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PURPOSE A challenge in the diagnosis of renal cell carcinoma (RCC) is to distinguish chromophobe RCC (chRCC) from benign renal oncocytoma, because these tumor types are histologically and morphologically similar, yet they require different clinical management. Molecular biomarkers could provide a way of distinguishing oncocytoma from chRCC, which could prevent unnecessary treatment of oncocytoma. Such biomarkers could also be applied to preoperative biopsy specimens such as needle core biopsy specimens, to avoid unnecessary surgery of oncocytoma. METHODS We profiled DNA methylation in fresh-frozen oncocytoma and chRCC tumors and adjacent normal tissue and used machine learning to identify a signature of differentially methylated cytosine-phosphate-guanine sites (CpGs) that robustly distinguish oncocytoma from chRCC. RESULTS Unsupervised clustering of Stanford and preexisting RCC data from The Cancer Genome Atlas (TCGA) revealed that of all RCC subtypes, oncocytoma is most similar to chRCC. Unexpectedly, however, oncocytoma features more extensive, overall abnormal methylation than does chRCC. We identified 79 CpGs with large methylation differences between oncocytoma and chRCC. A diagnostic model trained on 30 CpGs could distinguish oncocytoma from chRCC in 10-fold cross-validation (area under the receiver operating curve [AUC], 0.96 (95% CI, 0.88 to 1.00)) and could distinguish TCGA chRCCs from an independent set of oncocytomas from a previous study (AUC, 0.87). This signature also separated oncocytoma from other RCC subtypes and normal tissue, revealing it as a standalone diagnostic biomarker for oncocytoma. CONCLUSION This CpG signature could be developed as a clinical biomarker to support differential diagnosis of oncocytoma and chRCC in surgical samples. With improved biopsy techniques, this signature could be applied to preoperative biopsy specimens.

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to parts per million. This resulted in median area under the curve (AUC) values of 0.98 in advanced cancers and 0.80 in early-stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that INVAR may be applied without requiring personalized sequencing panels so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.

Cancer remains a leading cause of morbidity and mortality worldwide. Its evolutionary nature and resultant complex interactions with the tumour micro-environment and the host immune system engender heterogeneity, make developing interventions difficult. Usually detected at the advanced stages of disease, metastatic cancer accounts for 90% of cancer-associated deaths. Therefore early detection of cancer, combined with current therapies, would have a significant impact on survival and treatment of this insidious disease. Epigenetic changes such as DNA methylation are some of the early events in carcinogenesis. Here, we report on a machine learning model that can classify 13 types of cancer as well as non-cancer tissue samples using only DNA methylome data, with an accuracy of 98.2%. We utilise the features identified by this model to develop a robust deep neural network that can generalise to independent data sets. We also demonstrate that the methylation associated genomic loci detected by the classifier are associated with genes involved in cancer, providing insights into the epigenomic regulation of carcinogenesis.

// Dagmar Quandt 1 , Hans Dieter Zucht 2 , Arno Amann 3 , Anne Wulf-Goldenberg 4 , Carl Borrebaeck 5 , Michael Cannarile 6 , Diether Lambrechts 7 , Herbert Oberacher 8 , James Garrett 9 , Tapan Nayak 10 , Michael Kazinski 11 , Charles Massie 12 , Heidi Schwarzenbach 13 , Michele Maio 14 , Robert Prins 15 , Bjorn Wendik 16 , Richard Hockett 17 , Daniel Enderle 18 , Mikkel Noerholm 18 , Hans Hendriks 19 , Heinz Zwierzina 3 and Barbara Seliger 1 1 Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle, Germany 2 Protagen, Dortmund, Germany 3 Department of Haematology and Oncology, Medical University Innsbruck, Innsbruck, Austria 4 Experimental Pharmacology and Oncology, Berlin-Buch GmbH, Berlin, Germany 5 Create Health Translational Cancer Center, Lund University, Lund, Sweden 6 Roche Pharmaceutical Research and Early Development, Roche Innovation Center, Munich, Germany 7 Laboratory for Translational Genetics, VIB Center for Cancer Biology, Leuven, Belgium 8 Institute of Legal Medicine and Core Facility Metabolomics, Medical University of Innsbruck, Innsbruck, Austria 9 Global Drug Development, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA 10 Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland 11 Qiagen, Hilden, Germany 12 CRUK Cambridge Institute, Cambridge, UK 13 University Medical Center Hamburg-Eppendorf, Department of Tumor Biology Hamburg, Hamburg, Germany 14 Medical Oncology and Immunotherapy, University Hospital of Siena, Siena, Italy 15 Department of Neurosurgery, David Geffen School of Medicine at UCLA, Los Angeles, California, USA 16 Perkin Elmer, Hamburg, Germany 17 Biodesix, Boulder, Colorado, USA 18 Exosome Diagnostics GmbH, Martinsried, Germany 19 Hendriks Pharmaceutical Consulting, Purmerend, The Netherlands Correspondence to: Barbara Seliger, email: Barbara.Seliger@uk-halle.de Keywords: liquid biopsy, biomarker, tumor, high throughput analysis, immunotherapy Received: February 08, 2017 Accepted: April 04, 2017 Published: April 24, 2017 ABSTRACT During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies. Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients’ outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Patients with small tumors have few copies of ctDNA in plasma, resulting in limited sensitivity to detect low-volume or residual disease. We show that sampling limitations can be overcome and sensitivity for ctDNA detection can be improved by massively parallel sequencing when hundreds to thousands of mutations are identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) analysis pipeline, which combines patient-specific mutation lists with both custom error-suppression methods and signal enrichment based on biological features of ctDNA. In this framework, the sensitivity can be estimated independently for each sample based on the number of informative reads, which is the product of the number of mutations analyzed and the average depth of unique sequencing reads. We applied INVAR to deep sequencing data generated by custom hybrid-capture panels, and showed that when ~106 informative reads were obtained INVAR allowed detection of tumor-derived DNA fractions to parts per million (ppm). In serial samples from patients with advanced melanoma on treatment, we detected ctDNA when imaging confirmed tumor volume of ~1cm3. In patients with resected early-stage melanoma, ctDNA was detected in 40% of patients who later relapsed, with higher rates of detection when more informative reads were obtained. We further demonstrated that INVAR can be generalized and allows improved detection of ctDNA from whole-exome and low-depth whole-genome sequencing data.

Cell-free tumour-derived DNA (ctDNA) allows non-invasive monitoring of cancers but its utility in renal cell cancer (RCC) has not been established. Here, untargeted and targeted sequencing methods, applied to two independent cohorts of renal tumour patients (n=90), were used to determine ctDNA content in plasma and urine. Our data revealed lower plasma ctDNA levels in RCC relative to other cancers, with untargeted detection of ∼33%. A sensitive personalised approach, applied to plasma and urine from select patients improved detection to ∼50%, including in patients with early-stage and even benign lesions.A machine-learning based model predicted detection, potentially offering a means of triaging samples for personalised analysis. In addition, with limited data we observed that plasma, and for the first time, urine ctDNA may better represent tumour heterogeneity than tissue biopsy. Furthermore, longitudinal sampling of >200 plasma samples revealed that ctDNA can track disease course. Additional datasets will be required to validate these findings.Overall, our data highlight RCC as a ctDNA-low malignancy, but indicate potential clinical utility provided improvement in detection approaches.One sentence summaryComplementary sequencing methods show that cell-free tumour DNA levels are low in renal cancer though, via various strategies, may still be informative.

Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62 targets of compounds that may be active and should be considered candidates for future clinical trials. Postprint

Ten-eleven translocation (TET) proteins are recently characterized dioxygenases that regulate demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further derivatives. The recent finding that 5hmC is also a stable and independent epigenetic modification indicates that these proteins play an important role in diverse physiological and pathological processes such as neural and tumor development. Both the genomic distribution of (hydroxy)methylation and the expression and activity of TET proteins are dysregulated in a wide range of cancers including prostate cancer. Up to now it is still unknown how changes in TET and 5(h)mC profiles are related to the pathogenesis of prostate cancer. In this review, we explore recent advances in the current understanding of how TET expression and function are regulated in development and cancer. Furthermore, we look at the impact on 5hmC in prostate cancer and the potential underlying mechanisms. Finally, we tried to summarize the latest techniques for detecting and quantifying global and locus-specific 5hmC levels of genomic DNA. ispartof: Molecular and Cellular Endocrinology vol:462 issue:Pt A pages:41-55 ispartof: location:Ireland status: published

Muscle Invasive Bladder Cancer (MIBC) has a poor prognosis. Whilst patients can achieve a 6% improvement in overall survival with Neo-Adjuvant Chemotherapy (NAC), many do not respond. Body fluid mutant DNA (mutDNA) may allow non-invasive identification of treatment failure. We collected 248 liquid biopsy samples including plasma, cell pellet (UCP) and supernatant (USN) from spun urine, from 17 patients undergoing NAC. We assessed single nucleotide variants and copy number alterations in mutDNA using Tagged-Amplicon- and shallow Whole Genome- Sequencing. MutDNA was detected in 35.3%, 47.1% and 52.9% of pre-NAC plasma, UCP and USN samples respectively, and urine samples contained higher levels of mutDNA (p =

Objective: We have previously identified peroxiredoxin-3 (PRDX-3) as a cell-surface protein that is androgen regulated in the LNCaP prostate cancer (PCa) cell line. PRDX-3 is a member of the peroxiredoxin family that are responsible for neutralising reactive oxygen species. Experimental design: PRDX-3 expression was examined in tissue from 32 patients using immunohistochemistry. Subcellular distribution was determined using confocal microscopy. PRDX-3 expression was determined in antiandrogen-resistant cell lines by western blotting and quantitative RT–PCR. The pathways of PRDX-3 overexpression and knockdown on apoptosis and response to oxidative stress were investigated using protein arrays. Results: PRDX-3 is upregulated in a number of endocrine-regulated tumours; in particular in PCa and prostatic intraepithelial neoplasia. Although the majority of PRDX-3 is localised to the mitochondria, we have confirmed that PRDX-3 at the cell membrane is androgen regulated. In antiandrogen-resistant LNCaP cell lines, PRDX-3 is upregulated at the protein but not RNA level. Resistant cells also possess an upregulation of the tricarboxylic acid (TCA) pathway and resistance to H2O2-induced apoptosis through a failure to activate pro-apoptotic pathways. Knockdown of PRDX-3 restored H2O2 sensitivity. Conclusion: Our results suggest that PRDX-3 has an essential role in regulating oxidation-induced apoptosis in antiandrogen-resistant cells. PRDX-3 may have potential as a therapeutic target in castrate-independent PCa.

Two independent regions within HNF1B are consistently identified in prostate and ovarian cancer genome-wide association studies (GWAS); their functional roles are unclear. We link prostate cancer (PC) risk SNPs rs11649743 and rs3760511 with elevated HNF1B gene expression and allele-specific epigenetic silencing, and outline a mechanism by which common risk variants could effect functional changes that increase disease risk: functional assays suggest that HNF1B is a pro-differentiation factor that suppresses epithelial-to-mesenchymal transition (EMT) in unmethylated, healthy tissues. This tumor-suppressor activity is lost when HNF1B is silenced by promoter methylation in the progression to PC. Epigenetic inactivation of HNF1B in ovarian cancer also associates with known risk SNPs, with a similar impact on EMT. This represents one of the first comprehensive studies into the pleiotropic role of a GWAS-associated transcription factor across distinct cancer types, and is the first to describe a conserved role for a multi-cancer genetic risk factor.

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)–marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.

After briefly reviewing the nature of DNA methylation, its general role in cancer and the tools available to interrogate it, we consider the literature surrounding DNA methylation as relating to prostate cancer. Specific consideration is given to recurrent alterations. A list of frequently reported genes is synthesized from 17 studies that have reported on methylation changes in malignant prostate tissue, and we chart the timing of those changes in the diseases history through amalgamation of several previously published data sets. We also review associations with genetic alterations and hormone signalling, before the practicalities of investigating prostate cancer methylation using cell lines are assessed. We conclude by outlining the interplay between DNA methylation and prostate cancer metabolism and their regulation by androgen receptor, with a specific discussion of the mitochondria and their associations with DNA methylation.

To identify clinically relevant downstream effectors of androgen signaling, the androgen-regulated kinome was defined in prostate cancer (PCa). Within this study, choline kinase α (CHKA) was identified as an androgen receptor chaperone that is both a biomarker of progression and a potential therapeutic target for PCa.

JAK2 exon 12 mutations are associated with more marked and isolated erythrocytosis than JAK2V617F. We analyzed expression profiles of JAK2 exon 12-mutant and wild-type erythroid colonies from patients with polycythemia vera (PV). Exon 12 mutations were associated with interferon-target gene upregulation, STAT1 activation and additional gene expression changes that were quantitatively and qualitatively similar to those in JAK2V617F-heterozygous cells from essential thrombocythemia (ET) patients. These results demonstrate that JAK2 exon 12-mutated PV does not reflect attenuated STAT1 signaling, and that transcriptional consequences of JAK2 mutations are remarkably similar in JAK2 exon 12-mutated PV and JAK2V617F-positive ET.

Somatic mutations in JAK2 , CALR and MPL are found in the majority of myeloproliferative neoplasms (MPN) but many patients also harbor somatic mutations in epigenetic regulators of DNA methylation ( TET2 , DNMT3A and IDH1/2 ) or chromatin structure ( ASXL1 and EZH2 ). In MPN patients, mutations in

© 2015 Nature America, Inc. All rights reserved.Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases.

BACKGROUND: Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired. METHODS:/nWe determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells. RESULTS: The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as "TET2-first patients"), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first ("JAK2-first patients") had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2-TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation. CONCLUSIONS: The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research and others.). Work in the Green lab is supported by Leukemia and Lymphoma Research, Cancer Research UK, the Kay Kendall Leukaemia Fund, the NIHR Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre, and the Leukemia & Lymphoma Society of America. DGK was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research (Ottawa, ON), and a Lady Tata Memorial Trust International Award for Research in Leukaemia (London, UK). CAO was supported by a research fellowship from the Deutsche Forschungsgemeinschaft (DFG, OR255/1-1).

To the editor: The JAK2V617F mutation is found in most patients with polycythemia vera (PV) and 50% to 60% of those with essential thrombocythemia (ET). JAK2V617F -homozygous precursors arise through mitotic recombination, form larger clones in PV compared with ET,[1][1] and may play a causal role

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium.-- et al., The myelodysplastic syndromes are a heterogeneous group of hematologic cancers characterized by low blood counts, most commonly anemia, and a risk of progression to acute myeloid leukemia.1 These disorders have increased in prevalence and are expected to continue to do so. Blood films and bone marrow¿biopsy specimens from patients with myelodysplastic syndromes show dysplastic changes in myeloid cells, with abnormal proliferation and differentiation of one or more lineages. Target genes of recurrent chromosomal aberrations have been mapped,2,3 and several genes have been identified as recurrently mutated in these disorders, including NRAS (encoding neuroblastoma RAS viral oncogene homologue), TP53 (encoding tumor protein p53), RUNX1 (encoding runt-related transcription factor 1), CBL (encoding Cas-Br-M ecotropic retroviral transforming sequence),4,5 TET2 (encoding tet oncogene family member 2),6,7 ASXL1 (encoding additional sex combs¿like protein 1),8,9 and EZH2 (encoding enhancer of zeste homologue 2).10 With the exception of TET2, most of these genes are mutated in no more than 5 to 15% of cases, and generally the mutation rates are lower in the more benign subtypes of the disease. The myelodysplastic syndromes can be divided into several categories on the basis of bone marrow and peripheral-blood morphologic characteristics and cytogenetic changes.11 In low-risk disease, such as refractory anemia, cytopenias are the major clinical challenge, whereas high-risk disease, such as refractory anemia with excess blasts, is characterized by both cytopenias and a high rate of transformation to acute myeloid leukemia. More than a quarter of patients with myelodysplastic syndromes have large numbers of ring sideroblasts in the bone marrow,12 a sufficiently distinctive morphologic abnormality to warrant a separate designation. Ring sideroblasts are characteristically seen on iron staining of bone marrow aspirates as differentiating erythroid cells with a complete or partial ring of iron-laden mitochondria surrounding the nucleus. Several genetic lesions underpinning inherited sideroblastic anemias have been identified,13 including loss-of-function mutations in the genes ALAS2 (encoding delta aminolevulinate synthase 2), ABCB7 (encoding ATP-binding cassette, subfamily B, member 7), and SLC25A38 (solute carrier family 25, member 38). The pathogenesis of ring sideroblasts in myelodysplastic syndromes, however, remains obscure, although gene-expression studies have revealed up-regulation of genes involved in heme synthesis (including ALAS2) and down-regulation of ABCB7.14,15 We reasoned that the identification of recurrently mutated cancer genes in low-grade myelodysplastic syndromes could prove useful for the diagnosis of these disorders and provide new insights into the molecular pathogenesis of these syndromes, Supported by grants from the Wellcome Trust (077012/Z/05/Z, for the overall study, as well as WT088340MA, to Dr. Campbell), the Kay Kendall Leukaemia Fund, Leukemia Lymphoma Research (for the overall study and to Drs. Boultwood, Green, Vyas, and Wainscoat), the Adenoid Cystic Carcinoma Research Foundation, the Medical Research Council (MRC) (to Dr. Warren), the Oxford National Institutes for Health Research Biomedical Research Centre (to Drs. Boultwood, Vyas, and Wainscoat), the Swedish Cancer Society (to Dr. Hellstrom-Lindburg), the International Human Frontier Science Program Organization (to Dr. Varela), the Department of Veterans Affairs and the National Institutes of Health (R01-124929, P01-155249, P50- 100007, and P01-78378, to Drs. Munshi and Anderson), the Association for International Cancer Research and the Leukemia Lymphoma Society (to Drs. Warren and Green), Associazione Italiana per la Ricerca sul Cancro (to the University of Pavia, the University of Milan Bicocca, and Dr. Cazzola), and Fondazione Cariplo (to the University of Pavia and the University of Milan Bicocca).

Background The aberrant transcription in cancer of genes normally associated with embryonic tissue differentiation at various organ sites may be a hallmark of tumour progression. For example, neuroendocrine differentiation is found more commonly in cancers destined to progress, including prostate and lung. We sought to identify proteins which are involved in neuroendocrine differentiation and differentially expressed in aggressive/metastatic tumours. Results Expression arrays were used to identify up-regulated transcripts in a neuroendocrine (NE) transgenic mouse model of prostate cancer. Amongst these were several genes normally expressed in neural tissues, including the pro-neural transcription factors Ascl1 and Hes6. Using quantitative RT-PCR and immuno-histochemistry we showed that these same genes were highly expressed in castrate resistant, metastatic LNCaP cell-lines. Finally we performed a meta-analysis on expression array datasets from human clinical material. The expression of these pro-neural transcripts effectively segregates metastatic from localised prostate cancer and benign tissue as well as sub-clustering a variety of other human cancers. Conclusion By focussing on transcription factors known to drive normal tissue development and comparing expression signatures for normal and malignant mouse tissues we have identified two transcription factors, Ascl1 and Hes6, which appear effective markers for an aggressive phenotype in all prostate models and tissues examined. We suggest that the aberrant initiation of differentiation programs may confer a selective advantage on cells in all contexts and this approach to identify biomarkers therefore has the potential to uncover proteins equally applicable to pre-clinical and clinical cancer biology.