Disruption of DNA damage response pathways in tumorigenesis: investigating the role of ATM and BAP1 in hereditary cancers.

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.)

Combining molecular and immunohistochemical analyses of key drivers in primary melanomas: Interplay between germline and somatic variations

Due to the high mutational somatic burden of Cutaneous Malignant Melanoma (CMM) a thorough profiling of the driver mutations and their interplay is necessary to explain the timing of tumorigenesis or for the identification of actionable genetic events. The aim of this study was to establish the mutation rate of some of the key drivers in melanoma tumorigenesis combining molecular analyses and/or immunohistochemistry in 93 primary CMMs from an Italian cohort also characterized for germline status, and to investigate an interplay between germline and somatic variants. BRAF mutations were present in 68% of cases, while CDKN2A germline mutations were found in 16 % and p16 loss in tissue was found in 63%. TERT promoter somatic mutations were detected in 38% of cases while the TERT -245T > C polymorphism was found in 51% of cases. NRAS mutations were found in 39% of BRAF negative or undetermined cases. NF1 was expressed in all cases analysed. MC1R variations were both considered as a dichotomous variable or scored. While a positive, although not significant association between CDKN2A germline mutations, but not MC1R variants, and BRAF somatic mutation was found, we did not observe other associations between germline and somatic events. A yet undescribed inverse correlation between TERT -245T > C polymorphism and the presence of BRAF mutation was found. It is possible to hypothesize that -245T > C polymorphism could be included in those genotypes which may influence the occurrence of BRAF mutations. Further studies are needed to investigate the role of -245T > C polymorphism as a germline predictor of BRAF somatic mutation status

Heterogeneity and frequency of BRAF mutations in primary melanoma: Comparison between molecular methods and immunohistochemistry

Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy. BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used. Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable. Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid - PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples. Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques. Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered. Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples. Combining different methods excludes that the observed heterogeneity is technique-based. We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results. Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered

Circulating MicroRNAs as easy-to-measure aging biomarkers in older breast cancer patients: Correlation with chronological age but not with fitness/frailty status

Circulating microRNAs (miRNAs) hold great promise as easily accessible biomarkers for diverse (patho)physiological processes, including aging. We have compared miRNA expression profiles in cell-free blood from older versus young breast cancer patients, in order to identify "aging miRNAs" that can be used in the future to monitor the impact of chemotherapy on the patient's biological age. First, we assessed 175 miRNAs that may possibly be present in serum/plasma in an exploratory screening in 10 young and 10 older patients. The top-15 ranking miRNAs showing differential expression between young and older subjects were further investigated in an independent cohort consisting of another 10 young and 20 older subjects. Plasma levels of miR-20a-3p, miR-30b-5p, miR106b, miR191 and miR-301a were confirmed to show significant age-related decreases (all p 640.004). The remaining miRNAs included in the validation study (miR-21, miR-210, miR-320b, miR-378, miR-423-5p, let-7d, miR-140-5p, miR-200c, miR-374a, miR376a) all showed similar trends as observed in the exploratory screening but these differences did not reach statistical significance. Interestingly, the age-associated miRNAs did not show differential expression between fit/healthy and non-fit/frail subjects within the older breast cancer cohort of the validation study and thus merit further investigation as true aging markers that not merely reflect frailty

Biological ageing and frailty markers in breast cancer patients

Older cancer patients are a highly heterogeneous population in terms of global health and physiological reserves, and it is often difficult to determine the best treatment. Moreover, clinical tools currently used to assess global health require dedicated time and lack a standardized end score. Circulating markers of biological age and/or fitness could complement or partially substitute the existing screening tools. In this study we explored the relationship of potential ageing/frailty biomarkers with age and clinical frailty. On a population of 82 young and 162 older non-metastatic breast cancer patients, we measured mean leukocyte telomere length and plasma levels of interleukin-6 (IL-6), regulated upon activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein 1 (MCP-1), insulin-like growth factor 1 (IGF-1). We also developed a new tool to summarize clinical frailty, designated Leuven Oncogeriatric Frailty Score (LOFS), by integrating GA results in a single, semi-continuous score. LOFS' median score was 8, on a scale from 0=frail to 10=fit. IL-6 levels were associated with chronological age in both groups and with clinical frailty in older breast cancer patients, whereas telomere length, IGF-1 and MCP-1 only correlated with age. Plasma IL-6 should be further explored as frailty biomarker in cancer patients

Computerized scoring protocol for identification and quantification of different immune cell populations in breast tumor regions

status: accepte

The impact of adjuvant chemotherapy in older breast cancer patients on clinical and biological aging parameters

Purpose. This prospective observational study aimed to evaluate the impact of adjuvant chemotherapy on biological and clinical markers of aging and frailty. Methods. Women 65 70 years old with early breast cancer were enrolled after surgery and assigned to a chemotherapy (Docetaxel and Cyclophosphamide) group (CTG, n=57) or control group (CG, n=52) depending on their planned adjuvant treatment. Full geriatric assessment (GA) and Quality of Life (QoL) were evaluated at inclusion (T0), after 3 months (T1) and at 1 year (T2). Blood samples were collected to measure leukocyte telomere length (LTL), levels of interleukin-6 (IL-6) and other circulating markers potentially informative for aging and frailty: Interleukin-10 (IL- 10), Tumor Necrosis Factor Alpha (TNF-\u3b1), Insulin-like Growth Factor 1 (IGF-1), Monocyte Chemotactic Protein 1 (MCP-1) and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES). Results. LTL decreased significantly but comparably in both groups, whereas IL-6 was unchanged at T2. However, IL-10, TNF-\u3b1, IGF-1 and MCP-1 suggested a minor biological aging effect of chemotherapy. Clinical frailty and QoL decreased at T1 in the CTG, but recovered at T2, while remaining stable in the CG. Conclusion. Chemotherapy (TC) is unlikely to amplify clinical aging or induce frailty at 1 year. Accordingly, there is no impact on the most established aging biomarkers (LTL, IL-6)

First international workshop of the ATM and cancer risk group (4-5 December 2019).

The first International Workshop of the ATM and Cancer Risk group focusing on the role of Ataxia-Telangiectasia Mutated (ATM) gene in cancer was held on December 4 and 5, 2019 at Institut Curie in Paris, France. It was motivated by the fact that germline ATM pathogenic variants have been found to be associated with different cancer types. However, due to the lack of precise age-, sex-, and site-specific risk estimates, no consensus on management guidelines for variant carriers exists, and the clinical utility of ATM variant testing is uncertain. The meeting brought together epidemiologists, geneticists, biologists and clinicians to review current knowledge and on-going challenges related to ATM and cancer risk. This report summarizes the meeting sessions content that covered the latest results in family-based and population-based studies, the importance of accurate variant classification, the effect of radiation exposures for ATM variant carriers, and the characteristics of ATM-deficient tumors. The report concludes that ATM variant carriers outside of the context of Ataxia-Telangiectasia may benefit from effective cancer risk management and therapeutic strategies and that efforts to set up large-scale studies in the international framework to achieve this goal are necessary

First international workshop of the ATM and cancer risk group (4-5 December 2019)

The first International Workshop of the ATM and Cancer Risk group focusing on the role of Ataxia-Telangiectasia Mutated (ATM) gene in cancer was held on December 4 and 5, 2019 at Institut Curie in Paris, France. It was motivated by the fact that germline ATM pathogenic variants have been found to be associated with different cancer types. However, due to the lack of precise age-, sex-, and site-specific risk estimates, no consensus on management guidelines for variant carriers exists, and the clinical utility of ATM variant testing is uncertain. The meeting brought together epidemiologists, geneticists, biologists and clinicians to review current knowledge and on-going challenges related to ATM and cancer risk. This report summarizes the meeting sessions content that covered the latest results in family-based and population-based studies, the importance of accurate variant classification, the effect of radiation exposures for ATM variant carriers, and the characteristics of ATM-deficient tumors. The report concludes that ATM variant carriers outside of the context of Ataxia-Telangiectasia may benefit from effective cancer risk management and therapeutic strategies and that efforts to set up large-scale studies in the international framework to achieve this goal are necessary