Project description:Somatically acquired chromosomal translocation is a common mechanism of oncogene activation in many haematopoietic tumours and sarcomas. However, very few recurrent chromosomal translocations have been reported in more common epithelial tumours such as lung carcinomas.We established a cell line HCC2429 from an aggressive, metastatic lung cancer arising in a young, non-smoking woman, demonstrating a t(15;19)(q13.2;p13.1). The breakpoints on chromosomes 15 and 19 were cloned using long distance inverse PCR and we determined by RT-PCR that the translocation results in a novel fusion transcript in which the 3' end Brd4 on chromosome 19p is fused to the 5' end of NUT on chromosome 15q.In total, 128 lung cancer tissues were screened using fluorescent in situ hybridisation, but none of the tumours screened demonstrated t(15;19), suggesting that this translocation is not commonly found in lung cancer. Consistent with previous literature, ectopic expression of wild type Brd4 was shown to inhibit G(1) to S progression. However, we also found that the Brd4-NUT fusion augments the inhibition of progression to S phase compared with wild type Brd4.Alteration in cell cycle kinetics is important in tumorigenesis, although the exact role of Brd4-NUT fusion protein in the pathogenesis of lung cancers remains unclear and need to be further elucidated.
Project description:Chromosomal rearrangements resulting in gene fusions are frequently involved in carcinogenesis. Here, we describe a semiautomatic procedure for identifying fusion gene transcripts by using publicly available mRNA and EST databases. With this procedure, we have identified 96 transcript sequences that are derived from 60 known fusion genes. Also, 47 or more additional sequences appear to be derived from 20 or more previously unknown putative fusion genes. We have experimentally verified the presence of a previously unknown IRA1/RGS17 fusion in the breast cancer cell line MCF7. The fusion gene encodes the full-length RGS17 protein, a regulator of G protein-coupled signaling, under the control of the IRA1 gene promoter. This study demonstrates that databases of ESTs can be used to discover fusion genes resulting from structural rearrangement of chromosomes.
Project description:ENDOGLIN/CD105 (ENG) is a transmembrane glycoprotein and an auxiliary unit of the transforming growth factor-β (TGF-β); receptor, expressed predominantly in vascular endothelium. Noteworthy, Eng mRNA expression has been reported also in Kit(+) interstitial cells of Cajal (ICC) in the mouse intestine. Gastrointestinal stromal tumours (GIST) are thought to derive from ICC. Here we have investigated Eng expression in the Kit(K641E) mouse GIST model, in human GIST and in the Ba/F3 cell model. In wild type (WT) mouse antrum, Eng immunoreactivity (-ir) was detected in CD34(+) /CD31(+) endothelium and in Kit(+) ICC. In Kit(K641E) mice, hyperplasia of Kit(+) cells made Eng-ir even more evident. Quantitative PCR confirmed the increased expression of Eng transcript in Kit(K641E) mice. On human GIST TMA, 26/49 cases stained positive for ENG. Strong ENG staining was associated with malignant and high-risk tumours. ENG negative cases were predominantly of the epithelioid type or harboured PDGFRA mutation. In vitro, Eng mRNA was up-regulated in Ba/F3 cell lines stably expressing various oncogenic Kit mutations (K641E, del559, del814). This effect appeared to be independent of Kit activation, as neither the stimulation of WT Kit by its ligand SCF, nor the inhibition of Kit autophosphorylation by imatinib mesylate in oncogenic mutants, altered Eng expression. Elevated Eng expression in Kit oncogenic mutants appeared rather to be indirectly mediated by DNA hypomethylation, because treatment with the demethylating agent 5-Aza/dC increased Eng mRNA expression in Kit(WT) cells. ENG expression in ICC and in GIST deserves further consideration as ENG is emerging as a potential target for cancer therapy.
Project description:BACKGROUND:Entrectinib is a potent inhibitor of tropomyosin receptor kinase (TRK) A, B, and C, which has been shown to have anti-tumour activity against NTRK gene fusion-positive solid tumours, including CNS activity due to its ability to penetrate the blood-brain barrier. We present an integrated efficacy and safety analysis of patients with metastatic or locally advanced solid tumours harbouring oncogenic NTRK1, NTRK2, and NTRK3 gene fusions treated in three ongoing, early-phase trials. METHODS:An integrated database comprised the pivotal datasets of three, ongoing phase 1 or 2 clinical trials (ALKA-372-001, STARTRK-1, and STARTRK-2), which enrolled patients aged 18 years or older with metastatic or locally advanced NTRK fusion-positive solid tumours who received entrectinib orally at a dose of at least 600 mg once per day in a capsule. All patients had an Eastern Cooperative Oncology Group performance status of 0-2 and could have received previous anti-cancer therapy (except previous TRK inhibitors). The primary endpoints, the proportion of patients with an objective response and median duration of response, were evaluated by blinded independent central review in the efficacy-evaluable population (ie, patients with NTRK fusion-positive solid tumours who were TRK inhibitor-naive and had received at least one dose of entrectinib). Overall safety evaluable population included patients from STARTRK-1, STARTRK-2, ALKA-372-001, and STARTRK-NG (NCT02650401; treating young adult and paediatric patients [aged ?21 years]), who received at least one dose of entrectinib, regardless of tumour type or gene rearrangement. NTRK fusion-positive safety evaluable population comprised all patients who have received at least one dose of entrectinib regardless of dose or follow-up. These ongoing studies are registered with ClinicalTrials.gov, NCT02097810 (STARTRK-1) and NCT02568267 (STARTRK-2), and EudraCT, 2012-000148-88 (ALKA-372-001). FINDINGS:Patients were enrolled in ALKA-372-001 from Oct 26, 2012, to March 27, 2018; in STARTRK-1 from Aug 7, 2014, to May 10, 2018; and in STARTRK-2 from Nov 19, 2015 (enrolment is ongoing). At the data cutoff date for this analysis (May 31, 2018) the efficacy-evaluable population comprised 54 adults with advanced or metastatic NTRK fusion-positive solid tumours comprising ten different tumour types and 19 different histologies. Median follow-up was 12.9 months (IQR 8·77-18·76). 31 (57%; 95% CI 43·2-70·8) of 54 patients had an objective response, of which four (7%) were complete responses and 27 (50%) partial reponses. Median duration of response was 10 months (95% CI 7·1 to not estimable). The most common grade 3 or 4 treatment-related adverse events in both safety populations were increased weight (seven [10%] of 68 patients in the NTRK fusion-positive safety population and in 18 [5%] of 355 patients in the overall safety-evaluable population) and anaemia (8 [12%] and 16 [5%]). The most common serious treatment-related adverse events were nervous system disorders (three [4%] of 68 patients and ten [3%] of 355 patients). No treatment-related deaths occurred. INTERPRETATION:Entrectinib induced durable and clinically meaningful responses in patients with NTRK fusion-positive solid tumours, and was well tolerated with a manageable safety profile. These results show that entrectinib is a safe and active treatment option for patients with NTRK fusion-positive solid tumours. These data highlight the need to routinely test for NTRK fusions to broaden the therapeutic options available for patients with NTRK fusion-positive solid tumours. FUNDING:Ignyta/F Hoffmann-La Roche.
Project description: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.
Project description:Objectives:To investigate the added value of assessing transcripts for the long cAMP phosphodiesterase-4D (PDE4D) isoforms, PDE4D5 and PDE4D9, regarding the prognostic power of the 'CAPRA & PDE4D7' combination risk model to predict longitudinal postsurgical biological outcomes in prostate cancer. Patients and Methods:RNA was extracted from both biopsy punches of resected tumours (606 patients; RP cohort) and diagnostic needle biopsies (168 patients; DB cohort). RT-qPCR was performed in order to determine PDE4D5, PDE4D7, and PDE4D9 transcript scores in both study cohorts. By RNA sequencing, we determined the TMPRSS2-ERG fusion status of each tumour sample in the RP cohort. Kaplan-Meier survival analyses were then applied to correlate the PDE4D5, PDE4D7 and PDE4D9 scores with postsurgical patient outcomes. Logistic regression was then used to combine the clinical CAPRA score with PDE4D5, PDE4D7, and PDE4D9 scores in order to build a 'CAPRA & PDE4D5/7/9' regression model. ROC and decision curve analysis was used to estimate the net benefit of the 'CAPRA & PDE4D5/7/9' risk model. Results:Kaplan-Meier survival analysis, on the RP cohort, revealed a significant association of the PDE4D7 score with postsurgical biochemical recurrence (BCR) in the presence of the TMPRSS2-ERG gene rearrangement (logrank p<0.0001), compared to the absence of this gene fusion event (logrank p=0.08). In contrast, the PDE4D5 score was only significantly associated with BCR in TMPRSS2-ERG fusion negative tumours (logrank p<0.0001 vs. logrank p=0.4 for TMPRSS2-ERG+ tumours). This was similar for the PDE4D9 score although less pronounced compared to that of the PDE4D5 score (TMPRSS2ERG- logrank p<0.0001 vs. TMPRSS2ERG+ logrank p<0.005). In order to predict BCR after primary treatment, we undertook ROC analysis of the logistic regression combination model of the CAPRA score with the PDE4D5, PDE4D7, and PDE4D9 scores. For the DB cohort, this demonstrated significant differences in the AUC between the CAPRA and the PDE4D5/7/9 regression model vs. the CAPRA and PDE4D7 risk model (AUC 0.87 vs. 0.82; p=0.049) vs. the CAPRA score alone (AUC 0.87 vs. 0.77; p=0.005). The CAPRA and PDE4D5/7/9 risk model stratified 19.2% patients of the DB cohort to either 'no risk of biochemical relapse' (NPV 100%) or the 'start of any secondary treatment (NPV 100%)', over a follow-up period of up to 15 years. Decision curve analysis presented a clear, net benefit for the use of the novel CAPRA & PDE4D5/7/9 risk model compared to the clinical CAPRA score alone or the CAPRA and PDE4D7 model across all decision thresholds. Conclusion:Association of the long PDE4D5, PDE4D7, and PDE4D9 transcript scores to prostate cancer patient outcome, after primary intervention, varies in opposite directions depending on the TMPRSS2-ERG genomic fusion background of the tumour. Adding transcript scores for the long PDE4D isoforms, PDE4D5 and PDE4D9, to our previously presented combination risk model of the combined 'CAPRA & PDE4D7' score, in order to generate the CAPRA and PDE4D5/7/9 score, significantly improves the prognostic power of the model in predicting postsurgical biological outcomes in prostate cancer patients.
Project description:BACKGROUND:RNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5-35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data. METHODS:We describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization. RESULTS:We demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance. CONCLUSIONS:The approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis.