DNA copy number profiling reveals extensive genomic loss in hereditary BRCA1 and BRCA2 ovarian carcinomas.

BACKGROUND: Few studies have attempted to characterise genomic changes occurring in hereditary epithelial ovarian carcinomas (EOCs) and inconsistent results have been obtained. Given the relevance of DNA copy number alterations in ovarian oncogenesis and growing clinical implications of the BRCA-gene status, we aimed to characterise the genomic profiles of hereditary and sporadic ovarian tumours. METHODS: High-resolution array Comparative Genomic Hybridisation profiling of 53 familial (21 BRCA1, 6 BRCA2 and 26 non-BRCA1/2) and 15 sporadic tumours in combination with supervised and unsupervised analysis was used to define common and/or specific copy number features. RESULTS: Unsupervised hierarchical clustering did not stratify tumours according to their familial or sporadic condition or to their BRCA1/2 mutation status. Common recurrent changes, spanning genes potentially fundamental for ovarian carcinogenesis, regardless of BRCA mutations, and several candidate subtype-specific events were defined. Despite similarities, greater contribution of losses was revealed to be a hallmark of BRCA1 and BRCA2 tumours. CONCLUSION: Somatic alterations occurring in the development of familial EOCs do not differ substantially from the ones occurring in sporadic carcinomas. However, some specific features like extensive genomic loss observed in BRCA1/2 tumours may be of clinical relevance helping to identify BRCA-related patients likely to respond to PARP inhibitors.

Small bowel adenocarcinoma copy number profiles are more closely related to colorectal than to gastric cancers.

BACKGROUND: Small bowel adenocarcinoma (SBA) is a rare cancer and consequently, the options for clinical trials are limited. As they are treated according to either a colorectal or a gastric cancer regimen and the molecular biology of a tumor is a pivotal determinant for therapy response, chromosomal copy number aberrations were compared with the colorectal and gastric adenocarcinomas. MATERIALS AND METHODS: A total of 85 microsatellite stable (MSS) adenocarcinomas from the stomach, colorectum and small bowel were selected from existing array comparative genomic hybridization (aCGH) datasets. We compared the aCGH profiles of the three tumor sites by supervised analysis and hierarchical clustering. RESULTS: Hierarchical clustering revealed substantial overlap of 27 SBA copy number profiles with matched colorectal adenocarcinomas but less overlap with profiles of gastric adenocarcinomas. DNA copy number aberrations located at chromosomes 1p36.3-p34.3, 4p15.3-q35.2, 9p24.3-p11.1, 13q13.2-q31.3 and 17p13.3-p13.2 were the strongest features discriminating SBAs and colorectal adenocarcinomas from gastric adenocarcinomas. CONCLUSIONS: We show that MSS SBAs are more similar to colorectal than to gastric cancer, based on the 27 genome-wide DNA copy number profiles that are currently available. These molecular similarities provide added support for treatment of MSS small bowel cancers according to colorectal cancer regimens.

Fecal amine metabolite analysis before onset of severe necrotizing enterocolitis in preterm infants: a prospective case-control study.

Infants developing necrotizing enterocolitis (NEC) have a different metabolomic profile compared to controls. The potential of specific metabolomics, i.e. amino acids and amino alcohols (AAA), as early diagnostic biomarkers for NEC is largely unexplored. In this multicenter prospective case-control study, longitudinally collected fecal samples from preterm infants (born <30 weeks of gestation) from 1-3 days before diagnosis of severe NEC (Bell's stage IIIA/IIIB), were analyzed by targeted high-performance liquid chromatography (HPLC). Control samples were collected from gestational and postnatal age-matched infants. Thirty-one NEC cases (15 NEC IIIA;16 NEC IIIB) with 1:1 matched controls were included. Preclinical samples of infants with NEC were characterized by five increased essential amino acids-isoleucine, leucine, methionine, phenylalanine and valine. Lysine and ethanolamine ratios were lower prior to NEC, compared to control samples. A multivariate model was rendered based on isoleucine, lysine, ethanolamine, tryptophan and ornithine, modestly discriminating cases from controls (AUC 0.67; p < 0.001). Targeted HPLC pointed to several specific AAA alterations in samples collected 1-3 days before NEC onset, compared to controls. Whether this reflects metabolic alterations and has a role in early biomarker development for NEC, has yet to be elucidated.

Detection of colorectal cancer in urine using DNA methylation analysis.

Colorectal cancer (CRC) is the second leading cause for cancer-related death globally. Clinically, there is an urgent need for non-invasive CRC detection. This study assessed the feasibility of CRC detection by analysis of tumor-derived methylated DNA fragments in urine. Urine samples, including both unfractioned and supernatant urine fractions, of 92 CRC patients and 63 healthy volunteers were analyzed for DNA methylation levels of 6 CRC-associated markers (SEPT9, TMEFF2, SDC2, NDRG4, VIM and ALX4). Optimal marker panels were determined by two statistical approaches. Methylation levels of SEPT9 were significantly increased in urine supernatant of CRC patients compared to controls (p < 0.0001). Methylation analysis in unfractioned urine appeared inaccurate. Following multivariate logistic regression and classification and regression tree analysis, a marker panel consisting of SEPT9 and SDC2 was able to detect up to 70% of CRC cases in urine supernatant at 86% specificity. First evidence is provided for CRC detection in urine by SEPT9 methylation analysis, which combined with SDC2 allows for an optimal differentiation between CRC patients and controls. Urine therefore provides a promising liquid biopsy for non-invasive CRC detection.

Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression.

OBJECTIVE: This study aimed to identify the oncogenes at 20q involved in colorectal adenoma to carcinoma progression by measuring the effect of 20q gain on mRNA expression of genes in this amplicon. METHODS: Segmentation of DNA copy number changes on 20q was performed by array CGH (comparative genomic hybridisation) in 34 non-progressed colorectal adenomas, 41 progressed adenomas (ie, adenomas that present a focus of cancer) and 33 adenocarcinomas. Moreover, a robust analysis of altered expression of genes in these segments was performed by microarray analysis in 37 adenomas and 31 adenocarcinomas. Protein expression was evaluated by immunohistochemistry on tissue microarrays. RESULTS: The genes C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5, mapping at 20q, were significantly overexpressed in carcinomas compared with adenomas as a consequence of copy number gain of 20q. CONCLUSION: This approach revealed C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5 genes to be important in chromosomal instability-related adenoma to carcinoma progression. These genes therefore may serve as highly specific biomarkers for colorectal cancer with potential clinical applications.

MiR-17-92 cluster is associated with 13q gain and c-myc expression during colorectal adenoma to adenocarcinoma progression.

BACKGROUND: MicroRNAs are small non-coding RNA molecules, which regulate central mechanisms of tumorigenesis. In colorectal tumours, the combination of gain of 8q and 13q is one of the major factors associated with colorectal adenoma to adenocarcinoma progression. Functional studies on the miR-17-92 cluster localised on 13q31 have shown that its transcription is activated by c-myc, located on 8q, and that it has oncogenic activities. We investigated the contribution of the miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression. METHODS: Expression levels of the miR-17-92 cluster were determined in 55 colorectal tumours and in 10 controls by real-time RT-PCR. Messenger RNA c-myc expression was also determined by real-time RT-PCR in 48 tumours with array comparative genomic hybridisation (aCGH) data available. RESULTS: From the six members of the miR-17-92 cluster, all except miR-18a, showed significant increased expression in colorectal tumours with miR-17-92 locus gain compared with tumours without miR-17-92 locus gain. Unsupervised cluster analysis clustered the tumours based on the presence of miR-17-92 locus gain. Significant correlation between the expression of c-myc and the six miRNAs was also found. CONCLUSION: Increased expression of miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression is associated to DNA copy number gain of miR17-92 locus on 13q31 and c-myc expression.

Expression of Pla2g2a prevents carcinogenesis in Muc2-deficient mice.

Goblet cell depletion and down-regulation of MUC2 expression are observed in a significant percentage of human non-mucinous colorectal adenocarcinomas. Direct evidence for the role of MUC2 in gastrointestinal tumor formation was demonstrated by a knockout of Muc2 in mice that resulted in the development of adenocarcinomas in the small and large intestine. The secretory phospholipase Pla2g2a is a protein that confers resistance to Apc(Min/+)-induced intestinal tumorigenesis. Like Muc2, in the large intestine Pla2g2a is exclusively expressed by the goblet cells and Pla2g2a's tumor resistance is also strongest in the large intestine. Possible genetic interactions between Muc2 and Pla2g2a were examined by creating C57BL/6-Muc2(-/-)Pla2g2a transgenic mice. Expression of a Pla2g2a transgene reduced tumorigenesis in the large intestine by 90% in male Muc2(-/-) mice and by nearly 100% in female Muc2(-/-) mice. Expression of Pla2g2a also inhibited tumor progression. Microarray gene expression studies revealed Pla2g2a target genes that modulate intestinal energy metabolism, differentiation, inflammation, immune responses and proliferation. Overall, results of the present study demonstrate an Apc-independent role for Pla2g2a in tumor resistance and indicate that Pla2g2a plays an important role, along with Muc2, in protection of the intestinal mucosa.

Genome-wide DNA copy number alterations in head and neck squamous cell carcinomas with or without oncogene-expressing human papillomavirus.

Oncogene-expressing human papillomavirus type 16 (HPV16) is found in a subset of head and neck squamous cell carcinomas (HNSCC). HPV16 drives carcinogenesis by inactivating p53 and pRb with the viral oncoproteins E6 and E7, paralleled by a low level of mutations in TP53 and allelic loss at 3p, 9p, and 17p, genetic changes frequently found in HNSCCs of nonviral etiology. We hypothesize that two pathways to HNSCC exist: one determined by HPV16 and the other by environmental carcinogens. To define the critical genetic events in these two pathways, we now present a detailed genome analysis of HNSCC with and without HPV16 involvement by employing high-resolution microarray comparative genomic hybridization. Four regions showed alterations in HPV-negative tumors that were absent in HPV-positive tumors: losses at 3p11.2-26.3, 5q11.2-35.2, and 9p21.1-24, and gains/amplifications at 11q12.1-13.4. Also, HPV16-negative tumors demonstrated loss at 18q12.1-23, in contrast to gain in HPV16-positive tumors. Seven regions were altered at high frequency (>33%) in both groups: gains at 3q22.2-qter, 5p15.2-pter, 8p11.2-qter, 9q22-34.1, and 20p-20q, and losses at 11q14.1-qter and 13q11-33. These data show that HNSCC arising by environmental carcinogens are characterized by genetic alterations that differ from those observed in HPV16-induced HNSCC, and most likely occur early in carcinogenesis. A number of genetic changes are shared in both tumor groups and can be considered crucial in the later stages of HNSCC progression.

MiR expression profiles of paired primary colorectal cancer and metastases by next-generation sequencing.

MicroRNAs (miRs) have been recognized as promising biomarkers. It is unknown to what extent tumor-derived miRs are differentially expressed between primary colorectal cancers (pCRCs) and metastatic lesions, and to what extent the expression profiles of tumor tissue differ from the surrounding normal tissue. Next-generation sequencing (NGS) of 220 fresh-frozen samples, including paired primary and metastatic tumor tissue and non-tumorous tissue from 38 patients, revealed expression of 2245 known unique mature miRs and 515 novel candidate miRs. Unsupervised clustering of miR expression profiles of pCRC tissue with paired metastases did not separate the two entities, whereas unsupervised clustering of miR expression profiles of pCRC with normal colorectal mucosa demonstrated complete separation of the tumor samples from their paired normal mucosa. Two hundred and twenty-two miRs differentiated both pCRC and metastases from normal tissue samples (false discovery rate (FDR) <0.05). The highest expressed tumor-specific miRs were miR-21 and miR-92a, both previously described to be involved in CRC with potential as circulating biomarker for early detection. Only eight miRs, 0.5% of the analysed miR transcriptome, were differentially expressed between pCRC and the corresponding metastases (FDR <0.1), consisting of five known miRs (miR-320b, miR-320d, miR-3117, miR-1246 and miR-663b) and three novel candidate miRs (chr 1-2552-5p, chr 8-20656-5p and chr 10-25333-3p). These results indicate that previously unrecognized candidate miRs expressed in advanced CRC were identified using NGS. In addition, miR expression profiles of pCRC and metastatic lesions are highly comparable and may be of similar predictive value for prognosis or response to treatment in patients with advanced CRC.

Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis.

Little is known about the alterations in microRNA (miRNA) expression patterns during the consecutive stages of cervical cancer development and their association with chromosomal instability. In this study, miRNA expression in normal cervical squamous epithelium, high-grade precancerous lesions (cervical intraepithelial neoplasia (CIN2-3)), squamous cell carcinomas (SCCs) and adenocarcinomas (AdCAs) was integrated with previously generated chromosomal profiles of the same samples. Significantly differential expression during the consecutive stages of cervical SCC development was observed for 106 miRNAs. Of these differentially expressed miRNAs, 27 showed early transiently altered expression in CIN2-3 lesions only, 46 miRNAs showed late altered expression in SCCs only and 33 showed continuously altered expression in both CIN2-3 and SCCs. Altered expression of five significantly differentially expressed miRNAs, hsa-miR-9 (1q23.2), hsa-miR-15b (3q25.32), hsa-miR-28-5p (3q27.3), hsa-miR-100 and hsa-miR-125b (both 11q24.1), was directly linked to frequent chromosomal alterations. Functional analyses were performed for hsa-miR-9, representing a potential oncogene with increased expression linked to a chromosomal gain of 1q. Hsa-miR-9 overexpression was found to increase cell viability, anchorage-independent growth and migration in vitro. Upon organic raft culturing, hsa-miR-9 hampered differentiation and induced proliferation in all strata of the epithelial layer. These findings support a potential oncogenic function of hsa-miR-9 in cervical cancer. In summary, differential expression of 106 miRNAs, partly associated with chromosomal alterations, was observed during cervical SCC development. Altered expression of hsa-miR-9 associated with a chromosomal gain of chromosome 1q was shown to be functionally relevant, underlining the importance of deregulated miRNA expression in cervical carcinogenesis.

Increased gene copy numbers at chromosome 20q are frequent in both squamous cell carcinomas and adenocarcinomas of the cervix.

Genome-wide microarray-based comparative genomic hybridization (array CGH) was used to identify common chromosomal alterations involved in cervical carcinogenesis as a first step towards the discovery of novel biomarkers. The genomic profiles of nine squamous cell carcinomas (SCCs) and seven adenocarcinomas (AdCAs), as well as four human papillomavirus (HPV)-immortalized keratinocyte cell lines, were assessed. On a genome-wide scale, SCCs showed significantly more gains than AdCAs. More specifically, there was a striking and highly significant difference between the two histological types for gain at 3q12.1-28, which was predominantly observed in SCC. Other frequent alterations included gains of 1q21.1-31.1 and 20q11.21-13.33, and losses of 11q22.3-25 and 13q14.3-21.33. Subsequent FISH analysis for hTR, located at 3q26, confirmed the presence of 3q gain in SCCs and HPV-immortalized cell lines. Fine mapping of chromosome 20q using multiplex ligation-dependent probe amplification (MLPA) showed copy number increases for a number of genes located at 20q11-q12, including DNMT3B and TOP1. For DNMT3B, this correlated with elevated mRNA expression in 79% of cases. In conclusion, the assessment of frequent genomic alterations resulted in the identification of potential novel biomarkers, which may ultimately enable a better risk stratification of high-risk (hr)-HPV-positive women.

Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1).

The hypoxia-inducible factor 1 (HIF-1) plays a critical role in cellular responses to hypoxia. The aim of the present study was to evaluate which genes are induced by hypoxia, and whether this induction is mediated by HIF-1, by expression microarray analysis of wt and HIF-1alpha null mouse fibroblasts. Forty-five genes were up-regulated by hypoxia and 40 (89%) of these were regulated by HIF-1. Of the 114 genes down-regulated by hypoxia, 19 (17%) were HIF-1-dependent. All glycolytic enzymes were strongly up-regulated by hypoxia in a HIF-1-dependent manner. Genes already known to be related to hypoxia, such as glucose transporter 1, BNIP3, and hypoxia-induced gene 1, were induced. In addition, multiple new HIF-1-regulated genes were identified, including genes involved in metabolism (adenylate kinase 4, galactokinase), apoptosis (galectin-3 and gelsolin), and invasion (RhoA). Genes down-regulated by hypoxia were involved in cytoskeleton maintenance (Rho kinase), mRNA processing (heterogeneous nuclear ribonucleoprotein H1 and splicing factor), and DNA repair (REV3). Furthermore, seven cDNAs from genes with unknown function or expressed sequence tags (ESTs) were up-regulated and 27 such cDNAs were down-regulated. In conclusion, hypoxia causes down- rather than up-regulation of gene expression and HIF-1 seems to play a major role in the regulation of hypoxia-induced genes.