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Background In many cancers, microRNAs (miRs) contribute to metastatic progression by modulating phenotypic reprogramming processes such as epithelial-mesenchymal plasticity. This can be driven by miRs targeting multiple mRNA transcripts, inducing regulated changes across large sets of genes. The miR-target databases TargetScan and DIANA-microT predict putative relationships by examining sequence complementarity between miRs and mRNAs. However, it remains a challenge to identify which miR-mRNA interactions are active at endogenous expression levels, and of biological consequence. Methods We developed a workflow to integrate TargetScan and DIANA-microT predictions into the analysis of data-driven associations calculated from transcript abundance (RNASeq) data, specifically the mutual information and Pearson’s correlation metrics. We use this workflow to identify putative relationships of miR-mediated mRNA repression with strong support from both lines of evidence. Applying this approach systematically to a large, published collection of unique melanoma cell lines – the Ludwig Melbourne melanoma (LM-MEL) cell line panel – we identified putative miR-mRNA interactions that may contribute to invasiveness. This guided the selection of interactions of interest for further in vitro validation studies. Results Several miR-mRNA regulatory relationships supported by TargetScan and DIANA-microT demonstrated differential activity across cell lines of varying matrigel invasiveness. Strong negative statistical associations for these putative regulatory relationships were consistent with target mRNA inhibition by the miR, and suggest that differential activity of such miR-mRNA relationships contribute to differences in melanoma invasiveness. Many of these relationships were reflected across the skin cutaneous melanoma TCGA dataset, indicating that these observations also show graded activity across clinical samples. Several of these miRs are implicated in cancer progression (miR-211, -340, -125b, −221, and -29b). The specific role for miR-29b-3p in melanoma has not been well studied. We experimentally validated the predicted miR-29b-3p regulation of LAMC1 and PPIC and LASP1, and show that dysregulation of miR-29b-3p or these mRNA targets can influence cellular invasiveness in vitro. Conclusions This analytic strategy provides a comprehensive, systems-level approach to identify miR-mRNA regulation in high-throughput cancer data, identifies novel putative interactions with functional phenotypic relevance, and can be used to direct experimental resources for subsequent experimental validation. Computational scripts are available: http://github.com/uomsystemsbiology/LMMEL-miR-miner Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0554-y) contains supplementary material, which is available to authorized users.

Elevated levels of pregnancy-associated plasma protein-A (PAPP-A) have been implicated in the pathogenesis of various malignancies, including breast cancers. Breast cancer is one of the most frequent carcinomas and is the second most common cancer type detected in women of child-bearing age. Throughout pregnancy PAPP-A is produced and secreted by the placental syncytiotrophoblast cells; co-incidentally pregnancy-associated breast cancers often have an aggressive clinical course. The components of the PAPP-A/IGF axis was assessed in a panel of breast cancer cell lines. Using neutralising antibodies the impact of PAPP-A/IGF axis on cell motility was evaluated. PAPP-A was expressed in four of the twelve breast cancer cell lines tested. Blocking PAPP-A and IGFBP4 with neutralising antibodies significantly decreased motiliy of MDA-MB-231 cells. Upregulation of PAPP-A expression in breast tumours resulted in a trend towards worse overall survival. Notably, PAPP-A expression also positively correlated with epithelial-to-mesenchymal transition markers. In conclusion, these results indicate that PAPP-A plays an important role in breast cancer progression and it may be a promising therapeutic target in breast cancer.

PURPOSE: Goals of care (GoC) designations are an important part of advanced care planning (ACP) for patients with incurable cancers. Studies of outpatient oncology records show that most patients do not have GoC documented. We performed a retrospective analysis of changes in GoC designations in patients with advanced pancreatic cancer in Northern Alberta, Canada, during a system-wide ACP quality improvement initiative. METHODS: Four hundred seventy-one patients with newly diagnosis of advanced, non-neuroendocrine pancreatic cancer between 2010 and 2015 in Northern Alberta, Canada, were included. The ACP initiation launched April 2014, and included educational materials for patients and families, and a coded system of GoC designations describing care philosophies and preferences for resuscitation and medical interventions. Data sources included the Alberta Cancer Registry and oncology-specific electronic medical records. RESULTS: 25.5% of patients had a documented GoC, which increased over the study period (Mantel-Haenszel test-of-trend P < .001; increased from 7.8% in 2010 to 50.0% in 2015). GoC designations occurred later in patients who received palliative chemotherapy versus those who did not (median 130 days from diagnosis [95% CI, 76.019 to 183.981] v 36 days [95% CI, 28.107 to 43.893]; P < .001), and coincided with the end of treatment (median 4.5 days from last treatment). 64.8% of GoC designations were documented by palliative care physicians, but the proportion documented by medical oncologists increased with time (Mantel-Haenszel test-of-trend P = .020; increased from 0% in 2010 to 52.1% in 2015). CONCLUSION: GoC documentation increased in the outpatient records of patients with advanced pancreatic cancer during the system-wide, multifactorial ACP initiative. GoC documentation by medical oncologists also increased. These data provide real-world evidence supporting the impact of a specific ACP initiative to improve rates of GoC designation in patients with advanced cancer.

Highlights • Segmented peer review is a new process for multidisciplinary research submissions • It explicitly allocates segments of a manuscript to appropriate expert reviewers • Reviewers focus on segments of the manuscript within their specialty • Editors integrate segmented reviews for final decision (publish, revise, or reject) • Reduces barriers to publication inherent in multidisciplinary research, We propose a new process for peer review of multidisciplinary journal submissions called ‘segmented peer review’. The current translational research environment increasingly requires complex and multidisciplinary studies that span multiple distinct specialties within a single paper. Such papers present logistic and practical barriers to effective peer review. To address these barriers, papers undergoing segmented peer review require authors to explicitly i) identify each of the areas of expertise required to review the paper, ii) direct each reviewer to the relevant portions of the manuscript, and iii) suggest in-field reviewers. This segmentation of the paper is then followed by a ‘segmented peer review request’ tailored to the expertise of each potential reviewer, with a request to confine his / her review to those ‘in-scope’ aspects of the paper, while de-emphasizing any optional ‘out-of-scope’ comments. Each reviewer indicates the fitness for publication, or suitability for revision, of their particular segment of the manuscript. The segmented peer review process is completed when the editors integrate the segmented peer reviews. We propose segmented peer review as a fit-for-purpose process with tangible advantages for authors, reviewers, and journal editors. It should reduce the specific barriers to publication inherent in the evaluation of multidisciplinary research efforts., Graphical abstract Image, graphical abstract

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy associated with steadily increasing incidence and poor prognosis. Ongoing clinical trials are assessing the effectiveness and safety of a few immune checkpoint inhibitors (ICIs) in CCA patients. However, these ICI treatments as monotherapies may be effective for a proportion of patients with CCA. The prevalence and distribution of other immune checkpoints (ICs) in CCA remain unclear. In this pilot study, we screened databases of CCA patients for the expression of 19 ICs and assessed the prognostic significance of these ICs in CCA patients. Notably, expression of immune modulator IDO1 and PD-L1 were linked with poor overall survival, while FASLG and NT5E were related to both worse overall survival and progression-free survival. We also identified immune modulators IDO1, FASLG, CD80, HAVCR2, NT5E, CTLA-4, LGALS9, VTCN1 and TNFRSF14 that synergized with PD-L1 and correlated with worse patient outcomes. In vitro studies revealed that the expression of ICs was closely linked with aggressive CCA subpopulations, such as cancer stem cells and cells undergoing TGF-β and TNF-α-mediated epithelial-to-mesenchymal transition. These findings suggest that the aforementioned IC molecules may serve as potential prognostic biomarkers and drug targets in CCA patients, leading to lasting and durable treatment outcomes.

Purpose: Optimal first-line systemic therapy for patients with advanced nonsquamous (nonsq) non–small-cell lung cancer (NSCLC) requires confirmation of EGFR/ ALK status, which can delay treatment. We evaluated the impact of reflex testing, defined as pathologists initiating EGFR/ ALK testing at the time of diagnosis of nonsq NSCLC, on time to treatment (TTT). Methods: We conducted a retrospective review of patients with nonsq NSCLC with medical oncology consultation at Sunnybrook Odette Cancer Centre between March 18, 2010 and April 30, 2014. Data were compared during routine and reflex testing. TTT was defined as the interval between the first medical oncology visit with advanced NSCLC and the initiation of systemic therapy. Results: A total of 306 patients were included (n = 232 for routine testing, n = 74 for reflex testing). There was a trend to improvement in median TTT with reflex testing (36 days [interquartile range {IQR}, 16 to 71 days v 26 days [IQR, 8 to 41 days], P = .071). Omitting patients with intentional delays in systemic therapy for low-volume disease, poor performance status, comorbidity management, and/or radiation therapy, median TTT improved (34 days [IQR, 15 to 67 days] v 22 days [IQR, 8 to 42 days], P = .049). Time to optimal first-line systemic therapy according to published guidelines improved (median, 36 days [IQR, 16 to 91 days] v 24 days [IQR, 8 to 43 days], P = .036). There was no impact on receipt of any first-line systemic therapy (55% v 59%, P = .66). The quality of biomarker testing improved, with fewer unsuccessful tests ( EGFR, 14% v 4%, P = .039; and ALK, 17% v 3%, P = .037). Conclusion: Reflex testing of EGFR/ ALK improved the time to optimal systemic therapy and the quality of biomarker testing for patients with advanced nonsq NSCLC.

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

INTRODUCTION Gene fusions are often disease-defining events in cancer. The mutational processes that give rise to fusions, their timing relative to initial diagnosis, and whether they change at relapse are largely unknown. Mutational processes leave distinct marks in the tumor genome, meaning that DNA sequencing can be used to reconstruct how fusions are generated. A prototypical fusion-driven tumor is Ewing sarcoma (ES), a bone cancer predominantly affecting children and young adults. ES is defined by fusions involving EWSR1 , a gene encoding an RNA binding protein, and genes encoding E26 transformation-specific (ETS) transcription factors such as FLI1 . We sought to reconstruct the genomic events that give rise to EWSR1-ETS fusions in ES and chart their evolution from diagnosis to relapse. RATIONALE We studied the processes underpinning gene fusions in ES using the whole-genome sequences of 124 primary tumors. We determined the timing of the emergence of EWSR1 fusions relative to other mutations. To measure ongoing mutation rates and evolutionary trajectories of ES, we studied the genomes of primary tumors, tumors at relapse, and metastatic tumors. RESULTS We found that EWSR1-ETS , the key ES fusion, arises in 42% of cases via complex, loop-like rearrangements called chromoplexy, rather than by simple reciprocal translocations. Similar loops forming canonical fusions were found in three other sarcoma types. Timing the emergence of loops revealed that they occur as bursts in early replicating DNA, as a primary event in ES development. Additional gene disruptions are generated concurrently with the fusions within the loops. Chromoplexy-generated EWSR1 fusions appear to be associated with an aggressive form of the disease and a higher chance of relapse. Numerous mutations present in every cell of the primary were absent at relapse, demonstrating that the primary and relapsed diseases evolved independently. This divergence occurs after formation of an ancestral clone harboring EWSR1 fusions. Importantly, we determined that divergence of the primary tumor and the future relapsed tumor occurs 1 to 2 years before initial diagnosis, as estimated from the number of cell division–associated mutations. CONCLUSION Our findings provide insights into the pathogenesis and natural history of human sarcomas. They reveal complex DNA rearrangements to be a mutational process underpinning gene fusions in a large proportion of ES. Similar observations in other fusion-defined sarcoma types indicate that this process operates more generally. Such complex rearrangements occur preferentially in early replicating and transcriptionally active genomic regions, as evidenced by the additional genes disrupted. EWSR1 fusions arising from chromoplexy correlated with worse clinical outcomes. Formation of the EWSR1 fusion genes is a primary event in the life history of ES. We found evidence of a latency period between this seeding event and diagnosis. This is in keeping with the often-indolent nature of symptoms before clinical disease presentation.

Individuals with a 46,XX/46,XY karyotype are categorized as ovotesticular disorder of sexual development (ODSD) and have gonads with either an ovary on one side and a testis on the other side or a mixed ovotestis. To examine the distribution of 46,XX and 46,XY cells in gonads of 3 patients with ODSD, FISH for X and Y chromosomes and immunohistochemistry for SOX9 and FOXL2 were carried out. FISH analysis showed that XX signals were present in Sertoli cells in the seminiferous tubules, while cells containing Y signals were seen in epithelia of ovarian follicles. The immunolabeling of SOX9 and FOXL2 in the seminiferous tubules and ovarian follicles was mutually exclusive, irrespective of the presence of reversed sex chromosomes. We therefore suggest that the fate of individual gonadal epithelial cells is determined not only by the sex chromosomes but also by local environmental factors.

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.

Wilms tumour (WT) is an embryonal tumour that recapitulates kidney development. The normal kidney is formed from two distinct embryological origins: the metanephric mesenchyme (MM) and the ureteric bud (UB). It is generally accepted that WT arises from precursor cells in the MM; however whether UB-equivalent structures participate in tumorigenesis is uncertain. To address the question of the involvement of UB, we assessed 55 Wilms tumours for the molecular features of MM and UB using gene expression profiling, immunohistochemsitry and immunofluorescence. Expression profiling primarily based on the Genitourinary Molecular Anatomy Project data identified molecular signatures of the UB and collecting duct as well as those of the proximal and distal tubules in the triphasic histology group. We performed immunolabeling for fetal kidneys and WTs. We focused on a central epithelial blastema pattern which is the characteristic of triphasic histology characterized by UB-like epithelial structures surrounded by MM and MM-derived epithelial structures, evoking the induction/aggregation phase of the developing kidney. The UB-like epithelial structures and surrounding MM and epithelial structures resembling early glomerular epithelium, proximal and distal tubules showed similar expression patterns to those of the developing kidney. These observations indicate WTs can arise from a precursor cell capable of generating the entire kidney, such as the cells of the intermediate mesoderm from which both the MM and UB are derived. Moreover, this provides an explanation for the variable histological features of mesenchymal to epithelial differentiation seen in WT.

683 Background: Discussion of goals of care (GoC) is a key part of quality care for patients with palliative cancer. Numerous studies have shown that documentation of GoC in this population remains low. In 2014, Alberta Health Services launched a health-system wide initiative to provide patients with physical copies of their GoC designation intended to be available at all health-system interactions. Here we describe rates of GoC documentation in the period surrounding this initiative. Methods: This is a retrospective cohort analysis of 240 patients with locally advanced or metastatic pancreatic cancer treated with palliative chemotherapy from 2012-2015 in Alberta, Canada. Data were obtained from outpatient electronic medical record documentation and the provincial cancer registry. Results: 63.8% (153/240) of patients had a documented GoC discussion, with 60.4% (145/240) receiving a specific GoC designation. 59.6% (143/240) of patients were referred to palliative care, with 32.5% (78/240) seen by palliative care physician. Of 334 individual GoC discussions documented, 38.6% (129/334) were by medical oncologists, 2.3% (10/334) were by radiation oncologists, 27.2% (91/334) were by palliative care, and 19.2% (64/334) were by other inpatient physicians during hospital admissions. At least 9.6% (32/334) referenced discussions that occurred prior to initial consultation with an oncology physician. Conclusions: The majority of pancreatic cancer patients undergoing palliative chemotherapy had a documented GoC designation during the study period. Providing patients with physical copies of their GoC designation may therefore represent a simple but effective means of increasing GoC documentation in the outpatient oncology setting.

163 Background: Cancer care in Northern Alberta (Canada) is delivered at a single tertiary cancer centre, and 11 regional and community cancer centres (RCCC). We compared outcomes and care patterns for patients with advanced pancreatic cancer (APC; locally advanced or metastatic) in Northern Alberta treated with palliative chemotherapy at either the tertiary centre or an RCCC. Methods: This is a retrospective cohort analysis of APC patients treated with palliative chemotherapy from 2012-2015 in Northern Alberta (Canada). Data were obtained from outpatient medical oncology and palliative care notes and the provincial cancer registry. Survival analysis used a multivariate Cox-regression model. All other tests were Chi-squared/Fisher’s Exact. Results: We identified 106 patients, 90 treated in the tertiary centre, and 16 in a RCCC. Baseline characteristics were not significantly different. There were significant differences in first line chemotherapy regimen use (P = 0.037), with patients treated at RCCC more likely to receive gemcitabine during the study period (68.8% vs 36.6%), and less likely gemcitabine/nab-paclitaxel (12.5% vs 36.5%) or FOLFIRINOX (18.8% vs 28.7%). Patients treated at RCCC were less likely to see an outpatient palliative care physician (P = 0.020, 6.3% vs 35.6%), or have a documented goals of care designation (P = 0.005, 12.5% vs 52.2%). There was no significant difference in overall survival in a multivariate analysis (median 199 vs 232 days, HR = 1.080, 95% CI 0.594 – 1.966). Conclusions: We found that survival was not different for APC patients treated at the tertiary vs RCCC in Northern Alberta. However there were significant differences in the use of palliative care resources and 1st line chemotherapy regimens, which represent important disparities that should be the focus for future quality improvement.

11532 Background: Discussion of goals of care (GoC) is a key part of quality care for pts with palliative cancer. Numerous studies have shown that documentation of GoC in this population remains low. Here we describe changes in GoC documentation and other indicators of quality end-of-life care in PC pts undergoing palliative chemotherapy during a health-system wide initiative to improve advanced care planning (ACP). Methods: This is a retrospective cohort analysis of 106 pts with locally advanced or metastatic PC treated with palliative chemotherapy from 2012-2015 in Northern Alberta (Canada). In 2014, an initiative was launched to provide pts with hard copies of their GoC designation intended to be available at all health-system interactions. Data were obtained from outpatient medical oncology (MO) and palliative care (PAL) notes and the provincial cancer registry. Survival analysis used a multivariate Cox-regression. All other tests were Chi-squared. Results: 50% (53/106) of pts had a documented GoC discussion, with 45% (48/106) receiving a specific GoC designation. In 2012, 31% (6/19) of pts had a GoC designation, which increased to 61% (20/33) in 2015. Of 84 individual GoC discussions documented, 34% (29/84) were by MO, 62% (52/84) were by PAL, and at least 8% (7/84) referenced prior discussions with a family physician or discussion while admitted to hospital. Pts with a GoC designation had increased median overall survival (287 vs 216 days; HR = 0.62; p = 0.041), and were less likely to receive chemotherapy in the last two weeks of life (p = 0.016). Conclusions: Rates of GoC discussions for PC pts undergoing palliative chemotherapy increased during a health-system wide ACP initiative. Having a GoC designation was associated with greater overall survival and indicators of higher quality end-of-life care.

// BeeShin Tan 1 , Matthew Anaka 1 , Siddhartha Deb 2 , Claudia Freyer 1 , Lisa M. Ebert 3 , Anderly C. Chueh 1 , Sheren Al-Obaidi 1 , Andreas Behren 1 , Aparna Jayachandran 1 , Jonathan Cebon 1 , Weisan Chen 4 and John M. Mariadason 1 1 Ludwig Institute for Cancer Research Ltd. Melbourne-Austin Branch, Heidelberg, Victoria, Australia. 2 Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 3 Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, Australia. 4 School of Molecular Science, LaTrobe University, Bundoora, Victoria, Australia. Correspondence: John M. Mariadason, email: // Weisan Chen, email: // Keywords : FOXP3, melanoma, proliferation, apoptosis Received : November 12, 2013 Accepted : December 20, 2013 Published : December 20, 2013 Abstract The Forkhead box P3 (FOXP3) transcription factor is the key driver of regulatory T cell (Treg cells) differentiation and immunosuppressive function. In addition, FOXP3 has been reported to be expressed in many tumors, including melanoma. However, its role in tumorigenesis is conflicting, with both tumor suppressive and tumor promoting functions described. The aim of the current study was to characterize the expression and function of FOXP3 in melanoma. FOXP3 expression was detected by immunohistochemistry (IHC) in 12% (18/146) of stage III and IV melanomas. However expression was confined to fewer than 1% of cells in these tumors. Stable over-expression of FOXP3 in the SK-MEL-28 melanoma cell line reduced cell proliferation and clonogenicity in vitro , and reduced xenograft growth in vivo . FOXP3 over-expression also increased pigmentation and the rate of apoptosis of SK-MEL-28 cells. Based on its infrequent expression in human melanoma, and its growth inhibitory and pro-apoptotic effect in over-expressing melanoma cells, we conclude that FOXP3 is not likely to be a key tumor suppressor or promoter in melanoma.

Background The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. Methods We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. Results We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. Conclusion We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2759-2) contains supplementary material, which is available to authorized users.

Wilms tumor (WT) is the most frequent renal neoplasm of childhood; a myogenic component is observed in 5% to 10% of tumors. We demonstrate for the first time that myogenic WTs are associated with expression of PAX3, a transcription factor known to specify myoblast cell fate during muscle development. In a panel of 20 WTs, PAX3 was identified in 13 of 13 tumor samples with myogenic histopathology but was absent in 7 of 7 tumors lacking a myogenic component. Furthermore, we show that PAX3 is expressed in the metanephric mesenchyme and stromal compartment of developing mouse kidney. Modulation of endogenous PAX3 expression in human embryonic kidney (HEK293) cells influenced cell migration in in vitro assays. Mutations of WT1 were consistently associated with PAX3 expression in WTs, and modulation of WT1 expression in HEK293 cells was inversely correlated with the level of endogenous PAX3 protein. We demonstrate abundant PAX3 and absence of PAX2 expression in a novel cell line (WitP3) isolated from the stromal portion of a WT bearing a homozygous deletion of the WT1 gene. We hypothesize that PAX3 sets stromal cell fate in developing kidney but is normally suppressed by WT1 during the mesenchyme-to-epithelium transition leading to nephrogenesis. Loss of WT1 permits aberrant PAX3 expression in a subset of WTs with myogenic phenotype.

Wilms tumours (WTs) have two distinct types of histology with or without ectopic mesenchymal elements, suggesting that WTs arise from either the mesenchymal or epithelial nephrogenic lineages. Regardless of the presence or absence of CTNNB1 mutations, nuclear accumulation of beta-catenin is often observed in WTs with ectopic mesenchymal elements. Here, we addressed the relationship between the WNT-signalling pathway and lineage in WTs by examining CTNNB1 and WT1 mutations, nuclear accumulation of beta-catenin, tumour histology and gene expression profiles. In addition, we screened for mutations in WTX, which has been proposed to be a negative regulator of the canonical WNT-signalling pathway. Unsupervised clustering analysis identified two classes of tumours: mesenchymal lineage WNT-dependent tumours, and epithelial lineage WNT-independent tumours. In contrast to the mesenchymal lineage specificity of CTNNB1 mutations, WTX mutations were surprisingly observed in both lineages. WTX-mutant WTs with ectopic mesenchymal elements had nuclear accumulation of beta-catenin, upregulation of WNT target genes and an association with CTNNB1 mutations in exon 7 or 8. However, epithelial lineage WTs with WTX mutations had no indications of active WNT signalling, suggesting that the involvement of WTX in the WNT-signalling pathway may be lineage dependent, and that WTX may have an alternative function to its role in the canonical WNT-signalling pathway.

Current models of Wilms tumour development propose that histological features of the tumours are programmed by the underlying molecular aberrations. For example, tumours associated with WT1 mutations arise from intralobar nephrogenic rests (ILNR), concur with CTNNB1 mutations and have distinct histology, whereas tumours with IGF2 loss of imprinting (LOI) often arise from perilobar nephrogenic rests (PLNR). Intriguingly, ILNR and PLNR are found simultaneously in Wilms tumours in children with overgrowth who have constitutional IGF2 LOI. We therefore examined whether the precursor lesions or early epigenetic changes are the primary determinant of Wilms tumour histology. We examined the histological features and gene expression profiles of IGF2 LOI tumours and WT1-mutant tumours which are associated with PLNR and/or ILNR. Two distinct types of IGF2 LOI tumours were identified: the first type had a blastemal-predominant histology associated with PLNR, while the second subtype had a myogenic histology, increased expression of mesenchymal lineage genes and an association with ILNR, similar to WT1-mutant tumours. These ILNR-associated IGF2 LOI tumours also showed signatures of activation of the WNT signalling pathway: differential expression of beta-catenin targets (MMP2, RARG, DKK1) and WNT antagonist genes (DKK1, WIF1, SFRP4). Unexpectedly, the majority of these tumours had CTNNB1 mutations, which are normally only seen in WT1-mutant tumours. The absence of WT1 mutations in tumours with IGF2 LOI indicated that CTNNB1 mutations occur predominantly in tumours arising from ILNR independent of the presence or absence of WT1 mutations. Thus, even though these two classes of tumours with IGF2 LOI have the same underlying predisposing epigenetic error, the tumour histology and the gene expression profiles are determined by the nature of the precursor cells within the nephrogenic rests and subsequent CTNNB1 mutations.

Background The metabolism of cancer cells is often reprogrammed by dysregulation of metabolic enzymes. Transketolase-like 1 (TKTL1) is a homodimeric transketolase linking the pentose-phosphate pathway with the glycolytic pathway. It is generally silenced at a transcriptional level in somatic tissues. However, in human cancers its expression is associated with the acquisition of a glycolytic phenotype (the Warburg effect) by cancer cells that contributes to the progression of malignant tumors. In melanoma, defective promoter methylation results in the expression of genes and their products that can affect the tumor cell’s phenotype including the modification of immune and functional characteristics. The present study evaluates the role of TKTL1 as a mediator of disease progression in melanoma associated with a defective methylation phenotype. Methods The expression of TKTL1 in metastatic melanoma tumors and cell lines was analysed by qRT-PCR and immunohistochemistry. The promoter methylation status of TKTL1 in melanoma cells was evaluated by quantitative methylation specific PCR. Using qRT-PCR, the effect of a DNA demethylating agent 5-aza-2’-deoxycytidine (5aza) on the expression of TKTL1 was examined. Biochemical and molecular analyses such as glucose consumption, lactate production, invasion, proliferation and cell cycle progression together with ectopic expression and siRNA mediated knockdown were used to investigate the role of TKTL1 in melanoma cells. Results Expression of TKTL1 was highly restricted in normal adult tissues and was overexpressed in a subset of metastatic melanoma tumors and derived cell lines. The TKTL1 promoter was activated by hypomethylation and treatment with 5aza induced TKTL1 expression in melanoma cells. Augmented expression of TKTL1 in melanoma cells was associated with a glycolytic phenotype. Loss and gain of function studies revealed that TKTL1 contributed to enhanced invasion of melanoma cells. Conclusions Our data provide evidence for an important role of TKTL1 in aerobic glycolysis and tumor promotion in melanoma that may result from defective promoter methylation. This epigenetic change may enable the natural selection of tumor cells with a metabolic phenotype and thereby provide a potential therapeutic target for a subset of melanoma tumors with elevated TKTL1 expression. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2185-5) contains supplementary material, which is available to authorized users.

Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

The ability of cell lines to accurately represent cancer is a major concern in preclinical research. Culture of glioma cells as neurospheres in stem cell media (SCM) has been shown to better represent the genotype and phenotype of primary glioblastoma in comparison to serum cell lines. Despite the use of neurosphere-like models of many malignancies, there has been no robust analysis of whether other cancers benefit from a more representative phenotype and genotype when cultured in SCM. We analyzed the growth properties, transcriptional profile, and genotype of melanoma cells grown de novo in SCM, as while melanocytes share a common precursor with neural cells, melanoma frequently demonstrates divergent behavior in cancer stem cell assays. SCM culture of melanoma cells induced a neural lineage gene expression profile that was not representative of matched patient tissue samples and which could be induced in serum cell lines by switching them into SCM. There was no enrichment for expression of putative melanoma stem cell markers, but the SCM expression profile did overlap significantly with that of SCM cultures of glioma, suggesting that the observed phenotype is media-specific rather than melanoma-specific. Xenografts derived from either culture condition provided the best representation of melanoma in situ. Finally, SCM culture of melanoma did not prevent ongoing acquisition of DNA copy number abnormalities. In conclusion, SCM culture of melanoma does not provide a better representation of the phenotype or genotype of metastatic melanoma, and the resulting neural bias could potentially confound therapeutic target identification. Disclosure of potential conflicts of interest is found at the end of this article.

Genomic imprinting is a process that marks DNA, causing a change in gene or chromosome behavior, depending on the sex of the transmitting parent. In mammals, most examples of genomic imprinting affect the transcription of individual or small clusters of genes whereas in insects, genomic imprinting tends to silence entire chromosomes. This has been interpreted as evidence of independent evolutionary origins for imprinting. To investigate how these types of imprinting are related, we performed a phenotypic, molecular, and cytological analysis of an imprinted chromosome in Drosophila melanogaster. Analysis of this chromosome reveals that the imprint results in transcriptional silencing. Yet, the domain of transcriptional silencing is very large, extending at least 1.2 Mb and encompassing over 100 genes, and is associated with decreased somatic polytenization of the entire chromosome. We propose that repression of somatic replication in polytenized cells, as a secondary response to the imprint, acts to extend the size of the imprinted domain to an entire chromosome. Thus, imprinting in Drosophila has properties of both typical mammalian and insect imprinting which suggests that genomic imprinting in Drosophila and mammals is not fundamentally different; imprinting is manifest as transcriptional silencing of a few genes or silencing of an entire chromosome depending on secondary processes such as differences in gene density and polytenization.

The white gene of Drosophila melanogaster has been extensively studied, yet it is still not understood how its ectopic overexpression induces male-male courtship. To investigate the cellular basis of this behavior, we examined the sexual behavior of several classes of mutants. We find that male-male courtship is seen not only in flies overexpressing the white gene, but also in mutants expected to have mislocalized White protein. This finding confirms that mislocalizing White transporter in the cells in which it is normally expressed will produce male-male courtship behaviors; the courtship behavior is not an indirect consequence of aberrant physiological changes elsewhere in the body. Male-male courtship is also seen in some mutants with altered monoamine metabolism and deficits in learning and memory, but can be distinguished from that produced by White mislocalization by its reduced intensity and locomotor activity. Double mutants overexpressing white and with mutations in genes for serotonergic neurons suggest that male-male courtship produced by mislocalizing White may not be mediated exclusively by serotonergic neurons. We also find decreased olfactory learning in white mutants and in individuals with mutations in the genes for White's binding partners, brown and scarlet. Finally, in cultured Drosophila and mammalian cells, the White transporter is found in the endosomal compartment. The additional genes identified here as being involved in male-male courtship increase the repertoire of mutations available to study sexual behavior in Drosophila.

93 Background: EGFR mutation and ALK rearrangement testing is standard in the management of advanced non-sq NSCLC patients (pts). Previously at our centre, EGFR/ALK biomarker testing was requested by medical oncologists (MO). In June 2013 we implemented biomarker RT, defined as pathologists requesting EGFR/ALK at time of diagnosis of non sq-NSCLC irrespective of stage. The objective of this study was to test the hypothesis that if pathologists requested biomarker testing, appropriate tissue would be preserved and selected for testing, which would improve success rates of biomarker testing. Methods: Retrospective review of advanced non-sq NSCLC pts seen by MO at Sunnybrook Odette Cancer Centre from March 2010 to April 2014. Pt and EGFR/ALK test characteristics were compared before and after RT using Chi-square tests of association. Time outcomes were compared using Mann-Whitney U tests. Results: Of the 310 pts included, median age was 68, 44% female, 47% Caucasian, 93% adenocarcinoma, 22% EGFR+, 1% ALK+ and 84% either presented with or developed stage IV. Samples tested for EGFR and ALK were respectively: 53%, 51% core biopsies; 25%, 32% surgical resections; 20%, 16% cytology. The number of biomarker tests across all stages increased with RT (EGFR 70% vs 95%, p < 0.001 / ALK 44% vs 83%, p < 0.001). RT improved the rate of successfully completed tests (EGFR 86% vs 96%, p = 0.04 / ALK 83% vs 97%, p = 0.04). The remainder of tests were unsuccessful due to inconclusive results (EGFR 9% vs 4%, p = 0.25 / ALK 7% vs 2%, p = 0.25), insufficient tissue (EGFR 3% vs 0%, p = 0.33 / ALK 10% vs 0%, p = 0.03) or cancellation due to appropriate tissue not being sent from holding lab to testing lab (EGFR 2% vs 0, p = NS / ALK 0% vs 2%, p = NS). From core biopsies, there was trend to improved success of EGFR testing with RT (82% vs 97%, p = 0.06) and significant improvement of ALK testing (82% vs 100%, p = 0.04), with no impact on success from cytology samples. Rebiopsy rate for biomarker testing was low in both cohorts. Turnaround time for EGFR testing decreased [19 days (IQR 15-25) vs 17 days (IQR 12-21), p = 0.02]; ALK was unchanged. Conclusions: Implementation of RT improved successful completion of EGFR/ALK testing.

Degradation of cellular proteins by the proteasome is critical for the generation of MHC-associated peptides. The constitutive proteasome and the IFNγ-induced immunoproteasome (IP) differ in the use of three catalytic β subunits, which alters production of MHC class I epitopes. The potential for a disparate repertoire of epitopes produced between inflammatory/non-inflammatory tumours therefore arises. We have extensively investigated this phenomenon both broadly, in terms of the cancer cell as a whole, and specifically, by evaluating presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes. We profiled the immunopeptidome of a melanoma cell line under steady state or IFNγ-treated conditions; by mass spectrometry analysis of HLA Class I bound peptides. Profound changes in the overall epitope profile presented on HLA Class I molecules, leading to extensive alteration in the overall immunogenicity of the cell, were observed. We studied processing and presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes by melanoma cell lines. Using specific T-lymphocyte clones, we examined presentation of each epitope following processing through proteasome subtypes. We further investigated processing of these epitopes by selective inhibition of the IP catalytic subunit LMP7 by siRNA knockdown, or small molecule inhibition. In vivo, we demonstrate that melanoma cells can be induced to switch proteasome subtype following treatments which induce IFNγ at the tumour site. Our data demonstrate broad changes in epitopes presented by melanoma cells under inflammatory versus non-inflammatory conditions. These results illustrate a little-studied mechanism of immune escape by tumour cells. Awareness of how individual cancer epitopes are processed by melanoma cells is therefore critical to inform development of future therapies which involve cancer vaccination, adoptive T-lymphocyte transfer, or combination treatments including these.

Resistance to anti-cancer therapies arising from genetic heterogeneity is well understood. Less well characterised is the resistance to therapy proposed to occur via the plastic changes in melanoma cells generated by the impact of the tumor microenvironment. In melanoma, phenotypic-switching as means of plasticity has been well described and resembles, at least partially, an epithelial-to-mesenchymal transition (EMT). The presence of mesenchymal-like, invasive and slow-proliferating cells in melanoma, a highly aggressive cancer with intrinsic resistance to chemotherapy, is controversial and their potential contribution to therapeutic resistance has yet to be determined. Methods: We examined melanoma cell lines for the presence of mesenchymal-like cells using CM-Dil, a membrane dye evenly distributed to daughter cells with each cell division as a marker of reduced cell division. Functional assays included invasion and motility assays, in vivo mouse xenograft models as well as an in vivo chicken neural crest transplantation assay. Genome-wide gene-expression profiling was performed using Illumina HT12 arrays. Results: Here we show that mesenchymal-like cells are present in cell lines and in xenograft tumors, survive exposure to cytotoxic drugs and are invasive. Importantly, resistance was associated with a gene expression signature characterized by the expression of thrombospondin, TGFBI, genes associated with the extracellular matrix, and acquisition of increased invasiveness. This gene-expression pattern is reminiscent of an epithelial-to-mesenchymal transition in other solid cancers and a similar phenotype was detected after acquired resistance to the BRAF inhibitors PLX4720 and dabrafenib. Moreover, targeting thrombospondin in an in vivo model of melanoma cell plasticity and invasion led to the complete abrogation of the plastic and invasive potential of melanoma cells. Conclusion: The results link therapeutic resistance to the presence of mesenchymal-like and invasive cells, and identify potential targets for novel therapies directed at eradicating this key melanoma subpopulation. Citation Format: Andreas Behren, Aparna Jayachandran, Matthew Anaka, Christopher Hudson, Jonathan Cebon. Thrombospondin-1 is a targetable marker of invasive, mesenchymal- like melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2015. doi:10.1158/1538-7445.AM2014-2015

9063 Background: At 45/105pregnancies, Malignant Melanoma (MM) is the commonest cancer diagnosed in pregnant women. An aggressive course & poor outcomes are recognised to occur during pregnancy. IG...

8542 Background: The acquisition of new mutations can explain resistance to targeted therapies; however resistance also develops without demonstrable new mutations. To better understand potential mechanisms for treatment failure we studied subpopulations of chemo-resistant cells that can be identified in early passage cell lines and the cellular plasticity that yields these. Methods: Melanoma subpopulations were identified and sorted on the basis of functional assays. Gene expression was evaluated with Illumina HT12 arrays and qPCR to identify potential mechanisms and targets. Sorted cells were evaluated in vitro for the induction of phenotype switching and effects of target inhibition. Results: A subset of slow proliferating label-retaining cells (LRC) was identified using a membrane dye. Direct isolation of LRC from a pt showed these were not an in vitro artefact. LRC isolated from multiple cell lines comprised the majority of cells that resisted cytotoxic drugs and could invade through an artificial extracellular membrane. Gene expression profiling identified a network of over-expressed genes related to epithelial-to-mesenchymal transition (EMT) notable for the expression of Thrombospondin-1 (TSP-1), Transforming-Growth Factor, Beta Induced, (TGFBI) and other extracellular matrix molecules. Initial studies show that LRC shared gene expression characteristics with PLX4032-resistant cells. Although interrogating LRC by qPCR showed up-regulation of the putative melanoma stem cell marker ABCB5 and the lysine specific demethylase Jarid1B, cell sorting and serial re-labelling experiments revealed a dynamic and interchangeable phenotype for these cells, challenging the hierarchical stem cell model for melanoma. The observed slow-cycling and chemo-resistant phenotype could be induced in vitro through TGF beta-mediated phenotype switching and cellular invasion was blocked using an antibody against TSP-1. Conclusions: Cytokine-induced plasticity in melanoma yields invasive, therapy-resistant cells which express EMT-related genes and pathways. We hypothesize that these cells are a source of treatment resistance in vivo and blocking their emergence may prevent treatment failure.

// Aparna Jayachandran 1 , 2 , Matthew Anaka 1 , 2 , Prashanth Prithviraj 1 , 2 , Christopher Hudson 1 , Sonja J McKeown 3 , Pu-Han Lo 1 , Laura J Vella 1 , 2 , Colin R Goding 4 , Jonathan Cebon 1 , 2 , Andreas Behren 1 , 2 1 Ludwig Institute for Cancer Research, Melbourne-Austin Branch, Cancer Immunobiology Laboratory, Heidelberg, VIC 3084, Australia. 2 Department of Medicine, University of Melbourne, Victoria, 3010, Australia 3 Department of Anatomy and Neuroscience, University of Melbourne, Victoria, 3010, Australia 4 Ludwig Institute for Cancer Research, University of Oxford, Oxford, OX3 7DQ, UK Correspondence to: Andreas Behren, e-mail: andreas.behren@ludwig.edu.au Keywords: Thrombospondin 1, melanoma, epithelial-to-mesenchymal transition, chick embryo, invasion, drug resistance Received: April 29, 2014 Accepted: June 23, 2014 Published: July 08, 2014 ABSTRACT Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 ( THBS1 ) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.

Melanoma is the most common cancer diagnosed in pregnant women and an aggressive course with poorer outcomes is commonly described during pregnancy or shortly after childbirth. The underlying mechanisms for this are not understood. Here, we report that melanoma migration, invasiveness and progression are promoted by Pregnancy-Associated Plasma Protein-A (PAPPA), a pregnancy-associated metalloproteinase produced by the placenta that increases the bioavailability of IGF1 by cleaving it from a circulating complex formed with IGFBP4. We show that PAPPA is widely expressed by metastatic melanoma tumors and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes. Notably, inhibition of PAPPA significantly reduced invasion and migration of melanoma cells in vitro and in vivo within the embryonic chicken neural tube. PAPPA-enriched pregnancy serum treatment enhanced melanoma motility in vitro. Furthermore, we report that IGF1 can induce the phenotypic and functional effects of epithelial-to-mesenchymal transition (EMT) in melanoma cells. In this study, we establish a clear relationship between a pregnancy-associated protein PAPPA, melanoma and functional effects mediated through IGF1 that provides a plausible mechanism for accelerated melanoma progression during pregnancy. This opens the possibility of targeting the PAPPA/IGF1 axis therapeutically.