Lorenza Pastorino

A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.

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.

Genome-wide association study identifies three new melanoma susceptibility loci

We report a genome-wide association study for melanoma that was conducted by the GenoMEL Consortium. Our discovery phase included 2,981 individuals with melanoma and 1,982 study-specific control individuals of European ancestry, as well as an additional 6,426 control subjects from French or British populations, all of whom were genotyped for 317,000 or 610,000 single-nucleotide polymorphisms (SNPs). Our analysis replicated previously known melanoma susceptibility loci. Seven new regions with at least one SNP with P < 10−5 and further local imputed or genotyped support were selected for replication using two other genome-wide studies (from Australia and Texas, USA). Additional replication came from case-control series from the UK and The Netherlands. Variants at three of the seven loci replicated at P < 10−3: an SNP in ATM (rs1801516, overall P = 3.4 × 10−9), an SNP in MX2 (rs45430, P = 2.9 × 10−9) and an SNP adjacent to CASP8 (rs13016963, P = 8.6 × 10−10). A fourth locus near CCND1 remains of potential interest, showing suggestive but inconclusive evidence of replication (rs1485993, overall P = 4.6 × 10−7 under a fixed-effects model and P = 1.2 × 10−3 under a random-effects model). These newly associated variants showed no association with nevus or pigmentation phenotypes in a large British case-control series.

Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown. Using whole-exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin gene POT1 (chromosome 7, g.124493086C>T; p.Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere lengths and numbers of fragile telomeres, suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two separate Italian families, one variant per family, yielding a frequency for POT1 variants comparable to that for CDKN2A mutations in this population. These variants were not found in public databases or in 2,038 genotyped Italian controls. We also identified two rare recurrent POT1 variants in US and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

Identification of a SUFU germline mutation in a family with Gorlin syndrome.

Gorlin syndrome (GS) is inherited in an autosomal dominant pattern with high‐penetrance and is characterized by a range of developmental anomalies and increased risk of developing basal cell carcinoma and medulloblastoma. Between 50% and 85% of patients with GS harbor germ line mutations in the only susceptibility gene identified to date, PTCH1, a key component in the Sonic Hedgehog signaling pathway. Another component in this pathway, SUFU, is known to be involved in susceptibility to medulloblastoma but has never been reported in GS patients to date. We have identified the known c.1022 + 1G>A SUFU germ line splicing mutation in a family that was PTCH1‐negative and who had signs and symptoms of GS, including medulloblastoma. This is the first report of a germ line SUFU mutation associated with GS.

Medulloblastoma Variants: Age-Dependent Occurrence and Relation to Gorlin Syndrome—A New Clinical Perspective

Purpose: We aimed to test the hypothesis that medulloblastoma (MB) variants show a different age distribution and clinical behavior reflecting their specific biology, and that MB occurring at very young age is associated with cancer predisposition syndromes such as Gorlin syndrome (GS). Experimental Design: We investigated the frequency, age distribution, location, response to treatment, outcome, and association with familial cancer predisposition syndromes in a series of 82 cases of MB in patients ages <14 years diagnosed at the Giannina Gaslini Childrens Hospital, Genoa, between 1987 and 2004. Results: Desmoplastic MB and MB with extensive nodularity (MBEN), were present in 22 of 82 cases (27%) and were more frequent in children ages ≤3 years (13 of 25; 52%). In this age group, MBEN was significantly more frequent than desmoplastic MB and classic MB (P < 0.001) and had a good prognosis. MBEN was associated with GS in 5 of 12 cases. Overall, 8 cases occurred in the context of familial tumor predisposition syndromes (5 GS, 1 each NF1, Li-Fraumeni, and Fragile X) and 7 of these patients were ages ≤3 years at diagnosis. Desmoplastic histology and a more intensive treatment represented independent favorable prognostic factors in multivariate analysis (P = 0.003 and P = 0.0139, respectively). Metastasis was a predictor of bad outcome (P = 0.0001). Conclusions: Our data indicate that biologically different MB entities warrant risk-adapted treatment and that MBEN is strongly associated with GS. Patients, ages ≤3 years, with MB and their families should be investigated for tumor predisposition syndromes such as GS.

Association of MC1R Variants and Host Phenotypes With Melanoma Risk in CDKN2A Mutation Carriers: A GenoMEL Study

Background Carrying the cyclin-dependent kinase inhibitor 2A (CDKN2A) germline mutations is associated with a high risk for melanoma. Penetrance of CDKN2A mutations is modified by pigmentation characteristics, nevus phenotypes, and some variants of the melanocortin-1 receptor gene (MC1R), which is known to have a role in the pigmentation process. However, investigation of the associations of both MC1R variants and host phenotypes with melanoma risk has been limited. Methods We included 815 CDKN2A mutation carriers (473 affected, and 342 unaffected, with melanoma) from 186 families from 15 centers in Europe, North America, and Australia who participated in the Melanoma Genetics Consortium. In this family-based study, we assessed the associations of the four most frequent MC1R variants (V60L, V92M, R151C, and R160W) and the number of variants (1, ≥2 variants), alone or jointly with the host phenotypes (hair color, propensity to sunburn, and number of nevi), with melanoma risk in CDKN2A mutation carriers. These associations were estimated and tested using generalized estimating equations. All statistical tests were two-sided. Results Carrying any one of the four most frequent MC1R variants (V60L, V92M, R151C, R160W) in CDKN2A mutation carriers was associated with a statistically significantly increased risk for melanoma across all continents (1.24 × 10−6 ≤ P ≤ .0007). A consistent pattern of increase in melanoma risk was also associated with increase in number of MC1R variants. The risk of melanoma associated with at least two MC1R variants was 2.6-fold higher than the risk associated with only one variant (odds ratio = 5.83 [95% confidence interval = 3.60 to 9.46] vs 2.25 [95% confidence interval = 1.44 to 3.52]; Ptrend = 1.86 × 10−8). The joint analysis of MC1R variants and host phenotypes showed statistically significant associations of melanoma risk, together with MC1R variants (.0001 ≤ P ≤ .04), hair color (.006 ≤ P ≤ .06), and number of nevi (6.9 × 10−6 ≤ P ≤ .02). Conclusion Results show that MC1R variants, hair color, and number of nevi were jointly associated with melanoma risk in CDKN2A mutation carriers. This joint association may have important consequences for risk assessments in familial settings.

BRAF-mutant melanoma: treatment approaches, resistance mechanisms, and diagnostic strategies

BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy and have been approved for the treatment of BRAF-mutated metastatic melanoma. More recently, the combination of BRAF inhibitor dabrafenib with MEK inhibitor trametinib has shown improved progression-free survival, compared to dabrafenib monotherapy, in a Phase II study and has received approval by the US Food and Drug Administration. However, even when treated with the combination, most patients develop mechanisms of acquired resistance, and some of them do not achieve tumor regression at all, because of intrinsic resistance to therapy. Along with the development of BRAF inhibitors, immunotherapy made an important step forward: ipilimumab, an anti-CTLA-4 monoclonal antibody, was approved for the treatment of metastatic melanoma; anti-PD-1 agents achieved promising results in Phase I/II trials, and data from Phase III studies will be ready soon. The availability of such drugs, which are effective regardless of BRAF status, has made the therapeutic approach more complex, as first-line treatment with BRAF inhibitors may not be the best choice for all BRAF-mutated patients. The aim of this paper is to review the systemic therapeutic options available today for patients affected by BRAF V600-mutated metastatic melanoma, as well as to summarize the mechanisms of resistance to BRAF inhibitors and discuss the possible strategies to overcome them. Moreover, since the molecular analysis of tumor specimens is now a pivotal and decisional factor in the treatment strategy of metastatic melanoma patients, the advances in the molecular detection techniques for the BRAF V600 mutation will be reported.

Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants

Background CDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma. Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a systematic investigation of their phenotype. Methods All known families with CDK4 germline mutations (n=17) were recruited for the study by contacting the authors of published papers or by requests via the Melanoma Genetics Consortium (GenoMEL). Phenotypic data related to primary melanoma and pigmentation characteristics were collected. The CDK4 exon 2 and the complete coding region of the MC1R gene were sequenced. Results Eleven families carried the CDK4 R24H mutation whereas six families had the R24C mutation. The total number of subjects with verified melanoma was 103, with a median age at first melanoma diagnosis of 39 years. Forty-three (41.7%) subjects had developed multiple primary melanomas (MPM). A CDK4 mutation was found in 89 (including 62 melanoma cases) of 209 tested subjects. CDK4 positive family members (both melanoma cases and unaffected subjects) were more likely to have clinically atypical nevi than CDK4 negative family members (p<0.001). MPM subjects had a higher frequency of MC1R red hair colour variants compared with subjects with one tumour (p=0.010). Conclusion Our study shows that families with CDK4 germline mutations cannot be distinguished phenotypically from CDKN2A melanoma families, which are characterised by early onset of disease, increased occurrence of clinically atypical nevi, and development of MPM. In a clinical setting, the CDK4 gene should therefore always be examined when a melanoma family tests negative for CDKN2A mutation.

Prevalence of the E318K MITF germline mutation in Italian melanoma patients: associations with histological subtypes and family cancer history

A French and an Australian study have recently identified a rare germline functional variant in the microphthalmia‐associated transcription factor (MITF) (E318K) that predisposes to familial and sporadic melanoma and to renal cell carcinoma (RCC), showing a new link between two tumour types with different risk factors and between deregulated sumoylation and cancer. The aim of this study was to test the prevalence of the MITF E318K mutation in 667 Italian melanoma patients. We observed significant associations between histological subtypes and family cancer history. Carriers exhibited a nearly threefold higher risk of developing melanoma compared with controls. Carriers were also more likely to have developed multiple primary melanomas (6.40‐fold), compared with wt patients. Carriers with a personal and/or family history of pancreatic cancer and kidney cancer had a nearly 31‐ and eightfold higher risk of developing melanoma compared with wt patients. Our findings further support MITF as a medium‐penetrance melanoma susceptibility gene, highlight a potential association with histological subtypes and suggest that MITF may predispose to pancreatic cancer.