Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers

Background: Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported. Results: We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker. Conclusions: This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research

The Fetal Hypothalamus Has the Potential to Generate Cells with a Gonadotropin Releasing Hormone (GnRH) Phenotype

Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus. Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation. This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation

HELPing older people with very severe chronic obstructive pulmonary disease (HELP-COPD):mixed-method feasibility pilot randomised controlled trial of a novel intervention

BACKGROUND: Extending palliative care to those with advanced non-malignant disease is advocated, but the implications in specific conditions are poorly understood. AIMS: We piloted a novel nurse-led intervention, HELPing older people with very severe chronic obstructive pulmonary disease (HELP-COPD), undertaken 4 weeks after discharge from hospital, which sought to identify and address the holistic care needs of people with severe COPD. METHODS: This 6-month mixed-method feasibility pilot trial randomised (ratio 3:1) patients to HELP-COPD or usual care. We assessed the feasibility of using validated questionnaires as outcome measures and analysed the needs/actions recorded in the HELP-COPD records. Semi-structured interviews with a purposive sample of patients, carers and professionals explored the perceptions of HELP-COPD. Verbatim transcriptions and field notes were analysed using Normalisation Process Theory as a framework. RESULTS: We randomised 32 patients (24 to HELP-COPD); 19 completed the study (death=3, ill-health=4, declined=6). The HELP-COPD record noted a mean of 1.6 actions/assessment, mostly provision of information or self-help actions: only five referrals were made. Most patients were positive about HELP-COPD, discussing their concerns and coping strategies in all domains, but the questionnaires were burdensome for some patients. Adaptation to their slowly progressive disability and a strong preference to rely on family support was reflected in limited acceptance of formal services. Professionals perceived HELP-COPD as addressing an important aspect of care, although timing overlapped with discharge planning. CONCLUSIONS: The HELP-COPD intervention was well received by patients and the concept resonated with professionals, although delivery post discharge overlapped with existing services. Integration of brief holistic care assessments in the routine primary care management of COPD may be more appropriate

Genomic Profiling Identifies GATA6 as a Candidate Oncogene Amplified in Pancreatobiliary Cancer

Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies

Using qualitative synthesis to explore heterogeneity of complex interventions

Background: Including qualitative evidence on patients' perspectives in systematic reviews of complex interventions may reveal reasons for variation in trial findings. This is particularly the case when the intervention is for a long-term disease, as management may rely heavily on the efforts of the patient. Inclusion though seldom happens, possibly because of methodological challenges, and when it does occur the different forms of evidence are often kept separate. To explore heterogeneity in trial findings, we tested a novel approach to integrate qualitative review evidence on patients' perspectives with evidence from a Cochrane systematic review.Methods: We used, as a framework for a matrix, evidence from a qualitative review on patients' perspectives on helping them manage their disease. We then logged in the matrix whether the interventions identified in a Cochrane review corresponded with the patient perspectives on how to help them. We then explored correspondence. The Cochrane review we used included 19 trials of interventions to improve adherence to therapy in HIV/AIDS patients. The qualitative review we used included 23 studies on HIV/AIDS patients' perspectives on adherence; it translated the themes identified across the studies into recommendations in how to help patients adhere. Both reviews assessed quality. In the qualitative review they found no difference in findings between the better quality studies and the weaker ones. In the Cochrane review they were unable to explore the impact of quality in subgroup analysis because so few studies were of good quality.Results: Matrix tabulation of interventions and patients' perspectives identified a range of priorities raised by people infected with HIV-1 that were not addressed in evaluated interventions. Tabulation of the more robust trials revealed that interventions that significantly improved adherence contained more components considered important by patients than interventions where no statistically significant effect was found.Conclusions: This simple approach breaks new ground in cross tabulating qualitative evidence with the characteristics of trialled interventions. In doing so it tests the assumption that patients are more likely to adhere to interventions that match more closely with their concerns. The potential of this approach in exploring varying content and rates of success in trialled complex interventions deserves further evaluation

Integrative genomic and functional profiling of the pancreatic cancer genome

Abstract Background Pancreatic cancer is a deadly disease with a five-year survival of less than 5%. A better understanding of the underlying biology may suggest novel therapeutic targets. Recent surveys of the pancreatic cancer genome have uncovered numerous new alterations; yet systematic functional characterization of candidate cancer genes has lagged behind. To address this challenge, here we have devised a highly-parallel RNA interference-based functional screen to evaluate many genomically-nominated candidate pancreatic cancer genes simultaneously. Results For 185 candidate pancreatic cancer genes, selected from recurrently altered genomic loci, we performed a pooled shRNA library screen of cell growth/viability across 10 different cell lines. Knockdown-associated effects on cell growth were assessed by enrichment or depletion of shRNA hairpins, by hybridization to barcode microarrays. A novel analytical approach (COrrelated Phenotypes for On-Target Effects; COPOTE) was used to discern probable on-target knockdown, based on identifying different shRNAs targeting the same gene and displaying concordant phenotypes across cell lines. Knockdown data were integrated with genomic architecture and gene-expression profiles, and selected findings validated using individual shRNAs and/or independent siRNAs. The pooled shRNA library design delivered reproducible data. In all, COPOTE analysis identified 52 probable on-target gene-knockdowns. Knockdown of known oncogenes (KRAS, MYC, SMURF1 and CCNE1) and a tumor suppressor (CDKN2A) showed the expected contrasting effects on cell growth. In addition, the screen corroborated purported roles of PLEKHG2 and MED29 as 19q13 amplicon drivers. Most notably, the analysis also revealed novel possible oncogenic functions of nucleoporin NUP153 (ostensibly by modulating TGFβ signaling) and Kruppel-like transcription factor KLF5 in pancreatic cancer. Conclusions By integrating physical and functional genomic data, we were able to simultaneously evaluate many candidate pancreatic cancer genes. Our findings uncover new facets of pancreatic cancer biology, with possible therapeutic implications. More broadly, our study provides a general strategy for the efficient characterization of candidate genes emerging from cancer genome studies

Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types

<div><p>Gene fusions, like <i>BCR/ABL1</i> in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a “breakpoint analysis” pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving <i>ROS1</i> tyrosine kinase in angiosarcoma (<i>CEP85L/ROS1</i>), <i>SLC1A2</i> glutamate transporter in colon cancer (<i>APIP/SLC1A2</i>), <i>RAF1</i> kinase in pancreatic cancer (<i>ATG7/RAF1</i>) and anaplastic astrocytoma (<i>BCL6/RAF1</i>), <i>EWSR1</i> in melanoma (<i>EWSR1/CREM</i>), <i>CDK6</i> kinase in T-cell acute lymphoblastic leukemia (<i>FAM133B/CDK6</i>), and <i>CLTC</i> in breast cancer (<i>CLTC/VMP1</i>). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including <i>EGFRvIII</i> and <i>FIP1L1/PDGFRA</i>. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis.</p></div

Discovery of new cell line models for the known rearrangements, <i>EGFRvIII</i> and <i>FIP1L1/PDGFRA</i>.

<p>(<i>A</i>) Heatmap depicting genomic breakpoints within <i>EGFR</i> in the glioblastoma cell lines, CAS-1 and DKMG. (<i>B</i>) Identification of <i>EGFRvIII</i> in DKMG cells by paired-end RNA-seq. Paired-end reads supporting the rearrangement are depicted. (<i>C</i>) Verification of <i>EGFRvIII</i> expression by RT-PCR (top panel) and Western blotting (bottom panel) in DKMG. RT-PCR was done using primers flanking the exon 1/exon 8 junction of <i>EGFRvIII</i>, and Western blotting was done using an antibody specific to the EGFRvIII isoform. Control samples include U87 glioblastoma cells without <i>EGFR</i> rearrangement, U87-vIII cells engineered to express exogenous <i>EGFRvIII</i>, and A431 epidermoid carcinoma cells with <i>EGFR</i> amplification. (<i>D</i>) RBA identification of expression-level breakpoint within <i>PDGFRA</i> in SUPT13 T-ALL cells. ***<i>P<10⁻¹¹</i> (Student's t-test). (<i>E</i>) RNA-seq identification of <i>FIP1L1/PDGFRA</i>. (<i>F</i>) RT-PCR validation of <i>FIP1L1/PDGFRA</i> expression in SUPT13. (<i>G</i>) SUPT13 cells are sensitive to imatinib (IC₅₀ = 0.036 µM). K562 is a positive control CML cell line harboring BCR/ABL1 with known sensitivity to imatinib (IC₅₀ = 0.18 µM).</p