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Background Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents. Methods Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets. Results Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset. Conclusions The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches.

Supplemental Digital Content is available in the text., Gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract. We provide in the present article the molecular characterization of a series of primary GISTs in a cohort of Sardinian patients (Italy), with the aim to describe the patterns of KIT and PDGFRa mutations and the corresponding clinical features. Ninety-nine Sardinian patients with histologically-proven diagnosis of GIST were included in the study. Medical records and pathology reports were used to assess the demographic and clinical features of the patients and the disease at the time of the diagnosis. Formalin-fixed, paraffin-embedded tissue samples were retrieved for each case, and mutation analysis of the KIT and PDGFRa genes was performed. KIT and PDGFRa mutations were detected in 81.8% and 5% of the cases, respectively. The most common KIT mutation was W557_K558del in exon 11, while D842V in exon 18 was the most common PDGFRa genetic alteration; V561D was the only PDGFRa mutation found in exon 12. The global “wild-type” cases, with no mutations in either the KIT or PDGFRa genes, were 13 (13.1%). The mean survival of those patients was approximately 46.9 (±43.9) months. Globally, 86.9% of Sardinian patients with GIST had a KIT or PDGFRa mutation; the former were more frequent in comparison with other Italian cohorts, while PDGFRa mutations were rare. No statistical differences in survival between mutated and wild-type cases, and between KIT and PDGFRa mutated cases were detected in our study.

The improvement of the immunotherapeutic potential in most human cancers, including melanoma, requires the identification of increasingly detailed molecular features underlying the tumor immune responsiveness and acting as disease-associated biomarkers. In recent past years, the complexity of the immune landscape in cancer tissues is being steadily unveiled with a progressive better understanding of the plethora of actors playing in such a scenario, resulting in histopathology diversification, distinct molecular subtypes, and biological heterogeneity. Actually, it is widely recognized that the intracellular patterns of alterations in driver genes and loci may also concur to interfere with the homeostasis of the tumor microenvironment components, deeply affecting the immune response against the tumor. Among others, the different events linked to genetic instability—aneuploidy/somatic copy number alteration (SCNA) or microsatellite instability (MSI)—may exhibit opposite behaviors in terms of immune exclusion or responsiveness. In this review, we focused on both prevalence and impact of such different types of genetic instability in melanoma in order to evaluate whether their use as biomarkers in an integrated analysis of the molecular profile of such a malignancy may allow defining any potential predictive value for response/resistance to immunotherapy.

Simple Summary The recent WHO classification of melanocytic tumors requires the implementation of combined phenotypic–genotypic diagnostics. For rare tumors, such as atypical deep penetrating nevi, there is insufficient information regarding genetic status, and it is not yet clear whether the observed unusual morphological cyto-architectures reflect a distinct genomic profile or are associated with an increased metastatic potential and aggressive clinical behavior. We report herein a comprehensive next-generation sequencing (NGS) analysis of a series of atypical DPNs, showing their mutational profile with some specific signatures for these rare and diagnostically challenging tumors. Abstract Deep penetrating nevi (DPNs) are rare melanocytic neoplasms consisting of pigmented spindled or epithelioid melanocytes with a distinctive wedge-shaped configuration showing activation of the WNT pathway, with unusual cyto-architectural features. It is unclear whether they show a distinct genomic profile associated with a diverse metastatic potential. We describe herein a cohort of 21 atypical DPNs analyzed by next-generation sequencing using the Ion AmpliSeq™ Comprehensive Cancer Panel. We found that β-catenin exon 3 was mutated in 95% and MAP kinase pathway genes in 71% of the cases. Less frequent mutations were observed in HRAS (19%) and MAP2K1 (24%). Isocitrate dehydrogenases 1 (IDH1) mutations, including R132C, V178I, and S278L, were identified in 38% of cases and co-existed with BRAF/HRAS mutations. The only case with progressive nodal disease carried alterations in the β-catenin pathway and mutations in IDH1 and NRAS (codon 61). By a comprehensive mutation analysis, we found low genetic heterogeneity and a lack of significant associations between specific gene mutations and histopathological features, despite atypical features. Whether the acquisition of an NRAS or IDH1 mutation in an atypical DPN may represent a molecular evolution implying a pathway to melanoma progression should be confirmed in a larger series.

Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation status plays a predominant role in the management of MM patients. The aim of this study was to compare BRAF mutational testing performed by conventional nucleotide sequencing approaches with either real-time polymerase chain reaction (rtPCR) or next-generation sequencing (NGS) assays in a real-life, hospital-based series of advanced MM patients. Consecutive patients with AJCC (American Joint Committee on Cancer) stage IIIC and IV MM from Sardinia, Italy, who were referred for molecular testing, were enrolled into the study. Initial screening was performed to assess the mutational status of the BRAF and NRAS genes, using the conventional methodologies recognized by the nationwide guidelines, at the time of the molecular classification, required by clinicians: at the beginning, Sanger-based sequencing (SS) and, after, pyrosequencing. The present study was then focused on BRAF mutation detecting approaches only. BRAF wild-type cases with available tissue and adequate DNA were further tested with rtPCR (Idylla&trade, ) and NGS assays. Globally, 319 patients were included in the study, pathogenic BRAF mutations were found in 144 (45.1%) cases examined with initial screening. The rtPCR detected 11 (16.2%) and 3 (4.8%) additional BRAF mutations after SS and pyrosequencing, respectively. NGS detected one additional BRAF-mutated case (2.1%) among 48 wild-type cases previously tested with pyrosequencing and rtPCR. Our study evidenced that rtPCR and NGS were able to detect additional BRAF mutant cases in comparison with conventional sequencing methods, therefore, we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy of BRAF detection.

In the present study, the phenotype of melanoma cells resistant to dabrafenib (a B-RAF inhibitor) was investigated, to shed more light on melanoma resistance to B-RAF inhibition. Melanoma cells resistant to dabrafenib were generated using 3 different cell lines, A375, 397 and 624.38, all carrying B-RAFV600E, and they were characterized by cytofluorometric analysis, Ion Torrent technology, immunofluorescence and biochemistry. All dabrafenib-resistant cells showed, in addition to a re-activation of MAPK signaling, morphological changes compared to their sensitive counterparts, accompanied by an increase in CD90 (mesenchymal marker) expression and a decrease in E-cadherin (epithelial marker) expression, suggesting an epithelial-to-mesenchymal-like phenotypic transition. However, melanoma cells with TGF-β1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGF‑β1‑induced EMT melanoma cells, suggesting that TGF-β1-induced EMT was not associated with dabrafenib resistance. Although dabrafenib-resistant cells exhibited increased cell motility and E-cadherin/vimentin reorganization, as expected in EMT, all of them showed unvaried E-cadherin mRNA and unchanged Snail protein levels, while Twist1 protein expression was decreased with the exception of A375 dabrafenib-resistant melanoma cells, where it was unaffected. These findings suggest a distinct active EMT-like process adopted by melanoma cells under drug exposure. Furthermore, dabrafenib-resistant cells exhibited stem cell-like features, with Oct4 translocation from the cytoplasm to peri-nuclear sites and nuclei, and increased CD20 expression. In conclusion, our data, in addition to confirming that resistance to dabrafenib is dependent on re-activation of MAPK signaling, suggest that this resistance is linked to a distinct active EMT-like process as well as stem-cell features adopted by melanoma cells.

SUMMARY Among human cancers, melanoma remains one of the malignancies with an ever-growing incidence in white populations. Recent advances in biological and immunological therapeutic approaches as well as increased efforts for secondary prevention are contributing to improve the survival rates. It is likely that a significant fall in mortality rates for melanoma will be achieved by further increase of the early detection through a more accurate selection of the higher-risk individuals (i.e., carriers of predisposing genetic alterations). A similar scenario occurs in Italy. In the present review, we have considered data on incidence, survival and mortality rates of melanoma in Italian population, including evaluation of the main risk factors and genetic mutations underlying disease susceptibility.

Purpose of Review Conventional clinico-pathological features in melanoma patients should be integrated with new molecular diagnostic, predictive, and prognostic factors coming from the expanding genomic profiles. Cutaneous melanoma (CM), even differing in biological behavior according to sun-exposure levels on the skin areas where it arises, is molecularly heterogeneous. The next-generation sequencing (NGS) approaches are providing data on mutation landscapes in driver genes that may account for distinct pathogenetic mechanisms and pathways. The purpose was to group and classify all somatic driver mutations observed in the main NGS-based studies. Recent Findings Whole exome and whole genome sequencing approaches have provided data on spectrum and distribution of genetic and genomic alterations as well as allowed to discover new cancer genes underlying CM pathogenesis. Summary After evaluating the mutational status in a cohort of 686 CM cases from the most representative NGS studies, three molecular CM subtypes were proposed: BRAFmut , RASmut , and non-BRAFmut /non-RASmut .

Cutaneous melanoma is a common and aggressive human skin cancers. Much is actually known about the molecular mechanisms underlying melanoma pathogenesis. The aim of the study was to evaluate any possible correlation between mutations in main growth-controlling genes (BRAF, NRAS, CDKN2A) and copy number variations in frequently amplified candidate genes (MITF, EGFR, CCND1, cMET, and cKIT) during melanoma initiation and progression. A large series of primary and secondary melanoma tissue samples (N = 274) from 232 consecutively-collected patients of Italian origin as well as 32 tumor cell lines derived from primary and metastatic melanomas underwent mutation screening and fluorescence in situ hybridization (FISH) analysis. Overall, BRAF, NRAS, and CDKN2A were found mutated in 62.5%, 12.5% and 59% cell lines and in 47%, 16%, 12% tumor tissues, respectively. Quite identical mutation patterns between primary tumors and metastatic lesions were found for BRAF and NRAS genes; mutations of CDKN2A gene appeared to be instead selected during tumor progression. In cell lines, high rates of gene amplifications were observed (varying from 12.5% for cKIT to 50% for MITF); vast majority of cell lines (75%) presented at least one amplified gene. Conversely, prevalence of gene amplification was significantly and progressively decreasing in melanoma metastases (12%) and primary melanomas (4%). Our findings suggest that gene amplifications may be acquired during the late phases of melanoma evolution and mostly act as "passenger" or "non-causative" alterations.

Long non-coding RNAs cover large part of the non-coding information of the human DNA, which represents more than 90% of the whole genome. They constitute a wide and complex group of molecules with more than 200 nucleotides, which generally lack an open reading frame, and are involved in various ways in the pathophysiology of cancer. Their roles in the regulation of gene expression, imprinting, transcription, and post-translational processing have been described in several types of cancer. CASC2 was discovered in 2004 in patients with endometrial carcinoma as a potential tumor suppressor. Since then, additional studies in other types of neoplasia have been carried out, and both mechanisms and interactions of CASC2 in cancer have been better elucidated. In this review, we summarize the current knowledge on the role of CASC2 in the genesis, progression, and clinical management of human cancer.

Background Activation of oncogenes downstream the EGFR gene contributes to colorectal tumorigenesis and determines the sensitivity to anti-EGFR treatments. The aim of this study was to evaluate the prognostic value of KRAS, BRAF, NRAS and PIK3CA mutations in a large collection of CRC patients from genetically-homogeneous Sardinian population. Methods A total of 1284 Sardinian patients with histologically-proven diagnosis of colorectal carcinoma (CRC) and presenting with metastatic disease were included into the study. Genomic DNA was isolated from formalin-fixed, paraffin-embedded primary tumour tissue samples of CRC patients and screened for mutations in RAS and BRAF genes, using pyrosequencing assays, and in PIK3CA gene, using automated DNA sequencing assays. Results Overall, mutation rates were 35.6 % for KRAS, 4.1 % for NRAS, and 2.1 % for BRAF. Among available DNA samples, 114/796 (14.3 %) primary CRCs were found to carry a mutation in the PIK3CA gene. In this subset of patients analysed in all four genes, a pathogenetic mutation of at least one gene was discovered in about half (378/796; 47.5 %) of CRC cases. A mutated BRAF gene was found to steadily act as a negative prognostic factor for either time to progression as metastatic disease (from detection of primary CRC to diagnosis of first distant metastasis; p = 0.009) or partial survival (from diagnosis of advanced disease to the time of death or last control; p = 0.006) or overall survival (p

e21554Background: The prevalence of mutations in main genes involved in tumorigenesis during melanoma progression remains a controversial issue. Different melanoma tissues from the same patients we...

Molecular mechanisms involved in pathogenesis of malignant melanoma have been widely studied and novel therapeutic treatments developed in recent past years. Molecular targets for therapy have mostly been recognized in the RAS-RAF-MEK-ERK and PI3K-AKT signaling pathways; small-molecule inhibitors were drawn to specifically target key kinases. Unfortunately, these targeted drugs may display intrinsic or acquired resistance and various evidences suggest that inhibition of a single effector of the signal transduction cascades involved in melanoma pathogenesis may be ineffective in blocking the tumor growth. In this sense, a wider comprehension of the multiple molecular alterations accounting for either response or resistance to treatments with targeted inhibitors may be helpful in assessing, which is the most effective combination of such therapies. In the present review, we summarize the known molecular mechanisms underlying either intrinsic and acquired drug resistance either alternative roads to melanoma pathogenesis, which may become targets for innovative anticancer approaches.

Background: Assessment of the mutational status in EGFR, KRAS, BRAF has become crucial in recent years for the molecular classification of patients with non small cell lung cancer (NSCLC). In this population-based study, we evaluated the incidence rates and distribution of such somatic mutations among NSCLC patients in genetically isolated population from Sardinia. Material and methods: From July 2010, a total of 1,047 formalin-fixed paraffin-embedded tumor tissues from patients with NSCLC and ascertained Sardinian origin was prospectively collected at clinics across the entire island. Genomic DNA was isolated from tissue sections and screened for somatic mutations in EGFR, KRAS, and BRAF genes by automated DNA sequencing. Results: Overall, 112 (10.7%) analyzed patients carried an EGFR mutation. Somatic mutations in EGFR gene were quite equally distributed between exon 19 (57/112; 51%) and exon 21 (52/112; 46%), with few mutations (3/112; 3%) in exon 18 of EGFR. No significant difference in distribution of EGFR mutations according to the age at diagnosis was observed [EGFR mutant: median age, 67 (range, 37-85); EGFR wild-type: median age, 65 (range, 35-89)]. Females presented a significantly higher frequency of EGFR mutations in comparison to males (23.6% vs. 5.7%, respectively] (p = 0.003). According to the smoking history, a significant preponderance of EGFR mutations were observed in never smokers (43.2%) as compared to former smokers (5.8%) and smokers (3.6%) (p < 0.001). Among 634 patients whose somatic DNA was available for further analyses [71 (11.2%) cases carried EGFR mutations], we detected 138 cases (21.8%) with KRAS mutations and 3 (0.5%) with BRAF mutation (V600E). KRAS and BRAF mutations were significantly more prevalent in males than females (22.4% vs. 10.3%, respectively; p = 0.012) as well as in smokers (31.8%) than in former smokers (17.6%) or never smokers (4.5%) (p = 0.023). In this series, no concomitant mutations in EGFR, KRAS, and BRAF genes were detected. Therefore, two thirds (422/ 634; 66.6%) of such patients lacked somatic mutations in all three analyzed genes. Conclusions: Although the prevalence of mutations in the three main candidate gens among NSCLC patients from Sardinia was consistent with that reported in literature for Western populations, their distribution varied into the different clinical subgroups

Purpose Oncogenic activation of the BRAF gene has been demonstrated to be involved in the pathogenesis of malignant melanoma (MM). In this study, we investigated the contribution of BRAF to melanoma susceptibility, also making a comparison with frequency of CDKN2A germline mutations in MM patients from different areas in Italy. Patients and Methods Using a combination of denaturing high-performance liquid chromatography analysis and automated sequencing on genomic DNA from peripheral blood or tumor tissue samples, 569 MM patients (211 from northern Italy and 358 from southern Italy) were screened for BRAF mutations. Results Three BRAF germline sequence variants (M116R, V599E, and G608H) were identified in four (0.7%) of 569 MM patients. The most common BRAF mutation, V599E, was detected in one germline DNA sample only; M116R and G608H were newly described mutations. A high frequency (59%) of BRAF mutations was instead observed in tumor samples from patients also undergoing germline DNA analysis; at the somatic level, substitution of valine 599 was found to account for the majority (88%) of BRAF mutations. We then estimated the germline mutation rates in BRAF and CDKN2A among 358 consecutively collected patient samples originating in southern Italy; a low (2.5%) or very low (0.29%) prevalence of CDKN2A and BRAF mutations, respectively, was detected. Conclusion Mutation analysis of either blood DNA from a large collection of MM patients or matched MM tissues from a subset of such patients revealed that BRAF is somatically mutated and does not play a major role in melanoma susceptibility. The present study further suggests that patient origin may account for different mutation rates in candidate genes.

e21047 Background: The prevalence of mutations in driver genes during progression in cutaneous and mucosal melanomas remains inconclusive. We investigated the prevalence and distribution of mutations in main candidate genes involved in melanomagenesis among different melanoma tissues using a next-generation sequencing (NGS) approach. Methods: Forty-eight tumor samples from 36 patients with mucosal melanoma (MM) and fifty-two tumor samples from 34 patients with cutaneous melanoma (CM) were collected, after obtaining patients’ written informed consent for tissue sampling. Genomic DNA was isolated from macrodissected tumor tissues containing at least 80% neoplastic cells and analyzed for mutations in 25 most common melanoma-associated oncogenes and tumor suppressor genes, using the IMI Diagnostic Melanoma Panel on the Ion Torrent platform (Life Technologies, USA). Results: A total of 100 tumor tissues from 70 melanoma patients were analyzed. BRAF mutations were detected in 21/34 (62%) CM patients and 13/36 (36%) mm patients. The second most prevalent mutations were found in K-/N-RAS (6/34; 18%) and cKIT (6/36; 17%) genes among CM and mm patients, respectively. No concomitant mutations of BRAF, RAS, and cKIT genes were detected. Among others, mutations were more frequently found in CCND1 (20%), ARID2 (16%), and NF1(12%) genes, considering the entire series of patients. Vast majority of patients who had paired samples of primary and secondary melanomas showed consistent mutation patterns between primary tumors and metastatic lesions. Similar frequencies of mutations in driver genes were seen across metastatic sites. Conclusions: In the era of targeted therapies, assessment of the spectrum and distribution of mutations in main molecular targets among patients with melanoma is needed. Our findings about the prevalence of mutations in driver genes in paired tumor lesions from patients with cutaneous and mucosal melanoma may be useful in the management of such diseases. The Italian Melanoma Intergroup (IMI) includes the following additional members who participated as investigators in this study: Mario Mandalà, Paola Queirolo, Ignazio Stanganelli, Vanna Chiarion Sileni, Pietro Quaglino, Anna Maria Di Giacomo.

Background: Alterations in key-regulator genes of disease pathogenesis (BRAF, cKIT, CyclinD1) have been evaluated in patients with multiple primary melanoma (MPM). Methods: One hundred twelve MPM patients (96 cases with two primary melanomas, 15 with three, and 1 with four) were included into the study. Paired synchronous/asynchronous MPM tissues (N = 229) were analyzed for BRAF mutations and cKIT/CyclynD1 gene amplifications. Results: BRAF mutations were identified in 109/229 (48%) primary melanomas, whereas cKIT and CyclinD1 amplifications were observed in 10/216 (5%) and 29/214 (14%) tumor tissues, respectively. While frequency rates of BRAF mutations were quite identical across the different MPM lesions, a significant increase of cKIT (p < 0.001) and CyclinD1 (p = 0.002) amplification rates was observed between first and subsequent primary melanomas. Among the 107 patients with paired melanoma samples, 53 (49.5%) presented consistent alteration patterns between first and subsequent primary tumors. About one third (40/122; 32.8%) of subsequent melanomas presented a discrepant pattern of BRAF mutations as compared to incident primary tumors. Conclusions: The low consistency in somatic mutation patterns among MPM lesions from same patients provides further evidence that melanomagenesis is heterogeneous and different cell types may be involved. This may have implications in clinical practice due to the difficulties in molecularly classifying patients with discrepant primary melanomas.

BACKGROUND Loss of heterozygosity (LOH) at chromosome 10q25-q26 has been reported previously in endometrial carcinoma (EC), suggesting the presence of tumor suppressor gene(s). Nevertheless, frequency of genome-wide microsatellite instability (MSI) has been demonstrated higher in EC than in other common malignancy, mostly due to defective DNA mismatch repair. The authors further evaluated the role of the chromosome 10q25-q26 in endometrial tumorigenesis as well as the clinical significance of any observed genetic alteration in sporadic EC. METHODS Paired normal and tumor samples from 94 Sardinian patients with sporadic EC at various stages of disease were screened by polymerase chain reaction (PCR)–based microsatellite analysis. Genomic DNA was isolated from paraffin embedded tissues and amplified by PCR using microsatellite markers spanning approximately 14 cM at 10q25-q26. Microsatellite instability was studied at four loci mapping to different chromosomal locations. RESULTS Thirty-two (34%) EC patients were found negative for genetic alterations within the 10q25-q26 region. Among the remaining 62 (66%) EC cases, the authors identified 1) a minimum consensus region of LOH of approximately 1 cM, between D10S610 and D10S542 markers; and 2) a subset of tumors with prevalence of instability at 10q25-q26 (10qMI+), as expression of the presence of a MSI+ phenotype. CONCLUSIONS The authors' data establish the existence of significant correlations between disease stages and 10qMI+ (with or without MSI+). However, longer follow-up and additional studies are required to define the clinical significance of these findings as prognostic factors. Moreover, the minimum region of LOH at 10q25-q26 will be further analyzed for identifying the putative tumor suppressor gene involved in EC pathogenesis. Cancer 2000;89:1773–82. © 2000 American Cancer Society.

Purpose The prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues. Patients and Methods In all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites. Results BRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples. Conclusion In the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.

Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

Defective DNA mismatch repair and nonfunctional mechanisms controlling the proper progression of the cell cycle have been proposed as being responsible for the genomic instability and accumulation of karyotypic alterations in endometrial cancer (EC). To assess whether numerical chromosomal anomalies (aneuploidy) and microsatellite instability (MSI) might be representative of distinctive tumour behaviour, paraffin-embedded tissue samples from 86 patients with sporadic EC were evaluated by both fluorescence in situ hybridisation (FISH) and microsatellite analysis, using free nuclei and genomic DNAs (respectively). Approximately one-third of the tumours analysed (24/74; 32%) exhibited MSI, whereas 38/86 (44%) of the EC samples displayed aneuploidy. The majority of the unstable cases (15/24; 63%) were from advanced-stage patients. Conversely, 23 (61%) out of the 38 tumours with aneuploidy were from early-stage patients. No apparent correlation was found between MSI and aneuploidy, whereas the immunohistochemical (IHC) analysis revealed that inactivation of the MLH1 mismatch repair gene may be involved in the majority of the MSI+ sporadic ECs. No genetic or cytogenetic alteration analysed here seems to add any significant predictive value to the stage of disease. © 2002 Elsevier Science Ltd. All rights reserved.

e20013 Background: Genetic factors predisposing to melanoma at germline level have been demonstrated to be geographically heterogeneous. We here evaluated the spectrum of NRAS and BRAF mutations at somatic level, in a large subset of melanoma tissues from patients originating from different Italian geographical areas: Sardinia, whose population is genetically homogeneous, and Middle-South Italy, with a genetically heterogeneous population. Methods: Patients were enrolled consecutively between June 2008 and December 2012. Genomic DNA was isolated from tumor tissues [primary melanomas (N=439) and melanoma metastases (N=269)] or melanoma cell lines (N=32). Paired samples of primary melanomas (n=140) and synchronous or asynchronous metastases from the same patients (n=203) were included. The full coding sequences and splice junctions of NRAS (exons 2-3) and BRAF (exon 15) genes were screened for mutations through automated sequencing. Results: BRAF/NRAS mutations were identified in 63% of primary melanomas (48% BRAF; 15% NRAS), 67% melanoma metastases (51% BRAF; 16% NRAS), and 68% melanoma cell lines (56% BRAF; 12% NRAS). A non-significant increase in mutation frequency after progression from primary melanoma was observed. However, distribution of BRAF/NRAS mutations varied between in vivo tumors and melanoma cell lines, suggesting a preponderant role for BRAF activation in highly proliferating cultured melanoma cells. Among paired samples, consistency of BRAF/NRAS mutation patterns between metastatic and primary melanomas ranged from 71% (skin metastases) to about 90% (lymph node and visceral metastases). A significant inverse distribution of BRAF/NRAS mutation rates was observed in our series on the basis of the geographical origin of patients: for BRAF, 58% Sardinian vs. 42% non-Sardinian cases (p=0.045); for NRAS, 2% Sardinian vs. 21% non-Sardinian cases (p

Background: Mutations of NRAS and BRAF genes have been identified with high frequency in nevi, cutaneous melanomas, and melanoma metastases. Prevalence of such mutations during the disease progression phases and among the different types of metastasis still remains inconclusive. Methods: A total of 275 tumour tissues from 116 melanoma patients were screened for mutations; among them, paired samples of microdissected primary melanomas (N=92) and synchronous or asynchronous metastases (N=156) from same patients were included. Tissue samples underwent mutation analysis by automated DNA sequencing. Secondary lesions were from: lymph nodes (LM; N=77), skin (SM; N=36), visceral (VM; N=23) and brain (BM; N= 44) sites. The full coding sequences and splice junctions of p16CDKN2A (exons 1, 2, and 3) and NRAS (exons 2 and 3) genes as well as the entire sequence at the exon 15 of the BRAF gene were screened for mutations by direct sequencing on automated fluorescence-cycle sequencer (ABIPRISM 3130, Applied Biosystems, Foster City, CA). Results: Overall, mutations were identified in 56/95 (59%) primary melanomas [43% BRAF - 16% NRAS], 49/77 (64%) lymph nodes [48% BRAF - 16% NRAS], 22/36 (61%) subcutaneous metastases [53% BRAF - 8% NRAS], 13/23 (57%) visceral metastases [43% BRAF - 13% NRAS], and 31/44 (70%) brain metastases [48% BRAF - 23% NRAS]. Overall, a slight and not significant increase in mutation frequency after progression from primary melanoma was observed in our series: 115/180 (64%) mutated metastases [48% BRAF - 16% NRAS]. Considering the paired samples from the same patients, LM (92% consistency) and VM (96% consistency) presented a highly similar prevalence and distribution of BRAF/NRAS mutations in comparison with primary melanomas, whereas a discontinuous pattern of mutations was detected in BM (80% consistency) or, mostly, SM (75% consistency). Occurrence of distinct mutation distribution between primary melanomas and correspondent metastases suggests that independent subclones have been generated in a limited fraction of patients. The rate of mutations in p16CDKN2A gene was found to steadily increase moving from primary melanomas (5/69; 7%) to melanoma metastases (21/144; 15%), with the highest prevalence of p16CDKN2A alterations (18/29; 62%) in melanoma cell lines Conclusions: Our results provide further clues about the impact of NRAS and BRAF mutations among the different stages of melanoma progression. Moreover, we confirmed that p16CDKN2A mutation rates are increasing during disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3677. doi:1538-7445.AM2012-3677

8574 Background: Mutations of NRAS and BRAF genes have been identified with high frequency in nevi, cutaneous melanomas, and melanoma metastases. Prevalence of such mutations during the disease pro...

Background: Arginine deiminase (ADI), an enzyme that catalize the conversion of arginine to citrulline, has been used in clinical trials for the treatment of tumors which have been shown to be sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). We here evaluated the effect of ADI treatment on ASS expression in a series of melanoma cell lines. Methods: Twenty-five melanoma cell lines (6 derived from primary melanomas and 19 from MM metastases) and normal fibroblast cell as control underwent cell proliferation assays in presence or absence of ADI. The percentage of cell proliferation was estimated on day 6 of the ADI treatment by a colorimetric assay; the IC50 value was assessed for each cell line. Western blot analysis on lysates from treated and untreated cells were performed for evaluating the expression levels of both the ASS and the ornithine carbamoyl transferase (OTC) proteins. A subset of tissue sections from primary melanomas (N=10) and hepatocellular carcinomas (N=20) was investigated by immunohistochemistry for ASS expression. Results: Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition with a median IC50 value of 1.3 mU/mL (range, 0.4–2.0); growth of the remaining 4 (16%) MM cell lines was not inhibited and the IC50 was not reached. As expected, proliferation of the control fibroblasts was not inhibited. Expression analysis revealed that none of the 21 ADI-responsive cell lines presented detectable levels of the ASS protein before the ADI treatment; conversely, the 4 melanoma cell lines and the normal fibroblasts, which did not reached an IC50 value, showed a constitutive ASS expression in both treated and untreated cells. After treatment with ADI at a concentration corresponding to the IC50 values, 7/21 (33%) melanoma cell lines presented a markedly increased expression levels of the ASS protein; interestingly, six of them were from paired primary and secondary lesions of three patients. The median value of IC50 in this subset was of 1.8 mU/mL (range, 1.2–2.0). Nearly all samples (13/14; 93%) with absence of the ASS expression in both treated and untreated cells originated from MM metastases; among them, three cell lines were from paired primary and secondary lesions of the same patient. The median value of IC50 in this subset was of 1.2 mU/mL (range, 0.4–1.7). A constitutive expression of the OTC protein was found in all melanoma cell lines and no change in OTC expression was observed after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens (10 melanomas and 20 hepatocellular carcinomas). Conclusions: In our series, the most effective cell growth inhibition was observed in melanoma cells with absence of ASS expression either before or after the ADI treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A79.

8522 Background: Cytogenetic and molecular studies indicated the chromosome 9p21 and its CDKN locus, with the p16/CDKN2A tumor suppressor genes, as the genomic regions involved into the pathogenesis of malignant melanoma (MM). To further elucidate the role of such regions in melanoma, we evaluated the 9p21-linked molecular alterations during the different phases of melanocytic tumorigenesis. Methods: Paraffin-embedded tissue sections from common nevi, dysplastic nevi, and melanomas, including primary and metastatic MM lesion, as well as melanoma cell lines were evaluated by both fluorescence in situ hybridization (FISH) using probes spanning the entire 9p21 region and immunohistochemistry (IHC) for p16/CDKN2A expression. Results: Allelic deletion at the CDKN locus and p16/CDKN2A gene silencing were both observed at increased rates moving from nevi to early or advanced primary melanomas and to correspondent metastases. Dysplastic nevi, primary and secondary melanomas were instead found to be heterozygously deleted at one or more loci within the 9p21 chromosome in a quite similar fraction (55–62%) of cases. Inactivation of the p16/CDKN2A gene was found at rates higher in melanoma cell lines (67%) than in vivo melanomas (62% secondary and 12.5% primary melanomas), with a normal p16/CDKN2A IHC staining in all analyzed nevi. Conclusions: Our findings indicate a model of sequential accumulation of genetic alterations from normal melanocytes to metastatic melanoma, with a) 9p21 allelic loss playing a role in melanocytic transformation and tumor initiation; b) deletions at CDKN locus and p16/ CDKN2A gene inactivation participating to tumor dissemination. Presence of such alterations could be further evaluated as markers of the different phases of melanoma progression. No significant financial relationships to disclose.

3629 Background: Fanconi Anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and high incidence of leukemia and solid tumors. Eight FA associated genes have been cloned, and their products are thought to function in an integrated pathway which includes BRCA genes and maintains genomic stability. A molecular mechanism involved in the development and progression of some human malignancies, including colorectal cancer, is represented by a defective replication fidelity. Here we tested the role of the main FA genes in colorectal cancer (CRC). Methods: One hundred consecutively-collected patients with histologically-proven diagnosis of CRC and no mutation in the main mismatch repair genes (MLH1 and MSH2) were included into the study. Genomics DNA was screened using DHPLC for FANC-C/G/E/F genes and using MPLA for FANC-A/D2 genes. All PCR products with abnormal DHPLC profiles were sequenced on an automated sequencer. For 28 cases, tumor tissue samples were also analyzed. To date, total RNA was isolated from paired normal and tumor samples of 12 CRC patients. Gene expression was quantified by real-time RT-PCR using the Taqman approach. Results: At germline level, 2 cases presented FANC-C sequence variations (Asp267Ser, Val449Ser) and 5 cases showed a exon 26 deletion in FANC-A as detected by MLPA analysis. Among the 28 tumor samples available from patients negative for mutations in germline DNA, 2 (7%) cases presented a FANC-E mutation (Ala552Thr). Moreover, one of such somatic samples presented a BRCA2–8765delAG mutation (BRCA2 is also recognized as FANC-D1 gene). Assuming as significant difference in gene expression the presence of a variation of more than 50% in mRNA levels between normal and corresponding tumor samples, all tested FA genes (FANC-C/G/E/F) were found overexpressed in 5/12 (42%) tumor samples. Conclusions: Although preliminary, our findings seem to identify a subset of CRC patients with alterations in the FA/BRCA pathway which is required for the activation of DNA repair. Ongoing molecular analyses will better clarify the real frequency of each alteration in FA genes and their relationship with the histological and/or clinical features in colorectal cancer. No significant financial relationships to disclose.

Background: In our previous experience (JCO 2012;30:2522-9), we demonstrated a high consistency of BRAF and NRAS mutations between primary tumors and lymph node metastases in melanoma patients. A subset of paired samples of multiple asynchronous lymph node metastases from that series was here screened mainly using NGS technologies in order to better elucidate the existence of a real genetic homogeneity during clonal expansion to lymph node sites. Methods: Genomic DNA was isolated from frozen tissues of paired asynchronous lymph nodes, ascertained for presence of melanoma metastases (at least 80% of metastatic cells), using standard methods. Specimens from 12 patients were analyzed for mutations in 50 most common cancer genes with the Ion Torrent AmpliSeq Cancer Panel HotSpot.V2 (CHPv2) on the Ion PGMsequencer. All variants detected by NGS were confirmed through PCR-based Sanger sequencing. Paraffinembedded tumor tissues from the same series were evaluated by fluorescence in situ hybridization (FISH) assays, using probes specific for CyclinD1/EGFR genes or 9p21 locus and correspondent control centromeres. Results: Overall, only one (8%) of the 12 analyzed patients presented discrepancies in mutation patterns between the two distinct lymph nodal metastases developed in different times. In particular, the differences were represented by the BRAF-V600E and the CDKN2A-R80X mutations, suggesting that changes, when occur (though in a limited fraction of cases), they affect the main genes controlling cell proliferation and survival involved in melanomagenesis. No discrepancy was observed by FISH analysis into the same series. Considering the total number of analyzed samples, we detected the following mutations: BRAF-V600E (54% of cases), KDR-Q472H (50%), TP53-P72R (42%), cKIT-M541L (25%), NRAS-Q61R/K (25%). Conclusions: Our findings indicated a very low genetic heterogeneity during lymph node dissemination in melanoma patients, confirming the previously published data. Mutations in BRAF, NRAS, and cKIT genes were further confirmed to play a predominant role in melanoma pathogenesis.

Background A series of patients with multiple primary melanoma (MPM) has been studied for assessing frequency and distribution of alterations in candidate genes involved in susceptibility (CDKN2A) and pathogenesis (BRAF, cKIT, CyclinD1) of such a disease. Methods Two hundred and twenty-seven genomic DNA samples from paired synchronous and/or asynchronous tumour tissues of 106 MPM patients (92 cases with two, 13 with three, and 1 with four primary melanomas) were screened for somatic mutations in BRAF gene and FISHbased amplifications in cKIT and CyclynD1 genes. For 87 (84%) MPM patients from our series, peripheral blood was available and germline DNAs was analyzed for mutations in p16CDKN2A and p14CDKN2A genes. All mutation analyses we performed by direct automated DNA sequencing. Family history for melanoma was defined according to standardized criteria. Results At somatic level, BRAF mutations were identified in 108/ 227 (48%) primary melanoma tissues, whereas amplification of cKIT and CyclinD1 genes was observed in 8/208 (4%) and 28/204 (14%) analyzed tissue samples, respectively. Considering all types of genetic events, paired samples presented a poorly consistent distribution of somatic alterations in same patients [55/106 (52%) discrepant cases). Among them, 35/106 (33%) patients presented discrepant MPM lesions according to the BRAF mutation status. Among the 87 MPM patients whose germline DNA was available, 8 (9%) of them showed different CDKN2A germline mutations: 7 in p16CDKN2A and 1 in p14CDKN2A. Assessment of family history for melanoma revealed that 13/87 (15%) patients presented at least one additional family member affected; a total of ≥ 3 melanomas in family was observed in 20/87 (23%) cases of our series. The CDKN2A germline mutations were found significantly more frequent in patients with familial history of melanoma (7/13; 54%) compared with patients without (1/74; 1.4%) (P