Clinical genetic testing for familial melanoma in Italy: a cooperative study.

BACKGROUND: The Italian Society of Human Genetics' (SIGU) recommendations on genetic counseling and testing for hereditary melanoma state that clinical genetic testing can be offered to Italian melanoma families with at least two affected members. OBJECTIVE: In the framework of a cooperative study, we sought to establish the frequency of cyclin-dependent kinase inhibitor 2A mutations in melanoma families that underwent clinical genetic counseling and testing in accordance with the SIGU recommendations at 9 centers in different Italian regions. METHODS: Cyclin-dependent kinase inhibitor 2A testing was conducted by direct sequencing and multiplex ligation-dependent probe amplification analysis in melanoma families with at least two affected members. RESULTS: A total of 33% (68/204) of the families harbored cyclin-dependent kinase inhibitor 2A mutations. In the 145 families with two affected members the mutation frequency was 25%. Three novel mutations, L94P, A86T, and c.407dupG, were identified among the cases and not in 200 controls. LIMITATIONS: We were unable to perform separate analyses for individual centers, as in some cases the number of families was too small. CONCLUSIONS: The availability of clinical genetic testing for melanoma to families with just two affected members in the same branch is justified in Italy in terms of the likelihood of identifying a mutation.

Impact of E27X, a novel CDKN2A germ line mutation, on p16 and p14ARF expression in Italian melanoma families displaying pancreatic cancer and neuroblastoma.

Mutations in the CDKN2A gene underlie melanoma susceptibility in as many as 50% of melanoma kindreds in selected populations, and several CDKN2A founder mutations have been described. Inherited mutations in CDKN2A have been found to be associated with other, non-melanoma cancers including pancreatic cancer (PC) and neural system tumors (NST). Here we report a novel germline mutation in exon 1 of the CDKN2A gene, E27X, which we first detected in melanoma patients living in or originally from a small geographic area bordering Liguria in north-western Italy. A subset of melanoma kindreds positive for this mutation displayed PC and neuroblastoma. E27X generates a premature stop codon, leading to dramatically reduced protein levels of p16 and leaving p14ARF unaltered. As PC and NSTs have been postulated to be preferentially associated with CDKN2A mutations located in exon 2 and/or affecting p14ARF alone, the position of E27X in exon 1alpha provides interesting insights towards clarifying the mechanisms by which the CDKN2A/ARF locus is involved in cancer predisposition.

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.

Novel MC1R variants in Ligurian melanoma patients and controls.

Several variant forms of the melanocortin-1 receptor gene (MC1R) have been associated with red hair, fair skin and an increased risk for melanoma. Their involvement in melanoma susceptibility is apparently linked both to skin sensitivity and to non-pigmentary pathways. We investigated the frequency of the MC1R variants in the Italian region of Liguria, where the occurrence and penetrance of melanoma are low and primary susceptibility is characterized by prevalence of the CDKN2A c.301G>T [p.G101W] founder mutation. Additionally, we attempted to establish the frequency of the red hair/fair skin phenotype in our region. As predicted by anecdotal evidence, the frequency of red hair/phototype I was very low (0.7%). Screening of 17 red-haired individuals and their red-haired relatives, 207 controls and 214 melanoma patients unselected for hair color but all of Ligurian descent, led to the detection of 8 novel substitutions (c.133T>C [p.F45L], c.248C>T [p.S83L], c.332C>T [p. A111V], c.479G>A [p.R160Q], c.637C>T [p.R213W], c.793G>A [p. V265I], c.923C>T [p. T308M], c.943T>C [p.C315R]), 1 novel deletion (c.520_523delGTC [p.V174del]) and 3 novel synonymous variants (c.366G>C [p. V122V], c.684G>A [p. Q228Q], c.726C>T [p.T241T]). Preliminary genotype/phenotype correlation seems to indicate that other genes involved in the regulation of human pigmentation may mask the recessive action of high-penetrance MC1R alleles, thus determining the low frequency of at-risk phototypes and of incidence and/or penetrance of melanoma in Liguria.

Sentinel lymph node biopsy in patients with Stage I/II melanoma: Clinical experience and literature review.

BACKGROUND: The sentinel lymph node (sN) represents one of the most powerful predictors of the outcome of patients with Stages I and II cutaneous melanoma, and may be relevant for the therapeutic planning of early-stage melanoma patients. Since adopting the technique of lymphatic mapping with vital blue dye (Patent Blue-V) in July 1993, we have periodically up-dated the methodology and revised our results in order to define the contribution of radio-guided surgery (RGS) to the detection of the sN as well as the role of intraoperative frozen section examination of the sN. MATERIALS AND METHODS: Between July 1993 and December 1997, 180 patients with clinically node-negative primary cutaneous melanoma (Stages I-II) underwent sN biopsy followed by "selective lymph node dissection" (SLND) whenever sN metastasis was detected. Presently, complete data are available in 165 patients who were divided into two consecutive subsets of 39 and 126 patients, based on the technique for the identification of the sN: Patent Blue-V only or Patent Blue-V associated to RGS. Moreover, in this second subset of patients intraoperative frozen section findings were compared with definitive pathologic examination. RESULTS: As regards the first subset of 39 patients (17 males and 22 females; mean age 51.3 years), the sN was identified in 35 patients (89.7%); 8 patients (22.8%) were found to have metastatic melanoma cells in their sN, and they all underwent SLND of the affected basin. As regards the second set of 126 patients (54 males and 72 females; mean age 53.5 years), the sN was detected in every case by means of the combined technique (Patent Blue-V and RGS): in 4 of 126 patients (3.2%), the sN was detected by means of RGS only whereas in no patient was the sN detected by Patent Blue-V only. Frozen section examination was performed in 123 of 126 patients who had sN detection by Patent Blue-V and RGS, and the intraoperative examination had a sensitivity of 66.6% (22 of 33), specificity of 100% (90 of 90), negative predictive value of 89.1% (90 of 101), and accuracy of 91% (112 of 123). The benefit of frozen section examination in avoiding a two-stage procedure was 17.9% (22 of 123 patients). In patients with thicker lesions (pT(3)-pT(4)), the sensitivity and the benefit of intraoperative examination were 76% (19 of 25) and 32% (19 of 59 patients), respectively. CONCLUSIONS: Sentinel node lymphadenectomy can be better accomplished when both procedures (lymphatic mapping with Patent Blue-V and RGS) are used because the two methods look quite complementary. In fact, the use of the radiocolloid mapping allows to detect a hot spot in the regional basin prior to making the skin incision in order to perform a minimal invasive access, and it may also more accurately differentiate the true sN from a secondary echelon node (non-sN). The use of frozen section examination should be restricted to patients with pT(3)-pT(4) primary melanoma, due to the higher sensitivity and benefit in terms of avoiding a two-stage operative procedure.