Genetic modification of primary human B cells to model high-grade lymphoma
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ABSTRACT:
PROVIDER: EGAS00001003560 | EGA |
REPOSITORIES: EGA
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Project description:Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models.
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Project description:The use of stem cells for regenerative medicine has captured the imagination of the public, with media attention contributing to rising expectations of clinical benefits. Human embryonic stem cells (hESCs) are the best model for capital investment in stem cell therapy and there is a clear need for their robust genetic characterization before scaling-up cell expansion for that purpose. We have to be certain that the genome of the starting material is stable and normal, but the limited resolution of conventional karyotyping is unable to give us such assurance. Advanced molecular cytogenetic technologies such as array comparative genomic hybridization for identifying chromosomal imbalances, and single nucleotide polymorphism analysis for identifying ethnic background and loss of heterozygosity should be introduced as obligatory diagnostic tests for each newly derived hESC line before it is deposited in national stem cell banks. If this new quality standard becomes a requirement, as we are proposing here, it would facilitate and accelerate the banking process, since end-users would be able to select the most appropriate line for their particular application, thus improving efficiency and streamlining the route to manufacturing therapeutics. The pharmaceutical industry, which may use hESC-derived cells for drug screening, should not ignore their genomic profile as this may risk misinterpretation of results and significant waste of resources.
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Project description:Mature NK and T-cell lymphomas are occasionally encountered in Asia but are very rare in Western populations. In part due to its rarity, little is known about this group of neoplasms, and despite being rather different disease entities, they are all treated similarly but with diverse clinical outcomes. Novel biomarkers (at both the genetic and protein levels) are needed to resolve diagnostic difficulties, improve prognostication and develop targeted therapies. To rectify this deficiency, we interrogated the transcriptome of several NK and mature T-cell lymphomas by whole-genome expression profiling for new markers that may further stratify this diverse group of conditions. Our initial efforts have identified a promising candidate marker that appears to differentiate NKTL lymphoma from other forms of T-cell neoplasms, and this finding has been validated by immunohistochemistry on archival material in a large number of patient cases.
2011-03-01 | GSE20874 | GEO
Project description:Fallopian tube secretory epithelial cells (FTSECs) have been suggested to be the source of high-grade serous ovarian carcinoma (HGSOC). Although several genetic alterations are known to be involved in HGSOC development, the minimal requirements remain unclear. We aimed to identify oncogenic mutations indispensable for HGSOC development in a stepwise model, using immortalized FTSECs. FTSECs were isolated from clinical samples and immortalized by overexpression of cyclin D1, CDK4R24C, and hTERT. Oncogenic mutations in the p53, c-Myc, and RAS/PI3K pathways were mimicked by lentiviral transduction. We found two distinct patterns of gene alteration essential for HGSOC development: p53/KRAS/AKT and p53/KRAS/c-Myc. Dominant-negative p53, alone or combined with oncogenic KRAS (KRASV12), constitutively active AKT (CA-AKT), and c-Myc, did not induce tumorigenesis in immortalized cells; however, overexpression of CA-AKT or c-Myc, along with dominant-negative p53 and KRASV12, conferred tumorigenic potential. Transformed FTSECs formed tumors in nude mice that were grossly, histologically, and immunohistochemically similar to human HGSOCs. Interestingly, mice harboring tumors with c-Myc amplifications displayed extensive metastases, consistent with the increased dissemination in their human counterparts. Thus, aberrant p53/KRASV12/c-Myc or p53/KRASV12/PI3K-AKT signaling was the minimum requirement for FTSEC carcinogenesis. The model based on this evidence could shed light on the early stages of HGSOC development.
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2012-06-01 | GSE36982 | GEO
Project description:Mature NK and T-cell lymphomas are occasionally encountered in Asia but are very rare in Western populations. In part due to its rarity, little is known about this group of neoplasms, and despite being rather different disease entities, they are all treated similarly but with diverse clinical outcomes. Novel biomarkers (at both the genetic and protein levels) are needed to resolve diagnostic difficulties, improve prognostication and develop targeted therapies. To rectify this deficiency, we interrogated the transcriptome of several NK and mature T-cell lymphomas by whole-genome expression profiling for new markers that may further stratify this diverse group of conditions. Our initial efforts have identified a promising candidate marker that appears to differentiate NKTL lymphoma from other forms of T-cell neoplasms, and this finding has been validated by immunohistochemistry on archival material in a large number of patient cases. Correlating gene expression status with tumor type. Gene expression profiling was performed on 8 cases of angioimmunoblastic T-cell lymphoma (AITL), 4 cases of natural killer T-cell lymphoma (NKTL), and 6 cases of peripheral T-cell lymphoma - unspecified (PTCL-U). In addition, expression profiling was performed on one xenograft tumor, which was derived from a primary NKTL, and its cultured line.
2011-03-01 | E-GEOD-20874 | biostudies-arrayexpress
Project description:PURPOSE OF REVIEW:Rituximab-based chemoimmunotherapy has resulted in a marked improvement in the survival of diffuse large B cell lymphoma (DLBCL). We reflect upon the history front-line (1L) therapy and highlight advances in management. RECENT FINDINGS:Since the introduction of R-CHOP, the majority of randomized studies in the front-line treatment of DLBCL have failed to show a benefit. Such studies have involved treatment intensification, adding novel agents to the R-CHOP backbone and targeting such novel agents to biologically defined subgroups. R-CHOP therefore remains standard-of-care for most but new insights into the molecular biology of these diseases, and the development of active targeted molecules offers promise for the future. Accumulating evidence in the very elderly suggests dose attenuation does not compromise survival. Intensification in primary mediastinal B cell lymphoma may avoid the need for radiotherapy, but must be balanced against the risks. PET-CT- and ctDNA-based response assessment may now enable response adapted therapy and early prognostication, improving patient selection and potentially outcomes. Novel technologies and therapies in combination with novel molecular diagnostics will likely become the standard-of-care approach for the personalized therapy of DLBCL but need to be proven in well-designed and conducted randomized trials.
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Project description:The pathways responsible for tumorigenesis of high grade serous ovarian cancer (HGSOC) from the fallopian tube epithelium (FTE) are still poorly understood. A human prolactin (PRL) like gene, Prl2c2 was amplified >100 fold in a spontaneous model of FTE-derived ovarian cancer (MOEhigh - murine oviductal epithelium high passage). Prl2c2 stable knockdown in MOEhigh cells demonstrated a significant reduction in cell proliferation, 2-dimensional foci, anchorage independent growth, and blocked tumor formation. The overall survival of ovarian cancer patients from transcriptome analysis of 1868 samples was lower when abundant PRL and prolactin receptors (PRL-R) were expressed. A HGSOC cell line (OVCAR3) and a tumorigenic human FTE cell line (FT33-Tag-Myc) were treated with recombinant PRL and a significant increase in cellular proliferation was detected. A CRISPR/Cas9 mediated PRL-R deletion in OVCAR3 and FT33-Tag-Myc cells demonstrated significant reduction in cell proliferation and eliminated tumor growth using the OVCAR3 model. PRL was found to phosphorylate STAT5, m-TOR and ERK in ovarian cancer cells. This study identified Prl2c2 as a driver of tumorigenesis in a spontaneous model and confirmed that prolactin signaling supports tumorigenesis in high grade serous ovarian cancer.
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