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Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination.Significance:ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance.

Supplementary Figures. Supplementary Figure S1: Zeb2-wildtype cells outcompete Zeb2-negative cells in terms of expansion and differentiation. Supplementary Figure S2: Zeb2 does not accelerate melanoma development on a p53-wildtype background. Supplementary Figure S3: ZEB2 affects the phenotypical switch between proliferation and invasion in mouse and human melanoma cells. Supplementary Figure S4: ZEB2 affects the phenotypical switch between proliferation and invasion in human melanoma. Supplementary Figure S5: TGF-ß treatment mediates the switch from ZEB2 to ZEB1 and induces an invasive gene signature.

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination. Significance: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance.

Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments or in case of disease relapse, with no reliable molecular markers for progression-free survival (PFS) available. As chemotherapy primarily eliminates cancer cells by apoptosis, we here evaluated if the expression of key apoptosis regulators (Bax, Bak, Bcl-2, Bcl-xL, Smac, Procaspase-9, Apaf-1, Procaspase-3 and XIAP) allows prognosticating PFS in stage III/IV melanoma patients. Following antibody validation, marker expression was determined by automated and manual scoring of immunohistochemically stained tissue microarrays (TMAs) constructed from treatment-naive metastatic melanoma biopsies. Interestingly and counter-intuitively, low expression of the pro-apoptotic proteins Bax, Bak and Smac indicated better prognosis (log-rank p 12 months) on a case-by-case basis (area under the receiver operating characteristic curve (ROC AUC) = 0.79). Taken together, our results therefore suggest that Bax, Bak and Smac jointly define a signature with potential clinical utility in chemotherapy-treated metastatic melanoma. ispartof: CELL DEATH & DISEASE vol:11 issue:2 ispartof: location:England status: published

Background: Combining bevacizumab and chemotherapy produced superior response rates compared with chemotherapy alone in metastatic breast cancer. As bevacizumab may cause hypertension (HTN) and increase the risk of cardiac failure, we performed a pilot study to evaluate the feasibility and toxicity of a non-anthracycline-containing combination of docetaxel with cyclophosphamide and bevacizumab in early stage breast cancer patients. Methods: Treatment consisted of four 3-weekly cycles of docetaxel and cyclophosphamide (75/600 mg/m2). Bevacizumab was administered 15 mg/kg intravenously on day 1, and then every 3 weeks to a total of 18 cycles of treatment. Serum biomarker concentrations of vascular endothelial growth factor (VEGF), cardiac troponin-I (cTnI), myeloperoxidase (MPO), and placental growth factor (PlGF) were quantified using enzyme-linked immunosorbent assay (ELISA) in 62 patients at baseline and whilst on treatment to determine their utility as biomarkers of cardiotoxicity, indicated by left ventricular ejection fraction (LVEF). Results: A total of 106 patients were accrued in nine sites. Median follow up was 65 months (1–72 months). Seventeen protocol-defined relapse events were observed, accounting for an overall disease-free survival (DFS) rate of 84%. The DFS rates for hormone receptor positive (HR+) and triple-negative (TN) patients were 95% versus 43%, respectively. The median time to relapse was 25 (12–54) months in TN patients versus 38 (22–71) months in HR+ patients. There have been 13 deaths related to breast cancer . The overall survival (OS) rate was 88%. The 5-year OS rate in HR+ versus TN was 95% versus 57%. None of the measured biomarkers predicted the development of cardiotoxicity. Conclusions: We observed a low relapse rate in node-positive, HR+ patients; however, results in TN breast cancer were less encouraging. Given the negative results of three large phase III trials, it is unlikely that this approach will be investigated further. Trial Registration ClinicalTrials.gov Identifier: NCT00911716.

PURPOSE Dynamic network models predict clinical prognosis and inform therapeutic intervention by elucidating disease-driven aberrations at the systems level. However, the personalization of model predictions requires the profiling of multiple model inputs, which hampers clinical translation. PATIENTS AND METHODS We applied APOPTO-CELL, a prognostic model of apoptosis signaling, to showcase the establishment of computational platforms that require a reduced set of inputs. We designed two distinct and complementary pipelines: a probabilistic approach to exploit a consistent subpanel of inputs across the whole cohort (Ensemble) and a machine learning approach to identify a reduced protein set tailored for individual patients (Tree). Development was performed on a virtual cohort of 3,200,000 patients, with inputs estimated from clinically relevant protein profiles. Validation was carried out in an in-house stage III colorectal cancer cohort, with inputs profiled in surgical resections by reverse phase protein array (n = 120) and/or immunohistochemistry (n = 117). RESULTS Ensemble and Tree reproduced APOPTO-CELL predictions in the virtual patient cohort with 92% and 99% accuracy while decreasing the number of inputs to a consistent subset of three proteins (40% reduction) or a personalized subset of 2.7 proteins on average (46% reduction), respectively. Ensemble and Tree retained prognostic utility in the in-house colorectal cancer cohort. The association between the Ensemble accuracy and prognostic value (Spearman ρ = 0.43; P = .02) provided a rationale to optimize the input composition for specific clinical settings. Comparison between profiling by reverse phase protein array (gold standard) and immunohistochemistry (clinical routine) revealed that the latter is a suitable technology to quantify model inputs. CONCLUSION This study provides a generalizable framework to optimize the development of network-based prognostic assays and, ultimately, to facilitate their integration in the routine clinical workflow.

Because of advances in targeted therapies, the clinical evaluation of cutaneous melanoma is increasingly based on a combination of traditional histopathology and molecular pathology. Therefore, it is necessary to expand our knowledge of the molecular events that accompany the development and progression of melanoma to optimize clinical management. The central objective of this study was to increase our knowledge of the mutational events that complement melanoma progression. High-throughput genotyping was adapted to query 159 known single nucleotide mutations in 33 cancer-related genes across two melanoma cohorts from Ireland (n = 94) and Belgium (n = 60). Results were correlated with various clinicopathological characteristics. A total of 23 mutations in 12 genes were identified, that is - BRAF, NRAS, MET, PHLPP2, PIK3R1, IDH1, KIT, STK11, CTNNB1, JAK2, ALK, and GNAS. Unexpectedly, we discovered significant differences in BRAF, MET, and PIK3R1 mutations between the cohorts. That is, cases from Ireland showed significantly lower (P

Aims Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer (BC) deaths, owing to its intrinsic aggressiveness and a lack of treatment options, especially targeted therapies. Thus, there is an urgent need for the development of better targeted treatments for TNBC. Molecular alteration of AKT-3 was previously reported in oestrogen receptor (ER)-positive BC. AKT-3 has also been suggested to play a role in hormone-unresponsive BC. The aim of this study was to investigate molecular alterations of AKT-3 in TNBC, to perform associated survival analysis, and to compare these findings with the incidence of AKT-3 molecular alterations in ER-positive BC. Results Our study revealed AKT-3 amplification and deletions in 11% (9/82) and 13% (11/82) of TNBCs, respectively. In contrast, 1% (2/209) of ER-positive BCs were found to have AKT-3 amplifications and deletions. A higher prevalence of AKT-3 copy number gains was observed in TNBC [26% (21/82)] than in ER-positive BC [9% (19/209)]. AKT-3 amplification together with Akt-3 protein expression was negatively associated with recurrence-free survival in TNBC. Furthermore, a negative association between high AKT-3 copy number and recurrence-free survival was observed. Conclusion AKT-3 amplification could represent a potentially relevant oncogenic event in a subset of TNBCs that may, in turn, select cells sensitive to Akt-3 inhibitors.

Despite advances in our knowledge of the disease, malignant melanoma remains an unpredictable entity. The revolution in molecular biological techniques, such as DNA sequencing and gene-expression profiling, has uncovered many potential protein targets and biomarkers relevant to melanoma progression. Successful clinical application would be aided significantly by downstream proteomic validation of those candidate markers using a combination of immunohistochemistry and tissue microarrays. Yet, research in this context seems to lag behind the output of genomic data relating to melanoma. In this article, we look at the strengths and pitfalls of tissue microarrays in malignant melanoma. We will show how tissue microarrays have become a vital step in the transition from molecular techniques to useful clinical assays and interventions and look at likely future developments for advances in this field.

Background: The homeobox containing transcription factor MSX2 is a key regulator of embryonic development and has been implicated to have a role in breast and pancreatic cancer. Methods: Using a selection of two- and three-dimensional in vitro assays and tissue microarrays (TMAs), the clinical and functional relevance of MSX2 in malignant melanoma was explored. A doxycyline-inducible over-expression system was applied to study the relevance of MSX2 in vitro. For TMA construction, tumour material from 218 melanoma patients was used. Results: Ectopic expression of MSX2 resulted in the induction of apoptosis and reduced the invasive capacity of melanoma cells in three-dimensional culture. MSX2 over-expression was shown to affect several signalling pathways associated with cell invasion and survival. Downregulation of N-Cadherin, induction of p21 and inhibition of both BCL2 and Survivin were observed. Cytoplasmic MSX2 expression was found to correlate significantly with increased recurrence-free survival (P=0.008). Nuclear expression of MSX2 did not result in significant survival correlations, suggesting that the beneficial effect of MSX2 may be independent of its DNA binding activity. Conclusions: MSX2 may be an important regulator of melanoma cell invasion and survival. Cytoplasmic expression of the protein was identified as biomarker for good prognosis in malignant melanoma patients.

DNA methylation plays a major role in cancer by silencing tumour suppressor genes. In melanoma, only a discrete number of methylated genes have been identified so far. After the treatment of melanoma cells with a DNA methyltransferase inhibitor and subsequent transcriptomic profiling, we had identified earlier a cohort of melanoma progression-associated genes regulated by methylation. Here, we identified which of these genes are directly methylated in melanoma cell lines and tissues. First, we examined 16 genes by bisulphite sequencing in the WM793 isogenic cell line model series. Five of these genes (CYBA, FABP5, MT1E, TSPY1 and TAC1) displayed increased methylation in several invasive cell lines compared with the parental WM793 cells, indicating their involvement in progression. Next, we analyzed several matched primary/metastatic tumours using methylation-specific PCR, which revealed that MT1E (one of the five genes assessed) was methylated in the largest proportion of tumours. Examination of a larger cohort of samples showed that 1 of 17 (6%) of the benign naevi, 16 of 43 (37%) primary tumours and 6 of 13 (46%) of the metastases displayed MT1E methylation. In addition, ectopic over-expression of MT1E mediated sensitization to cisplatin-induced apoptosis. Overall, these studies suggest that MT1E is a potential tumour suppressor gene, whose loss may promote resistance to apoptosis-inducing therapies.

In this study, we used array-comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to examine genetic aberrations in melanoma cell lines and tissues. Array-comparative genomic hybridization revealed that the most frequent genetic changes found in melanoma cell lines were amplifications on chromosomes 7p and 20q, along with disruptions on Chr 9, 10, 11, 12, 22 and Y. Validation of the results using FISH on tissue microarrays (TMAs) identified TOP1 as being amplified in melanoma tissues. TOP1 amplification was detected in a high percentage (33%) of tumours and was associated with thicker, aggressive tumours. These results show that TOP1 amplification is associated with advanced tumours and poor prognosis in melanoma. These observations open the possibility that TOP1-targeted therapeutics may be of benefit in a particular subgroup of advanced stage melanoma patients.

Despite remarkable effort, malignant melanoma still remains a potent killer. Millions of dollars have been spent on clinical trials that have not succeeded in achieving significant patient benefit. The thorough validation of drug targets at an earlier stage is, therefore, an essential step in the development of new therapies. Since the development of microarray experiments, putative drug targets are being identified in a high-throughput manner. Though high-throughput functional validation methods are currently being established, a more specific, pre-clinical analysis of promising target genes remains inevitable. For this, a broad range of increasingly sophisticated functional models is available. In vitro, the microenvironment of skin can be simulated through various two or three-dimensional models. In vivo assays range from xenograft studies to the establishment of transgenic organisms. Here, we provide a summary of functional interrogation approaches in melanoma research, focusing on the application of these strategies to the development of new effective therapies.

Cancer may arise because the developmental programs that create the dramatic alterations in form and structure in embryonic development are potentially corrupted. The cells in our bodies retain memories of these processes and cancer can occur later in life if imperfections occur in the fidelity of these pathways. This article is particularly interested in the phenomenon of epithelial to mesenchymal transition, which occurs in embryogenesis. Also reviewed are the small molecules and pathways that are involved both in homeostasis in adult epithelium and embryogenesis in utero. There are five such pathways in particular selected for review in this article: the Wnt pathway, Hedgehog, Notch, PAR and Bone morphogenetic peptide/TGF beta. These are usually conserved throughout mammalian evolution. Though they have been arbitrarily separated in this article they are not exclusive from one another. Their pathologically altered expression is found especially frequently in childhood tumours where they may recapitulate their developmental role, and in tumours that resemble primitive precursor cells. These pathways are important for selecting cell fates, cellular rearrangements, cytological context and morphologic design in embryology as well as participating in epithelial function in adults.

The incidence of melanoma is increasing rapidly, with advanced lesions generally failing to respond to conventional chemotherapy. Here, we utilized DNA microarray-based gene expression profiling techniques to identify molecular determinants of melanoma progression within a unique panel of isogenic human melanoma cell lines. When a poorly tumorigenic cell line, derived from an early melanoma, was compared with two increasingly aggressive derivative cell lines, the expression of 66 genes was significantly changed. A similar pattern of differential gene expression was found with an independently derived metastatic cell line. We further examined these melanoma progression-associated genes via use of a tailored TaqMan Low Density Array (LDA), representing the majority of genes within our cohort of interest. Considerable concordance was seen between the transcriptomic profiles determined by DNA microarray and TaqMan LDA approaches. A range of novel markers were identified that correlated here with melanoma progression. Most notable was TSPY, a Y chromosome-specific gene that displayed extensive down-regulation in expression between the parental and derivative cell lines. Examination of a putative CpG island within the TSPY gene demonstrated that this region was hypermethylated in the derivative cell lines, as well as metastatic melanomas from male patients. Moreover, treatment of the derivative cell lines with the DNA methyltransferase inhibitor, 2'-deoxy-5-azacytidine (DAC), restored expression of the TSPY gene to levels comparable with that found in the parental cells. Additional DNA microarray studies uncovered a subset of 13 genes from the above-mentioned 66 gene cohort that displayed re-activation of expression following DAC treatment, including TSPY, CYBA and MT2A. DAC suppressed tumor cell growth in vitro. Moreover, systemic treatment of mice with DAC attenuated growth of melanoma xenografts, with consequent re-expression of TSPY mRNA. Overall, our data support the hypothesis that multiple genes are targeted, either directly or indirectly, by DNA hypermethylation during melanoma progression.

Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-to-mesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival. ispartof: Cell Death and Differentiation vol:21 issue:8 pages:1250-1261 ispartof: location:England status: published

Background: Breast cancer is the leading cause of cancer in women, with 1.8 million patients diagnosed globally each year. In early stage disease, anthracycline regimens are commonly employed, but the risk of cardiotoxicity is high. Replacing anthracyclines with taxanes in combination with cyclophosphamide may improve response [1] and Bevacizumab (BVZ) may provide addtional benefit. However, as BVZ can cause hypertension and increased risk of cardiac failure, a rationale exists for studying biomarkers of cardiotoxicity in this setting. Methods: We assessed a panel of biomarkers in n = 57 early stage breast cancer patients whom underwent docetaxel/cyclophosphamide/BVZ therapy on the TC-Avastin Trial (NCT00911716). Serum was collected prior to treatment and at 3 months. Cardiotoxicity was defined as decline in left ventricular ejection fraction ≥ 10% to Results: Changes in serum MPO or cTnI were not related to cardiotoxicity when analysed in the larger cohort. However, a “cut-off” PlGF fold change of 0.99 could differentiate patients who developed cardiotoxicity from those that did not. 74% of cardiotoxicity patients, compared to only 35% of non-cardiotoxicity fell below the 0.99 cut-off. Conversely, 65% of non-cardiotoxicity patients, compared to 25% of cardiotoxicity patients fell above the cut-off (p = 0.0042). Conclusions: Our data suggest that decreased PlGF at 3 months may predict cardiotoxicity. These observations require further validation. This work was funded by AngioTox, an EC FP7 IAPP Marie Curie Award (Grant Agreement 251528). [1]Jones et al., J. Clin. Oncol.27, 1177-1183 (2009); [2] Putt et al., Clin. Chem 61, 1164-1172 (2015). Citation Format: David W. Murray, Alice C. O’Farrell, Giuseppe Gullo, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Bonnie Ky, William M. Gallagher, Norma O’Donovan, John Crown, Annette T. Byrne. Analysis of putative serum biomarker candidates for cardiotoxicity prediction in a cohort of early stage breast cancer patients treated with docetaxel, cyclophosphamide, and bevacizumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1828.

Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.

DNA methylation plays a major role in cancer by silencing tumour suppressor genes. In melanoma, only a discrete number of methylated genes have been identified so far. After the treatment of melanoma cells with a DNA methyltransferase inhibitor and subsequent transcriptomic profiling, we had identified earlier a cohort of melanoma progression-associated genes regulated by methylation. Here, we identified which of these genes are directly methylated in melanoma cell lines and tissues. First, we examined 16 genes by bisulphite sequencing in the WM793 isogenic cell line model series. Five of these genes (CYBA, FABP5, MT1E, TSPY1 and TAC1) displayed increased methylation in several invasive cell lines compared with the parental WM793 cells, indicating their involvement in progression. Next, we analyzed several matched primary/metastatic tumours using methylation-specific PCR, which revealed that MT1E (one of the five genes assessed) was methylated in the largest proportion of tumours. Examination of a larger cohort of samples showed that 1 of 17 (6%) of the benign naevi, 16 of 43 (37%) primary tumours and 6 of 13 (46%) of the metastases displayed MT1E methylation. In addition, ectopic over-expression of MT1E mediated sensitization to cisplatin-induced apoptosis. Overall, these studies suggest that MT1E is a potential tumour suppressor gene, whose loss may promote resistance to apoptosis-inducing therapies.

In this study, we used array-comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to examine genetic aberrations in melanoma cell lines and tissues. Array-comparative genomic hybridization revealed that the most frequent genetic changes found in melanoma cell lines were amplifications on chromosomes 7p and 20q, along with disruptions on Chr 9, 10, 11, 12, 22 and Y. Validation of the results using FISH on tissue microarrays (TMAs) identified TOP1 as being amplified in melanoma tissues. TOP1 amplification was detected in a high percentage (33%) of tumours and was associated with thicker, aggressive tumours. These results show that TOP1 amplification is associated with advanced tumours and poor prognosis in melanoma. These observations open the possibility that TOP1-targeted therapeutics may be of benefit in a particular subgroup of advanced stage melanoma patients.

Melanoma is an aggressive disease that spreads quickly and is resistant to most therapeutic agents. In an effort to provide insight into the molecular basis of melanoma progression, the expression of 94 genes in 20 metastatic melanomas using a high-throughput real-time quantitative RT-PCR assay was analysed.A TaqMan low density array (LDA) was designed containing probes/primers directed towards a cohort of genes previously found to be differentially expressed in an isogenic cell line model of melanoma progression. For each sample, cDNA was prepared and added to the quantitative assay. The resulting data were then analysed for correlations with clinical data.Clustering analysis divided the melanomas into two major subgroups based on gene expression patterns. When analysed individually, several genes were associated with overall survival, depth and type of the primary tumour.We have identified a selection of genes linked to melanoma progression and patient outcome.

Summary Background Tyrosine phosphate is abnormally elevated in malignant melanoma, and this has been interpreted to reflect the activity of oncogenic protein tyrosine kinases. However, elevation may also arise due to decreased protein tyrosine phosphatase (PTP) expression. Objectives To survey phosphatase gene expression in melanoma cell lines, a benign naevus and normal melanocytes: we searched for downregulation of phosphatase gene expression in malignant cells that may indicate a role as melanoma suppressor genes. Methods Microarray analysis was used to compare gene expression for 133 phosphatase genes, comprising 39 PTPs, 16 dual-specificity phosphatases (DSPs), 47 serine/threonine phosphatases and 31 acid/alkaline and lipid-based phosphatases. Northern blotting analysis was used to study gene expression in human melanoma biopsies. Results There was decreased expression of four DSP genes (including PTEN); eight receptor PTP genes were downregulated in melanoma, among which were PTP-KAPPA and PTP-PI (consistent with our previous data). In addition, PTP-RF/LAR was downregulated in 13 of 22 metastatic melanomas. Conclusions The expression of multiple PTP receptors is decreased in melanoma; this may be a mechanism which stimulates autonomous growth in advanced melanoma.

Background: Aberrant activity of the mitogen-activated protein kinase (MAPK) signalling pathway represents a critical factor in the initiation and development of melanoma. Activation can be the result of largely mutually exclusive somatic mutations of KIT, NRAS, or more frequently, BRAF(V600E). The FDA and EMA approval of a BRAF inhibitor (vemurafenib) that blocks the function of the BRAFV600E protein established a new paradigm for targeted drug development. Currently, BRAF inhibitors are one of the most promising approaches to treat metastatic melanoma. Methods: In this study, we applied mass spectrometry-based SNP genotyping (Sequenom) to detect single nucleotide mutations in 33 cancer-related genes including BRAF, NRAS, KIT, CTNNB1, GNAS, and MET. PCR and extension primers for the multiplexed assay were designed with the Sequenom Assay Design software. Samples were processed as recommended by the manufacturer. Matrix chips were assayed on a Sequenom MassArray MALDI-TOF Mass Array system. Visual inspection and Sequenom Typer software were used to perform genotyping based on mass spectra. We compared two independent melanoma cohorts, one from Ireland (SVUH cohort; n=97) and the other from Belgium (Leuven cohort; n=60). Results: Intriguingly, patients from Ireland showed significant lower BRAFV600E mutation rates (19.0%) when compared to the mutation frequency seen with the Belgian cohort (43.3%) or with the mutation frequency reported by other studies on non-Irish populations (50-70%). In addition, BRAFV600K mutation is rare in the SVUH cohort (1.0%) compared with the Leuven cohort (6.7%). Mutations in BRAFV600M and V600R were Conclusion: Our observation of reduced BRAFV600E mutation rates in Irish patients will directly impact future treatment strategies for these patients. The identification of substantial mutation rates in MET opens up opportunities for targeting a significant subset of melanoma patients with MET inhibitors. Citation Format: Balazs Balint, Karin van den Hurk, Sinead Toomey, Louise Unwin, Kieran Sheahan, Enda McDermott, Ian Murphy, Joost van den Oord, Mairin Rafferty, Bryan Hennessy, William M. Gallagher. Low incidence of BRAFV600E mutation among melanoma patients in Ireland. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 23. doi:10.1158/1538-7445.AM2013-23

Background: P-Rex1 is a Rac-specific Rho GTPase guanine nucleotide exchange factor that has recently been implicated in cancer for the first time. As Rac has a central role in control of cell motility, we assessed the role of P-Rex1 in melanoma, a cancer where metastasis is the major cause of death. This was the first evaluation of P-Rex1 in a genetically modified animal model of cancer. We also investigated the function of P-Rex1 in melanoblasts, as the molecular pathways that drive their developmental migration are believed to underpin the movement of metastatic melanoma cells. Methods: P-Rex1−/− mice were crossed to mice carrying the DCT-lacZ transgene, a melanoblast reporter line, and a Tyr::NrasQ61K/°; INK4a−/− metastatic melanoma mouse model. Allograft tail vein (TV) injections were performed to further assess the endogenous function of P-Rex1 in metastasis. Panels of human melanoma cell lines and tissue were investigated with microarray, tissue culture, and immunohistochemical techniques. Results: P-Rex1−/− mice have a melanoblast migration defect during development (p=0.01), evidenced by a white belly. They were resistant to metastasis when crossed to the Tyr::NrasQ61K/°; INK4a−/− murine model of melanoma (1/30 mice with metastases vs 13/30 in control cohort; p=0.001). When melanocytes derived from this model were TV-injected into immune-competent allografts, endogenous P-Rex1 was found to facilitate metastatic incidence, growth, and organ spread following the intravasation stage of the metastatic cascade (p=0.02). Mechanistically, this was associated with P-Rex1 driving invasion in a Rac-dependent manner. In humans, P-Rex1 was elevated in the great majority of melanoma cell lines, with mRNA levels above the median statistically associated with metastasic propensity (p=0.005). Tissue immunohistochemistry revealed an association between tumor progression and P-Rex1 expression. Conclusions: P-Rex1 is a key component of melanoma progression, invasion and metastatic signaling, supporting the value of pharmacological inhibition of P-Rex1 activation of Rac for treatment of metastatic or high risk primary melanomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B147.

Digital pathology platforms with integrated artificial intelligence have the potential to increase the efficiency of the nonclinical pathologist’s workflow through screening and prioritizing slides with lesions and highlighting areas with specific lesions for review. Herein, we describe the comparison of various single- and multi-magnification convolutional neural network (CNN) architectures to accelerate the detection of lesions in tissues. Different models were evaluated for defining performance characteristics and efficiency in accurately identifying lesions in 5 key rat organs (liver, kidney, heart, lung, and brain). Cohorts for liver and kidney were collected from TG-GATEs open-source repository, and heart, lung, and brain from internally selected R&D studies. Annotations were performed, and models were trained on each of the available lesion classes in the available organs. Various class-consolidation approaches were evaluated from generalized lesion detection to individual lesion detections. The relationship between the amount of annotated lesions and the precision/accuracy of model performance is elucidated. The utility of multi-magnification CNN implementations in specific tissue subtypes is also demonstrated. The use of these CNN-based models offers users the ability to apply generalized lesion detection to whole-slide images, with the potential to generate novel quantitative data that would not be possible with conventional image analysis techniques.

Supplemental material, Supplementary_Table_2 for Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08–10 trial by Giuseppe Gullo, Alex J. Eustace, Alexandra Canonici, Denis M. Collins, Michael J. Kennedy, Liam Grogan, Oscar Breathhnach, John McCaffrey, Maccon Keane, Michael J. Martin, Rajnish Gupta, Gregory Leonard, Miriam O’Connor, Paula M. Calvert, Paul Donnellan, Janice Walshe, Enda McDermott, Kathleen Scott, Andres Hernando, Imelda Parker, David W. Murray, Alice C. O’Farrell, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Norma O’Donovan, William M. Gallagher, Bonnie Ky, Dimitrios Tryfonopoulos, Brian Moulton, Annette T. Byrne and John Crown in Therapeutic Advances in Medical Oncology

Supplemental material, Supplementary_Table_1 for Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08–10 trial by Giuseppe Gullo, Alex J. Eustace, Alexandra Canonici, Denis M. Collins, Michael J. Kennedy, Liam Grogan, Oscar Breathhnach, John McCaffrey, Maccon Keane, Michael J. Martin, Rajnish Gupta, Gregory Leonard, Miriam O’Connor, Paula M. Calvert, Paul Donnellan, Janice Walshe, Enda McDermott, Kathleen Scott, Andres Hernando, Imelda Parker, David W. Murray, Alice C. O’Farrell, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Norma O’Donovan, William M. Gallagher, Bonnie Ky, Dimitrios Tryfonopoulos, Brian Moulton, Annette T. Byrne and John Crown in Therapeutic Advances in Medical Oncology

Supplemental material, Supplementary_Table_3 for Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08–10 trial by Giuseppe Gullo, Alex J. Eustace, Alexandra Canonici, Denis M. Collins, Michael J. Kennedy, Liam Grogan, Oscar Breathhnach, John McCaffrey, Maccon Keane, Michael J. Martin, Rajnish Gupta, Gregory Leonard, Miriam O’Connor, Paula M. Calvert, Paul Donnellan, Janice Walshe, Enda McDermott, Kathleen Scott, Andres Hernando, Imelda Parker, David W. Murray, Alice C. O’Farrell, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Norma O’Donovan, William M. Gallagher, Bonnie Ky, Dimitrios Tryfonopoulos, Brian Moulton, Annette T. Byrne and John Crown in Therapeutic Advances in Medical Oncology

Supplemental material, Supplementary_Table_3 for Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08–10 trial by Giuseppe Gullo, Alex J. Eustace, Alexandra Canonici, Denis M. Collins, Michael J. Kennedy, Liam Grogan, Oscar Breathhnach, John McCaffrey, Maccon Keane, Michael J. Martin, Rajnish Gupta, Gregory Leonard, Miriam O’Connor, Paula M. Calvert, Paul Donnellan, Janice Walshe, Enda McDermott, Kathleen Scott, Andres Hernando, Imelda Parker, David W. Murray, Alice C. O’Farrell, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Norma O’Donovan, William M. Gallagher, Bonnie Ky, Dimitrios Tryfonopoulos, Brian Moulton, Annette T. Byrne and John Crown in Therapeutic Advances in Medical Oncology

Supplemental material, Supplementary_Table_1 for Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08–10 trial by Giuseppe Gullo, Alex J. Eustace, Alexandra Canonici, Denis M. Collins, Michael J. Kennedy, Liam Grogan, Oscar Breathhnach, John McCaffrey, Maccon Keane, Michael J. Martin, Rajnish Gupta, Gregory Leonard, Miriam O’Connor, Paula M. Calvert, Paul Donnellan, Janice Walshe, Enda McDermott, Kathleen Scott, Andres Hernando, Imelda Parker, David W. Murray, Alice C. O’Farrell, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Norma O’Donovan, William M. Gallagher, Bonnie Ky, Dimitrios Tryfonopoulos, Brian Moulton, Annette T. Byrne and John Crown in Therapeutic Advances in Medical Oncology

Supplemental material, Supplementary_Methods for Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08–10 trial by Giuseppe Gullo, Alex J. Eustace, Alexandra Canonici, Denis M. Collins, Michael J. Kennedy, Liam Grogan, Oscar Breathhnach, John McCaffrey, Maccon Keane, Michael J. Martin, Rajnish Gupta, Gregory Leonard, Miriam O’Connor, Paula M. Calvert, Paul Donnellan, Janice Walshe, Enda McDermott, Kathleen Scott, Andres Hernando, Imelda Parker, David W. Murray, Alice C. O’Farrell, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Norma O’Donovan, William M. Gallagher, Bonnie Ky, Dimitrios Tryfonopoulos, Brian Moulton, Annette T. Byrne and John Crown in Therapeutic Advances in Medical Oncology

Supplemental material, Supplementary_Table_2 for Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08–10 trial by Giuseppe Gullo, Alex J. Eustace, Alexandra Canonici, Denis M. Collins, Michael J. Kennedy, Liam Grogan, Oscar Breathhnach, John McCaffrey, Maccon Keane, Michael J. Martin, Rajnish Gupta, Gregory Leonard, Miriam O’Connor, Paula M. Calvert, Paul Donnellan, Janice Walshe, Enda McDermott, Kathleen Scott, Andres Hernando, Imelda Parker, David W. Murray, Alice C. O’Farrell, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Norma O’Donovan, William M. Gallagher, Bonnie Ky, Dimitrios Tryfonopoulos, Brian Moulton, Annette T. Byrne and John Crown in Therapeutic Advances in Medical Oncology

Supplemental material, Supplementary_Methods for Pilot study of bevacizumab in combination with docetaxel and cyclophosphamide as adjuvant treatment for patients with early stage HER-2 negative breast cancer, including analysis of candidate circulating markers of cardiac toxicity: ICORG 08–10 trial by Giuseppe Gullo, Alex J. Eustace, Alexandra Canonici, Denis M. Collins, Michael J. Kennedy, Liam Grogan, Oscar Breathhnach, John McCaffrey, Maccon Keane, Michael J. Martin, Rajnish Gupta, Gregory Leonard, Miriam O’Connor, Paula M. Calvert, Paul Donnellan, Janice Walshe, Enda McDermott, Kathleen Scott, Andres Hernando, Imelda Parker, David W. Murray, Alice C. O’Farrell, Ashwini Maratha, Patrick Dicker, Mairin Rafferty, Verena Murphy, Norma O’Donovan, William M. Gallagher, Bonnie Ky, Dimitrios Tryfonopoulos, Brian Moulton, Annette T. Byrne and John Crown in Therapeutic Advances in Medical Oncology