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1.  BRAF-mutant melanoma: treatment approaches, resistance mechanisms, and diagnostic strategies 
OncoTargets and therapy  2015;8:157-168.
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.
doi:10.2147/OTT.S39096
PMCID: PMC4303458  PMID: 25653539
melanoma; BRAF; vemurafenib; dabrafenib; resistance; BRAF inhibitor
2.  Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma 
Nature genetics  2014;46(5):482-486.
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 POT1 gene (g.7:124493086 C>T, Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere length and elevated fragile telomeres suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two other Italian families, yielding a frequency of POT1 variants comparable to that of 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 American and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.
doi:10.1038/ng.2941
PMCID: PMC4056593  PMID: 24686846
3.  Overcoming resistance to BRAF inhibition in BRAF-mutated metastatic melanoma 
Oncotarget  2014;5(21):10206-10221.
Almost 50% of metastatic melanoma patients harbor a BRAFV600 mutation andthe introduction of BRAF inhibitors has improved their treatment options. BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy and have been approved for the treatment of BRAF-mutated metastatic melanoma. However, most patients develop mechanisms of acquired resistance and about 15% of them do not achieve tumor regression at all, due to intrinsic resistance to therapy. Moreover, early adaptive responses limit the initial efficacy of BRAF inhibition, leading mostly to incomplete responses that may favor the selection of a sub-population of resistant clones and the acquisition of alterations that cause tumor regrowth and progressive disease.
The purpose of this paper is to review the mechanisms of resistance to therapy with BRAF inhibitors and to discuss the strategies to overcome them based on pre-clinical and clinical evidences.
PMCID: PMC4279367  PMID: 25344914
melanoma; BRAF; vemurafenib; dabrafenib; resistance; BRAF inhibitor; MEK inhibitor
4.  The Effect on Melanoma Risk of Genes Previously Associated With Telomere Length 
Telomere length has been associated with risk of many cancers, but results are inconsistent. Seven single nucleotide polymorphisms (SNPs) previously associated with mean leukocyte telomere length were either genotyped or well-imputed in 11108 case patients and 13933 control patients from Europe, Israel, the United States and Australia, four of the seven SNPs reached a P value under .05 (two-sided). A genetic score that predicts telomere length, derived from these seven SNPs, is strongly associated (P = 8.92x10-9, two-sided) with melanoma risk. This demonstrates that the previously observed association between longer telomere length and increased melanoma risk is not attributable to confounding via shared environmental effects (such as ultraviolet exposure) or reverse causality. We provide the first proof that multiple germline genetic determinants of telomere length influence cancer risk.
doi:10.1093/jnci/dju267
PMCID: PMC4196080  PMID: 25231748
5.  Genetic predisposition to pancreatic cancer 
World Journal of Gastroenterology : WJG  2014;20(31):10778-10789.
Pancreatic adenocarcinoma (PC) is the most deadly of the common cancers. Owing to its rapid progression and almost certain fatal outcome, identifying individuals at risk and detecting early lesions are crucial to improve outcome. Genetic risk factors are believed to play a major role. Approximately 10% of PC is estimated to have familial inheritance. Several germline mutations have been found to be involved in hereditary forms of PC, including both familial PC (FPC) and PC as one of the manifestations of a hereditary cancer syndrome or other hereditary conditions. Although most of the susceptibility genes for FPC have yet to be identified, next-generation sequencing studies are likely to provide important insights. The risk of PC in FPC is sufficiently high to recommend screening of high-risk individuals; thus, defining such individuals appropriately is the key. Candidate genes have been described and patients considered for screening programs under research protocols should first be tested for presence of germline mutations in the BRCA2, PALB2 and ATM genes. In specific PC populations, including in Italy, hereditary cancer predisposition genes such as CDKN2A also explain a considerable fraction of FPC.
doi:10.3748/wjg.v20.i31.10778
PMCID: PMC4138458  PMID: 25152581
Pancreatic adenocarcinoma; Susceptibility genes; CDKN2A; Melanoma; Hereditary cancer syndromes; Screening
6.  Association of genetic variants in CDK6 and XRCC1 with the risk of dysplastic nevi in melanoma-prone families 
Dysplastic nevi (DN) is a strong risk factor for cutaneous malignant melanoma (CMM), and it frequently occurs in melanoma-prone families. To identify genetic variants for DN, we genotyped 677 tagSNPs in 38 melanoma candidate genes that are involved in pigmentation, DNA repair, cell cycle control, and melanocyte proliferation pathways in a total of 504 individuals (310 with DN, 194 without DN) from 53 melanoma-prone families (23 CDKN2A mutation positive and 30 negative). Conditional logistic regression, conditioning on families, was used to estimate the association between DN and each SNP separately, adjusted for age, sex, CMM and CDKN2A status. P-values for SNPs in the same gene were combined to yield gene-specific p-values. Two genes, CDK6 and XRCC1, were significantly associated with DN after Bonferroni correction for multiple testing (P=0.0001 and 0.00025, respectively), whereas neither gene was significantly associated with CMM. Associations for CDK6 SNPs were stronger in CDKN2A mutation positive families (rs2079147, Pinteraction=0.0033), whereas XRCC1 SNPs had similar effects in mutation-positive and negative families. The association for one of the associated SNPs in XRCC1 (rs25487) was replicated in two independent datasets (random effect meta-analysis: P<0.0001). Our findings suggest that some genetic variants may contribute to DN risk independently of their association with CMM in melanoma-prone families.
doi:10.1038/jid.2013.316
PMCID: PMC3873368  PMID: 23892592
7.  Alcohol and Tobacco Lower the Age of Presentation in Sporadic Pancreatic Cancer in a Dose-Dependent Manner: A Multicenter Study 
OBJECTIVES
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).
METHODS
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.
RESULTS
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%, P < 0.0001). 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.
CONCLUSIONS
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.
doi:10.1038/ajg.2012.288
PMCID: PMC3923585  PMID: 22929760
8.  A variant in FTO shows association with melanoma risk not due to BMI 
Iles, Mark M | Law, Matthew H | Stacey, Simon N | Han, Jiali | Fang, Shenying | Pfeiffer, Ruth | Harland, Mark | MacGregor, Stuart | Taylor, John C | Aben, Katja K | Akslen, Lars A | Avril, Marie-Françoise | Azizi, Esther | Bakker, Bert | Benediktsdottir, Kristrun R | Bergman, Wilma | Scarrà, Giovanna Bianchi | Brown, Kevin M | Calista, Donato | Chaudru, Valerié | Fargnoli, Maria Concetta | Cust, Anne E | Demenais, Florence | de Waal, Anne C | Dębniak, Tadeusz | Elder, David E | Friedman, Eitan | Galan, Pilar | Ghiorzo, Paola | Gillanders, Elizabeth M | Goldstein, Alisa M | Gruis, Nelleke A | Hansson, Johan | Helsing, Per | Hočevar, Marko | Höiom, Veronica | Hopper, John L | Ingvar, Christian | Janssen, Marjolein | Jenkins, Mark A | Kanetsky, Peter A | Kiemeney, Lambertus A | Lang, Julie | Lathrop, G Mark | Leachman, Sancy | Lee, Jeffrey E | Lubiński, Jan | Mackie, Rona M | Mann, Graham J | Mayordomo, Jose I | Molven, Anders | Mulder, Suzanne | Nagore, Eduardo | Novaković, Srdjan | Okamoto, Ichiro | Olafsson, Jon H | Olsson, Håkan | Pehamberger, Hubert | Peris, Ketty | Grasa, Maria Pilar | Planelles, Dolores | Puig, Susana | Puig-Butille, Joan Anton | Randerson-Moor, Juliette | Requena, Celia | Rivoltini, Licia | Rodolfo, Monica | Santinami, Mario | Sigurgeirsson, Bardur | Snowden, Helen | Song, Fengju | Sulem, Patrick | Thorisdottir, Kristin | Tuominen, Rainer | Van Belle, Patricia | van der Stoep, Nienke | van Rossum, Michelle M | Wei, Qingyi | Wendt, Judith | Zelenika, Diana | Zhang, Mingfeng | Landi, Maria Teresa | Thorleifsson, Gudmar | Bishop, D Timothy | Amos, Christopher I | Hayward, Nicholas K | Stefansson, Kari | Bishop, Julia A Newton | Barrett, Jennifer H
Nature genetics  2013;45(4):428-432.
We report the results of an association study of melanoma based on the genome-wide imputation of the genotypes of 1,353 cases and 3,566 controls of European origin conducted by the GenoMEL consortium. This revealed a novel association between several single nucleotide polymorphisms (SNPs) in intron 8 of the FTO gene, including rs16953002, which replicated using 12,313 cases and 55,667 controls of European ancestry from Europe, the USA and Australia (combined p=3.6×10−12, per-allele OR for A=1.16). As well as identifying a novel melanoma susceptibility locus, this is the first study to identify and replicate an association with SNPs in FTO not related to body mass index (BMI). These SNPs are not in intron 1 (the BMI-related region) and show no association with BMI. This suggests FTO’s function may be broader than the existing paradigm that FTO variants influence multiple traits only through their associations with BMI and obesity.
doi:10.1038/ng.2571
PMCID: PMC3640814  PMID: 23455637
10.  Duplication of CXC chemokine genes on chromosome 4q13 in a melanoma-prone family 
Pigment cell & melanoma research  2012;25(2):243-247.
Summary
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.
doi:10.1111/j.1755-148X.2012.00969.x
PMCID: PMC3288577  PMID: 22225770
Familial melanoma; Germline copy number variations; disease susceptibility; CXC chemokines; chromosome 4q13
11.  On the Interplay of Telomeres, Nevi and the Risk of Melanoma 
PLoS ONE  2012;7(12):e52466.
The relationship between telomeres, nevi and melanoma is complex. Shorter telomeres have been found to be associated with many cancers and with number of nevi, a known risk factor for melanoma. However, shorter telomeres have also been found to decrease melanoma risk. We performed a systematic analysis of telomere-related genes and tagSNPs within these genes, in relation to the risk of melanoma, dysplastic nevi, and nevus count combining data from four studies conducted in Italy. In addition, we examined whether telomere length measured in peripheral blood leukocytes is related to the risk of melanoma, dysplastic nevi, number of nevi, or telomere-related SNPs. A total of 796 cases and 770 controls were genotyped for 517 SNPs in 39 telomere-related genes genotyped with a custom-made array. Replication of the top SNPs was conducted in two American populations consisting of 488 subjects from 53 melanoma-prone families and 1,086 cases and 1,024 controls from a case-control study. We estimated odds ratios for associations with SNPs and combined SNP P-values to compute gene region-specific, functional group-specific, and overall P-value using an adaptive rank-truncated product algorithm. In the Mediterranean population, we found suggestive evidence that RECQL4, a gene involved in genome stability, RTEL1, a gene regulating telomere elongation, and TERF2, a gene implicated in the protection of telomeres, were associated with melanoma, the presence of dysplastic nevi and number of nevi, respectively. However, these associations were not found in the American samples, suggesting variable melanoma susceptibility for these genes across populations or chance findings in our discovery sample. Larger studies across different populations are necessary to clarify these associations.
doi:10.1371/journal.pone.0052466
PMCID: PMC3531488  PMID: 23300679
12.  Novel PTCH1 Mutations in Patients with Keratocystic Odontogenic Tumors Screened for Nevoid Basal Cell Carcinoma (NBCC) Syndrome 
PLoS ONE  2012;7(8):e43827.
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.
doi:10.1371/journal.pone.0043827
PMCID: PMC3428295  PMID: 22952776
13.  Melanocortin-1 receptor, skin cancer and phenotypic characteristics (M-SKIP) project: study design and methods for pooling results of genetic epidemiological studies 
Background
For complex diseases like cancer, pooled-analysis of individual data represents a powerful tool to investigate the joint contribution of genetic, phenotypic and environmental factors to the development of a disease. Pooled-analysis of epidemiological studies has many advantages over meta-analysis, and preliminary results may be obtained faster and with lower costs than with prospective consortia.
Design and methods
Based on our experience with the study design of the Melanocortin-1 receptor (MC1R) gene, SKin cancer and Phenotypic characteristics (M-SKIP) project, we describe the most important steps in planning and conducting a pooled-analysis of genetic epidemiological studies. We then present the statistical analysis plan that we are going to apply, giving particular attention to methods of analysis recently proposed to account for between-study heterogeneity and to explore the joint contribution of genetic, phenotypic and environmental factors in the development of a disease. Within the M-SKIP project, data on 10,959 skin cancer cases and 14,785 controls from 31 international investigators were checked for quality and recoded for standardization. We first proposed to fit the aggregated data with random-effects logistic regression models. However, for the M-SKIP project, a two-stage analysis will be preferred to overcome the problem regarding the availability of different study covariates. The joint contribution of MC1R variants and phenotypic characteristics to skin cancer development will be studied via logic regression modeling.
Discussion
Methodological guidelines to correctly design and conduct pooled-analyses are needed to facilitate application of such methods, thus providing a better summary of the actual findings on specific fields.
doi:10.1186/1471-2288-12-116
PMCID: PMC3502117  PMID: 22862891
Genetic epidemiology; Melanoma; Meta-analysis; Pooled-analysis; Skin cancer; Study design
14.  Genome-wide association study identifies three new melanoma susceptibility loci 
Barrett, Jennifer H | Iles, Mark M | Harland, Mark | Taylor, John C | Aitken, Joanne F | Andresen, Per Arne | Akslen, Lars A | Armstrong, Bruce K | Avril, Marie-Francoise | Azizi, Esther | Bakker, Bert | Bergman, Wilma | Bianchi-Scarrà, Giovanna | Paillerets, Brigitte Bressac-de | Calista, Donato | Cannon-Albright, Lisa A | Corda, Eve | Cust, Anne E | Dębniak, Tadeusz | Duffy, David | Dunning, Alison | Easton, Douglas F | Friedman, Eitan | Galan, Pilar | Ghiorzo, Paola | Giles, Graham G | Hansson, Johan | Hocevar, Marko | Höiom, Veronica | Hopper, John L | Ingvar, Christian | Janssen, Bart | Jenkins, Mark A | Jönsson, Göran | Kefford, Richard F | Landi, Giorgio | Landi, Maria Teresa | Lang, Julie | Lubiński, Jan | Mackie, Rona | Malvehy, Josep | Martin, Nicholas G | Molven, Anders | Montgomery, Grant W | van Nieuwpoort, Frans A | Novakovic, Srdjan | Olsson, Håkan | Pastorino, Lorenza | Puig, Susana | Puig-Butille, Joan Anton | Randerson-Moor, Juliette | Snowden, Helen | Tuominen, Rainer | Van Belle, Patricia | van der Stoep, Nienke | Whiteman, David C | Zelenika, Diana | Han, Jiali | Fang, Shenying | Lee, Jeffrey E | Wei, Qingyi | Lathrop, G Mark | Gillanders, Elizabeth M | Brown, Kevin M | Goldstein, Alisa M | Kanetsky, Peter A | Mann, Graham J | MacGregor, Stuart | Elder, David E | Amos, Christopher I | Hayward, Nicholas K | Gruis, Nelleke A | Demenais, Florence | Newton Bishop, Julia A | Bishop, D Timothy
Nature Genetics  2011;43(11):1108-1113.
We report a genome-wide association study of melanoma, conducted by GenoMEL, of 2,981 cases, of European ancestry, and 1,982 study-specific controls, plus a further 6,426 French and UK population controls, all genotyped for 317,000 or 610,000 SNPs. The analysis confirmed previously known melanoma susceptibility loci. The 7 novel 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 Houston, Texas). Additional replication came from UK and Dutch case-control series. Three of the 7 regions replicated at p<10−3: an ATM missense polymorphism (rs1801516, overall p=3.4×10−9); a polymorphism within MX2 (rs45430, p=2.9×10−9) and a SNP adjacent to CASP8 (rs13016963, p=8.6×10−10). A fourth region near CCND1 remains of potential interest, showing suggestive but inconclusive evidence of replication. Unlike the previously known regions, the novel loci showed no association with nevus or pigmentation phenotypes in a large UK case-control series.
doi:10.1038/ng.959
PMCID: PMC3251256  PMID: 21983787
15.  PCCR: Pancreatic Cancer Collaborative Registry 
Cancer Informatics  2011;10:83-91.
The Pancreatic Cancer Collaborative Registry (PCCR) is a multi-institutional web-based system aimed to collect a variety of data on pancreatic cancer patients and high-risk subjects in a standard and efficient way. The PCCR was initiated by a group of experts in medical oncology, gastroenterology, genetics, pathology, epidemiology, nutrition, and computer science with the goal of facilitating rapid and uniform collection of critical information and biological samples to be used in developing diagnostic, prevention and treatment strategies against pancreatic cancer. The PCCR is a multi-tier web application that utilizes Java/JSP technology and has Oracle 10 g database as a back-end. The PCCR uses a “confederation model” that encourages participation of any interested center, irrespective of its size or location. The PCCR utilizes a standardized approach to data collection and reporting, and uses extensive validation procedures to prevent entering erroneous data. The PCCR controlled vocabulary is harmonized with the NCI Thesaurus (NCIt) or Systematized Nomenclature of Medicine-Clinical Terms (SNOMED-CT). The PCCR questionnaire has accommodated standards accepted in cancer research and healthcare. Currently, seven cancer centers in the USA, as well as one center in Italy are participating in the PCCR. At present, the PCCR database contains data on more than 2,700 subjects (PC patients and individuals at high risk of getting this disease). The PCCR has been certified by the NCI Center for Biomedical Informatics and Information Technology as a cancer Biomedical Informatics Grid (caBIG®) Bronze Compatible product. The PCCR provides a foundation for collaborative PC research. It has all the necessary prerequisites for subsequent evolution of the developed infrastructure from simply gathering PC-related data into a biomedical computing platform vital for successful PC studies, care and treatment. Studies utilizing data collected in the PCCR may engender new approaches to disease prognosis, risk factor assessment, and therapeutic interventions.
doi:10.4137/CIN.S6919
PMCID: PMC3085425  PMID: 21552494
biomedical informatics; pancreatic cancer; registry; caBIG® bronze compatible system
16.  A Novel Flexible Multiplex Bead-based Assay for Detecting Germline CDKN2A and CDK4 Variants in Melanoma-Prone Kindreds 
Background
The presence of recurrent high-risk mutations in CDKN2A and CDK4 among melanoma-prone kindreds suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. Moreover, with the emergence of new melanoma risk single nucleotide polymorphisms (SNPs) through genome-wide association studies, a flexible platform that can easily accommodate these new risk alleles is needed for more accurate genetic risk profiling. To this end, we have developed a novel melanoma-associated mutation detection method using a multiplex bead-based assay. This assay is suitable for high-throughput CDKN2A and CDK4 genotyping and can be eventually adapted to multiple loci across various constituent populations.
Methods
Genomic DNA from a 1603 subjects (1005 in training set, 598 in validation set) were amplified by multiplex PCR using five primer sets followed by multiplex allele-specific primer extension for 39 different known germline variants. The products were then sorted on an xMAP™ (formerly Tag-It™) array and detected by use of the Luminex xMAP™ system. Genotypes were compared to previously-determined sequence data.
Results
In the Toronto training cohort, variants were detected in 145 samples, giving complete concordance between the bead assay and direct sequencing results. Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77% concordance). Overall, the bead assay correctly genotyped 1540/1603 (96.07%) of all individuals in the study and 1540/1545 (99.68%) of individuals whose mutations were represented in the probe set. Out of a total of 62,512 SNP calls, 62,517 (99.99%) were correctly assigned.
Conclusions
In this initial evaluation, the multiplex bead-based assay for familial melanoma appears to be a highly accurate method for genotyping CDKN2A and CDK4 variants.
doi:10.1038/jid.2010.331
PMCID: PMC3045700  PMID: 21085193
Melanoma; CDKN2A; CDK4; p14ARF; familial; high-throughput
17.  Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma‐prone families from three continents 
Journal of Medical Genetics  2006;44(2):99-106.
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.
doi:10.1136/jmg.2006.043802
PMCID: PMC2598064  PMID: 16905682
melanoma;  CDKN2A ; multiple primary melanomas; pancreatic cancer
18.  Genome-wide association study identifies three loci associated with melanoma risk 
Nature genetics  2009;41(8):920-925.
We report a genome-wide association study of melanoma conducted by the GenoMEL consortium based on 317k tagging SNPs for 1650 genetically-enriched cases (from Europe and Australia) and 4336 controls and subsequent replication in 1149 genetically-enriched cases and 964 controls and a population-based case-control study of 1163 cases and 903 controls. The genome-wide screen identified five regions with genotyped or imputed SNPs reaching p < 5×10−7; three regions were replicated: 16q24 encompassing MC1R (overall p=2.54×10−27 for rs258322), 11q14-q21 encompassing TYR (p=2.41×10−14 for rs1393350) and 9p21 adjacent to MTAP and flanking CDKN2A (p=4.03×10−7 for rs7023329). MC1R and TYR are associated with pigmentation, freckling and cutaneous sun sensitivity, well-recognised melanoma risk factors, while the 9p21 locus is novel for common variants associated with melanoma. Despite wide variation in allele frequency, these genetic variants show notable homogeneity of effect across populations of European ancestry living at different latitudes and contribute independently to melanoma risk.
doi:10.1038/ng.411
PMCID: PMC2741419  PMID: 19578364
19.  Common sequence variants on 20q11.22 confer melanoma susceptibility 
Nature genetics  2008;40(7):838-840.
We conducted a genome-wide association pooling study for cutaneous melanoma and performed validation in samples totalling 2019 cases and 2105 controls. Using pooling we identified a novel melanoma risk locus on chromosome 20 (rs910873, rs1885120), with replication in two further samples (combined P <1 × 10-15). The odds ratio is 1.75 (1.53, 2.01), with evidence for stronger association in early onset cases.
doi:10.1038/ng.163
PMCID: PMC2755512  PMID: 18488026
20.  A comparison of CDKN2A mutation detection within the Melanoma Genetics Consortium (GenoMEL) 
CDKN2A is the major melanoma susceptibility gene so far identified, but only 40% of three or more case families have identified mutations. A comparison of mutation detection rates was carried out by “blind” exchange of samples across GenoMEL, the Melanoma Genetics Consortium, to establish the false negative detection rates. Denaturing high performance liquid chromatography (DHPLC) screening results from 451 samples were compared to screening data from nine research groups in which the initial mutation screen had been done predominantly by sequencing. Three samples with mutations identified at local centres were not detected by the DHPLC screen. No additional mutations were detected by DHPLC. Mutation detection across groups within GenoMEL is carried out to a consistently high standard. The relatively low rate of CDKN2A mutation detection is not due to failure to detect mutations and implies the existence of other high penetrance melanoma susceptibility genes.
doi:10.1016/j.ejca.2008.03.005
PMCID: PMC2494985  PMID: 18394881
CDKN2A; melanoma; mutation detection; sequencing; polymorphism; audit; DHPLC; False negative
21.  Sex hormone modulation of cell growth and apoptosis of the human monocytic/macrophage cell line 
Arthritis Research & Therapy  2005;7(5):R1124-R1132.
Sex hormones seem to modulate the immune/inflammatory responses by different mechanisms in female and male rheumatoid arthritis patients. The effects of 17β-oestradiol and of testosterone were tested on the cultured human monocytic/macrophage cell line (THP-1) activated with IFN-γ in order to investigate their role in cell proliferation and apoptosis. Activated human THP-1 cells were cultured in the presence of 17β-oestradiol and testosterone (final concentration, 10 nM). The evaluation of markers of cell proliferation included the NF-κB DNA-binding assay, the NF-κB 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 IκB-α and a decrease of the IκB-α phosphorylated form (ser 32), were found in testosterone-treated THP-1 cells. However, the NF-κB DNA binding was found increased in 17β-oestradiol-treated THP-1 cells. The treatment with staurosporine (enhancer of apoptosis) induced decreased NF-κB 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-κB complex.
doi:10.1186/ar1791
PMCID: PMC1257440  PMID: 16207329
22.  Inherited variants in the MC1R gene and survival from cutaneous melanoma: a BioGenoMEL study 
Pigment Cell & Melanoma Research  2012;25(3):384-394.
Summary
Inherited MC1R variants modulate MITF transcription factor signaling, which in turn affects tumor cell proliferation, apoptosis, and DNA repair. The aim of this BioGenoMEL collaborative study in 10 melanoma cohorts was to test the hypothesis that inherited variants thereby moderate survival expectation. A survival analysis in the largest cohort (Leeds) was carried out adjusting for factors known to impact on survival. The results were then compared with data from nine smaller cohorts. The absence of any consensus MC1R alleles was associated with a significantly lower risk of death in the Leeds set (HR, 0.64; 95% CI, 0.46–0.89) and overall in the 10 data sets (HR, 0.78; 95% CI, 0.65–0.94) with some support from the nine smaller data sets considered together (HR, 0.83; 95% CI, 0.67–1.04). The data are suggestive of a survival benefit for inherited MC1R variants in melanoma patients.
doi:10.1111/j.1755-148X.2012.00982.x
PMCID: PMC3490389  PMID: 22325793
MC1R; survival analysis; MITF; melanoma; forest plot
23.  Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants 
Journal of Medical Genetics  2013;50(4):264-270.
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.
doi:10.1136/jmedgenet-2012-101455
PMCID: PMC3607098  PMID: 23384855
Molecular genetics; Cancer: dermatological; Genetic screening/counselling

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