Project description:Comparative genomic hybridization analysis for detection of recurring gene copy number variation (CNV) among a set of lung cancer mestastatic brain tumors DNA was isolated and analyzed in a two-color experiment using Cancer CGH+SNP 180Kx4 arrays from Agilent and Agilent SureScan system: Cy5-labeled specimen DNA and Cy3-labeled Agilent characterized normal human reference DNA
Project description:Germline RUNX1 mutations are found in familial platelet disorders with predisposition to acute myelogenous leukemia (FPD/AML). This very rare disease is characterized by thrombocytopenia, platelet dysfunction and a 35% lifetime risk of developing MDS/AML and in rare cases also T-ALL. Here, we focus on a case of a man with a familial history of RUNX1 R174Q mutation who developed at the age of 42 years an EGIL T2-ALL and, two years after remission, an AML-M0. To investigate whether initial and relapsed leukemic blasts originated from the same clone, we performed CGH array and WES on both blasts populations. In both T2-ALL and AML-M0 samples, CGH array revealed loss of 1p36.32-23 and 17q11.2 and nine other small deletions. Both AML-M0 and T2-ALL blasts demonstrated clonal rearrangements of both TCRγ (Vγ9-Jγ1-1) and TCRδ (Dδ2-Jδ1 and Dδ2-Jδ3). 18 genes were found by WES to be mutated in both blasts at a frequency of more than 40%. Additional variants were identified only in T2-ALL or in AML-M0 evoking the existence of a common original clone, which gave rise to subclonal populations. MiSeq technology performed on peripheral blood-derived CD34+ cells five years prior T2-ALL development revealed only missense TET2 P1962T mutation at a frequency of 1% (which reaches a frequency of 50 % in fully transformed leukemic clone) suggesting that this mutation in association with germline RUNX1 R174Q mutation led to amplification of a hematopoietic clone susceptible to acquire other transforming alterations. Identification of clonal hematopoiesis with acquired mutations at low frequency in hematopoietic progenitors before leukemia development could clearly serve as a marker of pre-leukemic state and be helpful in patient care.
Project description:Expression of proteins regulating apoptosis (BCL-2, MCL-1, BCL-X and BAX) in acute myeloid leukemia (AML) blasts at diagnosis have been shown to be associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. This suggested a dynamic regulation of apoptosis. Herein, we further explored this aspect of apoptosis in AML. Firstly, the intra-individual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts showed a strong correlation, with expression values far beyond control lymphocytes. Secondly, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p=0.0067 and p=1.0 respectively). Using proteomics we further set out to identify novel proteins possibly engaged in apoptosis regulation. Proteomics analysis revealed that major functional protein clusters upregulated in secretomes of apoptosis-resistant AML, were presumably engaged in global gene regulation including mRNA splicing, protein translation and chromatin remodeling.
Project description:CD34-positive and negative fractions were used for genomic DNA preparation, which was hybridized to HGU133 Plus 2.0 arrays. Keywords: Disease state analysis
Project description:Expression of proteins regulating apoptosis (BCL-2, MCL-1, BCL-X and BAX) in acute myeloid leukemia (AML) blasts at diagnosis have been shown to be associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. This suggested a dynamic regulation of apoptosis. We hypothesized that expression of apoptosis-related proteins in AML blasts, and possibly also in bystander cells in the bone marrow, is regulated by extracellular factors present in the AML microenvironment. Tumor cell communication with its microenvironment is emerging as an important determinant playing multiple roles in cancer. Both soluble factors and extracellular vesicles (EVs), most notably exosomes, have been shown to influence cellular processes of malignant and normal cells in the tumor microenvironment. We performed a proteomics analysis of the whole secretome as well as of EVs secreted by AML blasts to pinpoint released protein factors that might mediate apoptosis-resistance.
Project description:MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and development of leukemia. Great interest emerged in modulating miRNA expression for therapeutic purposes. In order to identify miRNAs, which specifically suppress leukemic growth of AML with t(8;21), inv(16) or MLL-rearrangement by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases. Four miRNAs, specifically downregulated in MLL-rearranged, t(8;21) or inv(16) AMLs, were characterized by their tumor suppressive properties in cell lines representing those respective cytogenetic groups. Among those, forced expression of miR-9 reduced leukemic growth and induced monocytic differentiation of t(8;21) AML cell lines in vitro and in vivo. The tumor suppressive functions of miR-9 were specifically restricted to AML cell lines and primary leukemic blasts with t(8;21). On the other hand, these functions were not evident in AML blasts from patients with MLL-rearrangements. We showed that miR-9 exerts its effects through the cooperation with let-7 to repress the oncogenic LIN28B/HMGA2 axis. Thus, miR-9 is a tumor suppressor-miR which acts in a stringent cell context-dependent manner. In order to identify miRNAs, which specifically suppress leukemic growth of AML with t(8;21) (n=21), inv(16) (n=17) or MLL-rearrangement (n=35) by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases, which also included 12 t(15;17) and 5 t(7;12) samples.
Project description:Primary human AML bone marrow sample (newly diagnosed, prior to treatment initation) was obtained from donor after consent and AML blasts were isolated by standard Ficoll centrifugation. AML blasts were pulse-treated with daunorubicine (100 nM, 200 nM, 400 nM) or DMSO control for 1 hour, followed by wash-out and short-term cell culture to allow cellular responses to develop. 20,000 cells were analysed by LC-MS3, and 5,000 cells were subjected to flow cytometry apoptosis assay.
Project description:Expression of proteins regulating apoptosis (BCL-2, MCL-1, BCL-X and BAX) in acute myeloid leukemia (AML) blasts at diagnosis have been shown to be associated with disease-free survival. The concentrations of these proteins are combined in the Anti Apoptotic index (AAI). We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. This suggested a dynamic regulation of apoptosis. We hypothesized that expression of apoptosis-related proteins in AML blasts, and possibly also in bystander cells in the bone marrow, is regulated by extracellular factors present in the AML microenvironment. Tumor cell communication with its microenvironment is emerging as an important determinant playing multiple roles in cancer. Both soluble factors and extracellular vesicles (EVs), most notably exosomes, have been shown to influence cellular processes of malignant and normal cells in the tumor microenvironment. We performed a proteomics analysis of the whole secretome as well as of EVs secreted by AML blasts to pinpoint released protein factors that might mediate apoptosis-resistance.
Project description:Primary human AML cells (newly diagnosed, prior to treatment initation) were obtained from donor after consent and AML blasts were isolated by standard Ficoll centrifugation. AML blasts were treated with FLT3 inhibitor gilteritinib (or DMSO vehicle control) at 10 nM concentration for 6 h, followed by harvest for LC/MS.
Project description:Array Comparative Genomic Hybridization (aCGH) of 70 pancreatic ductal adenocarcinoma (PDAC) samples was performed on Agilent 244K CGH arrays in order to find common genomic aberrations for cancer gene discovery. Additionally, matched expression profiling on Agilent 44K arrays was performed. Common copy number aberrations were identified in order to identify a list of putative cancer genes. Expression profiling data was used to further enrich this list of putative cancer genes for more likely candidates. Last, the most promising candidates were functionally interrogated using RNA interference-mediated knockdown to mimic loss. Well-known PDAC cancer genes were observed as amplified (KRAS and MYC) and deleted (CDKN2A, TGFBR2, SMAD4, and MAP2K4).