.
Germline LOF variants in the BAP1 gene underlie the BAP1-Tumor Predisposition Syndrome (BAP1-TPDS), characterized by an increased susceptibility to melanoma (cutaneous and uveal) and other cancers, such as mesothelioma, clear cell renal cancer (CCRC), basal cell carcinoma, Melanocytic BAP1-associated intradermal tumor (MBAIT). However, the tumor spectrum of the BAP1-TPDS is not yet completely defined, as more types of cancers, including breast cancer, are being found associated with BAP1. The inclusion of a new type of cancer in the BAP1-TPDS is a complex task when it comes to cancers with a high incidence in the population, such as breast cancer. In fact, in the case of extremely rare syndromes such as BAP1-TPDS, it is difficult to determine if the occurrence of a high-incidence cancer is part of the syndrome or is merely due to chance.
During my research period at QIMR Berghofer (Brisbane, AU) from December 2018 to August 2019, we performed a study to assess if BAP1 is a breast cancer predisposition gene. First, we imputed and analyzed data from the BAP1 database of the recently established BAP1-Interest Group (BIG) Consortium, and we found an enrichment for breast cancer in 15% of families with BAP1 LOF variants.
To determine if germline BAP1 variants are enriched in breast cancer, we then assessed the frequency and type of BAP1 variants in 6088 BRCA1/2 negative high-risk breast cancer cases and 5847 healthy controls, who underwent germline testing via multi-gene panels.
We found four LOF in seven cases and one control, and 58 missense in 59 cases (0.9%) and 38 controls (0.6%). Five familial probands had the same c.783+2T>C splice site variant. We are currently performing a functional analysis to confirm that the variant affects splicing. Immunohistochemistry of tumor samples, only available for a small subset of patients, showed absence of BAP1 nuclear staining in three patients carrying the c.783+2T>C.
These data are in support of the hypothesis that BAP1 may be implicated in breast cancer predisposition. However, due to the scarcity of samples available for LOH assessment, and the fact that we don\u2019t have sufficient information to assess co-segregation in these families, there is still not enough supportive evidence to include breast cancer in the BAP1-TPDS.OTHER PROJECTS: CDKN2A AND SURVIVAL IN CUTANEOUS MELANOMA PATIENTS.
CDKN2A germline pathogenic variants have recently been associated with poor survival in melanoma patients. Despite the high mutation rate in melanoma patients from our centre (up to 10% in apparently sporadic patients), information on CDKN2A impact on survival in these patients is lacking. During the first year of my PhD course, I carried out a study to investigate whether poor survival associated with CDKN2A germline pathogenic variants recently observed in a Norwegian melanoma cohort could be confirmed in a high mutation prevalence Italian cohort of melanoma patients undergoing a mutation-based follow-up. Our study cohort consisred of one-thousand-two-hundred-thirty-nine cutaneous melanoma patients were tested for CDKN2A mutational status and then assigned to a follow-up scheme, according not only to family history but also to CDKN2A mutational status, as follow-up intervals were more frequent for CDKN2A mutation positive (MUT+) patients.
From this cohort, we selected 106 MUT+ patients (familial and apparently sporadic), and 199 CDKN2A mutation negative (MUT-) sporadic patients, matched by age and sex, and with similar tumor stage distribution. We found no difference in overall survival (Hazard Ratio (HR)=0.85, p=0.592, CI=0.48-1.52) and melanoma-specific survival (HR=0.86, p=0.718, CI=0.38-1.95) between MUT+ and MUT- patients. MUT+ patients were more likely to develop multiple melanomas and to undergo surgical excision of dysplastic nevi compared to MUT- patients.
These results show that CDKN2A pathogenic variants are not associated with survival in our cohort (project published: Dalmasso, Bruna, et al. "CDKN2A germline mutations are not associated with poor survival in an Italian cohort of melanoma patients." Journal of the American Academy of Dermatology 80.5 (2019): 1263-1271.)
