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Prostate cancer with a cribriform pattern, including invasive cribriform carcinoma (ICC) and/or intraductal carcinoma (IDC) is associated with a poor prognosis, and the underlying mechanisms are unclear. Therefore, we aimed to identify biomarkers for this feature. Using a radical prostatectomy cohort, we performed within-patient differential expression analyses with RNA sequencing data to compare samples with a cribriform pattern to those with non-cribriform Gleason pattern 4 (NcGP4; n=13). ACSM1, GRIN3A, PCDHB2, and REG4 were identified as differentially expressed, and validation was performed using real-time reverse transcription polymerase chain reaction (n=99; 321 RNA samples) and RNA in situ hybridization on tissue microarrays (n=479; 2047 tissue cores). GRIN3A was significantly higher expressed in cribriform pattern vs. NcGP4, when assessed within the same patient (n=27; p=0.005) and between different patients (n=83; p=0.001). Tissue cores with IDC more often expressed GRIN3A compared to ICC, NcGP4, and benign tissue (52 % vs. ≤ 32 %). When IDC and NcGP4 was compared within the same patient (173 pairs of tissue cores; 54 patients), 38 (22 %) of the tissue microarray core pairs had GRIN3A expression in only IDC, 33 (19 %) had expression in both IDC and NcGP4, 14 (8 %) in only NcGP4 and 88 (51 %) were negative in both entities (p=0.001). GRIN3A was as well associated with biochemical recurrence (log-rank, p=0.002). In conclusion, ectopic GRIN3A expression is an RNA-based biomarker for the presence of cribriform prostate cancer, particularly for IDC.

Prognostic biomarkers for prostate cancer are needed to improve prediction of disease course and guide treatment decisions. However, biomarker development is complicated by the common multifocality and heterogeneity of the disease. We aimed to determine the prognostic value of candidate biomarkers transcriptional regulator ERG and related ETS family genes, while considering tumor heterogeneity. In a multisampled, prospective, and treatment‐naïve radical prostatectomy cohort from one tertiary center (2010–2012, median follow‐up 8.1 years), we analyzed ERG protein (480 patients; 2047 tissue cores), and RNA of several ETS genes in a subcohort (165 patients; 778 fresh‐frozen tissue samples). Intra‐ and interfocal heterogeneity was identified in 29% and 33% (ERG protein) and 39% and 27% (ETS RNA) of patients, respectively. ERG protein and ETS RNA was identified exclusively in a nonindex tumor in 31% and 32% of patients, respectively. ERG protein demonstrated independent prognostic value in predicting biochemical (P = 0.04) and clinical recurrence (P = 0.004) and appeared to have greatest prognostic value for patients with Grade Groups 4–5. In conclusion, when heterogeneity is considered, ERG protein is a robust prognostic biomarker for prostate cancer.

Abstract Background Colorectal cancer is the 2nd leading cause of cancer-related deaths with few patients benefiting from biomarker-guided therapy. Mutation expression is essential for accurate interpretation of mutations as biomarkers, but surprisingly, little has been done to analyze somatic cancer mutations on the expression level. We report a large-scale analysis of allele-specific mutation expression. Methods Whole-exome and total RNA sequencing was performed on 137 samples from 121 microsatellite stable colorectal cancers, including multiregional samples of primary and metastatic tumors from 4 patients. Data were integrated with allele-specific resolution. Results were validated in an independent set of 241 colon cancers. Therapeutic associations were explored by pharmacogenomic profiling of 15 cell lines or patient-derived organoids. Results The median proportion of expressed mutations per tumor was 34%. Cancer-critical mutations had the highest expression frequency (gene-wise mean of 58%), independent of frequent allelic imbalance. Systematic deviation from the general pattern of expression levels according to allelic frequencies was detected, including preferential expression of mutated alleles dependent on the mutation type and target gene. Translational relevance was suggested by correlations of KRAS/NRAS or TP53 mutation expression levels with downstream oncogenic signatures (p < 0.03), overall survival among patients with stage II and III cancer (KRAS/NRAS: hazard ratio 6.1, p = 0.0070), and targeted drug sensitivity. The latter was demonstrated for EGFR and MDM2 inhibition in pre-clinical models. Conclusions Only a subset of mutations in microsatellite stable colorectal cancers were expressed, and the “expressed mutation dose” may provide an opportunity for more fine-tuned biomarker interpretations.

Testicular germ cell tumors are highly heritable, and the authors present the largest genome association study, identifying 22 novel loci, which account for a third of those identified to date. Implicated pathways include male germ cell development and differentiation, and chromosomal segregation.

In primary prostate cancer, the common multifocality and heterogeneity are major obstacles in finding robust prognostic tissue biomarkers. The long noncoding RNA SCHLAP1 has been suggested, but its prognostic value has not been investigated in the context of tumor heterogeneity. In the present study, expression of SCHLAP1 was investigated using real-time RT-PCR in a multisampled series of 778 tissue samples from radical prostatectomies of 164 prostate cancer patients (median follow-up time 7.4 y). The prognostic value of SCHLAP1 was evaluated with biochemical recurrence as endpoint.In total, 29% of patients were classified as having high expression of SCHLAP1 in at least one malignant sample. Among these, inter- and intrafocal heterogeneity was detected in 72% and 56%, respectively. High expression of SCHLAP1 was shown to be a predictor of biochemical recurrence in both uni- and multivariable cox regression analyses (P < 0.001 and P = 0.02). High expression of SCHLAP1 was also significantly associated with adverse clinicopathological characteristics, including grade group, high pT stage, invasive cribriform growth/intraductal carcinoma of the prostate, and reactive stroma. In conclusion, high expression of SCHLAP1 in at least one malignant sample is a robust prognostic biomarker in primary prostate cancer. For the first time, high SCHLAP1 expression has been associated with the aggressive histopathologic feature reactive stroma. The expression of SCHLAP1 is highly heterogeneous, and analysis of multiple samples is therefore crucial in determination of the SCHLAP1 status of a patient.

BACKGROUND Prostate cancer is multifocal with distinct molecular subtypes. The utility of genomic subtyping has been challenged due to inter- and intrafocal heterogeneity. We sought to characterize the subtype-defining molecular alterations of primary prostate cancer across all tumor foci within radical prostatectomy (RP) specimens and determine the prevalence of collision tumors.METHODS From the Early Detection Research Network cohort, we identified 333 prospectively collected RPs from 2010 to 2014 and assessed ETS-related gene (ERG), serine peptidase inhibitor Kazal type 1 (SPINK1), phosphatase and tensin homolog (PTEN), and speckle type BTB/POZ protein (SPOP) molecular status. We utilized dual ERG/SPINK1 immunohistochemistry and fluorescence in situ hybridization to confirm ERG rearrangements and characterize PTEN deletion, as well as high-resolution melting curve analysis and Sanger sequencing to determine SPOP mutation status.RESULTS Based on index focus alone, ERG, SPINK1, PTEN, and SPOP alterations were identified in 47.5%, 10.8%, 14.3%, and 5.1% of RP specimens, respectively. In 233 multifocal RPs with ERG/SPINK1 status in all foci, 139 (59.7%) had discordant molecular alterations between foci. Collision tumors, as defined by discrepant ERG/SPINK1 status within a single focus, were identified in 29 (9.4%) RP specimens.CONCLUSION Interfocal molecular heterogeneity was identified in about 60% of multifocal RP specimens, and collision tumors were present in about 10%. We present this phenomenon as a model for the intrafocal heterogeneity observed in previous studies and propose that future genomic studies screen for collision tumors to better characterize molecular heterogeneity.FUNDING Early Detection Research Network US National Cancer Institute (NCI) 5U01 CA111275-09, Center for Translational Pathology at Weill Cornell Medicine (WCM) Department of Pathology and Laboratory Medicine, US NCI (WCM SPORE in Prostate Cancer, P50CA211024-01), R37CA215040, Damon Runyon Cancer Research Foundation, US MetLife Foundation Family Clinical Investigator Award, Norwegian Cancer Society (grant 208197), and South-Eastern Norway Regional Health Authority (grant 2019016 and 2020063).

Abstract Background In breast cancer, activation of bone morphogenetic protein (BMP) signaling and elevated levels of BMP-antagonists have been linked to tumor progression and metastasis. However, the simultaneous upregulation of BMPs and their antagonist, and the fact that both promote tumor aggressiveness seems contradictory and is not fully understood. Methods We analyzed the transcriptomes of the metastatic 66cl4 and the non-metastatic 67NR cell lines of the 4T1 mouse mammary tumor model to search for factors that promote metastasis. CRISPR/Cas9 gene editing was used for mechanistic studies in the same cell lines. Furthermore, we analyzed gene expression patterns in human breast cancer biopsies obtained from public datasets to evaluate co-expression and possible relations to clinical outcome. Results We found that mRNA levels of the BMP-antagonist Grem1, encoding gremlin1, and the ligand Bmp4 were both significantly upregulated in cells and primary tumors of 66cl4 compared to 67NR. Depletion of gremlin1 in 66cl4 could impair metastasis to the lungs in this model. Furthermore, we found that expression of Grem1 correlated with upregulation of several stem cell markers in 66cl4 cells compared to 67NR cells. Both in the mouse model and in patients, expression of GREM1 associated with extracellular matrix organization, and formation, biosynthesis and modification of collagen. Importantly, high expression of GREM1 predicted poor prognosis in estrogen receptor negative breast cancer patients. Analyses of large patient cohorts revealed that amplification of genes encoding BMP-antagonists and elevation of the corresponding transcripts is evident in biopsies from more than half of the patients and much more frequent for the secreted BMP-antagonists than the intracellular inhibitors of SMAD signaling. Conclusion In conclusion, our results show that GREM1 is associated with metastasis and predicts poor prognosis in ER-negative breast cancer patients. Gremlin1 could represent a novel target for therapy.

Abstract Background The prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis. Methods We used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers. Results We identified 17 genes significantly differentially expressed between mFTC and wFTC. C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC. Conclusions We conclude that the underexpression of CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.

Abstract Background Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies. Methods We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays. Results The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA- and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFβ signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately ¼ of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene- and protein- expression was found for several oncogenes in individual cell lines, including MYC and ERBB2. Conclusions This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC.

Abstract Background Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a complex genome with thousands of small mutations—the microsatellite instability (MSI) phenotype. We investigated molecular heterogeneity and tumor immunogenicity in relation to clinical endpoints within this distinct subtype of colorectal cancers. Methods A total of 333 primary MSI+ colorectal tumors from multiple cohorts were analyzed by multilevel genomics and computational modeling—including mutation profiling, clonality modeling, and neoantigen prediction in a subset of the tumors, as well as gene expression profiling for consensus molecular subtypes (CMS) and immune cell infiltration. Results Novel, frequent frameshift mutations in four cancer-critical genes were identified by deep exome sequencing, including in CRTC1, BCL9, JAK1, and PTCH1. JAK1 loss-of-function mutations were validated with an overall frequency of 20% in Norwegian and British patients, and mutated tumors had up-regulation of transcriptional signatures associated with resistance to anti-PD-1 treatment. Clonality analyses revealed a high level of intra-tumor heterogeneity; however, this was not associated with disease progression. Among the MSI+ tumors, the total mutation load correlated with the number of predicted neoantigens (P = 4 × 10−5), but not with immune cell infiltration—this was dependent on the CMS class; MSI+ tumors in CMS1 were highly immunogenic compared to MSI+ tumors in CMS2-4. Both JAK1 mutations and CMS1 were favorable prognostic factors (hazard ratios 0.2 [0.05–0.9] and 0.4 [0.2–0.9], respectively, P = 0.03 and 0.02). Conclusions Multilevel genomic analyses of MSI+ colorectal cancer revealed molecular heterogeneity with clinical relevance, including tumor immunogenicity and a favorable patient outcome associated with JAK1 mutations and the transcriptomic subgroup CMS1, emphasizing the potential for prognostic stratification of this clinically important subtype. See related research highlight by Samstein and Chan 10.1186/s13073-017-0438-9

Intratubular germ cell neoplasia, the precursor of testicular germ cell tumors (TGCTs), is hypothesized to arise during embryogenesis from developmentally arrested primordial germ cells (PGCs) or gonocytes. In early embryonal life, the PGCs migrate from the yolk sac to the dorsal body wall where the cell population separates before colonizing the genital ridges. However, whether the malignant transformation takes place before or after this separation is controversial. We have explored the somatic exome-wide mutational spectra of bilateral TGCT to provide novel insight into the in utero critical time frame of malignant transformation and TGCT pathogenesis. Exome sequencing was performed in five patients with bilateral TGCT (eight tumors), of these three patients in whom both tumors were available (six tumors) and two patients each with only one available tumor (two tumors). Selected loci were explored by Sanger sequencing in 71 patients with bilateral TGCT. From the exome-wide mutational spectra, no identical mutations in any of the three bilateral tumor pairs were identified. Exome sequencing of all eight tumors revealed 87 somatic non-synonymous mutations (median 10 per tumor; range 5-21), some in already known cancer genes such as CIITA, NEB, platelet-derived growth factor receptor α (PDGFRA), and WHSC1. SUPT6H was found recurrently mutated in two tumors. We suggest independent development lineages of bilateral TGCT. Thus, malignant transformation into intratubular germ cell neoplasia is likely to occur after the migration of PGCs. We reveal possible drivers of TGCT pathogenesis, such as mutated PDGFRA, potentially with therapeutic implications for TGCT patients.

Several microRNAs (miRNAs) are known to be deregulated in colon cancer, but the mechanisms behind their potential involvement on proliferation and tumor cell survival are unclear. The present study aimed to identify miRNAs with functional implications for development of colon cancer. The cell proliferation and apoptosis were examined following perturbations of miRNA levels by employing a comprehensive miRNA library screen. miRNAs nominated for relevance to colon cancer were validated on expression and functional levels. By integrating the effect of miRNA up-regulation with the endogenous miRNA expression levels within the HT29, HCT116, and SW480 colon cancer cell lines, we identified miRNAs controlling cell proliferation (n = 53) and apoptosis (n = 93). From these functionally nominated miRNAs, we narrowed the list to 10 oncogene- and 20 tumor suppressor-like miRNAs that were also differentially expressed between colon cancer (n = 80) and normal colonic mucosa (n = 20). The differential expressions of miR-9, miR-31, and miR-182 were successfully validated in a series of colon carcinomas (n = 30) and polyps (n = 10) versus normal colonic mucosa (n = 10), whereas the functional effect was confirmed in an in-depth validation using different cell viability and apoptotic markers. Several transcription factors and genes regulating cell proliferation were identified as putative target genes by integrative miRNA/mRNA expression analysis obtained from the same colon cancer patient samples. This study suggests that deregulated expression of miR-9, miR-31, and miR-182 during carcinogenesis plays a significant role in the development of colon cancer by promoting proliferation and tumor cell survival.

This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa), namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1) and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2) was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B) was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

Alternative splicing is a key regulatory mechanism for gene expression, vital for the proper functioning of eukaryotic cells. Disruption of normal pre-mRNA splicing has the potential to cause and reinforce human disease. Owing to rapid advances in high-throughput technologies, it is now possible to identify novel mRNA isoforms and detect aberrant splicing patterns on a genome scale, across large data sets. Analogous to the genomic types of instability describing cancer genomes (eg, chromosomal instability and microsatellite instability), transcriptome instability (TIN) has recently been proposed as a splicing-related genome-wide characteristic of certain solid cancers. We present the R package TIN, available from Bioconductor, which implements a set of methods for TIN analysis based on exon-level microarray expression profiles. TIN provides tools for estimating aberrant exon usage across samples and for analyzing correlation patterns between TIN and splicing factor expression levels.

Introduction: Testicular germ cell tumors of adolescent and young adult men (TGCTs) generally have near triploid and complex karyotypes. The actual genes driving the tumorigenesis remain essentially to be identified. Materials and Methods: To determine the detailed DNA copy number changes, and investigate their impact on gene expression levels, we performed an integrated microarray profiling of TGCT genomes and transcriptomes. We analyzed 17 TGCTs, three precursor lesions, and the embryonal carcinoma cell lines, NTERA2 and 2102Ep, by comparative genomic hybridization microarrays (array-CGH), and integrated the data with transcriptome profiles of the same samples. Results: The gain of chromosome arm 12p was, as expected, the most common aberration, and we found CCND2, CD9, GAPD, GDF3, NANOG, and TEAD4 to be the therein most highly over-expressed genes. Additional frequent genomic aberrations revealed some shorter chromosomal segments, which are novel to TGCT, as well as known aberrations for which we here refined boundaries. These include gains from 7p15.2 and 21q22.2, and losses of 4p16.3 and 22q13.3. Integration of DNA copy number information to gene expression profiles identified that BRCC3, FOS, MLLT11, NES, and RAC1 may act as novel oncogenes in TGCT. Similarly, DDX26, ERCC5, FZD4, NME4, OPTN, and RB1 were both lost and under-expressed genes, and are thus putative TGCT suppressor genes. Conclusion: This first genome-wide integrated array-CGH and gene expression profiling of TGCT provides novel insights into the genome biology underlying testicular tumorigenesis.

By the use of high-throughput molecular technologies, the number of genes and proteins potentially relevant to testicular germ cell tumor (TGCT) and other diseases will increase rapidly. In a recent transcriptional profiling, we demonstrated the overexpression of GRB7 and JUP in TGCTs, confirmed the reported overexpression of CCND2. We also have recent evidences for frequent genetic alterations of FHIT and epigenetic alterations of MGMT. To evaluate whether the expression of these genes is related to any clinicopathological variables, we constructed a tissue microarray with 510 testicular tissue cores from 279 patients diagnosed with TGCT, covering various histological subgroups and clinical stages. By immunohistochemistry, we found that JUP, GRB7, CCND2 proteins were rarely present in normal testis, but frequently expressed at high levels in TGCT. Additionally, all premalignant intratubular germ cell neoplasias were JUP-immunopositive. MGMT and FHIT were expressed by normal testicular tissues, but at significantly lower frequencies in TGCT. Except for CCND2, the expressions of all markers were significantly associated with various TGCT subtypes. In summary, we have developed a high-throughput tool for the evaluation of TGCT markers, utilized this to validate five candidate genes whose protein expressions were indeed deregulated in TGCT.

Testicular germ cell tumor (TGCT) is the most common tumor type among adolescent and young adult males. Familial clustering and bilateral disease are suggestive of a genetic predisposition among a subgroup of these patients, but susceptibility genes for testicular cancer have not yet been identified. However, suggestive linkage between disease and genetic markers has been reported at loci on chromosome arms 3q, 5q, 12q, 18q, and Xq. We have analyzed primary familial/ bilateral (n=20) and sporadic (n=27) TGCTs, including 28 seminomas and 19 nonseminomas, for allelic imbalance (AI) within the autosomal regions. DNA from all tumors were analyzed by fluorescent polymerase chain reaction of 22 polymorphic loci at 3q27-ter, 5q13-35.1, 12q21-ter, and 18q12-ter. All tumor genotypes were evaluated against their corresponding constitutional genotypes. The percentages of TGCTs with genetic changes at 3q, 5q, 12q, and 18q, were 79%, 36%, 53% and 43%, respectively. The frequencies at 3q and 12q in nonseminomas were significantly higher than in seminomas (P=.003 and P=.004). In order to evaluate changes at hemizygous Xq loci, five loci were analyzed by co-amplification with an autosomal reference marker known to reveal retained heterozygosity in the tumor DNA. Gain of Xq sequences was seen in more than 50% of the tumors. The degree of amplification varied among the loci in each of five tumors, and based on these breakpoints, a common region of overlapping gains was found at X828. No significant differences were found between the frequencies of genetic changes in familial /bilateral versus sporadic tumors, an observation speaking in disfavor of the existence of a single susceptibility gene for TGCT in any of the analyzed regions. Our data suggest that gain of genetic material at distal Xq and losses at 5q and 18q contribute to establishment of seminomas, whereas imbalances at 3q as well as gain at distal part of 12q are associated with further progression into nonseminomas.

VTI1A-TCF7L2 was reported as a recurrent fusion gene in colorectal cancer (CRC), found to be expressed in three out of 97 primary cancers, and one cell line, NCI-H508, where a genomic deletion joins the two genes [1]. To investigate this fusion further, we analyzed high-throughput DNA and RNA sequencing data from seven CRC cell lines, and identified the gene RP11-57H14.3 (ENSG00000225292) as a novel fusion partner for TCF7L2. The fusion was discovered from both genome and transcriptome data in the HCT116 cell line. By triplicate nested RT-PCR, we tested both the novel fusion transcript and VTI1A-TCF7L2 for expression in a series of 106 CRC tissues, 21 CRC cell lines, 14 normal colonic mucosa, and 20 normal tissues from miscellaneous anatomical sites. Altogether, 42% and 45% of the CRC samples expressed VTI1A-TCF7L2 and TCF7L2-RP11-57H14.3 fusion transcripts, respectively. The fusion transcripts were both seen in 29% of the normal colonic mucosa samples, and in 25% and 75% of the tested normal tissues from other organs, revealing that the TCF7L2 fusion transcripts are neither specific to cancer nor to the colon and rectum. Seven different splice variants were detected for the VTI1A-TCF7L2 fusion, of which three are novel. Four different splice variants were detected for the TCF7L2-RP11-57H14.3 fusion. In conclusion, we have identified novel variants of VTI1A-TCF7L2 fusion transcripts, including a novel fusion partner gene, RP11-57H14.3, and demonstrated detectable levels in a large fraction of CRC samples, as well as in normal colonic mucosa and other tissue types. We suggest that the fusion transcripts observed in a high frequency of samples are transcription induced chimeras that are expressed at low levels in most samples. The similar fusion transcripts induced by genomic rearrangements observed in individual cancer cell lines may yet have oncogenic potential as suggested in the original study by Bass et al.

Sarcomas are relatively rare malignancies and include a large number of histological subgroups. Based on morphology alone, the differential diagnoses of sarcoma subtypes can be challenging, but the identification of specific fusion genes aids correct diagnostication. The presence of individual fusion products are routinely investigated in Pathology labs. However, the methods used are time-consuming and based on prior knowledge about the expected fusion gene and often the most likely break-point. In this study, 16 sarcoma samples, representing seven different sarcoma subtypes with known fusion gene status from a diagnostic setting, were investigated using a fusion gene microarray. The microarray was designed to detect all possible exon-exon breakpoints between all known fusion genes in a single analysis. An automated scoring of the microarray data from the 38 known sarcoma-related fusion genes identified the correct fusion gene among the top-three hits in 11 of the samples. The analytical sensitivity may be further optimised, but we conclude that a sarcoma-fusion gene microarray is suitable as a time-saving screening tool to identify the majority of the correct fusion genes.

Mesenchymal chondrosarcomas (MCs) account for 3-10% of primary chondrosarcomas. The cytogenetic literature includes only ten such tumours with karyotypic information and no specific aberrations have been identified. Using a purely molecular genetic approach a HEY1-NCOA2 fusion gene was recently detected in 10 of 15 investigated MCs. The fusion probably arises through intrachromosomal rearrangement of chromosome arm 8 q. We report a new case of MC showing a t(1;5)(q42;q32) as the sole karyotypic aberration. Through FISH and whole transcriptome sequencing analysis we found a novel fusion between the IRF2BP2 gene and the transcription factor CDX1 gene arising from the translocation. The IRF2BP2-CDX1 has not formerly been described in human neoplasia. In our hospital's archives three more cases of MC were found, and we examined them looking for the supposedly more common HEY1-NCOA2 fusion, finding it in all three tumours but not in the case showing t(1;5) and IRF2BP2-CDX1 gene fusion. This demonstrates that genetic heterogeneity exists in mesenchymal chondrosarcoma.

FLI1 and ERG, the major ETS transcription factors involved in rearrangements in the Ewing's sarcoma family of tumors (ESFT) and in prostate carcinomas (PCa), respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT) and choose four genes among the top-most differentially expressed between PCa with (PCa ERG+) and without (PCa ETS-) ETS fusion genes (HIST1H4L, KCNN2, ECRG4 and LDOC1), as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (NR0B1, CAV1, IGFBP3 and TGFBR2). Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS), we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa ERG+ and PCa ETS-, CAV1, IGFBP3 and TGFBR2 were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for ECRG4 and LDOC1, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both HIST1H4L and KCNN2 are significantly overexpressed in PCa ERG+ and that ERG binds to the promoter of these genes. Conversely, KCNN2 was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.

The tumor suppressor activity of Beclin 1 (BECN1), a subunit of class III phosphatidylinositol 3-kinase complex, has been attributed to its regulation of apoptosis and autophagy. Here, we identify FYVE-CENT (ZFYVE26), a phosphatidylinositol 3-phosphate binding protein important for cytokinesis, as a novel interacting protein of Beclin 1. A mutation in FYVE-CENT (R1945Q) associated with breast cancer abolished the interaction between FYVE-CENT and Beclin 1, and reduced the localization of these proteins at the intercellular bridge during cytokinesis. Breast cancer cells containing the FYVE-CENT R1945Q mutation displayed a significant increase in cytokinetic profiles and bi-multinuclear phenotype. Both Beclin 1 and FYVE-CENT were found to be downregulated in advanced breast cancers. These findings suggest a positive feedback loop for recruitment of FYVE-CENT and Beclin 1 to the intercellular bridge during cytokinesis, and reveal a novel potential tumor suppressor mechanism for Beclin 1.

Colorectal cancer (CRC) is one of the most common cancer types in developed countries. To identify molecular networks and biological processes that are deregulated in CRC compared to normal colonic mucosa, we applied Gene Set Enrichment Analysis to two independent transcriptome datasets, including a total of 137 CRC and ten normal colonic mucosa samples. Eighty-two gene sets as described by the Kyoto Encyclopedia of Genes and Genomes database had significantly altered gene expression in both datasets. These included networks associated with cell division, DNA maintenance, and metabolism. Among signaling pathways with known changes in key genes, the "Phosphatidylinositol signaling network", comprising part of the PI3K pathway, was found deregulated. The downregulated genes in this pathway included several members of the Phospholipase C protein family, and the reduced expression of two of these, PLCD1 and PLCE1, were successfully validated in CRC biopsies (n = 70) and cell lines (n = 19) by quantitative analyses. The repression of both genes was found associated with KRAS mutations (P = 0.005 and 0.006, respectively), and we observed that microsatellite stable carcinomas with reduced PLCD1 expression more frequently had TP53 mutations (P = 0.002). Promoter methylation analyses of PLCD1 and PLCE1 performed in cell lines and tumor biopsies revealed that methylation of PLCD1 can contribute to reduced expression in 40% of the microsatellite instable carcinomas. In conclusion, we have identified significantly deregulated pathways in CRC, and validated repression of PLCD1 and PLCE1 expression. This illustrates that the GSEA approach may guide discovery of novel biomarkers in cancer.

A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16) and without (n = 8) TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (r(s) = 0.65, p