Simona Frigerio

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.

Cutaneous Melanoma in Childhood and Adolescence Shows Frequent Loss of INK4A and Gain of KIT

Childhood cutaneous melanoma is a rare disease with increasing incidence. It is not clear whether it differs from adult melanoma in etiology and clinical evolution. To genetically characterize childhood melanoma, 21 pediatric patients were studied by germ-line analysis of CDKN2A, CDK4, and MC1R genes. In addition, alterations in CDKN2A, c-Kit, BRAF, and NRAS genes were evaluated at the somatic level by direct gene sequencing, fluorescence in situ hybridization analysis, and immunohistochemistry. As a control group of susceptible patients, we studied patients from 23 melanoma-prone families. At the germ-line level, CDKN2A and MC1R gene variants were detected in 2/21 and 12/21 pediatric patients and in 9/23 and 19/22 in familial patients. At the somatic level, most lesions (9/14) from pediatric patients showed CDKN2A locus homozygous deletions and a null p16 immunophenotype, whereas most lesions (5/8) from familial patients were disomic and immunoreactive. A c-Kit low-polysomy profile seems to parallel CDKN2A homozygous deletions in pediatric melanoma whereas the single activating mutation observed segregates with familial patients. Loss of KIT protein expression was frequent (7/14) in pediatric melanomas, where metastatic cases were prevalent. BRAF(V600E) mutation occurred at a similar rate (approximately 50%) in lesions from pediatric and familial patients, whereas no NRAS mutations were detected.

CDKN2A and MC1R variants influence dermoscopic and confocal features of benign melanocytic lesions in multiple melanoma patients

Non‐invasive diagnostic tools are effective in the histomorphological study of melanocytic lesions. The role of melanoma susceptibility genes on melanocytic nevi histopathological features is not clear. The current study aimed to correlate genetic alterations and histomorphological features of melanocytic nevi. Clinical, dermoscopic and confocal features of 34 multiple melanoma patients and 34 controls were compared. Among patients with melanoma, carriers of CDKN2A mutations and/or MC1R variants, and wild‐type genes were also compared. In patients with melanoma, a lighter phototype (P = 0.051), a higher number of nevi (P < 0.01) and clinically atypical nevi (P < 0.01) were observed. At dermoscopy, these nevi showed a complex pattern (P = 0.011), atypical network (P = 0.018) and irregular pigmentation (P = 0.037); at confocal, an irregular meshwork pattern (P = 0.026) with atypical nests (P = 0.016) and an inflammatory infiltrate (P = 0.048) were observed. Among patients with melanoma genetically tested, CDKN2A G101W mutation carriers were more frequently younger (P = 0.023), with clinically atypical nevi (P = 0.050), with cytological atypia (P = 0.033) at confocal. G101W mutation and MC1R variants carriers showed hypopigmented nevi (P = 0.002) and, at confocal, roundish cells infiltrating the junction (P = 0.019). These data suggest an influence of CDKN2A mutation and MC1R variants in the development of dysplastic melanocytic lesions. Non‐invasive histomorphological evaluation, together with genetic studies, improves melanoma risk identification and early diagnosis, for a patient‐tailored management.

Two new CHEK2 germ-line variants detected in breast cancer/sarcoma families negative for BRCA1 , BRCA2 , and TP53 gene mutations

CHEK2 gene mutations occur in a subset of patients with familial breast cancer, acting as moderate/low penetrance cancer susceptibility alleles. Although CHEK2 is no longer recognized as a major determinant of the Li-Fraumeni syndrome, a hereditary condition predisposing to cancer at multiple sites, it cannot be ruled out that mutations of this gene play a role in malignancies arising in peculiar multi-cancer families. To assess the contribution of CHEK2 to the breast cancer/sarcoma phenotype, we screened for germ-line sequence variations of the gene among 12 probands from hereditary breast/ovarian cancer families with one case of sarcoma that tested wild-type for mutations in the BRCA1, BRCA2, and TP53 genes. Two cases harbored previously unreported mutations in CHEK2, the c.507delT and c.38A>G, leading to protein truncation (p.Phe169LeufsX2) and amino acid substitution (p.His13Arg), respectively. These mutations were not considered common polymorphic variants, as they were undetected in 230 healthy controls of the same ethnic origin. While the c.38A>G encodes a mutant protein that behaves in biochemical assays as the wild-type form, the c.507delT is a loss-of-function mutation. The identification of two previously unreported CHEK2 variants, including a truncating mutation leading to constitutional haploinsufficiency, in individuals belonging to families selected for breast cancer/sarcoma phenotype, supports the hypothesis that the CHEK2 gene may act as a factor contributing to individual tumor development in peculiar familial backgrounds.

Association of microRNA 146a polymorphism rs2910164 and the risk of melanoma in an Italian population.

Association of microRNA 146a polymorphism rs2910164 and the risk of melanoma in an Italian population Macarena Gomez-Lira, Silvia Ferronato, Elisa Orlandi, Anna Dal Molin, Giovanni Malerba, Simona Frigerio, Monica Rodolfo* and Maria Grazia Romanelli* Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Verona, Italy; Unit of Immunotherapy, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Correspondence: Macarena Gomez-Lira, Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Strada Le Grazie, 8. 37134 Verona, Italy, Tel.: +39 0458027674, Fax: 0458027180, e-mail: macarena.gomezlira@univr.it *Rodolfo M and Romanelli MG, are both co-senior authors

Common Delayed Senescence of Melanocytes from Multiple Primary Melanoma Patients

766 into the dorsal skin of C57BL/6J mice 3 times a week for 1 week. Epidermal thickness was significantly increased in TNS4-overexpressing mice by 70% compared with that in control mice (P < 0.05) despite comparable inflammation (CD45and K6-positive cells) and infection efficacy (GFP-positive cells) (Figure 2c). Consistently, the number of Ki67-positive cells was significantly higher in TNS4-overexpressing mice than in control mice (TNS4 vs. GFP 1⁄4 63% vs. 21%, P < 0.05) (Figure 2d). TNS4 not only regulates several receptor tyrosine kinases but also functions as an important linker between integrins and receptor tyrosine kinase signaling pathways (Muharram et al., 2014). TNS4 interactswith ITGB1andMET, increasing the protein stability of two receptors and leading to increased survival and proliferation of cancer cells (Muharram et al., 2014). Although many downstream signaling cascades linking integrins to proliferation have been identified, the specific components conveying integrin signals across adhesion complexes have not been identified (Moreno-Layseca and Streuli, 2014). Although we could not exclude the possibility that TNS4 might be regulated by integrins other than ITGB4, our findings showed that TNS4induced keratinocyte proliferation is mediated by activation of the ITGB4, FAK, and ERK signaling pathway. Collectively, our results indicate that TNS4 associates with ITGB4 and transmits integrin signals (Muharram et al., 2014) to FAK and ERK and that this downstream signaling cascade promotes cell proliferation in normal

Malignant and benign tumors associated with multiple primary melanomas: just the starting block for the involvement of MITF, PTEN and CDKN2A in multiple cancerogenesis?

To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org Malignant and benign tumors associated with multiple primary melanomas: just the starting block for the involvement of MITF, PTEN and CDKN2A in multiple cancerogenesis? Annamaria Pollio, Aldo Tomasi, Stefania Seidenari, Giovanni Pellacani, Monica Rodolfo, Simona Frigerio, Andrea Maurichi, Daniela Turchetti, Sara Bassoli, Cristel Ruini and Giovanni Ponti