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The objective of this study was to examine the association between tobacco and alcohol dose and type and the age of onset of pancreatic adenocarcinoma (PancCa).Prospective data from the Pancreatic Cancer Collaborative Registry were used to examine the association between age of onset and variables of interest including: gender, race, birth country, educational status, family history of PancCa, diabetes status, and tobacco and alcohol use. Statistical analysis included logistic and linear regression, Cox proportional hazard regression, and time-to-event analysis.The median age to diagnosis for PancCa was 66.3 years (95% confidence intervals (CIs), 64.5-68.0). Males were more likely than females to be smokers (77% vs. 69%, P=0.0002) and heavy alcohol and beer consumers (19% vs. 6%, 34% vs. 19%, P0.0001). In univariate analysis for effects on PancCa presentation age, the following were significant: gender, alcohol and tobacco use (amount, status and type), family history of PancCa, and body mass index. Both alcohol and tobacco had dose-dependent effects. In multivariate analysis, alcohol status and dose were independently associated with increased risk for earlier PancCa onset with greatest risk occurring in heavy drinkers (HR 1.62, 95% CI 1.04-2.54). Smoking status had the highest risk for earlier onset pancreatic cancer with a HR of 2.69 (95% CI, 1.97-3.68) for active smokers and independent effects for dose (P=0.019). The deleterious effects for alcohol and tobacco appear to resolve after 10 years of abstinence.Alcohol and tobacco use are associated with a dose-related increased risk for earlier age of onset of PancCa. Although beer drinkers develop pancreatic cancer at an earlier age than nondrinkers, alcohol type did not have a significant effect after controlling for alcohol dose.

Copy number variations (CNVs) have been shown to contribute substantially to disease susceptibility in several inherited diseases including cancer. We conducted a genome-wide search for CNVs in blood-derived DNA from 79 individuals (62 melanoma patients and 17 spouse controls) of 30 high-risk melanoma-prone families without known segregating mutations using genome-wide comparative genomic hybridization (CGH) tiling arrays. We identified a duplicated region on chromosome 4q13 in germline DNA of all melanoma patients in a melanoma-prone family with three affected siblings. We confirmed the duplication using quantitative PCR and a custom-made CGH array design spanning the 4q13 region. The duplicated region contains 10 genes, most of which encode CXC chemokines. Among them, CXCL1 (melanoma growth-stimulating activity α) and IL8 (interleukin 8) have been shown to stimulate melanoma growth in vitro and in vivo. Our data suggests that the alteration of CXC chemokine genes may confer susceptibility to melanoma.

Host, environmental and genetic factors differently modulate cutaneous melanoma (CM) risk across populations. Currently, the main genetic risk determinants are germline mutations in the major known high-risk susceptibility genes, CDKN2A and CDK4, and variants of the low-risk gene MC1R, which is key in the pigmentation process. This case-control study aimed at investigating the influence of the main host and environmental risk factors and of MC1R variation on CM risk in 390 CDKN2A-negative and 49 CDKN2A-positive Italian individuals. Multivariate analysis showed that MC1R variation, number of nevi and childhood sunburns doubled CM risk in CDKN2A-negative individuals. In CDKN2A-positive individuals, family history of CM and presence of atypical nevi, rather than MC1R status, modified risk (20.75- and 2.83-fold, respectively). Occupational sun exposure increased CM risk (three to sixfold) in both CDKN2A-negative and CDKN2A-positive individuals, reflecting the occupational habits of the Ligurian population and the geographical position of Liguria.

Keratocystic odontogenic tumors (KCOTs) are cystic tumors that arise sporadically or associated with nevoid basal cell carcinoma syndrome (NBCCS). NBCCS is a rare autosomal dominantly inherited disease mainly characterized by multiple basal cell carcinomas, KCOTs of the jaws and a variety of other tumors. PTCH1 mutation can be found both in sporadic or NBCCS associated KCOTs. The aim of the current study was to assess whether a combined clinical and bio-molecular approach could be suitable for the detection of NBCCS among patients with a diagnosis of keratocystic odontogenic tumors (KCOTs). The authors collected keratocystic odontogenic tumors recorded in the database of the Pathology Department of the University of Modena and Reggio Emilia during the period 1991–2011. Through interviews and examinations, family pedigrees were drawn for all patients affected by these odontogenic lesions. We found out that 18 of the 70 patients with KCOTs and/or multiple basal cell carcinomas actually met the clinical criteria for the diagnosis of NBCCS. A wide inter- and intra-familial phenotypic variability was evident in the families. Ameloblastomas (AMLs) were reported in two probands that are also carriers of the PCTH1 germline mutations. Nine germline mutations in the PTCH1 gene, 5 of them novel, were evident in 14 tested probands. The clinical evaluation of the keratocystic odontogenic tumors can be used as screening for the detection of families at risk of NBCCS. Keratocystic odontogenic lesions are uncommon, and their discovery deserves the search for associated cutaneous basal cell carcinomas and other benign and malignant tumors related to NBCCS.

To the Editor Germline aberrations in the CDKN2A gene are observed in some melanoma-prone families and represent high penetrance mutations (Hussussian et al., 1994; Kamb et al., 1994). INK4A (p16) and ARF (p14) are two distinct proteins encoded by the CDKN2A locus. Loss of INK4A has been associated with unrestricted cell cycle progression through retinoblastoma (RB) protein inactivation, while loss of ARF has been linked to p53 inactivation with subsequent malfunction in cell cycle regulation, apoptosis and DNA repair (Chin et al., 2006). The NRAS and BRAF genes are commonly mutated in sporadic primary cutaneous melanomas, with mutation frequencies between 4–50% (Platz et al., 2008) and 25–80% (Platz et al., 2008), respectively. Codon 61 is the most common position of NRAS alterations in melanoma, with frequent glutamine changes to either lysine, Q61K (c.181C>A), or arginine, Q61R (c.182A>G) (Omholt et al., 2002). Mutations in this residue lock the Ras protein in the GTP-bound state with subsequent continuous activation of its downstream effectors (Platz et al., 2008) through the Ras-Raf-MEK-ERK and Ras-PI3K-Akt pathways. Approximately 90% of reported BRAF mutations occur at residue 600, which is located in the activation domain of this kinase (Thomas, 2006). Current results from melanoma cohorts show that mutations in these genes are almost always mutually exclusive (Edlundh-Rose et al., 2006; Omholt et al., 2003; Platz et al., 2008). Moreover, a high rate of BRAF mutation is found also in nevi, suggesting a role in early stages of the neoplastic process (Pollock et al., 2003). Previously, a high frequency of NRAS mutations (95%) has been reported in Swedish familial melanoma cases with germline CDKN2A alterations (Eskandarpour et al., 2003). The association of BRAF somatic mutations with MC1R germline variants indicates an influence of constitutive genotype on the preferential acquisition of specific mutations during melanoma development (Landi et al., 2006). In line with this, cooperation between RAS and CDKN2A has been shown in animal models of melanoma (Chin et al., 1997). Since there is limited information on NRAS and BRAF mutations in familial melanoma we sought to assess their mutation frequency in melanomas from patients with different CDKN2A germline alterations. The study was performed on formalin-fixed, paraffin-embedded (FFPE) primary familial cutaneous melanomas originating from Brisbane, Australia (16 samples from 15 patients) and Genoa, Italy (3 patients/samples). Clinical and pathological characteristics are shown in Table 1. One patient with a CDKN2A L32P mutation had two melanomas originating from the trunk and upper extremity, respectively, while a single melanoma was analyzed from each of the other patients. Eight different germline CDKN2A mutations were present in the patients from whom the melanomas were analyzed (Table 2). Laser capture microdissection, DNA extraction, polymerase chain reaction (PCR) amplification, single strand conformation polymorphism (SSCP) and nucleotide sequence analyses of NRAS exon 2 and BRAF exon 15 were carried out as previously described (Jovanovic et al., 2008; Omholt et al., 2002; Omholt et al., 2003). Each mutation was confirmed by two independent PCR/SSCP analyses followed by sequence analysis performed in both directions. The study was approved by the Ethics Committees of the Queensland Institute of Medical Research, University of Genoa and Karolinska Institutet. Table 1 Patient and tumor characteristics Table 2 NRAS and BRAF genotypes in familial melanomas harboring CDKN2A germline mutations Three (16%) samples had NRAS residue 61 alterations (one Q61R and two Q61K substitutions) while 7 (37%) samples had valine to glutamic acid changes in amino acid 600 of BRAF (V600E; Table 2). The NRAS mutation frequency in this study was lower than we reported previously in melanomas from Swedish families with germline CDKN2A mutations (Eskandarpour et al., 2003). The reason for this is unclear. It could possibly be attributed to different origins of studied cohorts (Sweden versus Australia/Italy) and to different CDKN2A germline alterations in these two studies (the 112Argdup founder mutation is predominant in Swedish families, which is in contrast to genotypes reported here). However, we cannot exclude that technical factors, possibly related to the fragmented nature of DNA extracted from FFPE samples, may play a role in different rates of mutation detection. Intriguingly, all 3 tumors with NRAS mutations also had BRAF V600E mutations. The presence of both NRAS and BRAF V600E mutations in the same lesions is contrary to the current consensus that such mutations are almost always mutually exclusive in melanomas and other tumor types (Davies et al., 2002; Omholt et al., 2003; Thomas et al., 2007). However, Pollock et al (2003) observed concomitant NRAS and BRAF V600E mutations in 9% of nevi and suggested this might be due to different clonal nests of cells within these tumors carrying distinct mutations, a possibility that could also explain our findings. Although intriguingly, the joint presence of both NRAS and BRAF mutations was found only in tumors from patients with CDKN2A L32P mutations. In two of these tumors, NRAS and BRAF mutations (NRAS/BRAF: Q61K/V600E and Q61R/V600E) were found in two different DNA extracts while in one case, alterations of both genes (NRAS/BRAF: Q61K/V600E) were identified in the same DNA extract. Thus far, it is recognized that a mutation in either NRAS or BRAF is sufficient for activation of the Ras-Raf-MEK-ERK pathway, with mutant RAS having a 50-fold higher activation effect than mutant BRAF (Davies et al., 2002). Although we do not have any evidence that the NRAS and BRAF mutations found in the same DNA extract were coexisting in the same cells, it is possible that the L32P mutation in CDKN2A somehow permits cellular tolerance of these dual mutations. In conclusion, the NRAS and BRAF mutation rates we observed in familial melanomas were generally lower than most previous reports in sporadic melanoma but equal to those reported for primary melanomas of similar thickness (Goel et al., 2006; Shinozaki et al., 2004). Samples that harbored INK4A L32P substitutions also had high frequency of coexisting mutations in both NRAS and BRAF. This suggests that in some instances constitutional CDKN2A mutations affect the occurrence of somatic mutations in NRAS and BRAF, although further work is needed to substantiate this hypothesis.

MDM2 is a major negative regulator of p53 function and is directly regulated by MYCN in neuroblastoma (NB) cells. MDM2 SNP309, a T-to-G substitution in the MDM2 promoter associated with higher gene expression compared to wild-type, may attenuate the p53 pathway in NB, in which p53 mutations are rare. We investigated its impact on NB development and survival in relation with major clinical and biological characteristics.A consecutive cohort of 497 NB children, diagnosed in Italy between 1995 and 2005, and a healthy control population of 471 adults were genotyped for MDM2 SNP309. NB patients were followed up until June 30, 2008.Patients and controls showed similar distribution of MDM2 SNP309 genotypes. In patients, the polymorphism was not associated with any characteristic at diagnosis. In localized stages no effect of the polymorphism on survival was evident. In stage 4 patients overall survival (OS), event free survival (EFS) and survival after relapse (SAR) were significantly poorer for TG/GG than for TT patients (P = 0.008; P = 0.013; P = 0.046, respectively). In this group, such an effect was more evident in patients with MYCN amplification (OS: P0.001; EFS: P = 0.028; SAR: P0.001).While MDM2 SNP309 status does not affect the risk of developing NB nor disease outcome for localized cancer cases, it significantly correlates with survival in stage 4 NB patients, particularly in the presence of MYCN amplification. The impact of small molecule inhibitors of MDM2 activity in the management of such patients could be usefully considered.

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.

Background Breast cancer patients have a cumulative lifetime risk of 2%–15% of developing a contralateral metastatic or ex novo primary cancer. From prognostic and therapeutic viewpoints, it is important to differentiate metastatic from second primary. To distinguish these entities, we investigated whether the pattern of X chromosome inactivation could determine whether the two tumors derived from different progenitor cells. Materials and methods The clonality of bilateral breast cancer was evaluated through the X-inactivation analysis using the human androgen receptor gene (HUMARA) polymorphism and the histopathologic and molecular results were compared. A different or an identical pattern of X inactivation was considered as indicator of a second primary cancer or not informative, respectively. We considered morphological indicators of a new primary cancer the absence of concordance in the histological type or a better histological differentiation. Results Ten patients with bilateral breast cancer were evaluated. Morphological criteria indicated that eight were second primary, a conclusion confirmed by the X-inactivation analysis. Two cases classified as recurrence according to morphological criteria were classified as second tumor by molecular analysis. Conclusion Our results show that the HUMARA clonality assay can improve the histological parameters in differentiating metastatic cancer from second primary cancer.

We conducted a genome-wide association pooling study for cutaneous melanoma and performed validation in samples totaling 2,019 cases and 2,105 controls. Using pooling, we identified a new melanoma risk locus on chromosome 20 (rs910873 and rs1885120), with replication in two further samples (combined P < 1 x 10(-15)). The per allele odds ratio was 1.75 (1.53, 2.01), with evidence for stronger association in early-onset cases.

IntroductionCalcitonin measurement is advised in the diagnosis of thyroid nodules, as it is an accurate marker of medullary thyroid carcinoma (MTC). C-cell hyperplasia (CCH)-induced hypercalcitoninemia cannot be distinguished from that induced by MTC, unless surgery is performed.CaseWe report the clinical and biological features of a patient with a family history of cancer, including melanoma and pancreatic cancer, who had previously undergone surgery for melanoma. He presented the unusual association of papillary thyroid carcinoma (PTC), normocalcemic hyperparathyroidism, and hypercalcitoninemia with a pathological response to pentagastrin, which was histologically deemed secondary to CCH. Multiple endocrine neoplasia (MEN) 2A was diagnosed. RET gene analysis showed a p.V804M missense mutation in exon 14, a low- but variably penetrant defect found in both sporadic and MEN2A-associated MTC/CCH, and a p.G691S polymorphism in exon 11. Furthermore, the germline P48T mutation was found in the CDKN2A gene exon 1, which is known to be associated with melanoma and pancreatic cancer. The patient showed the uncommon coexistence of a germline mutation in two suppressor genes, RET and CDKN2A; this finding, deemed to be a mere coincidence, did not modify the phenotype expected by each single mutation. CCH associated with V804M RET mutation is a precancerous condition and surgery is recommended. In order to exclude MTC, surgery is advised in patients with a pathological calcitonin response to pentagastrin, in the absence of thyroid autoimmunity. CCH-induced hypercalcitoninemia can be associated with thyroid cancers other than MTC (e.g., PTC). Family history is important in scheduling specific genetic screening in high-risk patients and their relatives.

We have investigated the frequency and spectrum of CDKN2A/CDK4 mutations in 23 cutaneous melanoma families from Central Italy (Tuscany). Three distinct mutations were identified in five families. One mutation, p.G23S, was present in three families. Several lines of evidence indicate that p.G23S is a pathogenic mutation: it is located in the functionally important first ankyrinic domain of p16, it was not detected in a sample of 100 control individuals, and it was present in all tested affected individuals from the three families. Haplotype analysis showed a common ancestral origin of the p.G23S mutation. Our data show that the p.G23S mutation is an important cause of hereditary melanoma in Tuscany.

The G101W founder mutation is the most common CDKN2A mutation in Italy, Spain, and France. As the background of modifying genes, environmental exposures, and sun behavior vary across countries, studying G101W carriers from distinct countries offers a unique opportunity to evaluate possible modifying factors in melanoma development. We evaluated 76 G101W cases and 59 carrier controls from France, Italy, Spain, and the United States. Hair color and dysplastic nevi distributions differed significantly in cases and controls across the 4 study groups. Cases also varied significantly for eye color, freckling, and nevi. The distribution of MC1R variants in cases differed significantly across study groups because 12% of Italian melanoma patients had ≥2 MC1R variants vs. >50% for the other case groups. Several MC1R covariates showed significant associations with melanoma risk in all groups combined and in the American, French, and Spanish samples; no significant findings were observed in the Italian sample. In multiple-case families, the number and type of MC1R variants varied significantly between multiple-primary-melanoma and single-primary-melanoma patients from the 4 groups; there was also a significant decrease in median age at melanoma diagnosis as the number or type of MC1R variants increased. The variation in the effects of the cutaneous phenotypic and MC1R factors across the study sample suggests that these factors differentially contribute to development of melanoma even on a common genetic background of a germline CDKN2A mutation. Differences in melanoma risk across geographic regions justify the need for individual studies in each country before counseling should be considered. © 2007 Wiley-Liss, Inc.

Cutaneous melanoma (CM) is rare in children, representing 1-3% of all paediatric malignancies and occurring at a frequency of 0.3-0.4% before puberty. MC1R is a key gene for skin pigmentation and is highly polymorphic in Caucasians. MC1R gene variants are associated with CM in different populations, and with congenital melanocytic naevi in children. The aim of this study is to evaluate whether the prevalence of MC1R variants differed among sporadic childhood and adolescent CM cases compared to adult patients. Data were gathered through the M-SKIP project, an international pooled-analysis on MC1R variants, skin cancer and phenotypic characteristics. CM cases with information on age at diagnosis were selected from the M-SKIP dataset and divided into three groups: childhood (age ≤14 years, N=13), adolescent (age 15 to 18 years, N=52) and adult (age > 18 years, N=7,696). The frequency of carrying specific MC1R variants as well as at least one MC1R variant were compared between childhood/adolescent and adult CM cases with Chi Square test. The prevalence of any MC1R variant was lower in children (≤14 years, 63%) than in adolescents (15-18 years, 71%) or adults (>18 years, 75%), although overall the difference was not statistically significant. A higher prevalence of the MC1R V92M variant was found in childhood and adolescent compared to adult CM cases (23% vs 5%, p=0.06). In contrast, the MC1R R151C variant was found less frequently in childhood and adolescent than in adult cases (9% vs 18%, p=0.06). Looking at rare variants in 5,983 cases with MC1R sequenced, 3 (9%) carriers of MC1R ins86A were found among 32 childhood and adolescent patients, while only 44 (1%) carriers of the same variant were found among 5,951 adult cases (p MC1R variants ins86A and V92M, but not R151C, may be specifically associated with childhood and adolescent melanoma. Further studies using a larger sample size is needed to validate the present findings. Citation Format: Sara Raimondi, Sara Gandini, Peter A. Kanetsky, Veronica Hoiom, Rajiv Kumar, Paola Ghiorzo, Tadeusz Debniak, Ricardo Fernandez de Misa, Giuseppe Palmieri, Jiali Han, Maria Teresa Landi, Terry Dwyer, Maria Concetta Fargnoli, Wojciech Branicki, Nelleke A. Gruis, Alexander Stratigos, Gloria Ribas, M. Laurin Council, Manfred Kayser, Philippe Autier, Jose Carlos García-Borrón, Julian Little, Julia Newton-Bishop, Francesco Sera, Eduardo Nagore. Role of MC1R variants in childhood and adolescent melanoma. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A38.

GenoMEL, comprising major familial melanoma research groups from North America, Europe, Asia, and Australia has created the largest familial melanoma sample yet available to characterize mutations in the high-risk melanoma susceptibility genes CDKN2A/alternate reading frames (ARF), which encodes p16 and p14ARF, and CDK4 and to evaluate their relationship with pancreatic cancer (PC), neural system tumors (NST), and uveal melanoma (UM). This study included 466 families (2,137 patients) with at least three melanoma patients from 17 GenoMEL centers. Overall, 41% (n = 190) of families had mutations; most involved p16 (n = 178). Mutations in CDK4 (n = 5) and ARF (n = 7) occurred at similar frequencies (2-3%). There were striking differences in mutations across geographic locales. The proportion of families with the most frequent founder mutation(s) of each locale differed significantly across the seven regions (P = 0.0009). Single founder CDKN2A mutations were predominant in Sweden (p.R112_L113insR, 92% of family's mutations) and the Netherlands (c.225_243del19, 90% of family's mutations). France, Spain, and Italy had the same most frequent mutation (p.G101W). Similarly, Australia and United Kingdom had the same most common mutations (p.M53I, c.IVS2-105A>G, p.R24P, and p.L32P). As reported previously, there was a strong association between PC and CDKN2A mutations (P < 0.0001). This relationship differed by mutation. In contrast, there was little evidence for an association between CDKN2A mutations and NST (P = 0.52) or UM (P = 0.25). There was a marginally significant association between NST and ARF (P = 0.05). However, this particular evaluation had low power and requires confirmation. This GenoMEL study provides the most extensive characterization of mutations in high-risk melanoma susceptibility genes in families with three or more melanoma patients yet available. (Cancer Res 2006; 66(20): 9818-28)

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.

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.

Expression of p16INK4A, the product of the melanoma susceptibility gene CDKN2A, has been shown to decrease in correlation with tumor progression. P16INK4A is a key regulator of cell-cycle function, and likely interacts with a variety of targets alongside cyclin-dependent kinases (CDKs). One such target is nuclear factor KB (NF-kappaB), a pleiotropic transcription factor that plays a crucial role in apoptosis, oncogenesis and cell cycle control. NF-kappaB p65 has been shown to be activated in melanoma cell lines but few studies decribe its expression in the tissue. In the present study we focused on synchronous expression of p16INK4A and NF-kappaB p65 and their functional activation in melanoma cell lines and biopsy tissue. Activation of NF-kappaB p65, as observed by electrophoretic mobility shift assay in cell lines, was correlated with expression and cellular localization of the active and inactive forms of its inhibitor, IkappaB-alpha. In melanocytic lesions, p16INK4A and NF-kappaB p65 expression were inversely correlated with levels of the nuclear component of NF-kappaB p65 increasing from nevi to primary melanomas and metastases.

Little is known about the correlation between the loss of p16 expression and tumor progression in familial melanoma; no systematic study has been conducted on p16 expression in melanocytic tumors from patients carrying germline CDKN2A mutations. We analyzed 98 early primary lesions from familial patients, previously tested for germline CDKN2A status, by quantitative immunohistochemistry using 3 p16 antibodies. We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas, including in situ lesions, than in nevi. Of other features analyzed, tumor thickness showed the most significant correlation with p16 levels. Lesions from mutation-negative patients displayed combined nuclear and cytoplasmic staining. However, some mutation-positive lesions (ie, G101W, 113insR, M53I, R24P, and 33ins24), including benign nevi, showed nuclear mislocalization, confirming previous studies suggesting that subcellular distribution indicates functional impairment of p16.

Background Roughly 40% of germinal mutations in melanoma families (MF) affect p16INK4a and p14ARF. We investigated the association between INK4/ARF alterations and the occurrence of pancreatic cancer in MF and in sporadic pancreatic cancer (SPC) patients. Patients and methods Forty-nine MF, 66 SPC cases and 54 controls were enrolled. The INK4/ARF locus was screened. Results As compared with the general population, the risk of pancreatic cancer (PC) was increased 9.4-fold [95% confidence interval (CI) 2.7–33.4] and 2.2-fold (95% CI 0.8–5.7) in G101W-positive and -negative MF, respectively, while mean ages at onset were 61 and 77 years, respectively. A 1.7 (95% CI 1.06–2.79) increased risk of cancer at any site was observed among first-degree relatives of SPC cases as compared with controls. The G101W founder mutation was detected in 4% of SPC cases but the rate increased to 13% when tumor clustering in either branch of families was taken into account. One G101W-positive PC patient with a melanoma in a first-degree relative harbored a germline deletion of the second allele, including exon 1B. Conclusions The presence of a deletion including exon 1B in two PC patients points to the involvement of p14ARF in the development of PC and may suggest that the increased risk of PC in MF is caused by impairment of both loci.

Background: Leflunomide and its active metabolite A77 1726 reversibly inhibits the enzyme dihydro-orotate dehydrogenase, the rate limiting step in de novo synthesis of pyrimidines and progression of the cell cycle in different cell lines, mainly activated T lymphocytes. Objective: To analyse in vitro the possible anti-inflammatory effects exerted by A77 1726, on cultured macrophages, obtained from the synovial tissues of patients with rheumatoid arthritis (RA). Methods: The effects of different doses of A77 1726 on intracytoplasmic expression and extracellular concentration of inflammatory cytokines (tumour necrosis factor α (TNFα), interleukin (IL) 1s, IL6), as well as the influence on production and expression of intercellular adhesion molecule-1 (ICAM-1) and cyclo-oxygenase 2 (COX-2) by primary cultures of synovial macrophages from patients with RA, were evaluated by immunocytochemistry and western blot analysis. The observations were made at four and 24 hours. Results: A progressive and significant time and dose dependent decrease of the number of positive macrophages for intracellular TNFα and IL1s, treated with different doses of A77 1726, was found in comparison with untreated cells. The extracellular concentration of TNFα was found to be significantly decreased in media containing cultured macrophages at 24 hours for all tested doses of A77 1726. At 24 hours, a significant time and dose dependent decrease of ICAM-1 and COX-2 expression by cultured macrophages after A77 1726 treatment was found. Conclusions: In conclusion, the mechanism of antiproliferative activity exerted by leflunomide on activated T lymphocytes seems to be the same mechanism (alteration of the cell cycle progression) which interferes with the functions of other activated cells—namely, the monocytes/macrophages, which are strongly involved in the inflammatory reaction in RA synovial tissue. The positive clinical results seem to confirm that leflunomide exerts an anti-inflammatory action on phagocytic cells in short and long term treatment of RA.

Background: Germline mutations in the CDKN2A gene, which encodes two proteins (p16INK4A and p14ARF), are the most common cause of inherited susceptibility to melanoma. We examined the penetrance of such mutations using data from eight groups from Europe, Australia and the United States that are part of The Melanoma Genetics Consortium Methods: We analyzed 80 families with documented CDKN2A mutations and multiple cases of cutaneous melanoma. We modeled penetrance for melanoma using a logistic regression model incorporating survival analysis. Hypothesis testing was based on likelihood ratio tests. Covariates included gender, alterations in p14APF protein, and population melanoma incidence rates. All statistical tests were two-sided. Results: The 80 analyzed families contained 402 melanoma patients, 320 of whom were tested for mutations and 291 were mutation carriers. We also tested 713 unaffected family members for mutations and 194 were carriers. Overall, CDKN2A mutation penetrance was estimated to be 0.30 (95% confidence interval (CI) = 0.12 to 0.62) by age 50 years and 0.67 (95% CI = 0.31 to 0.96) by age 80 years. Penetrance was not statistically significantly modified by gender or by whether the CDKN2A mutation altered p14ARF protein. However, there was a statistically significant effect of residing in a location with a high population incidence rate of melanoma (P = .003). By age 50 years CDKN2A mutation penetrance reached 0.13 in Europe, 0.50 in the United States, and 0.32 in Australia; by age 80 years it was 0.58 in Europe, 0.76 in the United States, and 0.91 in Australia. Conclusions: This study, which gives the most informed estimates of CDKN2A mutation penetrance available, indicates that the penetrance varies with melanoma population incidence rates. Thus, the same factors that affect population incidence of melanoma may also mediate CDKN2A penetrance.

CDKN2A germline mutation frequency estimates are commonly based on families with several melanoma cases. When we started counseling in a research setting on gene susceptibility analysis in northern and central Italy, however, we mostly found small families with few cases. Here we briefly characterize those kindred, estimate CDKN2A/CDK4 mutation test yields, and provide indications on the possibility of implementing formal DNA testing for melanoma-prone families in Italy. In September 1995 we started genetic counseling in a research setting at our Medical Genetics Center. Screening for CDKN2A/CDK4 mutations was performed on families with two melanoma patients, one of whom was younger than 50 years at onset, the other complying with one of the following: 1) being a first-degree relative, 2) having an additional relative with pancreatic cancer, or 3) having multiple primary melanomas. Sixty-two of 67 (80%) melanoma cases met our criteria. Four previously described CDKN2A mutations (G101W, R24P, V126D, and N71S) were found in 21 of the 62 families (34%) with a high prevalence of G101W (18/21). The percentage of families with two melanoma cases/family harboring a mutation was low (7%, 2/27), but rose to 45% (9/20) if one of the melanoma patients carried multiple melanomas or if pancreatic cancer was present in that family. In the 15 families with three melanoma cases the presence of a mutation was higher (67%, 10/15) and reached 100% in the 4 families with four or more melanoma cases. Our results suggest that CDKN2A/CDK4 counseling-based mutational analysis may be reasonably efficient also for families with two melanoma cases, if one patient carries multiple melanomas or if pancreatic cancer is present in the family.

In mammalian systems, controlled progression through the cell cycle is essential for normal proliferation and its loss is a hallmark of malignancy. p16INK4a inhibits the cyclin-dependent kinases CDK4 and CDK6, thereby keeping the retinoblastoma protein (pRB) in a hypo-phosphorylated state, which can lead to G1/S checkpoint activation. In addition p16INK4a plays a crucial role in the process of replicative senescence. p16INK4a loss or inactivation is associated with predisposition to melanoma, pancreatic cancer and other malignancies, highlighting its recognized function as a tumor suppressor. Given its critical role in cell homeostasis, there is much interest in understanding the molecular regulators of p16INK4a expression. Here we report that p16 belongs to the expanding group of proteins whose translation is influenced by sequence/structural features of the 5’UTR mRNA that are endowed of cellular Internal Ribosome Entry Site (IRES) activity. To study the potential for p16INK4a 5’UTR to drive cap-independent translation we developed a dual-luciferase assay using a bicistronic vector (named pRuF), where wild type or deletion mutants of the p16INK4a 5’UTRs were cloned as intervening sequence between Renilla and Firefly luciferase cDNAs. The p16INK4a 5’UTR sequence in inverted orientation was included as additional control. Results of reporters’ relative activity coupled to control analyses of actual bicistronic mRNA transcription, indicated that the wild type p16INK4a 5’UTR could stimulate Firefly luciferase translation. The IRES-like activity could not be mapped to a specific region of the 5’UTR based on results with deletion constructs, and was two-fold stronger compared to the cMYC 5’UTR, a known cellular IRES used as a control. Notably, hypoxic stress in particular, but also the treatment with mTOR inhibitors, enhanced the translation-stimulating property of the p16INK4a 5’UTR. RNA immuno-precipitation (RIP) assays performed in the p16-positive melanoma-derived cell line SK-Mel-28 suggest that the RNA-binding protein YBX-1, known to act in translation control, can participate in p16 INK4a translation. Experiments were YBX-1 was over-expressed or knocked-down by si-RNA confirmed its involvement in p16INK4a cap-independent translational regulation. Taken collectively, our results suggest that the 5’UTR region can modulate p16INK4a mRNA translation efficiency. Citation Format: Alessandra Bisio, Elisa Latorre, Virginia Andreotti, Alessandro Provenzani, Giovanna Bianchi- Scarrà, Paola Ghiorzo, Alberto Inga. An internal ribosomal entry site in the 5’-untranslated region of p16INK4a mRNA provides a novel mechanism for the regulation of its translation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3384. doi:10.1158/1538-7445.AM2014-3384

Approximately 10% of cutaneous malignant melanoma (CMM) cases occur in a familial setting and known high-penetrance melanoma susceptibility genes (CDKN2A, CDK4, BAP1, and TERT) account for melanoma susceptibility in a small proportion of melanoma-prone families. To identify additional high-penetrance susceptibility genes for familial CMM, we performed whole exome sequencing in 101 CMM cases/obligate carriers in 56 unrelated melanoma-prone families recruited from the Romagna area in Italy. We identified a germline founder mutation in Protection Of Telomeres 1(POT1) (g.7:124493086 C>T, p.S270N) in five unrelated melanoma-prone families. The mutation was also found in a single sporadic CMM case from Romagna out of 1,824 Italian cases examined, but it was not seen in public databases, our internal exome databases of over 1000 subjects, and 878 Spanish CMM cases and 3,489 controls (2,038 Italian and 1,451Spanish) genotyped. POT1 is a component of the telomeric shelterin complex that plays a critical role in maintaining telomere integrity and regulating telomere length. The p.S270N mutation is highly conserved among vertebrates and predicted to be deleterious by most computational programs we evaluated. Carriers of the p.S270N mutation had increased length and heterogeneity as well as increased average number of fragile telomeres in peripheral blood mononuclear cell (PBMC) DNA compared to age-matched CMM cases (controls) without the mutation. Exome sequencing analysis of Italian families also identified two other rare missense substitutions in POT1 (g.7:124464052 C>G [p.Q623H] and g.7:124503540 C>T [p.R137H]) that were found in all cases sequenced in two families. Both missense mutations are absent from public databases and 3,489 genotyped controls, and are predicted to be deleterious by most algorithms. The carriers of these two mutations showed slightly but significantly increased telomere intensity signals and telomere fragility in PBMCs compared to age-matched controls. We further sequenced POT1 exons in 768 CMM cases and 768 controls collected from Italy and we found that CMM cases showed a significant increase in the burden of rare exonic variants compared to controls (OR=5.4, 95% CI=1.5-29.2, P=0.0021). Subsequently, we identified two novel recurrent germline missense mutations in POT1 (g.7:124499043 C>T, p.D224N and g.7:124469308 C>G, p.A532P) in American and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations and that genes involved in telomere maintenance may play important roles in melanoma development. Citation Format: Jianxin Shi, Xiaohong R. Yang, Bari Ballew, Melissa Rotunno, Donato Calista, Maria C. Fargnoli, Paola Ghiorzo, Brigitte Bressac-de Paillerets, Eduardo Nagore, NCI DCEG Cancer Sequencing Working Group, Xing Hua, Paula Hyland, Jinhu Yin, Haritha Vallabhaneni, Weihang Chai, Sarangan Ravichandran, Alexander Eggermont, Mark Lathrop, Ketty Peris, Giovanna Bianchi-Scarra, Giorgio Landi, Sharon Savage, Joshua Sampson, Ji He, Meredith Yeager, Lynn Goldin, Florence Demenais, Stephen Chanock, Margaret Tucker, Alisa Goldstein, Yie Liu, Maria T. Landi. Exome sequencing identified POT1, a telomere shelterin gene, as a major susceptibility gene for familial cutaneous malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 941. doi:10.1158/1538-7445.AM2014-941

To further extend the previous analysis of cis-acting elements and cognate trans-acting factors that contribute to GM-CSF transcriptional regulations, we have examined a promoter region between -1742 and -2010. DNase I footprinting assays showed four protected sequences named A, B, C and D. DNA transfections in the T-lymphoid Mo cell line, which constitutively expresses GM-CSF, indicated that the A element, located between -2002 and -1984, has a positive role on transcription. Further characterization by electrophoretic mobility shift assays in the presence of different competitor oligonucleotides showed that this element binds a factor of the NF-kappa B/Rel family.

Background: CDKN2A (p16) and CDK4 are high-risk susceptibility genes for cutaneous melanoma. CDKN2A is the major identified high-risk gene and CDKN2A-mutated families have been extensively characterized. Melanoma families with CDK4 mutation are very rare. Only 10 such families have been published and a systematic investigation of their phenotype is lacking. Aim of study: We evaluated all known CDK4-mutated families with regard to phenotypic features of their melanocytic disease. We also examined if common variants of the melanocortin-1 receptor (MC1R) gene had an effect on age of melanoma onset. Methods: CDK4-melanoma families, previously ascertained by different research groups, were selected for this study either by contacting the authors of published papers or by requests via the GenoMEL Melanoma Genetics Consortium. Phenotypic data related to melanoma and pigmentation were collected via a standardized form. For all family members with available DNA, exon 2 of CDK4 and the complete coding region of the MC1R gene were sequenced. Results: Thirteen CDK4 families (including three unpublished) were enrolled in the study. Altogether, 83 subjects with malignant melanoma, having a total of 125 melanomas, were verified in the pedigrees. The mean and median age of melanoma diagnosis was 38.5 years and 36.5 years, respectively. Of the 83 melanoma-positive subjects, 29 (34.9%) had multiple primary melanoma. Superficially spreading melanoma was the most common histological diagnosis (52.2%). DNA was available from 198 members of the pedigrees, and 81 tested positive for either the CDK4 R24H or R24C mutation; 117 subjects including 39 spouses tested negative. Subjects with the CDK4 mutation or with verified melanoma had a much higher prevalence of atypical nevi (79.5% and 77.2%, respectively) than CDK4-negative family members (26.0%; p Conclusion: CDK4-positive melanoma families are phenotypically similar to families with a CDKN2A mutation with regard to age of melanoma diagnosis, histological tumor type and the presence of atypical nevi/multiple melanomas. In a clinical setting, the CDK4 gene should therefore be examined if a melanoma family is negative for a CDKN2A mutation. Although based on only 13 CDK4 families, mutation carriers do not appear to have an increased predisposition to pancreatic cancer as noted in some pedigrees with CDKN2A mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 869. doi:10.1158/1538-7445.AM2011-869

Up to 10% of all melanoma patients show a family history of disease. One main predisposing gene for familial melanoma development is CDKN2A, a tumor suppressor gene that encodes two distinct proteins, p16INK4A and p14ARF. CDKN2A germline mutations have been found in 20-40% of melanoma-prone families. So far, BRAF and NRAS are the most frequently altered oncogenes described in cutaneous melanoma. To date, little is known about the somatic changes that occur in familial melanoma. The aim of this study was to determine the prevalence of BRAF and NRAS somatic mutations in familial melanomas and to evaluate the associations between these mutations and clinicopathological factors. The study was performed as a collaboration within GenoMEL (The Melanoma Genetics Consortium). A total of 135 primary familial cutaneous melanomas (88 tumors from CDKN2A mutation-carriers and 47 from CDKN2A non-carriers) were investigated. Paraffin-blocks of tumors were collected from 8 GenoMEL centers in Australia, Italy, Latvia, the Netherlands, Spain, Sweden, and UK. Tumor cells were isolated by laser capture microdissection and mutation screening (BRAF exon 15 and NRAS exon 3) was performed by direct sequencing. Mutation analysis revealed BRAF and NRAS mutations in 41.6% and 11.4% of tumors, respectively. In no case were BRAF and NRAS mutations found to coexist in the same tumor. There was no difference in BRAF mutation frequency between tumors from patients with CDKN2A alterations and tumors from patients wild-type for CDKN2A (41.0% vs. 42.9%). Similarly, the frequency of NRAS mutations did not differ markedly when tumors from CDKN2A mutation-carriers and non-carriers were compared (9.0% vs. 15.6%). There was also no difference in the frequency of BRAF mutations between tumors with CDKN2A mutations disrupting both p16INK4A and p14ARF and tumors with CDKN2A mutations affecting p16INK4A only (40.6% vs. 36.4%). When tumors with different CDKN2A founder mutations were compared, BRAF mutations were seen in 50.0%, 45.8%, 37.5% and 35.0% of p.M53I, p.R112_L113insR, p.G101W and c.225_243del19 altered tumors, respectively. NRAS mutations were observed in 14.3% 12.5% and 10.5% of p.R112_L113insR, p.M53I and c.225_243del19 altered tumors, respectively, whereas no NRAS mutations were seen in tumors with the p.G101W founder mutation. Patients with BRAF-mutated tumors had a significantly lower median age at diagnosis compared to patients with NRAS-mutated or BRAF/NRAS wild-type tumors (43.5, 54 and 51.5 years, respectively). Moreover, melanomas with mutated BRAF or NRAS were thicker than BRAF/NRAS wild-type cases (median thickness 1.0, 1.4 and 0.7mm, respectively). Our results suggest a role for the BRAF and NRAS oncogenes in familial melanoma. Somatic changes of these genes occur in tumors from both CDKN2A mutation carriers and non-carriers. Presence of BRAF or NRAS mutations associated with patient and tumor characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 867. doi:10.1158/1538-7445.AM2011-867

10543 Background: 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 and neural system tumours. 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 pancreatic cancer and neuroblastoma. Methods: We investigated 70 pancreatic cancer patients from a case-control study living in or originally coming from a small geographic area bordering Liguria, in north-western Italy, for CDKN2A mutations and ARF. Results: 2 out of 70 patients displayed the E27X mutation and 1 of the 2 patients had a first degree relative affected by pancreatic cancer. Conclusions: E27X generates a premature stop codon, leading to dramatically reduced protein levels of p16 and leaving p14ARF unaltered. As pancreatic cancer and neural system tumours 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 1a provides interesting insights towards clarifying the mechanisms by which the CDKN2A/ARF locus is involved in cancer predisposition. Its finding in pancreatic cancer patients confirms common susceptibility to melanoma and pancreatic cancer due to this CDKN2A exon 1 mutation. No significant financial relationships to disclose.

To further clarify the complex transcriptional regulation of the human GM-CSF gene, which was extensively investigated in activated T cells, we have studied the role of an upstream NF-kappaB like site in the 5637 non-lymphoid cell line, which derives from a bladder carcinoma and constitutively produces GM-CSF. This sequence, named the A element, has an active role on GM-CSF transcription and is responsive to the tumor promoter PMA in transient transfection experiments. We describe here a heterodimeric binding complex of NF-kappaB subunits (c-Rel and p65) which is identical to the one obtained using the HIV-LTR-kappaB site as recognition sequence and different from the one (c-Rel and p50) observed with nuclear extracts from Mo T-lymphoid HTLV-II infected cells.

Sex hormones seem to modulate the immune/inflammatory responses by different mechanisms in female and male rheumatoid arthritis patients. The effects of 17beta-oestradiol and of testosterone were tested on the cultured human monocytic/macrophage cell line (THP-1) activated with IFN-gamma in order to investigate their role in cell proliferation and apoptosis. Activated human THP-1 cells were cultured in the presence of 17beta-oestradiol and testosterone (final concentration, 10 nM). The evaluation of markers of cell proliferation included the NF-kappaB DNA-binding assay, the NF-kappaB inhibition complex, the proliferating cell nuclear antigen expression and the methyl-tetrazolium salt test. Apoptosis was detected by the annexin V-propidium assay and by the cleaved poly-ADP ribose polymerase expression. Specific methods included flow analysis cytometry scatter analysis, immunocytochemistry and western blot analysis. Cell growth inhibition and increased apoptosis were observed in testosterone-treated THP-1 cells. Increased poly-ADP ribose polymerase-cleaved expression and decreased proliferating cell nuclear antigen expression, as well as an increase of IkappaB-alpha and a decrease of the IkappaB-alpha phosphorylated form (ser 32), were found in testosterone-treated THP-1 cells. However, the NF-kappaB DNA binding was found increased in 17beta-oestradiol-treated THP-1 cells. The treatment with staurosporine (enhancer of apoptosis) induced decreased NF-kappaB DNA binding in all conditions, but particularly in testosterone-treated THP-1 cells. Treatment of THP-1 by sex hormones was found to influence cell proliferation and apoptosis. Androgens were found to increase the apoptosis, and oestrogens showed a protective trend on cell death--both acting as modulators of the NF-kappaB complex.