Sara Gargiulo

Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents

Background: The major factors individually reported to be associated with an increased frequency of CDKN2A mutations are increased number of patients with melanoma in a family, early age at melanoma diagnosis, and family members with multiple primary melanomas (MPM) or pancreatic cancer. Methods: These four features were examined in 385 families with ⩾3 patients with melanoma pooled by 17 GenoMEL groups, and these attributes were compared across continents. Results: Overall, 39% of families had CDKN2A mutations ranging from 20% (32/162) in Australia to 45% (29/65) in North America to 57% (89/157) in Europe. All four features in each group, except pancreatic cancer in Australia (p = 0.38), individually showed significant associations with CDKN2A mutations, but the effects varied widely across continents. Multivariate examination also showed different predictors of mutation risk across continents. In Australian families, ⩾2 patients with MPM, median age at melanoma diagnosis ⩽40 years and ⩾6 patients with melanoma in a family jointly predicted the mutation risk. In European families, all four factors concurrently predicted the risk, but with less stringent criteria than in Australia. In North American families, only ⩾1 patient with MPM and age at diagnosis ⩽40 years simultaneously predicted the mutation risk. Conclusions: The variation in CDKN2A mutations for the four features across continents is consistent with the lower melanoma incidence rates in Europe and higher rates of sporadic melanoma in Australia. The lack of a pancreatic cancer–CDKN2A mutation relationship in Australia probably reflects the divergent spectrum of mutations in families from Australia versus those from North America and Europe. GenoMEL is exploring candidate host, genetic and/or environmental risk factors to better understand the variation observed.

CDKN2A mutations and MC1R variants in Italian patients with single or multiple primary melanoma.

We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital‐based study of single (SPM, n = 398) and multiple primary melanoma (MPM, n = 95). The overall frequency of CDKN2A mutations was 15.2%, and four‐fold higher in MPM than in SPM cases (OR = 4.27; 95% CI 2.43–7.53). The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases. Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The G101W and E27X founder mutations were the most common. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. One novel mutation, T77A, was identified. Several non‐coding variants with unknown functional significance were also found (5′UTR −25C > T, −21C > T, −67G > C, IVS1 +37G > C); the novel 5′UTR −21C > T variant was not detected in controls. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Compared to the SPM patients, MPM cases had a 2‐fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. We observed no interaction between CDKN2A status and the presence of MC1R variants. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.

CDKN2A is the main susceptibility gene in Italian pancreatic cancer families

Background Most familial pancreatic cancer (FPC) remains unexplained. The identification of individuals with a high genetic risk of developing pancreatic adenocarcinoma (PC) is important to elucidate its biological basis and is critical to better define emerging strategies for the detection of early pancreatic neoplasms. Patients and methods A series of 225 consecutively enrolled patients with PC were tested for CDKN2A mutations. After personal and family cancer histories of all the patients had been reviewed, a subset of the patients were classified as FPC and were also tested for mutations in PALLD, PALB2, BRCA1 and BRCA2 as FPC candidate genes. Results The CDKN2A mutation rate in the 225 PC cases was 5.7%. The CDKN2A founder mutations, p.E27X and p.G101W, were predominant, but the mutation spectrum also included p.L65P, p.G67R and two novel, potentially pathogenic variants, promoter variant c.-201ACTC>CTTT and p.R144C. None of the patients with FPC harboured germline mutations in PALLD, PALB2 or BRCA2. One family was positive for the BRCA1 UV variant p.P727L. Strikingly, five of 16 patients with FPC (31%) carried CDKN2A mutations. Conclusion These findings suggest that a sizeable subset of Italian FPC families may carry CDKN2A mutations. This result may be of value for identifying the best candidates for future PC screening trials in Italy.

Germline MLH1 and MSH2 mutations in Italian pancreatic cancer patients with suspected Lynch syndrome

Lynch syndrome is an inherited cancer syndrome caused by germline mutations in mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. LS predisposes to high risk of early-onset colorectal, endometrial and other tumors. Patients with Lynch syndrome have also been shown to have an elevated risk for pancreatic cancer (PC). In this study, we aimed to estimate the frequency of suspected Lynch syndrome among a series of 135 PC patients. Further, we wanted to determine the frequency of MMR gene mutations in the suspected Lynch syndrome cases. We also aimed to verify the pathogenicity of any novel non-truncating variants we might detect with a functional assay. Based on personal and/or familial cancer history, 19 patients were classified as suspected Lynch syndrome cases. DNA material for mutation analysis was available for eleven of them. Four patients were found to carry a total of five MLH1 or MSH2 variants. Of these, MSH2-Q402X, MSH2-G322D, and MLH1-K618A had been previously reported, while the MSH2-E205Q and MSH2-V367I variants were novel. MSH2-Q402X is a known stop mutation and reported here for the first time here in association with PC. MLH1-K618A was found in the unaffected branch of a kindred, suggesting that it may be a polymorphism or a low penetrance variant. MSH2-G322D likely does not cause a MMR defect, although this variant has also been associated with breast cancer as indeed seen in our patient. The novel variants MSH2-E205Q and MSH2-V367I were found in the same patient. Both novel variants were however functional in the applied MMR assay. Our findings suggest that only a small subset of pancreatic cancer patients carry pathogenic MMR mutations.

Early onset may predict G101W CDKN2A founder mutation carrier status in Ligurian melanoma patients

Although the presence of multiple cases of melanoma on the same side of a family is the best predictor of germline CDKN2A mutation, other features (i.e. early age at onset) may be useful to identify carriers. We analysed the records of 682 hospital-based Ligurian melanoma patients. Of these, 238 cases (34 familial, 14 non-familial multiple primary and 190 non-familial single primary melanomas) were consecutively enrolled for screening of the CDKN2A and CDK4 genes. Screening of the 34 familial patients revealed that nine were carriers of the CDKN2A G101W founder mutation. Of the 14 non-familial multiple primary melanoma patients, three carried the G101W founder mutation and one the P48T mutation. For the non-familial patients with a single melanoma, 17 of 190 carried germline CDKN2A mutations, with most (16/17) carrying the G101W Ligurian founder mutation and one a novel single base pair substitution, D74Y. The effect of mutation on age at diagnosis was significant (P=0.012) after correcting for melanoma type (familial or non-familial), number of primaries (single or multiple), gender and disease occurrence (incident or prevalent). Early age at onset may be a good predictor of CDKN2A mutation in Liguria, where the G101W founder mutation is prevalent among melanoma patients, independent of family history.

Contribution of germline mutations in the BRCA and PALB2 genes to pancreatic cancer in Italy

Pancreatic adenocarcinoma (PC) is the third most common cancer associated with BRCA mutations. Most notice has been given to BRCA2, while the association between BRCA1 and PC is less widely reported. Recently, PALB2 has been implicated in both PC and breast cancer (BC) susceptibility. We selected 29 Italian PC patients from a case–control study of PC according to their personal and family history of both PC and breast/ovarian cancer (BC/OC) and tested them for presence of germline mutations in BRCA1, BRCA2 and PALB2. We identified no germline mutations or deletions in PALB2, but detected 7 BRCA mutations (4 in BRCA1 and 3 in BRCA2). These findings suggest that PALB2 does not play a major role in PC susceptibility in our population. As we found an almost equal frequency of germline mutations in BRCA1 and BRCA2, germline alterations in either of these genes may explain a subset of Italian families presenting both PC and BC/OC. Moreover, as we began the observation of these families from probands who are affected by PC, we provide here a direct assessment of the role of PALB2 and BRCA mutations in PC susceptibility.

CDKN2A and MC1R analysis in amelanotic and pigmented melanoma

Amelanotic melanoma (AM) is a rare subtype of melanoma with little or no clinically visible pigment; it is more difficult to diagnose than pigmented melanoma (PM), and has a worse prognosis. In the attempt to find a genetic explanation for the distinction between AM and PM, we conducted a case–case study, matching AM and PM patients, and testing them for germline mutations in high- (p16INK4A, p14ARF, CDK4) and low-penetrance (MC1R) melanoma susceptibility genes. Similar CDKN2A mutations were found in both sets of melanomas. A p14ARF splice germline mutation was detected for the first time in an Italian family with AM. This rare mutation, which has been described only once previously, may be involved in predisposition to the amelanotic phenotype in combination with germline MC1R variants and coordinate somatic expression of pigmentation genes and their regulators.

Five novel germline function‐impairing mutations of CYLD in Italian patients with multiple cylindromas

To the Editor : The Brooke–Spiegler syndrome (BSS, OMIM 605041) – which predisposes to multiple cylindromas, trichoepitheliomas and spiradenomas – familial cylindromatosis (FC, OMIM 132700) and multiple familial trichoepithelioma (MFT, OMIM 601606) are autosomal dominant disorders with variable penetrance (1). Cylindromas also occur in individuals without a family history of the disease, usually as solitary lesions on the scalp or face (2), and occasionally undergo malignant transformation (3). The BSS, FC and MFT susceptibility gene is CYLD, a tumor suppressor gene that encodes a deubiquitinating enzyme and negatively regulates NF-KB signaling by deubiquitinating Tumor Necrosis Factor Receptor (TNFR) associated factor 2 (TRAF2) (4–7). Both germline and somatic CYLD mutations have been reported. Most are nonsense or frameshift mutations resulting in a truncated protein, only very few are missense or splicing mutations (2). We report here the results of the first molecular analysis of six consenting Italian patients with histologically confirmed multiple cylindromas, in which we identified five novel germline CYLD mutations (not found in 300 control chromosomes) (Table 1). Patient 1 is a 46-year-old man who began developing numerous small nodules on his scalp and thorax starting at the age of 42 (Fig. 1a). He had no offspring; his parents displayed no cutaneous lesions and consented to be tested. The patient harbored a single nucleotide duplication, c.561_562dupT, in exon 5 (Fig. 1f), which is predicted to change the reading frame resulting in a premature stop codon after two amino acids (p.Q188SfsX2). Neither of his parents carried the mutation. All of the mutations described by other studies so far are located in the 3′ two-thirds of the coding sequence in the mid-portion and C-terminal region. This is the first CYLD mutation lying far toward the N-terminal region in the first cytoskeleton-associated protein glycine-rich (CAPGLY) domain (Fig. 2). Interestingly, Gao et al. reported that this domain of CYLD is mainly responsible for the interaction of CYLD with microtubules (8). It has been suggested that, as a regulator of apoptosis and enhancer of mitotic entry, CYLD has both tumor-suppressing and tumor-promoting activity (9). This additional function of CYLD and the location of this mutation may explain the benign nature of most cylindromas, as seen in our patient, who, compared with the other patients we analyzed, had a milder phenotype. Patient 2 is a 79-year-old woman who began developing skin lesions at age 16 (Fig. 1b). Biopsies showed that most were cylindromas, some had combined features of cylindroma and spiradenoma and a number of the smaller nodules were trichoepitheliomas. A cutaneous carcinosarcoma on the trunk had been previously excised. She thus displayed the phenotypical features of BSS, as described in detail elsewhere (10). She referred that her mother had developed similar lesions. Her son, patient 3, aged 54, had developed six basal cell carcinomas and seven cylindromas. In both patients, we detected a novel single nucleotide duplication, c.1392_1393dupT, in exon 10 (Fig. 1g), which is predicted to change the reading frame resulting in a premature stop codon after 10 amino acids (p.G465WfsX10). This confirms that intra-familial phenotypic variability is a characterizing feature associated with mutations in CYLD. Patient 4 is a 70-year-old woman who began developing cylindromas in her early thirties (Fig. 1c). She did not refer having any affected relatives and had five healthy children. A 14 nucleotide deletion c.1893_1906delATATTATAGT GAAA in exon 13 was found (Fig. 1h). The deletion is predicted to change the reading frame resulting in a premature stop codon after 10 amino acids (p.E631_T636DfsX10).