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PURPOSE Actinic keratoses (AK) are premalignant tumors that can be clinically difficult to differentiate from skin cancer. An easy, quick, and reliable noninvasive alternative to biopsy is needed to definitively confirm the clinical diagnoses. This study evaluates Tape Stripping (TS) of stratum corneum (SC) for noninvasive biomarker analysis of AK. METHOD Lesional and nonlesional human SC samples are obtained by application of stripping tapes on the skin of five AK patients. Following sample preparation, protein digests are analyzed by LC-MS/MS. Bioinformatics analyses are performed using Funrich, Ingenuity Pathway Analysis (IPA), and Oncomine bioinformatics and analytical tools. RESULTS Of the total 613 unique proteins identified, 477 overlap with proteins identified in the proteomic analysis of formalin-fixed and paraffin-embedded (FFPE) AK samples. Additionally, 32 proteins are significantly increased and four proteins decreased in AK samples compared to the normal skin (p

Background: This study examined the prevalence and correlates of skin cancer screening behaviours among individuals at high risk of developing melanoma due to strong family history. Methods: A total of 120 individuals with a known family-specific CDKN2A mutation (72% response rate) completed a self-report questionnaire assessing annual frequency of skin self-examination (SSE), clinical skin examination (CSE) and a variety of potential demographic, clinical and psychosocial correlates. Results: In the past 12 months, 50% of participants reported engaging in SSE at least four times, and 43% of participants had undergone at least one CSE. Engagement in SSE was associated with doctor recommendation (β=1.77, P=0.001), confidence in one's ability to perform SSE (β=1.44, P

Importance There are limited data among young adults on sunscreen use during childhood and adulthood and on the association of sunscreen use with melanoma risk. Objective To assess correlates of early-life sunscreen use and the association between sunscreen use and risk of cutaneous melanoma before age 40 years. Design, Setting, and Participants This population-based, case-control family study analyzed Australian Melanoma Family Study data for persons with questionnaire data on sunscreen use collected by interview from 2001 to 2005 across 3 states in Australia, representing two-thirds of the country’s population. Case participants (aged 18-39 years) had confirmed first primary melanoma. Siblings of case participants were included, and case participants without a sibling control were excluded. Unrelated controls (aged 18-44 years) were recruited from the electoral roll or were a spouse, partner, or friend nominated by case participants. Data analyses were conducted from October 2017 to February 2018. Exposures Self- and parent-reported sunscreen use, sun exposure, and other candidate risk factors during childhood and adulthood. Main Outcomes and Measures Logistic regression analyses adjusted for potential confounders were used to estimate odds ratios (ORs) for melanoma and for correlates of sunscreen use. Results Participation was 629 of 830 contactable cases (76%) (629 of 1197 overall [53%]), 240 of 570 contactable controls (42%) from the electoral roll (240 of 1068 overall [23%]), and 295 of 371 nominated spouse or friend controls (80%); analysis incuded 603 cases and 1088 controls. The median (interquartile range) age was 32 (28-36) years for 603 cases, 35 (30-38) years for 478 unrelated controls, and 34 (29-38) years for 610 sibling controls. There were more women than men (range, 57%-62%) in all groups, approximately 40% (range, 39%-43%) of participants had a university education, and most participants (range, 58%-73%) had British/northern European ethnicity. Risk of melanoma was less with higher use of sunscreen in childhood (OR for highest vs lowest tertiles, 0.60; 95% CI, 0.42-0.87; P = .02 for trend) and across the lifetime (OR, 0.65; 95% CI, 0.45-0.93; P = .07 for trend). Subgroup analyses suggested that the protective association of sunscreen with melanoma was stronger for people reporting blistering sunburn, receiving a diagnosis of melanoma at a younger age, or having some or many nevi. Total lifetime sun exposure was unrelated to melanoma risk (OR for highest vs lowest tertile, 0.97; 95% CI, 0.66-1.43; P = .94 for trend). By contrast, total sun exposure inversely weighted by sunscreen use (as a measure of sun exposure unprotected by sunscreen) was significantly associated with melanoma risk (OR, 1.80; 95% CI, 1.22-2.65; P = .007 for trend) and appeared stronger for people having lighter pigmentation or some or many nevi or using sunscreen to stay longer in the sun. Regular users of sunscreen were more likely to be female, younger, and of British or northern European ancestry and to have higher educational levels, lighter skin pigmentation, and a stronger history of blistering sunburn. Conclusions and Relevance Our findings provided evidence that regular sunscreen use is significantly associated with reduced risk of cutaneous melanoma among young adults and identified several characteristics associated with less sunscreen use.

The heterogeneity of multiple case breast cancer families that do not carry mutations in BRCA1 or BRCA2 (non-BRCA1/2 families) poses a challenge to the identification of breast cancer susceptibility genes. The aim of this study was to determine whether intrafamilial concordance in breast cancer pathology could identify subgroups of non-BRCA1/2 families with consistent genotypic features. Invasive breast cancers were reviewed from 84 individuals belonging to 30 multiple-case families; BRCA1 (n = 9), BRCA2 (n = 10), and non-BRCA1/2 (n = 11). Hierarchical cluster analysis based on histopathology and age at first diagnosis was then used to specify three subgroups designated Clusters 1-3. The genomic features of non-BRCA1/2 families were examined by genome wide linkage and FGFR2 SNP genotyping, according to whether they showed cluster-concordant or cluster-mixed familial pathology. The majority of pathogenic BRCA1 mutation carriers (80%) fell into a single cluster. In contrast pathogenic BRCA2 mutation carriers were distributed across all three clusters and within families, cluster groups were also generally mixed. Most non-BRCA1/2 mutation carriers belonged to Cluster 3 (71%). Genome wide linkage data from five non-BRCA1/2 Cluster 3-concordant families were compared with four mixed cluster non-BRCA1/2 families. This revealed a number of distinct linkage peaks, including some regions previously associated with breast cancer susceptibility. The distribution of low risk alleles in FGFR2 was not different between these two subgroups (P = 0.237). The pattern of breast cancer pathology concordance amongst family members may assist the investigation of breast cancer susceptibility in multiple case families.

Sunbed use is associated with increased risk of melanoma. Younger people might be more susceptible to the carcinogenic effects of ultraviolet radiation. We investigated the association between sunbed use and risk of early-onset cutaneous malignant melanoma. From the Australian Melanoma Family Study, a multicentre, population-based, case-control-family study, we analysed data for 604 cases diagnosed between ages 18 and 39 years and 479 controls. Data were collected by interview. Associations were estimated as odds ratios (ORs) using unconditional logistic regression, adjusting for age, sex, city, education, family history, skin color, usual skin response to sunlight and sun exposure. Compared with having never used a sunbed, the OR for melanoma associated with ever-use was 1.41 (95% confidence interval (CI) 1.01-1.96), and 2.01 (95% CI 1.22-3.31) for more than 10 lifetime sessions (P(trend) 0.01 with cumulative use). The association was stronger for earlier age at first use (P(trend) 0.02). The association was also stronger for melanoma diagnosed when aged 18-29 years (OR for more than 10 lifetime sessions = 6.57, 95% CI 1.41-30.49) than for melanoma diagnosed when 30-39 years (OR 1.60, 95% CI 0.92-2.77; P(interaction) 0.01). Among those who had ever used a sunbed and were diagnosed between 18 and 29 years of age, three quarters (76%) of melanomas were attributable to sunbed use. Sunbed use is associated with increased risk of early-onset melanoma, with risk increasing with greater use, an earlier age at first use and for earlier onset disease.

An antibody microarray was developed for profiling the surface proteome of melanoma cells, which may facilitate melanoma sub-classification and provide important prognostic information useful in predicting the clinical behavior of the melanoma (e.g., likely sites of metastatic spread), patient outcome and treatment response. Forty-eight antibodies were selected based on their correlation with melanoma development, progression and/or prognosis and printed on nitrocellulose slides. The immobilised antibodies capture live cells expressing corresponding antigens to produce a cell binding dot pattern representing the surface antigen profile (immunophenotype) of the melanoma. Surface antigen signatures were determined for a normal melanocyte and 6 melanoma cell lines and cell suspensions prepared from 10 surgically excised melanoma lymph node metastases. A procedure for obtaining separate surface antigen profiles for melanoma cells and leukocytes from clinical lymph node samples was also developed using anti-CD45 magnetic beads. The capture of live, bead-bound leukocytes on these antibody microarrays provides a significant enhancement of this microarray technology. The antibody microarray will be used to profile panels of surgically excised melanoma lymph node metastases (melanoma and leukocyte fractions) to determine whether the immunophenotypes correlate with clinicopathological characteristics, disease progression and clinical outcome.

10082Background: People with melanoma want and need effective treatments for managing and living with fear of cancer recurrence (FCR). This study reports the long-term efficacy of a psycho-educatio...

PURPOSE To validate differences in protein levels between good and poor prognosis American Joint Committee on Cancer (AJCC) stage III melanoma patients and compile a protein panel to stratify patient risk. EXPERIMENTAL DESIGN Protein extracts from melanoma metastases within lymph nodes in patients with stage III disease with good (n = 16, >4 years survival) and poor survival (n = 14

The CDKN2A locus encodes for two distinct tumor suppressor proteins, p16 INK4A and p14 ARF , involved in cell cycle regulation. CDKN2A germline mutations have been associated with familial predisposition to melanoma and other tumor types. Besides bona-fide pathogenic mutations, many sequence variants have been identified, but their effect is not well known. We detected the p.Gly23Asp missense mutation in one of the two tested melanoma patients of a family with three melanoma cases. Even though the mutated amino acid is located in a conserved domain that specifically binds to and blocks the function of CDK4/6, its lack of segregation with disease suggested a series of functional assays to discriminate between a pathogenic variant and a neutral polymorphism. The effect of this mutation has been investigated exploiting four p16 INK4A properties: its ability (i) to bind CDK4, (ii) to inhibit pRb phosphorylation, (iii) to evenly localize in the cell, and (iv) to cause cell cycle arrest. The mutant protein properties were evaluated transfecting three different cell lines (U2-OS and NM-39, both p16-null, and SaOS 2, p53 and pRb-null) with plasmids expressing either p16 wt , p16 23Asp , or the p16 32Pro pathogenic variant. We found that p16 23Asp was less efficient than p16 wt in CDK4 binding, in inhibiting pRb phosphorylation, in inducing G1 cell cycle arrest; moreover, its pattern of distribution throughout the cell was suggestive of protein aggregation, thus assessing a pathogenic role for p16 23Asp in familial melanoma.

Discovering and understanding genetic risk factors for melanoma and their interactions with phenotype, sun exposure, and other risk factors could lead to new strategies for melanoma control. This paper describes the Australian Melanoma Family Study, which uses a multicenter, population-based, case-control-family design. From 2001 to 2005, the authors recruited 1,164 probands including 629 cases with histopathologically confirmed, first-primary cutaneous melanoma diagnosed before age 40 years, 240 population-based controls frequency matched for age, and 295 spouse/friend controls. Information on lifetime sun exposure, phenotype, and residence history was collected for probands and nearly 4,000 living relatives. More than 3,000 subjects donated a blood sample. Proxy-reported information was collected for childhood sun exposure and deceased relatives. Important features of this study include the population-based, family-based design; a focus on early onset disease; probands from 3 major cities differing substantially in solar ultraviolet exposure and melanoma incidence; a population at high risk because of high ultraviolet exposure and susceptible pigmentation phenotypes; population-based, spouse/friend, and sibling controls; systematic recruitment of relatives of case and control probands; self and parent reports of childhood sun exposure; and objective clinical skin examinations. The authors discuss methodological and analytical issues related to the study design and conduct, as well as the potentially novel insights the study can deliver.

We report a genome-wide association study of melanoma conducted by the GenoMEL consortium based on 317K tagging SNPs for 1,650 selected cases and 4,336 controls, with replication in an additional two cohorts (1,149 selected cases and 964 controls from GenoMEL, and a population-based case-control study in Leeds of 1,163 cases and 903 controls). The genome-wide screen identified five loci with genotyped or imputed SNPs reaching P < 5 x 10(-7). Three of these loci were replicated: 16q24 encompassing MC1R (combined P = 2.54 x 10(-27) for rs258322), 11q14-q21 encompassing TYR (P = 2.41 x 10(-14) for rs1393350) and 9p21 adjacent to MTAP and flanking CDKN2A (P = 4.03 x 10(-7) for rs7023329). MC1R and TYR are associated with pigmentation, freckling and cutaneous sun sensitivity, well-recognized melanoma risk factors. Common variants within the 9p21 locus have not previously been 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 show independent association to disease risk.

Summary Background Accurate classification of primary melanocytic tumours as benign or malignant is crucial for prognostic prediction and appropriate patient management. Several chromosomal aberrations have been frequently identified in melanomas, but are absent in melanocytic naevi. We performed four-colour fluorescence in situ hybridisation (FISH) analysis of melanocytic tumours to determine the accuracy of the technique in classifying melanocytic tumours as benign or malignant. Methods FISH was performed on paraffin-embedded tissue from 40 histologically unequivocal melanocytic tumours (10 metastatic melanomas, 10 primary melanomas and 20 benign melanocytic naevi) using the product Vysis LSI RREB1/LSI MYB/LSI CCND1/CEP 6 probes (Abbott Molecular Laboratories, USA), which is designed to detect the copy number of the RREB1 (6p25), MYB (6q23), and CCND1 (11q13) genes and FISH positivity is defined by means of a scoring algorithm. Results FISH distinguished the melanomas and the naevi with a sensitivity of 90% (10/10 primary melanoma cases and 8/10 metastatic melanoma cases, respectively), and a specificity of 95%. The most common abnormalities in the melanomas were increased copies of 11q (70%) and 6p (70%), followed by 6q loss relative to cep6 (50%). Fifteen of the 18 positive melanomas were positive by more than one criterion. Conclusions The results of this study show that FISH, using a panel of four probes, is a sensitive and specific method of classifying benign and malignant melanocytic tumours. The four-colour FISH technique has the potential to assist in the stratification of the subgroup of melanocytic tumours which are difficult to classify using conventional histology.

Purpose: The aim of this prospective cohort study was to examine uptake and psychological, behavioral, and cognitive outcomes of genetic testing for melanoma risk among individuals with a known family-specific CDKN2A mutation. Methods: A total of 119 individuals were ascertained via a genetic epidemiological study and completed a series of mailed, self-administered questionnaires at multiple time points, including notification of genetic test availability, and 2 weeks and 12 months after receipt of genetic test results (for “test participants”), or 12 months after notification (for “decliners”). Results: Since January 2005, 21% of participants (n = 25) have undergone genetic testing, with 75% of those who have received results identified as mutation carriers (n = 15). Factors associated with uptake of genetic counseling included perceived susceptibility to melanoma (odds ratio = 3.60, P = 0.0008), and fatalistic beliefs about melanoma (odds ratio = 0.57, P = 0.005). Compared with baseline, carriers reported significantly reduced anxiety scores at 2 weeks, and reduced depression scores at 2 weeks and 12 months, after receipt of genetic test results. Carriers also reported a significantly greater frequency of clinical skin examination at 12-month follow-up compared with decliners (χ2 = 5.70, P = 0.02). No hypothesis testing was carried out for noncarriers because of their limited number. Conclusion: These data provide preliminary evidence for healthy psychological, behavioral, and cognitive adjustment after participation in genetic testing for melanoma risk.

We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer. (Mol Cancer Res 2009;7(1):41–54)

Metastatic cutaneous melanoma is highly resistant to cytotoxic drugs, and this contributes to poor prognosis. In vivo studies on the chemosensitivity of metastatic melanoma are rare and hampered by poor response rates to systemic chemotherapeutics. Patients who undergo isolated limb infusion (ILI) with cytotoxic drugs show high response rates and are, therefore, a good cohort for studying chemosensitivity in vivo. We used tumors from patients who underwent ILI to study the role of melanoma tumor-suppressor genes and oncogenes on melanoma chemosensitivity. Prospectively acquired tumors from 30 patients who subsequently underwent ILI with melphalan and actinomycin-D for metastatic melanoma were investigated for mRNA expression levels of p14(ARF), p16(INK4a), and MITFm. The mutation status of B-RAF, N-RAS, and PTEN were also determined. A high percentage of tumors had activating mutations in either B-RAF (15/30) or N-RAS (10/30) and only two tumors carried altered PTEN. High expression of p16(INK4a) and absence of an activating B-RAF mutation independently predicted response to treatment. Further, inducible expression of p16(INK4a) sensitized a melanoma cell line to death induced by melphalan or actinomycin-D. This study shows that high expression of p16(INK4a) or the absence of activated B-RAF correlates with in vivo response of melanoma to cytotoxic drugs.

Despite expanding knowledge regarding the genetics of melanoma, there have been few attempts to define the psychosocial experiences of individuals with a family history of this disease. This study explored the ways in which individuals at varying levels of risk perceive, and respond to, melanoma. Forty semi-structured interviews were undertaken with affected (n = 20) and unaffected (n = 20) individuals with or without a family history of melanoma. Data were analysed for potential thematic differences between risk groups, genders, and intentions to pursue genetic testing for melanoma risk. Overall, participants with a family history were in acceptance of their increased risk status and had developed ways of coping without major disruption to their daily lives. However, some participants expressed ambiguity regarding the causes of melanoma and the effectiveness of health behaviours such as sun protection. Major thematic patterns identified for those intending to pursue genetic testing were: negative emotional associations with melanoma; an emphasis on screening and sun avoidance, but not sun protection; and heightened perceptions of personal susceptibility to melanoma. In contrast, thematic patterns identified for those likely to decline testing were: ready access to stories of melanoma survival; and an emphasis on the causal role of sun exposure, whilst still believing that genetic factors may contribute to melanoma susceptibility. Compared to males, females reported a greater tendency to completely avoid the sun in order to reduce their melanoma risk. The data provide preliminary evidence for the importance of identifying misconceptions that may impede informed decision-making about genetic testing for melanoma risk. Copyright © 2007 John Wiley & Sons, Ltd.

Despite rapid advancements in molecular genetics research, little is known about the psychological experiences of individuals with a family history of melanoma. The present study aimed to identify factors contributing to psychological distress among affected and unaffected individuals with a strong family history of melanoma. A total of 121 adults who had recently been informed of the identification of a family-specific mutation in the CDKN2A melanoma susceptibility gene, completed a self-report questionnaire assessing cancer-specific and generalized distress, and a variety of potential predictors. Having a personal history of melanoma (OR = 3.37, p = 0.033), perceiving greater family implications of melanoma (OR = 2.52, p0.0001), and the tendency to monitor for threatening information (OR = 3.12, p = 0.008) were associated with melanoma-specific distress. Being childless (beta = 2.09, p = 0.007), perceiving sun exposure as an important cause of melanoma (beta = 1.15, p = 0.015), and perceiving greater family implications of melanoma (beta = 1.02, p = 0.002) were associated with greater generalized anxiety, while monitoring moderated the relationship between endorsement of a genetic model of melanoma and generalized anxiety (p = 0.005). As in other common familial cancers, distress was relatively uncommon in this familial melanoma cohort, even after notification of the presence of a family mutation. Participants do not contemplate their melanoma risk in isolation, but evaluate their risk vis-à-vis the experiences of their relatives.

Mutations in the DNA mismatch repair gene MSH2 lead to increased replication error and microsatellite instability and account for a substantial proportion of hereditary non-polyposis colorectal cancer (Lynch syndrome). A recent international collaborative genome-wide linkage scan (GWS) for breast cancer susceptibility loci found some evidence for there being a breast cancer susceptibility gene in a genomic region on chromosome 2p close to MSH2. We sought to investigate the possibility that mutations in MSH2 might explain the multiple cases of breast cancer in some families that were included in the international GWS. DNA samples from the affected probands of 59 multiple-case breast cancer families, many of whom gave LOD scores >0.5 in the MSH2 region, were screened for large genomic alterations in MSH2 via the Multiplex Ligation-dependant Probe Amplification (MLPA) assay and for coding region mutations via exonic sequencing. Several of the families also contained cases of colorectal cancer in addition to breast cancer and had been included in the GWS that had identified a positive LOD score on chromosome 2p. Using MLPA, c.1236C > T was identified in one proband but this variant was not predicted to create an alternate acceptor/donor site within exon 7 MSH2 using in silico analyses. A c.1734T > C was identified in a second proband via exonic sequencing but testing of the variant in other family members did not support segregation of this variant with disease. Extensive screening of 59 multiple-case breast cancer families did not identify any coding region mutations or larger genomic alterations in MSH2 that might implicate MSH2 as a breast cancer susceptibility gene.

Melanocytic lesions, including Spitz nevi (SN), common benign nevi (CBN) and cutaneous metastatic melanoma (CMM), were analyzed for activating mutations in NRAS, HRAS and BRAF oncogenes, which induce cellular proliferation via the MAP kinase pathway. One of 22 (4.5%) SN tested showed an HRAS G61L mutation. Another lesion, a 'halo' SN, showed a BRAF V600E (T1796A) mutation. BRAF V600E mutations were found in two thirds (20/31) of CBN, while a further 19% (6/31) showed NRAS codon 61 mutations. One third of CMM (10/30) had various BRAF mutations of codon 600, and a further 6% (2/31) showed NRAS codon 61 mutations. Seventeen SN tested for loss of heterozygosity (LOH) at 9p and 10q regions, known to be frequently deleted in melanoma, showed LOH at the 9p loci D9S942 and IFNA. A further lesion was found with low-level microsatellite instability at one locus, D10S214. The low rate of RAS-RAF mutations (2/22, 9.1%) observed in SN suggests that these lesions harbor as yet undetected activating mutations in other components of the RAS-RAF-MEK-ERK-MAPK pathway. Germline DNA from members of 111 multiple-case melanoma families, representing a range of known (CDKN2A) and unknown predisposing gene defects, was analyzed for germline BRAF mutations, but none was found.

© 2015, National Academy of Sciences. All rights reserved. Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized in parallel to different anatomic sites, rather than sequentially from one site to the next. In five of these six patients, the metastasizing cells had themselves arisen from a common parental subpopulation in the primary, indicating that the ability to establish metastases is a late-evolving trait. Interestingly, we discovered that individual metastases were sometimes founded by multiple cell populations of the primary that were genetically distinct. Such establishment of metastases by multiple tumor subpopulations could help explain why identical resistance variants are identified in different sites after initial response to systemic therapy. One primary tumor harbored two subclones with different oncogenic mutations in CTNNB1, which were both propagated to the same metastasis, raising the possibility that activation of wingless-type mouse mammary tumor virus integration site (WNT) signaling may be involved, as has been suggested by experimental models.

Purpose: Understanding why some melanomas test negative for PD-L1 by IHC may have implications for the application of anti-PD-1 therapies in melanoma management. This study sought to determine somatic mutation and gene expression patterns associated with tumor cell PD-L1 expression, or lack thereof, in stage III metastatic melanoma to better define therapeutically relevant patient subgroups. Experimental Design: IHC for PD-L1 was assessed in 52 American Joint Committee on Cancer stage III melanoma lymph node specimens and compared with specimen-matched comprehensive clinicopathologic, genomic, and transcriptomic data. Results: PD-L1–negative status was associated with lower nonsynonymous mutation (NSM) burden (P = 0.017) and worse melanoma-specific survival [HR = 0.28 (0.12–0.66), P = 0.002] in stage III melanoma. Gene set enrichment analysis identified an immune-related gene expression signature in PD-L1–positive tumors. There was a marked increase in cytotoxic T-cell and macrophage-specific genes in PD-L1–positive melanomas. CD8Ahigh gene expression was associated with better melanoma-specific survival [HR = 0.2 (0.05–0.87), P = 0.017] and restricted to PD-L1–positive stage III specimens. NF1 mutations were restricted to PD-L1–positive tumors (P = 0.041). Conclusions: Tumor negative PD-L1 status in stage III melanoma lymph node metastasis is a marker of worse patient survival and is associated with a poor immune response gene signature. Lower NSM levels were associated with PD-L1–negative status suggesting differences in somatic mutation profiles are a determinant of PD-L1–associated antitumor immunity in stage III melanoma. Clin Cancer Res; 22(15); 3915–23. ©2016 AACR.

Background Despite a good prognosis for most melanoma survivors, many experience substantial fear of new or recurrent melanoma, worry and anxiety about the future, and unmet healthcare needs. In this protocol, we outline the design and methods of the Melanoma Care Study for melanoma survivors at high risk of developing new primary disease. The objective of this study is to evaluate the efficacy and cost-effectiveness of a psycho-educational intervention for improving psychological and behavioural adjustment to melanoma risk. Design The study design is a two-arm randomised controlled trial comparing a psycho-educational intervention to usual care. Methods The intervention is comprised of a newly-developed psycho-educational booklet and three telephone sessions delivered by a trained psychologist. A total of 154 melanoma survivors at high risk of developing new primary disease who are attending one of three melanoma high risk clinics in New South Wales, Australia, will be recruited. Participants will be assessed at baseline (6 weeks before their high risk clinic dermatological appointment), and then 4 weeks and 6 months after their appointment. If effectiveness of the intervention is demonstrated at 6 months, an additional assessment at 12 months is planned. The primary outcome is fear of new or recurrent melanoma, as assessed by the Fear of Cancer Recurrence Inventory (FCRI). Secondary outcomes include anxiety, depression, unmet supportive care needs, satisfaction with clinical care, knowledge, behavioural adjustment to melanoma risk, quality of life, and cost-effectiveness of the intervention from a health system perspective. Following the intention-to-treat principle, linear mixed models will be used to analyse the data to account for repeated measures. A process evaluation will also be carried out to inform and facilitate potential translation and implementation into clinical practice. Discussion This study will provide high quality evidence on the efficacy and cost-effectiveness of a psycho-educational intervention aimed at improving psychological and behavioural adjustment amongst melanoma survivors at high risk of new primary disease. Trial registration ACTRN 12613000304730

Although the effectiveness of many psychosocial interventions for people with cancer has been established, one barrier to implementation in routine clinical care is a lack of data on cost-effectiveness. We conducted a systematic review to assess the cost-effectiveness of psychosocial interventions for improving psychological adjustment among people with cancer.A systematic review of the literature, study appraisal and narrative synthesis.Eight studies involving 1668 patients were identified. Four of these reported outcomes in a cost per quality adjusted life year (QALY) framework. Six studies reported psychosocial interventions to be cost-effective for improving health-related quality of life, mood, pain, distress or fear of cancer progression, compared with usual care. Of the six psychosocial interventions identified as cost-effective, three were cognitive-behavioural therapy-based interventions, one was a nurse-delivered telephone follow-up plus educational group programme, one was a group-based exercise and psychosocial intervention and one was a series of 10 face-to-face or telephone-based individual support sessions delivered by a nurse. The quality of studies assessed according to the Consensus Health Economic Criteria-list criteria was good overall; however, some studies were limited by their choice of outcome measure and omission of important categories of costs.Several psychosocial interventions, particularly those based on cognitive-behavioural therapy, have been demonstrated to represent good value for money in cancer care. Future research should include a clear definition of the economic question, inclusion of all relevant costs, and consideration of utility-based quality of life measures for QALY estimation. Copyright © 2016 John WileySons, Ltd.

Summary We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF , mutant RAS , mutant NF1 , and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Background Inherited susceptibility genes have been associated with histopathologic characteristics of tumors. Objective We sought to identify associations between histology of melanomas and CDKN2A genotype. Methods This was a case-control study design comparing 28 histopathologic tumor features among individuals with sporadic melanomas (N = 81) and cases from melanoma families with (N = 123) and without (N = 120) CDKN2A germline mutations. Results Compared with CDKN2A − cases, mutation carriers tended to have histologic features of superficial spreading melanoma subtype including higher pigmentation ( P trend = .02) and increased pagetoid scatter ( P trend = .07) after adjusting for age at diagnosis, sex, and American Joint Committee on Cancer thickness category. Similar associations were observed when comparing mutation carriers with a combined group of CDKN2A − (wild type) and sporadic melanomas. The presence of spindle cell morphology in the vertical growth phase was also an important predictor of genotype. Of the 15 cases with this phenotype, none were observed to harbor a CDKN2A mutation. Limitations Our study examined rare mutations and may have been underpowered to detect small, but biologically significant associations between histology and genotype. Conclusion Familial melanomas with CDKN2A mutations preferentially express a histologic phenotype of dense pigmentation, high pagetoid scatter, and a non-spindle cell morphology in the vertical growth phase.

In metastatic melanoma, it is vital to identify and validate biomarkers of prognosis. Previous studies have systematically evaluated protein biomarkers or mRNA-based expression signatures. No such analyses have been applied to microRNA (miRNA)-based prognostic signatures. As a first step, we identified two prognostic miRNA signatures from publicly available data sets (Gene Expression Omnibus/The Cancer Genome Atlas) of global miRNA expression profiling information. A 12-miRNA signature predicted longer survival after surgery for resection of American Joint Committee on Cancer stage III disease (>4 years, no sign of relapse) and outperformed American Joint Committee on Cancer standard-of-care prognostic markers in leave-one-out cross-validation analysis (error rates 34% and 38%, respectively). A similar 15-miRNA biomarker derived from The Cancer Genome Atlas miRNA-seq data performed slightly worse (39%) than these current biomarkers. Both signatures were then assessed for replication in two independent data sets and subjected to systematic cross-validation together with the three other miRNA-based prognostic signatures proposed in the literature to date. Five miRNAs (miR-142-5p, miR-150-5p, miR-342-3p, miR-155-5p, and miR-146b-5p) were reproducibly associated with patient outcome and have the greatest potential for application in the clinic. Our extensive validation approach highlighted among multiple independent cohorts the translational potential and limitations of miRNA signatures, and pointed to future directions in the analysis of this emerging class of markers.

The shelterin complex protects chromosomal ends by regulating how the telomerase complex interacts with telomeres. Following the recent finding in familial melanoma of inactivating germline mutations in POT1, encoding a member of the shelterin complex, we searched for mutations in the other five components of the shelterin complex in melanoma families.Next-generation sequencing techniques were used to screen 510 melanoma families (with unknown genetic etiology) and control cohorts for mutations in shelterin complex encoding genes: ACD, TERF2IP, TERF1, TERF2, and TINF 2. Maximum likelihood and LOD [logarithm (base 10) of odds] analyses were used. Mutation clustering was assessed with χ(2) and Fisher's exact tests. P values under .05 were considered statistically significant (one-tailed with Yates' correction).Six families had mutations in ACD and four families carried TERF2IP variants, which included nonsense mutations in both genes (p.Q320X and p.R364X, respectively) and point mutations that cosegregated with melanoma. Of five distinct mutations in ACD, four clustered in the POT1 binding domain, including p.Q320X. This clustering of novel mutations in the POT1 binding domain of ACD was statistically higher (P = .005) in melanoma probands compared with population control individuals (n = 6785), as were all novel and rare variants in both ACD (P = .040) and TERF2IP (P = .022). Families carrying ACD and TERF2IP mutations were also enriched with other cancer types, suggesting that these variants also predispose to a broader spectrum of cancers than just melanoma. Novel mutations were also observed in TERF1, TERF2, and TINF2, but these were not convincingly associated with melanoma.Our findings add to the growing support for telomere dysregulation as a key process associated with melanoma susceptibility.

Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.

Most of the known breast cancer susceptibility genes (BRCA1, BRCA2, CHEK2 and ATM) are involved in the damage response pathway. Other members of this pathway are therefore good candidates for additional breast cancer susceptibility genes. ATR, along with ATM, plays a central role in DNA damage recognition and Chk1 relays checkpoint signals from both ATR and ATM. PPP2R1B and PPP2R5B code for subunits of protein phosphatase 2A (PP2A), which regulates autophosphorylation of ATM. In addition, EIF2S6/Int-6, which was originally identified as a common integration site for the mouse mammary tumour virus in virally induced mouse mammary tumours, is a candidate breast cancer susceptibility gene because of its putative role in maintaining chromosome stability. To investigate the role of ATR, CHK1, PPP2R1B, PPP2R5B and EIF2S6/Int-6, we carried out mutation analysis of these genes in the index cases from non-BRCA1/BRCA2 breast cancer families. We also screened sporadic breast tumours for somatic mutations in PPP2R1B and PPP2R5B. Although we identified many novel variants, we found no evidence that highly penetrant germline mutations in these five genes contribute to familial breast cancer susceptibility.

Purpose: The psychological consequences of genetic testing for mutations among individuals at increased risk of developing melanoma remain unexamined. The present study aimed to explore anticipated emotional, behavioral, cognitive, and familial responses to hypothetical genetic testing for melanoma susceptibility. Methods: Forty semi-structured interviews were undertaken with affected (n=20) and unaffected (n=20) individuals at either high or average risk of developing melanoma due to family history. Results: In-depth thematic analysis revealed that, in response to being identified as a mutation carrier, most participants with a family history anticipated calmly accepting their increased risk; either increasing precaution adoption or maintaining already vigilant behavioral practices; perceiving such information as important and valuable; and communicating genetic test results to family members, despite the acknowledgement of potential difficulties. In response to being identified as a non-carrier, the majority of participants expected to feel relieved; to maintain current precautionary health practices; to still perceive themselves at some risk of developing melanoma; and to be wary of the potential negative behavioral consequences of disclosing such information to family members. Women appeared more likely than men to acknowledge the potential for depression and worry following genetic testing. In contrast, more males than females expected to carry a gene mutation, and viewed their current preventive practices as optimum. Conclusion: Genetic testing for melanoma risk is likely to elicit a complex array of emotional, behavioral, cognitive, and familial responses for both testees and their family members, and these responses are likely to bear subtle differences for males and females.

Purpose Marked interindividual variation in drug disposition and toxicity pose an ongoing challenge to chemotherapy dosage individualization. The aim of this study was to evaluate pretreatment clinical features, genotype and functional indicators of drug clearance as predictors of vinorelbine clearance, and myelotoxicity that could inform dosage optimization. Patients and Methods Forty-one patients with cancer received a 60 mg intravenous dose of vinorelbine. Pretreatment routine body size measurements and blood tests were performed. Midazolam clearance and hepatic technetium labeled sestamibi (99mTc-MIBI) clearance were used to investigate CYP3A and ABCB1 (MDR1, P-glycoprotein) phenotype respectively and selected single nucleotide polymorphisms in CYP3A and ABCB1 were documented. A limited blood sampling strategy was employed and vinorelbine concentrations were determined by high-performance liquid chromatography. Posterior Bayesian estimates of vinorelbine clearance were obtained for each patient using population pharmacokinetic modeling. Myelotoxicity was estimated from the fractional survival of neutrophils post-treatment. Results There was 4.3-fold variation in vinorelbine clearance across the cohort. In a multivariable analysis, pretreatment estimated creatinine clearance (P < .01) and hepatic 99mTc-MIBI clearance (P = .01) were independent predictors of vinorelbine clearance. Fractional survival of neutrophils ranged from 1.3% to 100% and was significantly correlated with vinorelbine clearance (P < .01). Body-surface area was the only pretreatment predictor of fractional survival of neutrophils independent of vinorelbine clearance (P = .02). Conclusion Specific indicators of drug clearance provide predictive information about vinorelbine pharmacokinetics, and body-surface area, probably reflecting normal bone marrow reserve, provides an additional pharmacodynamic indicator. Use of a fixed dose of vinorelbine with modifications guided by pretreatment measures is worthy of prospective evaluation.

Introduction: The potential role of genetic testing in families with an inherited pattern of melanoma is a complex issue, and yet limited data exist on perceptions of predictive genetic testing for mutations among individuals at high risk of melanoma. Methodology: Forty semi-structured interviews were undertaken with affected and unaffected individuals at either high or average risk of developing melanoma due to family history. Interviews addressed key issues such as: the role of genetics in causal attributions for melanoma; genetic testing intentions and motivations; perceived accuracy of genetic testing in predicting melanoma onset, and the impact of varied accuracy on testing intentions; views on the testing of children; perceived benefits and limitations of testing; and information needs and communication preferences. Results: In-depth thematic analysis revealed a number of important qualitative differences between groups at varying risk of melanoma, and genders. Specifically, participants with a family history of melanoma believed genetic factors play an important role in melanoma causation; conveyed strong intentions to pursue genetic testing; and viewed the benefits of genetic testing as outweighing the limitations. Females appeared to endorse the testing of children more firmly than males, and males' intentions to pursue testing appeared more contingent on penetrance than females'. Across groups, the most preferred communication option was an informational video. Conclusion: Those at high risk of melanoma due to family history express a strong interest in predictive genetic testing. Copyright © 2006 John Wiley & Sons, Ltd.

Germ-line mutations of the tumor-suppressor gene CDKN2A predispose individuals to melanoma in families worldwide. However, coding mutations of CDKN2A have not been detected in a significant proportion of those affected. The identification of a disease-associated intronic mutation of CDKN2A in UK families, which has proved to be the most common CDKN2A mutation as yet identified in this population, has highlighted the possibility that additional causal mutations may lie within the intronic sequence of the gene. In this article, we describe the comprehensive screening of 109 English and 26 Australian melanoma pedigrees for intronic mutations of CDKN2A. In total, 24 sequence variants were identified across the two introns of the gene. We show evidence that two of the CDKN2A intronic variants (IVS1 + 1104 C > A and IVS1 - 1104 C > G) predispose to melanoma. IVS1 + 1104 was shown to result in the aberrant splicing of both p16(INK4a) and p14(ARF) mRNA. Overall, however, the proportion of English melanoma families with these variants is small.

Melanoma-associated germline mutations affecting the tumor suppressor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16INK4a, have been identified in over 100 melanoma-prone families worldwide. To predict the melanoma risk for carriers of specific mutations, mutant p16INK4a can be tested in biochemical and cellular assays. In most cases, p16INK4a mutations with predicted disease relation, due to segregation with melanoma, are functionally impaired in such assays. The N-terminal 24 base pair duplication of CDKN2A, however, encodes a p16INK4a variant previously shown to have wild-type function, despite segregating with melanoma in at least 5 melanoma families. To clarify whether the duplication mutation has a cell cycle regulatory defect or behaves like wild-type p16INK4a, we reanalyzed the cell cycle-inhibitory activity of this mutation. Stable cell clones of the p16-null WMM1175 melanoma cell line inducible for ectopic p16INK4a were used in this study. In these cells, p16INK4a expression can be controlled at physiologic levels. Our results show that in comparison to wild-type p16INK4a, the duplication mutant induced weaker S-phase inhibition and cells expressing this mutant form of p16INK4a retained colony formation ability. We also show that the cell cycle-regulatory defect of the p16INK4a duplication mutant was associated with decreased inhibition of pRb phosphorylation even though it retained significant binding to CDK4.

Background and Objective The adenosine triphosphate-binding cassette transporter ABCB1 (P-glycoprotein) mediates terminal excretion of many chemotherapeutic agents, and variable ABCB1 activity may be an important contributor to interpatient variability in the clearance of chemotherapeutic agents. Our objective was to determine the elimination constant (kH) for hepatic elimination of technetium Tc 99m-labeled sestamibi (99mTc-MIBI) in patients with cancer and to compare this putative indicator of ABCB1 phenotype with clinical features and common ABCB1 genetic variants. Methods 99mTc-MIBI kH was determined from the time-dependent elimination profile of 99mTc-MIBI over a 90-minute hepatic scanning period in 66 patients with cancer. Single nucleotide polymorphisms (SNPs) in ABCB1 exons 12 (C1236T), 21 (G2677T/A), and 26 (C3435T) were documented by polymerase chain reaction-restriction fragment length polymorphism analysis. Results: There was a 12-fold variation in 99mTc-MIBI kH across the cohort, which was not correlated with sex, age, conventional liver function test results, previous chemotherapy treatment, or history of liver metastasis. Mean 99mTc-MIBI kH was significantly reduced in patients with SNPs in exons 21 and 26 such that mean 99mTc-MIBI kH was 1.90 times (95% confidence interval, 1.14–2.66; P = .02) and 2.21 times (95% confidence interval, 1.47–2.97; P < .01) higher in subjects homozygous for the wild-type alleles than in those homozygous for these SNPs, respectively. Conclusion Hepatic elimination of 99mTc-MIBI is a potential in vivo probe of hepatic ABCB1 activity that is significantly associated with the presence of common SNPs in ABCB1. 99mTc-MIBI hepatic scanning may provide a useful pretreatment indicator of ABCB1-mediated drug clearance in cancer patients. Clinical Pharmacology & Therapeutics (2005) 77, 33–42; doi: 10.1016/j.clpt.2004.09.002

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs' and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.

Over the past 20 years, the incidence of cutaneous malignant melanoma (CMM) has increased dramatically worldwide. A positive family history of the disease is among the most established risk factors for CMM; it is estimated that 10% of CMM cases result from an inherited predisposition. Although mutations in two genes, CDKN2A and CDK4, have been shown to confer an increased risk of CMM, they account for only 20%-25% of families with multiple cases of CMM. Therefore, to localize additional loci involved in melanoma susceptibility, we have performed a genomewide scan for linkage in 49 Australian pedigrees containing at least three CMM cases, in which CDKN2A and CDK4 involvement has been excluded. The highest two-point parametric LOD score (1.82; recombination fraction [theta] 0.2) was obtained at D1S2726, which maps to the short arm of chromosome 1 (1p22). A parametric LOD score of 4.65 (theta=0) and a nonparametric LOD score of 4.19 were found at D1S2779 in nine families selected for early age at onset. Additional typing yielded seven adjacent markers with LOD scores3 in this subset, with the highest parametric LOD score, 4.95 (theta=0) (nonparametric LOD score 5.37), at D1S2776. Analysis of 33 additional multiplex families with CMM from several continents provided further evidence for linkage to the 1p22 region, again strongest in families with the earliest mean age at diagnosis. A nonparametric ordered sequential analysis was used, based on the average age at diagnosis in each family. The highest LOD score, 6.43, was obtained at D1S2779 and occurred when the 15 families with the earliest ages at onset were included. These data provide significant evidence of a novel susceptibility gene for CMM located within chromosome band 1p22.

Autosomal dominant inheritance of mutations in the INK4a/ARF locus or the CDK4 gene may confer a high risk of cutaneous melanoma development. The penetrance of INK4a/ARF mutations is influenced by UV exposure. Inherited variants in the melanocortin-1 receptor also confer increased risk of cutaneous melanoma. Features associated with increased genetic susceptibility to cutaneous melanoma include the presence of multiple affected first-degree relatives on one side of the family, multiple primary melanomas in the same individual, earlier age of onset, and the presence of multiple atypical nevi, but none of these factors reliably predicts for the presence of INK4a/ARF mutations. It is currently premature to offer predictive DNA testing for melanoma outside of defined research protocols. This is because of (1) the low likelihood of finding mutations in known melanoma susceptibility genes, even in more than 60% of melanoma-prone kindreds; (2) the broad confidence limits on current estimates of lifetime penetrance of INK4a/ARF mutations and the wide variation in this penetrance with locality; (3) a high “background” incidence of melanoma in non-mutation carriers in melanoma-prone families; (4) current uncertainties about the factors determining the functionality and phenotypic expression of the trait among carriers of these mutations (penetrance), even if found; and (5) the lack of proved efficacy of melanoma prevention and surveillance strategies, even for mutation carriers. Rather than singling out those deemed to be at high risk because of family history, all patients carrying risk factors for cutaneous melanoma should be subject to stringent programs of sun protection and skin surveillance.

Family history is a strong risk factor for the development of primary melanoma and is associated in a subset with inherited mutations in melanoma susceptibility genes. This study sought to determine whether differences in metastatic pattern exist between patients with a positive family history (FH+) and those with a negative family history (FH-). Such differences could have importance for clinical management and in the determination of the function of both known and putative melanoma susceptibility genes. A retrospective, nested case-controlled study was performed. Of the FH+ cohort (n = 38), 26 were from kindreds with two histologically verified affected first-degree relatives and 12 were from kindreds with three or more affected members, at least two of whom were first-degree relatives. Three FH- controls from the Sydney Melanoma Unit database were matched to each case for age, sex, stage at diagnosis, number of primary melanomas and year of diagnosis (n = 114). There were no statistically significant differences between the two groups with regard to overall survival from initial diagnosis (FH+, 57.4 months; FH-, 50.0 months; P = 0.99), median survival from time of first metastasis (FH+, 15.4 months; FH-, 15.9 months; P = 0.94), or median disease-free interval (FH+, 26.4 months; FH-, 29.7 months; P = 0.73). On multivariate conditional logistic regression analysis, there was no statistically significant difference between the two groups in the probability of developing initial metastases or of ever developing metastases at specific sites. Survival, disease-free interval and distribution of metastatic sites are similar in both familial and non-familial melanoma. Genetic susceptibility for melanoma may lower the threshold for entering an otherwise common molecular pathway for tumour development and evolution.

Introduction: Melanoma accounts for 5% of all skin cancers, yet is the most common cause of skin cancer-related deaths, and is the commonest lethal malignancy in young people ( Methods: Protein extracts from early passages of ten AJCC Stage III local lymph node metastatic melanoma cell lines with known MAPK mutational status (BRAFmut, NRASmut, MAPKwt,) were used. Further, 32 fresh frozen NRASmut or BRAFmut AJCC Stage IIIC metastatic melanoma (lymph node metastases) specimens were obtained from patients with varying survival. Lysed cells and tissue were digested with trypsin and analysed by LC-MS/MS using a TripleTOF 5600/6600 mass spectrometer. Data-independent acquisition (or SWATH-MS) was used to match multiplexed MS/MS spectra against a spectral ion library enabling relative protein quantification. Cell line viability in the presence of 2µM AZD6244 (MEKi) was determined using a PrestoBlue assay. Results: The SWATH-MS approach enabled quantification of ~2500 proteins across all 10 cell lines. Principal component analysis clearly segregated melanomas based on in vitro sensitivity to MEK inhibition, whereas genotype alone did not. In total, we show 57 proteins whose abundances correlate (r²>0.75) with response to MEKi, revealing roles in cell pigmentation biosynthesis, lipid metabolism, adherence and inter-cell communication. Survival analysis demonstrated that patients whose cell lines were responsive to MEKi had lower mortality than patients whose cells were resistant to MEKi (survival >7.5 years MEKi sensitive versus Conclusion: Proteomic profiling by mass spectrometry demonstrated a MEK inhibition phenotype in early passage cell lines and fresh frozen melanoma metastases which may provide utility for selecting patients to treat with MEK drugs. Citation Format: Christoph Krisp, Robert Parker, Dana Pascovici, James Wilmott, John F. Thompson, Graham J. Mann, Richard A. Scolyer, Nicholas K. Hayward, Mark P. Molloy. Proteome phenotype of stage III metastatic melanoma and response to MEK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2206. doi:10.1158/1538-7445.AM2017-2206

Melanoma is the deadliest form of human skin cancer. The incidence of melanoma continues to rise. Recent advances in knowledge of melanoma genetics, genomics and biology has led to an optimistic view of the therapeutic outlook for melanoma patients. We analyzed sequence data from >500 melanoma genomes/exomes to identify novel tumor suppressor genes in melanoma. RASA2 was identified as the most highly somatically mutated novel tumor suppressor gene. RASA2 was mutated in 5% of melanomas and deleted in an additional 16.4% of cases. RASA2 is a GTPase Activating Protein (GAP) that regulates RAS; which is one of the most highly mutated oncogenes in melanoma but drugs targeting RAS have as yet shown poor efficacy. The role of RASA2 has not been investigated in melanoma. NF1, which encodes another RAS- specific GAP, was found to be frequently mutated in melanoma. Interestingly, mutations in RASA2 and NF1 co-occur in the same patients with high frequency. We plan to elucidate the roles of RASA2 in melanomagenesis and to understand why RASA2 and NF1 mutations co-occur despite the fact that both proteins are RasGAPs. Ras includes three isoforms: NRas, KRas and HRas. Our preliminary data show that RASA2 is more specific to NRAS and that NF1 is more specific to KRAS and HRAS. This finding highlights the existence of a paradigm of cooperativity in which combined loss of multiple negative regulators (RASA2 and NF1) of the RAS pathway is required for melanoma development. Therefore, this type of enhancement of RAS signaling is possibly selected for in some melanomas. We will apply a proteomic screen using BioID to identify RASA2 and NF1 binding partners to provide insights into the functional effects and consequences of alterations in RASA2 and NF1. We expect that these studies will not only identify the cellular components that contribute to the Ras signaling pathway but will also identify potential novel therapeutic targets. Citation Format: Rand Arafeh, Nouar Qutob, Rafi Rafi Emmanuel, Jason Madore, Abdel Elkahloun, James S. James S. Wilmott, Jared J. Gartner, Antonella Di Pizio, Ron Rotkopf, Ken Dutton-Regester, Victoria Hill, Antonia Pritchard, Jimmy C. Lin, Steven A Rosenberg, Javed Khan, Shifra Ben-Dor, Masha Y. Masha Y. Niv, Igor Ulitsky, Graham J Mann, Richard A. Scolyer, Nicholas K. Hayward, Yardena Samuels. Deciphering distinct roles of RASA2 in melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-031. doi:10.1158/1538-7445.AM2017-LB-031

9541 Background: BRAF V600E and V600K/R mm have distinct clinicopathologic features suggesting different etiology. V600K/R mm appears less responsive to MAPK inhibitors (MAPKi) compared to V600E MM. We investigated potential mechanisms for this by comparing the gene expression and mutation profiles of these two melanoma subgroups. Methods: BRAF V600 mutant mm patients (pts) treated with MAPKi (BRAFi +/- MEKi) between July/2009 and July/2013 were selected. Demographics, clinicopathologic features and clinical outcomes were examined. Pre-treatment FFPE tumors underwent RNA expression profiling (795-gene nanostring panel) and DNA sequencing (239 gene NGS panel). Molecular results were validated using an independent cohort from the The Cancer Genome Atlas (TCGA). Results: 95 mm pts were included (78 V600E, 17 V600K/R), with median (med) follow-up of 18.4 months (mo). 74 (78%) had BRAFi, 21 (22%) had BRAF/MEKi. At MAPKi start, there were no differences between subgroups regarding age, gender, ECOG, AJCC stage or LDH level. V600K/R pts had a trend to less tumour regression by RECIST (med 30% vs 51%, p = 0.08) and shorter PFS (med 5.1 vs 7.1mo, p = 0.08) than V600E, with no difference in OS (20.8mo vs 17.9mo, p = 0.64). V600K/R had lower expression of the MAPK-pathway feedback regulator DUSP6 and glycosyltransferase GCNT1, compared to V600E (p < 0.05). Analysis of TCGA data (122 V600E, 21 V600K/R) confirmed these findings. There was a trend toward higher mutational load in V600K/R than V600E, confirmed with TCGA data (p < 0.05). V600K/R had a higher proportion of mutations in PIK3CA and several tumour suppressor genes (FBXW7, NF2, RB1 and SMAD4), with only FBXW7 confirmed using TCGA data. Conclusions: V600K/R mm has inferior response and shorter survival with MAPKi than V600E, potentially due to less reliance on MAPK pathway activation (lower DUSP6 expression) and greater use of alternative drivers of oncogenesis (higher mutational load, particularly in tumor suppressor genes). Further analyses will be performed, including comparison of MAPK and additional pathway signalling in cell models. Response to immunotherapy will also be examined.

Importance The identification of a subgroup at higher risk of melanoma may assist in early diagnosis. Objective To characterize melanoma patients and the clinical features associated with their melanomas according to patient risk factors: many nevi, history of previous melanoma, and family history of melanoma, to assist with improving the identification and treatment of a higher-risk subgroup. Design, Setting, and Participants The Melanoma Patterns of Care study was a population-based observational study of physicians’ reported treatment of 2727 patients diagnosed with an in situ or invasive primary melanoma over a 12-month period from October 2006 to 2007 conducted in New South Wales. Our analysis of these data took place from 2015 to 2016. Main Outcomes and Measures Age at diagnosis and body site of melanoma. Results Of the 2727 patients with melanoma included, 1052 (39%) were defined as higher risk owing to a family history of melanoma, multiple primary melanomas, or many nevi. Compared with patients with melanoma who were at lower risk (ie, without any of these risk factors), the higher-risk group had a younger mean age at diagnosis (62 vs 65 years, P

Background Classical approaches to predicting patient clinical outcome via gene expression information are primarily based on differential expression of unrelated genes (single-gene approaches) or genes related by, for example, biologic pathway or function (gene-sets). Recently, network-based approaches utilising interaction information between genes have emerged. An open problem is whether such approaches add value to the more traditional methods of signature modelling. We explored this question via comparison of the most widely employed single-gene, gene-set, and network-based methods, using gene expression microarray data from two different cancers: melanoma and ovarian. We considered two kinds of network approaches. The first of these identifies informative genes using gene expression and network connectivity information combined, the latter drawn from prior knowledge of protein-protein interactions. The second approach focuses on identification of informative sub-networks (small networks of interacting proteins, again from prior knowledge networks). For all methods we performed 100 rounds of 5-fold cross-validation under 3 different classifiers. For network-based approaches, we considered two different protein-protein interaction networks. We quantified resulting patterns of misclassification and discussed the relative value of each relative to ongoing development of prognostic biomarkers. Results We found that single-gene, gene-set and network methods yielded similar error rates in melanoma and ovarian cancer data. Crucially, however, our novel and detailed patient-level analyses revealed that the different methods were correctly classifying alternate subsets of patients in each cohort. We also found that the network-based NetRank feature selection method was the most stable. Conclusions Next-generation methods of gene expression signature modelling harness data from external networks and are foreshadowed as a standard mode of analysis. But what do they add to traditional approaches? Our findings indicate there is value in the way in which different subspaces of the patient sample are captured differently among the various methods, highlighting the possibility of 'combination' classifiers capable of identifying which patients will be more accurately classified by one particular method over another. We have seen this clearly for the first time because of our in-depth analysis at the level of individual patients.

Background Mutations in the CDKN2A and CDK4 genes predispose to melanoma. From three case-control studies of cutaneous melanoma, we estimated the prevalence and predictors of these mutations for people from regions with widely differing latitudes and melanoma incidence. Methods Population-based cases and controls from the United Kingdom (1586 cases, 499 controls) and Australia (596 early-onset cases, 476 controls), and a hospital-based series from Spain (747 cases, 109 controls), were screened for variants in all exons of CDKN2A and the p16INK4A binding domain of CDK4. Results The prevalence of mutations for people with melanoma was similar across regions: 2.3%, 2.5% and 2.0% for Australia, Spain and the United Kingdom respectively. The strongest predictors of carrying a mutation were having multiple primaries (odds ratio (OR) = 5.4, 95% confidence interval (CI: 2.5, 11.6) for 2 primaries and OR = 32.4 (95% CI: 14.7, 71.2) for 3 or more compared with 1 primary only); and family history (OR = 3.8; 95% CI:1.89, 7.5) for 1 affected first- or second-degree relative and OR = 23.2 (95% CI: 11.3, 47.6) for 2 or more compared with no affected relatives). Only 1.1% of melanoma cases with neither a family history nor multiple primaries had mutations. Conclusions There is a low probability (

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. publishedVersion

Background: Awareness of individual risk may encourage improved prevention and early detection of melanoma. Methods: We evaluated the accuracy of self-reported pigmentation and nevus phenotype compared with clinical assessment, and examined agreement between nevus counts from selected anatomical regions. The sample included 456 cases with invasive cutaneous melanoma diagnosed between ages 18 to 39 years and 538 controls from the population-based Australian Melanoma Family Study. Participants completed a questionnaire about their pigmentation and nevus phenotype, and attended a dermatologic skin examination. Results: There was strong agreement between self-reported and clinical assessment of eye color [κ, = 0.78; 95% confidence interval (CI), 0.74–0.81]; and moderate agreement for hair color (κ = 0.46; 95% CI, 0.42–0.50). Agreement between self-reported skin color and spectrophotometer-derived measurements was poor (κ = 0.12; 95% CI, 0.08–0.16) to moderate (Spearman correlation rs = −0.37; 95% CI, −0.32 to −0.42). Participants tended to underestimate their nevus counts and pigmentation; men were more likely to underreport their skin color. The rs was 0.43 (95% CI, 0.38–0.49) comparing clinical total body nevus counts with self-reported nevus categories. There was good agreement between total body nevus counts and site-specific nevus counts, particularly on both arms. Conclusions: Young adults have suboptimal accuracy when assessing important risk characteristics including nevus numbers and pigmentation. Measuring nevus count on the arms is a good predictor of full body nevus count. Impact: These results have implications for the likely success of targeted public health programs that rely on self-assessment of these factors. Cancer Epidemiol Biomarkers Prev; 24(4); 736–43. ©2015 AACR.

Objectives Prostate cancer screening using prostate-specific antigen (PSA) remains controversial. In deciding about screening, men must weigh the benefits and harms: little is known about benefit: harm trade-offs men are willing to accept. The objective of this study was to assess men's preferences for PSA screening, and the trade-offs between benefits and harms men are willing to accept when deciding about screening. Methods Preferences of 662 men aged 40–69 were assessed using a discrete choice experiment. PSA screening was described by six attributes: prostate cancer deaths, prostate cancer diagnoses, unnecessary biopsies from false-positive PSA tests, impotence, urinary incontinence/bowel problems and cost. A mixed logit model was used to examine the influence of attributes on men's preferences for PSA testing; benefit: harm trade-offs were also calculated. Results Men's preferences were significantly influenced by test characteristics, particularly potential mortality benefit, unnecessary biopsies and likelihood of urinary incontinence or bowel problems; preferences were also influenced by age, prior PSA testing experience and perceived risk of prostate cancer. Men were willing to accept between 65 and 233 of 10 000 extra men with unnecessary biopsies, and between 31 and 72 of 10 000 extra men with incontinence/bowel problems to avoid one prostate cancer death. Conclusions Differences in valuations of attributes and trade-offs acceptable to men of different ages suggest a one size fits all approach to PSA testing, regardless of age, may not reflect men's preferences. Our results can be used by policymakers to ensure screening programmes are in line with men's preferences and by clinicians and patients to facilitate informed discussions of the most relevant benefits and downsides of PSA screening for an individual man.

Although genetic studies have reported a number of loci associated with melanoma risk, the complex genetic architecture of the disease is not yet fully understood. We sought to identify common genetic variants associated with melanoma risk in a genome-wide association study (GWAS) of 2298 cases and 6654 controls. Thirteen of 15 known loci were replicated with nominal significance. A total of 69 single-nucleotide polymorphisms (SNPs) were selected for in silico replication in two independent melanoma GWAS datasets (a total of 5149 cases and 12 795 controls). Seven novel loci were nominally significantly associated with melanoma risk. These seven SNPs were further genotyped in 234 melanoma cases and 238 controls. The SNP rs4698934 was nominally significantly associated with melanoma risk. The combined odds ratio per T allele = 1.18; 95% confidence interval (1.10-1.25); combined P = 7.70 × 10(-) (7). This SNP is located in the intron of the TET2 gene on chromosome 4q24. In addition, a novel somatic mutation of TET2 was identified by next-generation sequencing in 1 of 22 sporadic melanoma cases. TET2 encodes a member of TET family enzymes that oxidizes 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). It is a putative epigenetic biomarker of melanoma as we previously reported, with observation of reduced TET2 transcriptional expression. This study is the first to implicate TET2 genetic variation and mutation in melanoma.