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Oesophageal adenocarcinoma is a poor prognosis cancer and the molecular features underpinning response to treatment remain unclear. We investigate whole genome, transcriptomic and methylation data from 115 oesophageal adenocarcinoma patients mostly from the DOCTOR phase II clinical trial (Australian New Zealand Clinical Trials Registry-ACTRN12609000665235), with exploratory analysis pre-specified in the study protocol of the trial. We report genomic features associated with poorer overall survival, such as the APOBEC mutational and RS3-like rearrangement signatures. We also show that positron emission tomography non-responders have more sub-clonal genomic copy number alterations. Transcriptomic analysis categorises patients into four immune clusters correlated with survival. The immune suppressed cluster is associated with worse survival, enriched with myeloid-derived cells, and an epithelial-mesenchymal transition signature. The immune hot cluster is associated with better survival, enriched with lymphocytes, myeloid-derived cells, and an immune signature including CCL5, CD8A, and NKG7. The immune clusters highlight patients who may respond to immunotherapy and thus may guide future clinical trials.

Figure S3. Inhibition of FGFR and PI3K pathways by BGJ398, GDC-0941 BYL719 and BKM120 in MFE296 cells.

Treatment for metastatic melanoma includes targeted and/or immunotherapy. Although many patients respond, only a subset has complete response. As late-stage patients often have multiple tumors in difficult access sites, non-invasive techniques are necessary for the development of predictive/prognostic biomarkers. PET/CT scans from 52 patients with stage III/IV melanoma were assessed and CT image parameters were evaluated as prognostic biomarkers. Analysis indicated patients with high standard deviation or high mean of positive pixels (MPP) had worse progression-free survival (P = 0.00047 and P = 0.0014, respectively) and worse overall survival (P = 0.0223 and P = 0.0465, respectively). Whole-exome sequencing showed high MPP was associated with BRAF mutation status (P = 0.0389). RNA-sequencing indicated patients with immune “cold” signatures had worse survival, which was associated with CT biomarker, MPP4 (P = 0.0284). Multiplex immunofluorescence confirmed a correlation between CD8 expression and image biomarkers (P = 0.0028).Implications:CT parameters have the potential to be cost-effective biomarkers of survival in melanoma, and reflect the tumor immune-microenvironment.

Supplementary Figure Legends 1-4.

Kaplan-Meier (log-rank) analysis of MPP4 and SD3 showing the PFS and OS for stage III patients only. a. PFS for MPP4, **p=0.0017, b. OS for MPP4, *p=0.0117, c. PFS for SD3, ***p=0.0003 and d. OS for SD3, **p=0.0094.

Multiplex immunofluorescence staining quantification by Visiopharm Image Analytical System. DAPI nuclei were segmented defining the areas. Then each marker was quantified within this DAPI area. MPP4 low (MelR062) and MPP4 high (MelR166) phenotypes are represented.

Table S1. Patient Characteristics. AJCC: American Joint Committee on Cancer Table S2. Clinical information including histology, stage and survival. Table S3. Univariate analysis of clinico-pathological and CT features. Log-rank (Mantel-Cox) test to determine optimal cut-offs for low/high groups. Table S4. RNAseq immune genes for multiplex analysis.

The association between TMB and SD3/MPP4 is not statistically significant. A. MPP4, not significant, Fisher's exact. B. SD3, not significant, Fisher's exact.

The incidence of oesophageal adenocarcinoma (OAC) is rising in Western countries, with a 5-year overall survival (OS) rate of 14%. Curative treatment based on oesophagectomy is only suitable for ~50% of patients due to late-stage diagnosis. While the addition of preoperative chemotherapy or chemoradiotherapy has improved OS in OAC patients, little is known about the molecular basis of treatment response and patient outcomes. We investigated multi-omics data including whole-genome sequencing, RNA sequencing, methylation profiles and immunohistochemistry from 115 OAC patients mostly from DOCTOR phase II clinical trial that utilized neoadjuvant therapy (ANZCTR-ACTRN12609000665235). We identified genomic features associated with poor OS, such as the APOBEC mutational signature. We also showed that positron emission tomography non-responders have more sub-clonal genomic copy number alterations. RNA sequencing and methylation profiles suggested four immune clusters associated with OS and progression-free survival. The immune-suppressed cluster was associated with worse survival and epithelial-mesenchymal transition signature and enriched with myeloid-derived cells. The immune hot cluster was associated with better survival and enriched with T-cells, myeloid-derived cells, interferon-gamma and alpha responses, and immune markers such as CCL5, CD8A, and NKG7. We investigated multi-omic features of OAC tumours from patients who were part of phase II clinical trial. We discovered prognostic features such as mutational signatures and complex genomic events. We identified distinct immune clusters associated with patient outcomes and the potential of immunotherapy for select clusters. We characterized molecular features of OAC patients which has the potential to predict responses to neoadjuvant therapy and develop better-selected therapy, monotherapy, or combination therapy in the future.

Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.

Background: The incidence of oesophageal adenocarcinoma (OAC) is rapidly increasing and despite improvements in treatment, the five-year survival rate remains poor. Prognostic biomarkers that address the genomic heterogeneity in this highly complex disease will aid the development of precision therapeutics and improve patient survival. The aim of this study was to determine whether circulating tumour DNA (ctDNA) has prognostic significance as a biomarker in OAC patients.Methods: We profiled 209 blood and tumour samples from 57 OAC patients. Using a panel of 77 cancer genes, we sequenced ctDNA in plasma samples (n=127) which were taken at multiple time points pre- and post-therapy. In parallel, we sequenced matched tumour samples from 39 patients using the same gene panel. To assess whether the ctDNA profile reflected the tumour heterogeneity, we sequenced additional multi-region primary tumour samples (n=17). In addition, we analysed whole-genome and whole-exome sequencing data from primary tumours for a subset of 18 patients. Results: Using a tumour agnostic approach, we found that detectable ctDNA variants in post-treatment plasma samples were associated with worse disease specific survival. To evaluate whether the ctDNA originated from the primary tumour, we performed a tumour informed analysis which confirmed post-treatment ctDNA variants were associated with worse survival. To determine whether ctDNA could be used as a clinical follow-up test, we assessed blood samples from multiple time points before and after treatment, in a subset of patients. Results showed that the variant allele frequency of ctDNA variants increased with disease recurrence. Conclusion: This study demonstrates that ctDNA variants can be detected in patients with OAC and this has potential clinical utility as a prognostic biomarker for survival.

Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. We previously identified activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression-free and cancer-specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release and impaired mitochondrial respiration in two FGFR2-mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; in addition, the pan-caspase inhibitor Z-VAD-FMK was unable to prevent cell death, suggesting that the cell death is caspase-independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial-dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly, we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR-dependent malignancies.

Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.

BackgroundEndometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprised of four molecular subtypes with differing etiology, prognoses, and response to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. Pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models.MethodsHere, we report whole exome or whole genome sequencing of 11 PDX models and the matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the different molecular subtypes of EC.ResultsPDX models were successfully generated from all four molecular subtypes of EC and uterine carcinosarcomas, and they recapitulated morphology and the molecular landscape of primary tumors without major genomic drift. We also observed a wide range of inter-tumor and intra-tumor heterogeneity, well captured by different PDX lineages, which could lead to different treatment responses. An in vivo response to talazoparib was detected in two p53mut models consistent with stable disease, however both lacked the HR deficiency genomic signature.ConclusionsEC PDX models represent the four molecular subtypes of disease and can capture intra-tumoral heterogeneity of the original primary tumor. PDXs of the p53mut molecular subtype showed sensitivity to PARPi, however, deeper and more durable responses will likely require combination of PARPi with other agents.

e16028 Background: : Prognosis of oesophageal adenocarcinoma (OAC) remains poor globally. Traditional histopathology classifications have minimal impact on clinical management and outcomes. To improve patient outcomes, prognostic and therapeutic stratification is required within this cohort of morphologically homogenous cancers. Identification and validation of candidate prognostic and therapeutic biomarkers may contribute to personalisation of therapy for patients with OAC. Methods: We used RNAseq of OAC tumour tissues to classify OAC into subgroups which were associated with patient survival and the immune infiltrate in the tumour micro-environment. We identified genes that discriminate each group and selected thirteen to perform IHC against the protein products of these genes using the same OAC samples. The IHC markers that best discriminate the immune-clusters were applied to an independent set of OAC in tissue micro-arrays (TMAs) to validate the prognostic significance of the immune-clusters. Results: Using RNA-seq we identified four candidate immune-clusters namely, immune hot, immune cold, immune suppressed and immune moderate, that correlate with progression free and overall patient survival. IHC revealed a correlation of five of thirteen of these IHC markers with the RNAseq data, and the IHC was able to corroborate the classification of OAC immune-clusters. The best markers predictive of these immune-clusters underwent IHC in an independent OAC TMA cohort to confirm the prognostic significance of these markers. The TMAs contain matched primary and nodal disease, so we will also report on the presence of the four subgroups within nodal disease. To date, two of the IHC markers are associated with survival benefit in the TMAs patient group. Conclusions: We anticipate that validating IHC markers to reliably identify the four immune-clusters of OAC will be useful in predicting prognosis, survival and therapeutic classification of OAC in precision oncology of the OAC.

Treatment for metastatic melanoma includes targeted and/or immunotherapy. Although many patients respond, only a subset has complete response. As late-stage patients often have multiple tumors in difficult access sites, non-invasive techniques are necessary for the development of predictive/prognostic biomarkers. PET/CT scans from 52 patients with stage III/IV melanoma were assessed and CT image parameters were evaluated as prognostic biomarkers. Analysis indicated patients with high standard deviation or high mean of positive pixels (MPP) had worse progression-free survival (P = 0.00047 and P = 0.0014, respectively) and worse overall survival (P = 0.0223 and P = 0.0465, respectively). Whole-exome sequencing showed high MPP was associated with BRAF mutation status (P = 0.0389). RNA-sequencing indicated patients with immune “cold” signatures had worse survival, which was associated with CT biomarker, MPP4 (P = 0.0284). Multiplex immunofluorescence confirmed a correlation between CD8 expression and image biomarkers (P = 0.0028). Implications: CT parameters have the potential to be cost-effective biomarkers of survival in melanoma, and reflect the tumor immune-microenvironment.

Background Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprises four molecular subtypes with differing etiology, prognoses, and responses to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. As pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models. Methods Here, we report whole exome or whole genome sequencing of 11 PDX models and their matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the copy number high/p53-mutant and mismatch-repair deficient molecular subtypes of EC. Results PDX models were successfully generated from grade 2/3 tumors, including three uterine carcinosarcomas. The models showed similar histomorphology to the primary tumors and represented all four molecular subtypes of EC, including five mismatch-repair deficient models. The different PDX lineages showed a wide range of inter-tumor and intra-tumor heterogeneity. However, for most PDX models, one arm recapitulated the molecular landscape of the primary tumor without major genomic drift. An in vivo response to talazoparib was detected in four copy number high models. Two models (carcinosarcomas) showed a response consistent with stable disease and two models (one copy number high serous EC and another carcinosarcoma) showed significant tumor growth inhibition, albeit one consistent with progressive disease; however, all lacked the HR deficiency genomic signature. Conclusions EC PDX models represent the four molecular subtypes of disease and can capture intra-tumor heterogeneity of the original primary tumor. PDXs of the copy number high molecular subtype showed sensitivity to PARPi; however, deeper and more durable responses will likely require combination of PARPi with other agents.

Patients with late stage resected cutaneous melanoma have poor overall survival (OS) and experience irreversible adverse events from systemic therapy. There is a clinical need to identify biomarkers to predict outcome. Performing germline/tumour whole-exome sequencing of 44 stage III/IV melanoma patients we identified pathogenic germline mutations in CDKN2A, CDK4, ATM, POLH, MRE11A, RECQL4 and XPC, affecting 7/44 patients. These mutations were associated with poor OS (p = 0.0082). We confirmed our findings in The Cancer Genome Atlas (TCGA) human skin cutaneous melanoma cohort where we identified pathogenic variants in 40/455 patients (p = 0.0203). Combining these cohorts (n = 499) further strengthened these findings showing germline carriers had worse OS (p = 0.0009). Additionally, we determined whether tumour mutation burden (TMB) or BRAF status were prognostic markers of survival. Low TMB rate (p = 0.0034) and BRAF p.V600 mutation (p = 0.0355) were associated with worse progression-free survival. Combining these biomarkers indicated that V600 mutant patients had significantly lower TMB (p = 0.0155). This was confirmed in the TCGA (n = 443, p = 0.0007). Integrative analysis showed germline mutation status conferred the highest risk (HR 5.2, 95% CI 1.72–15.7). Stage IV (HR 2.5, 0.74–8.6) and low TMB (HR 2.3, 0.57–9.4) were similar, whereas BRAF V600 status was the weakest prognostic biomarker (HR 1.5, 95% CI 0.44–5.2).

Endometrial cancer is a major gynaecological cancer with a high incidence resulting in over 500 Australian women dying every year. Good pre-clinical models are urgently needed to study the biology of this disease, and to develop and test novel treatment strategies. Most current pre-clinical research is conducted in cell lines that often do not resemble the disease on the molecular level and do not recapitulate tumour heterogeneity, which results in untranslatable research findings. Patient-derived xenograft (PDX) models overcome the issues of representing tumour molecular landscape and heterogeneity, making them a translatable pre-clinical cancer model. Here, we generated and genomically characterised 11 PDX models by performing whole genome or whole exome sequencing of primary patient tumours and matched PDX samples. We performed a detailed genomic characterisation of these models to determine their suitability as pre-clinical models, study tumour heterogeneity and identify biomarkers of response and resistance. PDX models were successfully generated from all four molecular subtypes of EC and uterine carcinosarcomas, and they recapitulated morphology and the molecular landscape of primary tumors without major genomic drift. We also observed a wide range of inter-tumor and intra-tumor heterogeneity, well captured by different PDX lineages, which could lead to different treatment responses. An in vivo response to talazoparib was detected in two p53mut models consistent with stable disease, however both lacked the HR deficiency genomic signature. EC PDX models represent the four molecular subtypes of disease and can capture intra-tumoral heterogeneity of the original primary tumor. PDXs of the p53mut molecular subtype showed sensitivity to PARPi, however, deeper and more durable responses will likely require combination of PARPi with other agents. Citation Format: Olga Kondrashova, Vanessa F. Bonazzi, Deborah Smith, Katia Nones, Asmerom Sengal, Robert Ju, Leisl M. Packer, Lambros T Koufariotis, Stephen H. Kazakoff, Aimee L. Davidson, Priya Ramarao-Milne, Vanessa Lakis, Felicity Newell, Rebecca Rogers, Claire Davies, Naven Chetty, Lewis Perrin, John Pearson, Ann-Marie Patch, Andrea Garrett, Nicola Waddell, Pamela Pollock, James Nicklin. Genomic characterization of endometrial cancer patient-derived xenografts reveals intratumor heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR007.

Drugs such as gemcitabine that increase replication stress are effective chemotherapeutics in a range of cancer settings. These drugs effectively block replication and promote DNA damage, triggering a cell cycle checkpoint response through the ATR-CHK1 pathway. Inhibiting this signaling pathway sensitises cells to killing by replication stress inducing drugs. Here, we investigated the effect of low level replication stress induced by low concentrations (>0.2 mM) of the reversible ribonucleotide reductase inhibitor hydroxyurea (HU), which slows S phase progression but has little effect on cell viability or proliferation. We demonstrate that HU effectively synergises with CHK1, but not ATR inhibition, in >70% of melanoma and non-small cell lung cancer cells assessed, resulting in apoptosis and complete loss of proliferative potential in vitro and in vivo. Normal fibroblasts and haemopoietic cells retain viability and proliferative potential following exposure to CHK1 inhibitor plus low doses of HU, but normal cells exposed to CHK1 inhibitor combined with sub-micromolar concentrations of gemcitabine exhibited complete loss of proliferative potential. The effects of gemcitabine on normal tissue correlate with irreversible ATR-CHK1 pathway activation, whereas low doses of HU reversibly activate CHK1 independently of ATR. Combined use of CHK1 inhibitor and subclinical HU also triggered an inflammatory response involving the recruitment of macrophages in vivo. These data indicate that combining CHK1 inhibitor with subclinical HU is superior to combination with gemcitabine, as it provides equal anti-cancer efficacy but with reduced normal tissue toxicity. These data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination. This article is protected by copyright. All rights reserved.

Background Oesophageal adenocarcinoma (EAC) incidence is increasing and has a poor survival rate. Barrett’s oesophagus (BE) is a precursor condition that is associated with EAC and often occurs in conjunction with chronic gastro-oesophageal reflux, however many individuals diagnosed with BE never progress to cancer. An understanding of the genomic features of BE and EAC may help with the early identification of at-risk individuals. Methods In this study, we assessed the genomic features of 16 BE samples using whole-genome sequencing. These included non-dysplastic samples collected at two time-points from two BE patients who had not progressed to EAC over several years. Seven other non-dysplastic samples and five dysplastic BE samples with high-grade dysplasia were also examined. We compared the genome profiles of these 16 BE samples with 22 EAC samples. Results We observed that samples from the two non-progressor individuals had low numbers of somatic single nucleotide variants, indels and structural variation events compared to dysplastic and the remaining non-dysplastic BE. EAC had the highest level of somatic genomic variations. Mutational signature 17, which is common in EAC, was also present in non-dysplastic and dysplastic BE, but was not present in the non-progressors. Many dysplastic samples had mutations in genes previously reported in EAC, whereas only mutations in CDKN2A or in the fragile site genes appeared common in non-dysplastic samples. Rearrangement signatures were used to identify a signature associated with localised complex events such as chromothripsis and breakage fusion-bridge that are characteristic of EACs. Two dysplastic BE samples had a high contribution of this signature and contained evidence of localised rearrangements. Two other dysplastic samples also had regions of localised structural rearrangements. There was no evidence for complex events in non-dysplastic samples. Conclusions The presence of complex localised rearrangements in dysplastic samples indicates a need for further investigations into the role such events play in the progression from BE to EAC. Electronic supplementary material The online version of this article (10.1186/s12920-019-0476-9) contains supplementary material, which is available to authorized users.

// Mitchell S. Stark 1,2 , Vanessa F. Bonazzi 3 , Glen M. Boyle 1 , Jane M. Palmer 1 , Judith Symmons 1 , Catherine M. Lanagan 1 , Christopher W. Schmidt 1 , Adrian C. Herington 2 , Robert Ballotti 3 , Pamela M. Pollock 2 and Nicholas K. Hayward 1 1 QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD, Australia 2 School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia 3 Inserm U1065, Centre Mediterraneen de Medecine Moleculaire, Equipe 1, Biologie et pathologies des melanocytes, Nice, France Correspondence to: Mitchell S. Stark, email: // Keywords : miRNA, microRNA, miR-514a, BRAFi, NF1 Received : March 16, 2015 Accepted : April 07, 2015 Published : April 23, 2015 Abstract To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1 , CDK2 , MC1R , and NF1 , previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E -targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies. Mitchell S. Stark

Melanoma is a complex multifactorial disease; therefore, a combination of various approaches is necessary to girt all aspects of its biology and identify the many different genes and factors involved in its etiology.Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. Here, we describe a new pipeline based on an integrative and comparative analysis of several array platform, post-demethylation treatment expression data, methylation array, and constitutive mRNA expression analysis to identify novel TSGs frequently methylated in melanoma.

Melanoma is a complex multifactorial disease; therefore, a combination of various approaches is necessary to girt all aspects of its biology and identify the many different genes and factors involved in its etiology.Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. Here, we describe a new pipeline based on an integrative and comparative analysis of several array platform, post-demethylation treatment expression data, methylation array, and constitutive mRNA expression analysis to identify novel TSGs frequently methylated in melanoma.

Improved therapeutic approaches are needed for the treatment of recurrent and metastatic endometrial cancer. Endometrial cancers display hyperactivation of the MAPK and PI3K pathways, the result of somatic aberrations in genes such as

To identify microRNAs potentially involved in melanomagenesis, we compared microRNA expression profiles between melanoma cell lines and cultured melanocytes. The most differentially expressed microRNA between the normal and tumor cell lines was miR-211. We focused on this pigment-cell-enriched miRNA as it is derived from the microphthalmia-associated transcription factor (MITF)-regulated gene, TRPM1 (melastatin). We find that miR-211 expression is greatly decreased in melanoma cells and melanoblasts compared to melanocytes. Bioinformatic analysis identified a large number of potential targets of miR-211, including POU3F2 (BRN2). Inhibition of miR-211 in normal melanocytes resulted in increased BRN2 protein, indicating that endogenous miR-211 represses BRN2 in differentiated cells. Over-expression of miR-211 in melanoma cell lines changed the invasive potential of the cells in vitro through directly targeting BRN2 translation. We propose a model for the apparent non-overlapping expression levels of BRN2 and MITF in melanoma, mediated by miR-211 expression.

Selumetinib (AZD6244, ARRY-142886) is a selective, non–ATP-competitive inhibitor of mitogen-activated protein/extracellular signal–regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR–based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies. Cancer Res; 70(6); 2264–73

Melanocytic behavior, survival, and proliferation are regulated through a complex system of cell-cell adhesion molecules. Pathologic changes leading to development of malignant melanoma, upset the delicate homeostatic balance between melanocytes and keratinocytes and can lead to altered expression of cell-cell adhesion and cell-cell communication molecules. Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression. We now show loss of another member of the superfamily of classical cadherins, H-cadherin (CDH13), which may be involved in the development of malignant melanoma. The provided data show that H-cadherin expression is lost in nearly 80% of the analyzed melanoma cell lines. Knockdown of H-cadherin using siRNA increases invasive capacity in melanocytes. Functional assays show that the re-expression of H-cadherin decreases migration and invasion capacity, as well as anchorage-independent growth in comparison to control melanoma cells. Furthermore, melanoma cells, which re-express H-cadherin via stable transfection show a reduction in rate of tumor growth in a nu/nu mouse tumor model in comparison to the parental control transfected cell lines. Our study presents for the first time the down-regulation of H-cadherin in malignant melanomas and its possible functional relevance in maintenance healthy skin architecture.

// Mitchell S. Stark 1, 2 , Lisa N. Tom 1 , Glen M. Boyle 2 , Vanessa F. Bonazzi 3 , H. Peter Soyer 1 , Adrian C. Herington 3 , Pamela M. Pollock 3 , Nicholas K. Hayward 2 1 Dermatology Research Centre, The University of Queensland, School of Medicine, Translational Research Institute, Brisbane, QLD, Australia 2 QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD, Australia 3 School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, at The Translational Research Institute, Brisbane, QLD, Australia Correspondence to: Mitchell S. Stark, email: m.stark@uq.edu.au Keywords: SSX, microRNA, melanoma, PMP22, miR-4731 Received: May 02, 2015 Accepted: June 01, 2016 Published: June 16, 2016 ABSTRACT We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanoma tissues from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines (HT144, MM96L and MM253). Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n=1092) had potential to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the ‘cell cycle’ pathway and the ‘melanosome’. Following miR-4731 overexpression, a selection (n=81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2 , ORC5L , and PCNA ) and the melanosome (e.g. RAB7A , CTSD , and GNA13 ). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2 , SSX4 , and SSX4B ) as a result of miR-4731 overexpression. Moreover, this down-regulation of mRNA expression resulted in ablation or reduction of SSX4 protein, which, in keeping with previous studies, resulted in loss of 2D colony formation. We therefore speculate that loss of miR-4731 expression in stage IV patient tumours supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels.

Endometrial cancer (EC) is the most commonly diagnosed malignancy of the female reproductive tract. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumors that are refractory to current chemotherapies with an associated poor prognosis. Our laboratory identified activating mutations in Fibroblast Growth Factor Receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid endometrial tumors and have since shown in a large (n=970) multi-institutional cohort they are associated with shorter progression free and cancer specific survival. Although FGFR inhibitors are in clinical trials in several cancer types, no detailed study of the mechanism of cell death has been published. We now show that treatment with BGJ398, AZD4547 and PD173074 leads to the induction of mitochondrial depolarization and changes in metabolic flux in two endometrial cancer cell lines (JHUEM2 and AN3CA) carrying activating mutations (C383R and N550K respectively). Despite this mitochondrial dysfunction, we have convincingly shown that the cell death following FGFR inhibition was caspase-independent, as evidenced by the lack of caspase-3, -7, and -9 activation, absence of PARP cleavage, and the inability of the broad-spectrum caspase inhibitor, Z-VAD-FMK, to prevent cell death. Knockdown of EndoG and AIF, common mediators of caspase-independent death, had no effect. Detailed quantification of LC3 positive puncta shows an increase in autophagy in JHUEM2 and AN3CA cells treated with all FGFR inhibitors. Knockdown of ATG3, ATG7 and ATG12 resulted in a slight increase in Annexin positive cell death indicating that the autophagy was cytoprotective in this context. We have now confirmed this novel caspase-independent cell death is mitochondrial dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly we have shown that the combination of FGFR inhibitors with the BH3 mimetic ABT737 can markedly augment this caspase-independent cell death which may have implications for the design of more effective clinical trials. Citation Format: Leisl Packer, Sara Byron, Samantha Stehbens, Vanessa Bonazzi, David Loch, Andreas Wortmann, Mike Gartside, Nigel Waterhouse, Jennifer Gunter, Pamela M. Pollock. FGFR inhibition in endometrial cancer induces caspase-independent cell death that can be augmented with ABT-737 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B31.

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.

// Mickael Ohanna 1,2,* , Yann Cheli 1,2,* , Caroline Bonet 1,2 , Vanessa F Bonazzi 4 , Marylin Allegra 1,3 , Sandy Giuliano 1,2 , Karine Bille 1,2 , Philippe Bahadoran 1,2,3 , Damien Giacchero 3 , Jean Philippe Lacour 2,3 , Glen M Boyle 4 , Nicholas F Hayward 4 , Corine Bertolotto 1,2,* and Robert Ballotti 1,2,3,* . 1 Inserm U1065, Centre Mediterraneen de Medecine Moleculaire, Equipe 1, Biologie et pathologies des melanocytes: de la pigmentation cutanee au melanome. Equipe labellisee Ligue 2013, Nice, F-06204, France 2 Universite de Nice Sophia-Antipolis, UFR Medecine, Nice, F-06107, France 3 Centre Hospitalier Universitaire, Service de Dermatologie, Nice, F-06204, France 4 Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane 4006, Australia * These authors equally contributed to the work Correspondence: Robert Ballotti, email: // Keywords : melanoma, senescence, secretome, STAT3 Received : June 30, 2013 Accepted : August 17, 2013 Published : August 19, 2013 Abstract Here, we showed that the secretome of senescent melanoma cells drives basal melanoma cells towards a mesenchymal phenotype, with characteristic of stems illustrated by increased level of the prototype genes FN1, SNAIL, OCT4 and NANOG. This molecular reprogramming leads to an increase in the low-MITF and slow-growing cell population endowed with melanoma-initiating cell features. The secretome of senescent melanoma cells induces a panel of 52 genes, involved in cell movement and cell/cell interaction, among which AXL and ALDH1A3 have been implicated in melanoma development. We found that the secretome of senescent melanoma cells activates the STAT3 pathway and STAT3 inhibition prevents secretome effects, including the acquisition of tumorigenic properties. Collectively, the findings provide insights into how the secretome of melanoma cells entering senescence upon chemotherapy treatments increases the tumorigenicity of naive melanoma cells by inducing, through STAT3 activation, a melanoma-initiating cell phenotype that could favor chemotherapy resistance and relapse.

We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanomas from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines. Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n = 1092) were predicted to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the ‘cell cycle’ and ‘melanosome’ pathways. Following miR-4731 overexpression, a selection (n = 81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as result of miR-4731 overexpression. We therefore speculate that loss of miR-4731 expression supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels together with members of the cell cycle pathway, which warrants further investigation. Citation Format: Mitchell S. Stark, Lisa Tom, Glen M. Boyle, Vanessa F. Bonazzi, Adrian C. Herington, Pamela M. Pollock, Nicholas K. Hayward. The melanoma-enriched microRNA miR-4731 regulates genes involved in cell cycle and the melanosome. [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 1093.

Family and twin studies indicate that melanoma susceptibility has a strong genetic component. Very rarely, melanoma runs in families in which there is an inherited mutation in a single ‘high penetrance’ gene, but in the general population melanoma susceptibility is thought to be governed by variation in a series of ‘low penetrance’ genes. We sought to identify new melanoma risk genes of both classes by conducting an Australian genome-wide association study (GWAS) of ~2200 melanoma cases and ~4300 matched controls (from the AMFS and Q-MEGA studies), in parallel with whole-genome sequencing of cases from densely affected melanoma families with follow up genotyping of interesting variants in the GWAS sample and other highly case-loaded melanoma families. Genotyping of the GWAS sample was carried out using Illumina Hap610K or OMNI 1M arrays. All 25 SNPs that reached genome-wide statistical significance (i.e. p

The aetiology of melanoma, the most lethal form of skin cancer, is complex, involving both genetic and environmental components. Over the past decade, many genetic alterations affecting melanoma development have been identified and more recently a new epigenetic level of regulation has increasingly been explored. MicroRNA (miRNA)-mediated epigenetic regulation of tumour suppressor genes and oncogenes has been shown to play a central role in melanomagenesis. Over the past few years, many studies combining miRNA expression arrays and quantitative reverse transcriptase-PCR assays have identified different miRNAs deregulated during melanoma progression. Several groups have focused their efforts on understanding the functional role of these different miRNAs in melanoma, identifying their direct targets and elucidating their mechanisms of regulation. This review summarizes the present knowledge of miRNA dysregulation in melanoma. On the basis of the current literature, we present a network of miRNA interactions involved in melanoma progression. Some of these key miRNAs may have utility as diagnostic markers or in targeted treatments.

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

To identify microRNAs potentially involved in melanomagenesis, we compared microRNA expression profiles between melanoma cell lines and cultured melanocytes. The most differentially expressed microRNA between the normal and tumor cell lines was miR-211. We focused on this pigment-cell-enriched miRNA as it is derived from the microphthalmia-associated transcription factor (MITF)-regulated gene, TRPM1 (melastatin). We find that miR-211 expression is greatly decreased in melanoma cells and melanoblasts compared to melanocytes. Bioinformatic analysis identified a large number of potential targets of miR-211, including POU3F2 (BRN2). Inhibition of miR-211 in normal melanocytes resulted in increased BRN2 protein, indicating that endogenous miR-211 represses BRN2 in differentiated cells. Over-expression of miR-211 in melanoma cell lines changed the invasive potential of the cells in vitro through directly targeting BRN2 translation. We propose a model for the apparent non-overlapping expression levels of BRN2 and MITF in melanoma, mediated by miR-211 expression.

So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families. Mutations in CDKN2A account for approximately 40% of familial cases, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds. Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma. We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF). Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant. Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case-control sample. Likewise, it was similarly associated in an independent case-control sample from the United Kingdom. In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both. The variant allele was also associated with increased naevus count and non-blue eye colour. Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.

Background: Our lab has identified FGFR2 mutations in 12% of over 1500 endometrioid endometrial cancers (ECs) analyzed. The majority of these tumors harbor genetic aberrations in the PI3K/AKT pathway resulting in constitutive activation. In other tissue types, PTEN loss has been shown to provide resistance to specific PIK3CA inhibition. We have previously reported that FGFR inhibition with PD173074 induced cell death despite loss of PTEN. We hypothesize that dual inhibition of FGFR2 and the PI3K pathway will lead to increased cell death and more effective tumor growth inhibition. Materials and Methods: Two FGFR2 mutant EC cell lines were treated with BGJ398 (pan FGFR inhibitor) alone or in combination with either a pan-PI3K inhibitor (GDC0941) or a specific PI3KA inhibitor (BYL719). Synergy was assessed using the fixed ratio method proposed by Chou and Talalay. Cell death was determined by Annexin V staining and colony formation assays using low clinically relevant drug concentrations. Downstream signaling pathways were analyzed by Western blot analysis and BGJ398 in combination with either GDC0941 or BYL719 was assessed in vivo. Results: Synergy was observed between BGJ398 and either the pan-PI3K inhibitor (GDC0941), or the specific PI3KA inhibitor (BYL719). Regression of tumor xenografts was observed in mice treated with BGJ398 and GDC0941 as well as BGJ398 and BYL719, despite PTEN inactivation. Conclusions: Dual targeting of the FGFR and PI3K pathways is more effective than targeting either of them alone. PTEN loss does not provide resistance to PIK3CA inhibition in our EC models suggesting context dependent differences in PI3K signaling. Citation Format: Pamela M. Pollock, Xinyan Geng, Vanessa F. Bonazzi, Clare Mahon, Sally Stephenson, Leisl Packer. Combination of BGJ398 with either a pan-PI3K inhibitor or a specific PIK3CA inhibitor shows synergy in FGFR2 mutant endometrial cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B114.

Inactivation on both alleles of the hSNF5/INI1 tumor suppressor gene which encodes a subunit of the human SWI/SNF chromatin remodelling complex occurs in most malignant rhabdoid tumors. No paralog of hSNF5/INI1 is identified in the human genome. In contrast, it has two homologs in the yeast Saccharomyces cerevisiae, SNF5 and SFH1 which encode core components of the ySWI/SNF and RSC complexes, respectively. The homology mainly concerns an approximately 200 amino acid region termed the SNF5 homology domain. We have tested the ability of the hSNF5/INI1-wild type gene product and of chimerical constructs in which the yeast SNF5 domains were replaced by that of the human protein, to complement yeast snf5 and sfh1 phenotypes. Neither growth deficiencies on different carbon sources of snf5 yeasts nor the lethality of the sfh1 phenotype could be rescued. This strongly suggests that the SNF5 homology domain presents species-specific functions.