DNA-Seq of human Acute Panmyelosis with Myelofibrosis (APMF) Samples
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ABSTRACT: FFPE Bone marrow core biopsis of a cohort of 16 APMF Samples were analysed by OncoScan copy number analysis (E-MTAB-7888). 5 out of this 16 Samples had enough DNA available for NGS using the Illumina TruSight Myeloid and/or Tumor 15 NGS Gene Panels.
Project description:Affymetrix Oncoscan assays were performed according to the manufacturer's instructions from DNA extracted from FFPE tissues. Copy number analysis using Affymetrix Oncoscan arrays was performed for 16 APMF Samples.
Project description:Upstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leucocyte antigen (HLA)-presented peptides and suggest their potential role in cancer cell development and survival. However, it is so far unclear if uORF-encoded peptides could serve as tumor-associated antigen targets and thus also play a role in cancer immune surveillance. Combining mass spectrometry-based immunopeptidome analysis in primary tumor and healthy tissues and evaluation of proto-oncogene-associated uORF-mediated translational control we here identified a panel of HLA-presented tumor-associated uORF-derived antigens. These uORF-derived tumor antigens were further shown to induce multifunctional antigen-specific T cells, validating their suitability as antigen targets for T cell-based cancer immunotherapy. Our data further unravel the role of uORF-encoded peptides in malignant disease, suggesting uORF-derived tumor-associated antigens as targets for anti-cancer immune surveillance and immunotherapy development.
Project description:Identification of physiologically relevant peptide vaccine targets calls for the direct analysis of the entirety of naturally presented human leukocyte antigen (HLA) ligands, termed the HLA ligandome. In this study, we implemented this direct approach using immunoprecipitation and mass spectrometry to define acute myeloid leukemia (AML)-associated peptide vaccine targets. Mapping the HLA class I ligandomes of 15 AML patients and 35 healthy controls, more than 25 000 different naturally presented HLA ligands were identified. Target prioritization based on AML exclusivity and high presentation frequency in the AML cohort identified a panel of 132 LiTAAs (ligandome-derived tumor-associated antigens), and 341 corresponding HLA ligands (LiTAPs (ligandome-derived tumor-associated peptides)) represented subset independently in >20% of AML patients. Functional characterization of LiTAPs by interferon-γ ELISPOT (Enzyme-Linked ImmunoSpot) and intracellular cytokine staining confirmed AML-specific CD8+ T-cell recognition. Of note, our platform identified HLA ligands representing several established AML-associated antigens (e.g. NPM1, MAGED1, PRTN3, MPO, WT1), but found 80% of them to be also represented in healthy control samples. Mapping of HLA class II ligandomes provided additional CD4+ T-cell epitopes and potentially synergistic embedded HLA ligands, allowing for complementation of a multipeptide vaccine for the immunotherapy of AML.
Project description:Patients with cytogenetically normal acute myeloid leukemia (CN-AML) show heterogeneous treatment outcomes. We used gene expression profiling to develop a gene signature that predicts overall survival (OS) in CN-AML. Based on data from 163 patients treated in the German AMLCG 1999 trial and analyzed on oligonucleotide microarrays, we used supervised principal component analysis to identify 86 probe sets (representing 66 different genes) which correlated with OS, and defined a prognostic score based on this signature. When applied to an independent cohort of 79 CN-AML patients, this continuous score remained a significant predictor for OS (hazard ratio [HR], 1.85; P=0.002), EFS (HR, 1.73; P=0.001), and RFS (HR, 1.76; P=0.025). It kept its prognostic value in multivariate analyses adjusting for age, FLT3 ITD and NPM1 status. In a validation cohort of 64 CN-AML patients treated on CALGB study 9621, the score also predicted OS (HR, 4.11; P<0.001), EFS (HR, 2.90; P<0.001), and RFS (HR, 3.14, P<0.001) and retained its significance in a multivariate model for OS. In summary, we present a novel gene expression signature that offers additional prognostic information for patients with CN-AML. Experiment Overall Design: Analysis of 163 samples of bone morrow or peripheral blood mononuclear cells from adult patients with untreated acute myeloid leukemia
Project description:Purpose: CEBPA mutations are found as either biallelic (biCEBPA) or monoallelic (moCEBPA). We set out to explore whether the kind of CEBPA mutation is of prognostic relevance in cytogenetically normal AML (CN-AML). Patients and Methods: 467 homogeneously treated CN-AML patients were subdivided into moCEBPA, biCEBPA and wildtype (wt) CEBPA patients. The subgroups were analyzed for clinical parameters and for additional mutations in the NPM1, FLT3 and MLL genes. Furthermore, we obtained gene expression profiles (GEP) for a subgroup of 61 patients using oligonucleotide microarrays. 61 bone-marrow samples from CN-AML patients were analyzed using Affymetrix HG-U133 oligonucleotide microarrays (Affymetrix, Santa Clara, CA). Sample preparation, hybridization and image acquisition were performed according to standard Affymetrix protocols. Custom chip definition files based on the GeneAnnot database were used for data analysis (Ferrari et al, BMC Bioinformatics 8:446). The twilight algorithm was used to comapre gene expression profiles of patients with wildtype CEBPA and mono- and biallelic CEBPA mutations.
Project description:Acute myeloid leukemia (AML) is a heterogeneous disease in respect of molecular aberrations and prognosis. We used gene expression profiling of 562 patients treated in the German AMLCG 1999 trial to develop a gene signature that predicts survival in AML. Analysis of 562 samples (140 HGU-133plus2; 422 HGU-133A; 422 HGU-133B) from adult patients with acute myeloid leukemia (AML).
Project description:In acute myeloid leukemia (AML) non-random clonal chromosome aberrations are detectable in ~55% of adults with AML. Translocation t(8;21)(q22;q22) resulting in the 5'RUNX1/3'RUNX1T1 fusion gene occurs in ~8% of acute myeloid leukemia (AML) cases. Also, insertions ins(8;21) and ins(21;8) have been described that show a broad heterogeneity at the molecular level with inserted fragment sizes ranging from 2.4 to 44 Mb. Microarray-based comparative genomic hybridization (arrayCGH) in 49 intermediate-risk AML and RT-PCR-based screening in 532 AML cases allowed the detection of ins(21;8)/ins(8;21) in three cases; arrayCGH and subsequent RT-PCR revealed an ~0.5 Mb sized inserted fragment generating the 5'RUNX1/3'RUNX1T1 fusion gene in one case with a submicroscopic ins(21;8)(q22;q22q22) whereas the other two cases were identified by banding analysis and RT-PCR, respectively. Gene expression profiling (GEP) and a detailed review of the literature highlighted similar biological features of AML cases with ins(21;8)/ins(8;21) and t(8;21)(q22;q22). Our study demonstrates the potential of high-resolution array-based analysis and GEP and provides further evidence that AML with insertions generating the 5'RUNX1/3'RUNX1T1 fusion not only biologically resemble the t(8;21)(q22;q22) AML subgroup, but might also share their prognostically favorable clinical behavior. Thus, similar treatment options should be considered in these patients. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
Project description:Acute myeloid leukemia with normal karyotype (NK-AML) represents a cytogenetic grouping with intermediate prognosis but substantial molecular and clinical heterogeneity. Within this subgroup, presence of FLT3 (FMS-like tyrosine kinase 3) internal tandem duplication (ITD) mutation predicts less favorable outcome. The goal of our study was to discover gene-expression patterns correlated with FLT3-ITD mutation, and to evaluate the utility of a FLT3 signature for prognostication. The dataset comprises gene-expression profiles of 137 normal karyotype acute myeloid leukemia (NK-AML) specimens carried out using Stanford cDNA microarrays, to accompany the study of L Bullinger et al. For each array, Channel 2 represents Cy5-labeled NK-AML RNA, and Channel 1 Cy3-labeled universal reference RNA. Keywords: Logical Set DNA microarrays were used to profile gene expression in a training set of 65 NK-AML cases. Supervised analysis was applied to build a gene expression-based predictor of FLT3-ITD mutation status. The predictor was then evaluated by classifying expression profiles from an independent test set of 72 NK-AML cases.
Project description:Despite improvement of current treatment strategies and novel targeted drugs, relapse and treatment resistance determine the major cause of death for acute myeloid leukemia (AML) patients. To identify the underlying molecular characteristics, numerous studies have been aimed to decipher the genomic- and transcriptomic landscape of AML. Nevertheless, further molecular changes allowing malignant cells to escape treatment are yet to be elucidated. Mass spectrometry is a powerful tool enabling detailed insights into proteomic changes that could explain AML relapse and resistance. Here, we investigated AML samples from 47 adult and 22 pediatric patients at serial time-points during disease progression using high resolution isoelectric focusing liquid chromatography mass spectrometry. We show that the proteomic profile at relapse is enriched for mitochondrial ribosomal proteins and subunits of the respiratory chain complex, indicative of reprogrammed energy metabolism from diagnosis to relapse. Further, higher levels of granzymes and lower levels of the anti-inflammatory protein CR1/CD35 suggest an inflammatory signature promoting disease progression. Finally, through a proteogenomic approach, we detected novel peptides, which present a promising repertoire in the search for biomarkers and tumor-specific druggable targets. Altogether, this study highlights the importance of proteomic studies in holistic approaches to improve treatment and survival of AML patients.
Project description:In this report we demonstrate that the ability to alter self-renewal in vitro and in vivo is a more generalized property of leukemia-associated oncogenes. We further demonstrate that disparate leukemia-associated oncogenes initiate early common and overlapping transformation and self-renewal gene expression programs to mediate these effects. Furthermore, elements of these programs can be detected in established leukemia stem cells from an animal model and across a large cohort of patients with differing acute myeloid leukemia (AML) subtypes, where they strongly predict for disease biology. Finally, individual genes from the programs are demonstrated to partially phenocopy the leukemia-associated oncogenes and themselves alter self-renewal in committed murine progenitors and generate AML when expressed in murine bone marrow. A total of 253 RNA samples derived from adult AML patients were provided by the German Austrian AML Study Group (AMLSG) [AMLSG trials AML HD98A (ClinicalTrials.gov Identifier: NCT00146120), and AML HD98B (Schlenk et al., 2009)]. Conventional cytogenetic banding, and FLT3, CEBPA and NPM1 mutational analysis were performed as previously described (Schlenk et al., N Engl J Med 2008). Detailed clinical, cytogenetic and molecular cytogenetic information are also provided in Table S3 along with the publication. Following enrichment, all samples contained at least 80% leukemic cells. Gene expression profiling (GEP) was performed as previously described (Bullinger et al., N Engl J Med 2004). Separate filtering and batch centering were performed for the group of 253 Samples (relative to GSE16432). Filtered data presented as a supplementary file at the foot of this record.