Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 25
Filter
1.
Bioinformatics ; 33(19): 2977-2985, 2017 Oct 01.
Article in English | MEDLINE | ID: mdl-28481978

ABSTRACT

MOTIVATION: Although sequencing-based technologies are becoming the new reference in genome analysis, comparative genomic hybridization arrays (aCGH) still constitute a simple and reliable approach for copy number analysis. The most powerful algorithms to analyze such data have been freely provided by the scientific community for many years, but combining them is a complex scripting task. RESULTS: The cghRA framework combines a user-friendly graphical interface and a powerful object-oriented command-line interface to handle a full aCGH analysis, as is illustrated in an original series of 107 Diffuse Large B-Cell Lymphomas. New algorithms for copy-number calling, polymorphism detection and minimal common region prioritization were also developed and validated. While their performances will only be demonstrated with aCGH, these algorithms could actually prove useful to any copy-number analysis, whatever the technique used. AVAILABILITY AND IMPLEMENTATION: R package and source for Linux, MS Windows and MacOS are freely available at http://bioinformatics.ovsa.fr/cghRA. CONTACT: mareschal@ovsa.fr or fabrice.jardin@chb.unicancer.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
Comparative Genomic Hybridization , Lymphoma, Large B-Cell, Diffuse/genetics , Algorithms , Genomics , Humans , Polymorphism, Genetic , Software
3.
Cell Mol Life Sci ; 74(20): 3851-3861, 2017 10.
Article in English | MEDLINE | ID: mdl-28601984

ABSTRACT

Enterovirus infections are implicated in the development of type 1 diabetes (T1D). MicroRNAs as regulators of gene expression are involved in many physiological and pathological processes. Given that viral infections dysregulate cellular microRNAs, we investigated the impact of persistent coxsackievirus B4 infection on microRNA expression of human pancreatic cells. Next-generation sequencing was used to determine microRNA expression in PANC-1 cells persistently infected (for several weeks) with coxsackievirus B4 and uninfected control cells. Target prediction restricted to T1D risk genes was performed with miRWalk2.0. Functional annotation analysis was performed with DAVID6.7. Expression of selected microRNAs and T1D risk genes was measured by quantitative reverse-transcription polymerase chain reaction. Eighty-one microRNAs were dysregulated in persistently infected PANC-1 cells. Forty-nine of the known fifty-five T1D risk genes were predicted as putative targets of at least one of the dysregulated microRNAs. Most functional annotation terms that were enriched in these 49 putative target genes were related to the immune response or autoimmunity. mRNA levels of AFF3, BACH2, and IL7R differed significantly between persistently infected cells and uninfected cells. This is the first characterization of the microRNA expression profile changes induced by persistent coxsackievirus B4 infection in pancreatic cells. The predicted targeting of genes involved in the immune response and autoimmunity by the dysregulated microRNAs as well as the dysregulated expression of diabetes risk genes shows that persistent coxsackievirus B4 infection profoundly impacts the host cell. These data support the hypothesis of a possible link between persistent coxsackievirus B4 infection and the development of T1D.


Subject(s)
Coxsackievirus Infections/genetics , Enterovirus B, Human/physiology , Gene Expression Regulation , MicroRNAs/genetics , Pancreas/cytology , Pancreas/virology , Cell Line , Coxsackievirus Infections/complications , Coxsackievirus Infections/virology , Diabetes Mellitus, Type 1/etiology , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/virology , Humans , Pancreas/metabolism
4.
Neurobiol Dis ; 96: 312-322, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27663142

ABSTRACT

CAG triplet expansions in Ataxin-2 gene (ATXN2) cause spinocerebellar ataxia type 2 and have a role that remains to be clarified in Parkinson's disease (PD). To study the molecular events associated with these expansions, we sequenced them and analyzed the transcriptome from blood cells of controls and three patient groups diagnosed with spinocerebellar ataxia type 2 (herein referred to as SCA2c) or PD with or without ATXN2 triplet expansions (named SCA2p). The transcriptome profiles of these 40 patients revealed three main observations: i) a specific pattern of pathways related to cellular contacts, proliferation and differentiation associated with SCA2p group, ii) similarities between the SCA2p and sporadic PD groups in genes and pathways known to be altered in PD such as Wnt, Ephrin and Leukocyte extravasation signaling iii) RNA metabolism disturbances with "RNA-binding" and "poly(A) RNA-binding" as a common feature in all groups. Remarkably, disturbances of ALS signaling were shared between SCA2p and sporadic PD suggesting common molecular dysfunctions in PD and ALS including CACNA1, hnRNP, DDX and PABPC gene family perturbations. Interestingly, the transcriptome profiles of patients with parkinsonian phenotypes were prevalently associated with alterations of translation while SCA2c and PD patients presented perturbations of splicing. While ATXN2 RNA expression was not perturbed, its protein expression in immortalized lymphoblastoid cells was significantly decreased in SCA2c and SCA2p versus control groups assuming post-transcriptional biological perturbations. In conclusion, the transcriptome data do not exclude the role of ATXN2 mutated alleles in PD but its decrease protein expression in both SCA2c and SCA2p patients suggest a potential involvement of this gene in PD. The perturbations of "RNA-binding" and "poly(A) RNA-binding" molecular functions in the three patient groups as well as gene deregulations of factors not yet described in PD but known to be deleterious in other neurological conditions, suggest the existence of RNA-binding disturbances as a continuum between spinocerebellar ataxia type 2 and Parkinson's disease.


Subject(s)
Parkinson Disease/etiology , Parkinson Disease/metabolism , RNA/metabolism , Spinocerebellar Ataxias/complications , Spinocerebellar Ataxias/metabolism , Adult , Aged , Ataxin-2/metabolism , Case-Control Studies , Disease Progression , Female , Humans , Male , Microarray Analysis , Middle Aged , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/metabolism , Transcriptome , Trinucleotide Repeat Expansion/genetics
5.
Br J Haematol ; 173(3): 413-20, 2016 05.
Article in English | MEDLINE | ID: mdl-26898266

ABSTRACT

High-throughput sequencing (HTS) is considered a technical revolution that has improved our knowledge of lymphoid and autoimmune diseases, changing our approach to leukaemia both at diagnosis and during follow-up. As part of an immunoglobulin/T cell receptor-based minimal residual disease (MRD) assessment of acute lymphoblastic leukaemia patients, we assessed the performance and feasibility of the replacement of the first steps of the approach based on DNA isolation and Sanger sequencing, using a HTS protocol combined with bioinformatics analysis and visualization using the Vidjil software. We prospectively analysed the diagnostic and relapse samples of 34 paediatric patients, thus identifying 125 leukaemic clones with recombinations on multiple loci (TRG, TRD, IGH and IGK), including Dd2/Dd3 and Intron/KDE rearrangements. Sequencing failures were halved (14% vs. 34%, P = 0.0007), enabling more patients to be monitored. Furthermore, more markers per patient could be monitored, reducing the probability of false negative MRD results. The whole analysis, from sample receipt to clinical validation, was shorter than our current diagnostic protocol, with equal resources. V(D)J recombination was successfully assigned by the software, even for unusual recombinations. This study emphasizes the progress that HTS with adapted bioinformatics tools can bring to the diagnosis of leukaemia patients.


Subject(s)
Computational Biology/methods , High-Throughput Nucleotide Sequencing/methods , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Adolescent , Adult , Child , Child, Preschool , Clone Cells , Diagnostic Errors/prevention & control , Gene Rearrangement, T-Lymphocyte , High-Throughput Nucleotide Sequencing/standards , Humans , Infant , Infant, Newborn , Neoplasm, Residual/diagnosis , Prospective Studies , Software , V(D)J Recombination/genetics , Young Adult
6.
BMC Genomics ; 15: 409, 2014 May 28.
Article in English | MEDLINE | ID: mdl-24885090

ABSTRACT

BACKGROUND: V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also useful markers of pathologies. In leukemia, they are used to quantify the minimal residual disease during patient follow-up. However, the full breadth of lymphocyte diversity is not fully understood. RESULTS: We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamed V(D)J junctions and gather them into clones for quantification. This analysis is based on a seed heuristic and is fast and scalable because in the first phase, no alignment is performed with germline database sequences. The algorithms were applied to TR γ HTS data from a patient with acute lymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified the main clone, as well as additional clones that were not identified with standard protocols. CONCLUSIONS: The proposed algorithms provide new insight into the analysis of high-throughput sequencing data for leukemia, and also to the quantitative assessment of any immunological profile. The methods described here are implemented in a C++ open-source program called Vidjil.


Subject(s)
Algorithms , High-Throughput Nucleotide Sequencing/methods , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Sequence Analysis, DNA/methods , V(D)J Recombination , Humans , Neoplasm, Residual/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Software
7.
Am J Hematol ; 89(6): 610-5, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24616160

ABSTRACT

Although acute myeloid leukemia (AML) with t(8;21) belongs to the favorable risk AML subset, relapse incidence may reach 30% in those patients. RUNX1-RUNX1T1 fusion transcript is a well-established marker for minimal residual disease (MRD) monitoring. In this study, we investigated the feasibility and performances of RUNX1-RUNX1T1 DNA as MRD marker in AML with t(8;21). In 17/22 patients with t(8;21)-positive AML treated in the French CBF-2006 trial, breakpoints in RUNX1 and RUNX1T1 were identified using long-range PCR followed by next-generation sequencing. RUNX1-RUNX1T1 DNA quantification was performed by real-time quantitative PCR using patient-specific primers and probe. MRD levels were evaluated in 71 follow-up samples from 16 patients, with a median of four samples [range 2-7] per patient. RUNX1 breakpoints were located in intron 5 in all cases. RUNX1T1 breakpoints were located in intron 1b in 15 cases and in intron 1a in two cases. RUNX1-RUNX1T1 MRD levels measured on DNA and RNA were strongly correlated (r = 0.8, P < 0.0001). Discordant MRD results were observed in 10/71 (14%) of the samples: in three samples from two patients who relapsed, RUNX1-RUNX1T1 was detectable only on DNA, while RUNX1-RUNX1T1 was detectable only on RNA in seven samples. MRD monitoring on genomic DNA is feasible, but with sensitivity variations depending on the patient breakpoint sequence and the qPCR assay efficiency. Although interpretation of the results is easier because it is closely related to the number of leukemic cells, this method greatly increases time, cost and complexity, which limits its interest in routine practice.


Subject(s)
Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , Core Binding Factor Alpha 2 Subunit/genetics , DNA, Neoplasm/genetics , Leukemia, Myeloid, Acute/genetics , Oncogene Proteins, Fusion/genetics , Translocation, Genetic , Adolescent , Adult , Core Binding Factor Alpha 2 Subunit/metabolism , Female , Genomics , Humans , Male , Middle Aged , Neoplasm, Residual , Oncogene Proteins, Fusion/metabolism , RUNX1 Translocation Partner 1 Protein , Tumor Cells, Cultured , Young Adult
8.
Am J Hematol ; 88(4): 306-11, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23460398

ABSTRACT

Germline heterozygous alterations of the tumor-suppressor gene neurofibromatosis-1 (NF1) lead to neurofibromatosis type 1, a genetic disorder characterized by a higher risk to develop juvenile myelomonocytic leukemia and/or acute myeloid leukemia (AML). More recently, somatic 17q11 deletions encompassing NF1 have been described in many adult myeloid malignancies. In this context, we aimed to define NF1 involvement in AML. We screened a total of 488 previously untreated de novo AML patients for the NF1 deletion using either array comparative genomic hybridization (aCGH) or real-time quantitative PCR/fluorescence in situ hybridization approaches. We also applied massively parallel sequencing for in depth mutation analysis of NF1 in 20 patients including five NF1-deleted patients. We defined a small ∼0.3 Mb minimal deleted region involving NF1 by aCGH and an overall frequency of NF1 deletion of 3.5% (17/485). NF1 deletion is significantly associated with unfavorable cytogenetics and with monosomal karyotype notably. We discovered six NF1 variants of unknown significance in 7/20 patients of which only one out of four disappeared in corresponding complete remission sample. In addition, only one out of five NF1-deleted patients has an acquired coding mutation in the remaining allele. In conclusion, direct NF1 inactivation is infrequent in de novo AML and may be a secondary event probably involved in leukemic progression.


Subject(s)
Gene Deletion , Leukemia, Myeloid, Acute/genetics , Neurofibromatosis 1/genetics , Neurofibromin 1/genetics , Adolescent , Adult , Aged , Alleles , Comparative Genomic Hybridization , Female , Gene Frequency , High-Throughput Nucleotide Sequencing , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Male , Middle Aged , Mutation Rate , Neurofibromin 1/deficiency , Real-Time Polymerase Chain Reaction
9.
bioRxiv ; 2023 Nov 07.
Article in English | MEDLINE | ID: mdl-37965202

ABSTRACT

In hereditary papillary renal cell carcinoma (HPRCC), the MET receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations recently identified in non-small cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: in this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed ten uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N-lobe of the MET kinase, proved able to induce cell transformation, further enhanced by HGF stimulation: His1086Leu, Ile1102Thr, Leu1130Ser, and Cis1125Gly. Similar to the variant resulting in MET exon14 skipping, the two N-lobe MET variants His1086Leu, Ile1102Thr further characterized were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation displayed also transforming potential, promoting tumor growth in a xenograft model. In addition, the N-lobe-mutated MET variants were found to trigger a common HGF-stimulation-dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N-lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET TKIs opens the way for use of targeted therapies for patients harboring the corresponding mutations.

10.
Blood Adv ; 7(15): 4024-4034, 2023 08 08.
Article in English | MEDLINE | ID: mdl-37205853

ABSTRACT

Whether the LSC17 gene expression can improve risk stratification in the context of next generation sequencing-based risk stratification and measurable residual disease (MRD) in patients with intensively treated AML has not been explored. We analyzed LSC17 in 504 adult patients prospectively treated in the ALFA-0702 trial. RUNX1 or TP53 mutations were associated with higher LSC1 scores while CEBPA and NPM1 mutations were associated with lower scores. Patients with high LSC17 scores had a lower rate of complete response (CR) in a multivariable analysis (odds ratio, 0.41; P = .0007), accounting for European LeukemiaNet 2022 (ELN22), age, and white blood cell count (WBC). LSC17-high status was associated with shorter overall survival (OS) (3-year OS: 70.0% vs 52.7% in patients with LSC17-low status; P < .0001). In a multivariable analysis considering ELN22, age, and WBC, patients with LSC17-high status had shorter disease-free survival (DFS) (hazard ratio [HR], 1.36; P = .048) than those with LSC17-low status. In 123 patients with NPM1-mutated AML in CR, LSC17-high status predicted poorer DFS (HR, 2.34; P = .01), independent of age, WBC, ELN22 risk, and NPM1-MRD. LSC-low status and negative NPM1-MRD identified a subset comprising 48% of patients with mutated NPM1 with a 3-year OS from CR of 93.1% compared with 60.7% in those with LSC17-high status and/or positive NPM1-MRD (P = .0001). Overall, LSC17 assessment refines genetic risk stratification in adult patients with AML treated intensively. Combined with MRD, LSC17 identifies a subset of patients with NPM1-mutated AML with excellent clinical outcome.


Subject(s)
Leukemia, Myeloid, Acute , Nucleophosmin , Adult , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/drug therapy , Remission Induction , Disease-Free Survival , Risk Factors , Neoplasm, Residual/genetics
11.
J Exp Clin Cancer Res ; 42(1): 260, 2023 Oct 07.
Article in English | MEDLINE | ID: mdl-37803448

ABSTRACT

BACKGROUND: In the era of personalized medicine, the establishment of preclinical models of cancer that faithfully recapitulate original tumors is essential to potentially guide clinical decisions. METHODS: We established 7 models [4 cell lines, 2 Patient-Derived Tumor Organoids (PDTO) and 1 Patient-Derived Xenograft (PDX)], all derived from the same Ovarian Clear Cell Carcinoma (OCCC). To determine the relevance of each of these models, comprehensive characterization was performed based on morphological, histological, and transcriptomic analyses as well as on the evaluation of their response to the treatments received by the patient. These results were compared to the clinical data. RESULTS: Only the PDX and PDTO models derived from the patient tumor were able to recapitulate the patient tumor heterogeneity. The patient was refractory to carboplatin, doxorubicin and gemcitabine, while tumor cell lines were sensitive to these treatments. In contrast, PDX and PDTO models displayed resistance to the 3 drugs. The transcriptomic analysis was consistent with these results since the models recapitulating faithfully the clinical response grouped together away from the other classical 2D cell culture models. We next investigated the potential of drugs that have not been used in the patient clinical management and we identified the HDAC inhibitor belinostat as a potential effective treatment based on PDTO response. CONCLUSIONS: PDX and PDTO appear to be the most relevant models, but only PDTO seem to present all the necessary prerequisites for predictive purposes and could constitute relevant tools for therapeutic decision support in the context of these particularly aggressive cancers refractory to conventional treatments.


Subject(s)
Carcinoma , Organoids , Humans , Xenograft Model Antitumor Assays , Cell Line, Tumor , Treatment Outcome
12.
PLoS One ; 17(4): e0267508, 2022.
Article in English | MEDLINE | ID: mdl-35486629

ABSTRACT

Acute myeloid leukemia (AML) is characterized by blocked differentiation and extensive proliferation of hematopoietic progenitors/precursors. Relapse is often observed after chemotherapy due to the presence of residual leukemic cells, which is also called minimal residual disease (MRD). Subclonal heterogeneity at diagnosis was found to be responsible for MRD after treatment. Patient xenograft mouse models are valuable tools for studying MRD after chemotherapy; however, the contribution of the immune system in these models is usually missing. To evaluate its role in leukemic persistence, we generated an immune-competent AML mouse model of persistence after chemotherapy treatment. We used well-characterized (phenotypically and genetically) subclones of the murine C1498 cell line stably expressing the ZsGreen reporter gene and the WT1 protein, a valuable antigen. Accordingly, these subclones were also selected due to their in vitro aracytidine (Ara-c) sensitivity. A combination of 3 subclones (expressing or not expressing WT1) was found to lead to prolonged mouse survival after Ara-c treatment (as long as 150 days). The presence of residual leukemic cells in the blood and BM of surviving mice indicated their persistence. Thus, a new mouse model that may offer insights into immune contributions to leukemic persistence was developed.


Subject(s)
Leukemia, Myeloid, Acute , Animals , Cytarabine/pharmacology , Cytarabine/therapeutic use , Disease Models, Animal , Disease Progression , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mice , Neoplasm, Residual/diagnosis
13.
Commun Biol ; 3(1): 101, 2020 03 05.
Article in English | MEDLINE | ID: mdl-32139796

ABSTRACT

The molecular mechanisms underlying caudal-to-rostral progression of Lewy body pathology in Parkinson's disease remain poorly understood. Here, we identified transcriptomic signatures across brain regions involved in Braak Lewy body stages in non-neurological adults from the Allen Human Brain Atlas. Among the genes that are indicative of regional vulnerability, we found known genetic risk factors for Parkinson's disease: SCARB2, ELOVL7, SH3GL2, SNCA, BAP1, and ZNF184. Results were confirmed in two datasets of non-neurological subjects, while in two datasets of Parkinson's disease patients we found altered expression patterns. Co-expression analysis across vulnerable regions identified a module enriched for genes associated with dopamine synthesis and microglia, and another module related to the immune system, blood-oxygen transport, and endothelial cells. Both were highly expressed in regions involved in the preclinical stages of the disease. Finally, alterations in genes underlying these region-specific functions may contribute to the selective regional vulnerability in Parkinson's disease brains.


Subject(s)
Brain/pathology , Gene Expression Profiling , Lewy Bodies/genetics , Lewy Bodies/pathology , Parkinson Disease/genetics , Parkinson Disease/pathology , Transcriptome , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Databases, Genetic , Disease Progression , Female , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Phenotype , Risk Assessment , Risk Factors , Young Adult
14.
Cell Death Dis ; 11(5): 360, 2020 05 12.
Article in English | MEDLINE | ID: mdl-32398681

ABSTRACT

Cellular stress response contributes to epithelial defense in adaptation to environment changes. Galectins play a pivotal role in the regulation of this response in malignant cells. However, precise underlying mechanisms are largely unknown. Here we demonstrate that Galectin-3, a pro and anti-apoptotic lectin, is required for setting up a correct cellular response to stress by orchestrating several effects. First, Galectin-3 constitutes a key post-transcriptional regulator of stress-related mRNA regulons coordinating the cell metabolism, the mTORC1 complex or the unfolded protein response (UPR). Moreover, we demonstrated the presence of Galectin-3 with mitochondria-associated membranes (MAM), and its interaction with proteins located at the ER or mitochondrial membranes. There Galectin-3 prevents the activation and recruitment at the mitochondria of the regulator of mitochondria fission DRP-1. Accordingly, loss of Galectin-3 impairs mitochondrial morphology, with more fragmented and round mitochondria, and dynamics both in normal and cancer epithelial cells in basal conditions. Importantly, Galectin-3 deficient cells also display changes of the activity of the mitochondrial respiratory chain complexes, of the mTORC1/S6RP/4EBP1 translation pathway and reactive oxygen species levels. Regarding the ER, Galectin-3 did not modify the activities of the 3 branches of the UPR in basal conditions. However, Galectin-3 favours an adaptative UPR following ER stress induction by Thapsigargin treatment. Altogether, at the ER-mitochondria interface, Galectin-3 coordinates the functioning of the ER and mitochondria, preserves the integrity of mitochondrial network and modulates the ER stress response.


Subject(s)
Blood Proteins/metabolism , Endoplasmic Reticulum/metabolism , Epithelial Cells/metabolism , Galectins/metabolism , Mitochondria/metabolism , Apoptosis/genetics , Endoplasmic Reticulum Stress/physiology , Humans , Mitochondrial Membranes/metabolism , Reactive Oxygen Species/metabolism , Thapsigargin/metabolism , Unfolded Protein Response/physiology
15.
Genes Chromosomes Cancer ; 47(12): 1110-7, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18767145

ABSTRACT

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence in leukemic stem cells of the Philadelphia chromosome (Ph) and the formation of the BCR-ABL1 fusion. Untreated, the disease progresses to accelerate phase and blast crisis in which hematopoietic differentiation has become arrested. CML progression is frequently associated with cytogenetic evidence of clonal evolution, defined as additional chromosomal aberrations. We here report a CML resistant to tyrosine kinase inhibitors that rapidly progressed to blastic phase. At this time, array CGH performed on CD34(+) cells revealed cryptic partial deletions of both PRDM16 and RUNX1 and duplication of the der(21) chromosome. These genomic rearrangements were confirmed by FISH with probes targeting the deletion on chromosome 21 (24 kb), and with BAC probes flanking the deletion on 1p36 (220 kb). However, no cryptic t(1;21)(p36;q22) and/or RUNX1-PRDM16 were detected, suggesting that these deletions are the residual hallmarks of a more complex mechanism of chromosomal rearrangement, as indicated by the additional inversion of the region bounded by 1p36.32 and 1p36.12 breaks. At the molecular level, these abnormalities lead to the overexpression of the PR-domain negative oncogenic isoform of PRDM16, associated with two deleted copies within the runt domain of C-teminal aberrant RUNX1. These events are not detectable by conventional cytogenetic and molecular strategies, and may be of underestimated frequency in disease progression.


Subject(s)
Chromosomes, Human, Pair 21/genetics , Core Binding Factor Alpha 2 Subunit/genetics , DNA-Binding Proteins/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Oncogene Proteins, Fusion/genetics , Sequence Deletion , Transcription Factors/genetics , Adult , Disease Progression , Female , Gene Duplication , Humans , Models, Genetic , Nucleic Acid Hybridization , Oncogene Proteins, Fusion/metabolism , Protein Isoforms , Recombinant Fusion Proteins/genetics , Translocation, Genetic , Tumor Cells, Cultured
16.
Mol Cancer Ther ; 18(6): 1137-1148, 2019 06.
Article in English | MEDLINE | ID: mdl-30926633

ABSTRACT

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.


Subject(s)
Colorectal Neoplasms/genetics , Mutation , Receptor Protein-Tyrosine Kinases/genetics , Adult , Aged , Animals , Base Sequence , Cell Transformation, Neoplastic/genetics , Colorectal Neoplasms/pathology , Colorectal Neoplasms/secondary , Colorectal Neoplasms/surgery , Female , Genome, Human/genetics , HCT116 Cells , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Male , Mice , Middle Aged , NIH 3T3 Cells , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Transfection
17.
Leukemia ; 33(2): 348-357, 2019 02.
Article in English | MEDLINE | ID: mdl-30089916

ABSTRACT

Despite constant progress in prognostic risk stratification, children with acute myeloid leukemia (AML) still relapse. Treatment failure and subsequent relapse have been attributed to acute myeloid leukemia-initiating cells (LSC), which harbor stem cell properties and are inherently chemoresistant. Although pediatric and adult AML represent two genetically very distinct diseases, we reasoned that common LSC gene expression programs are shared and consequently, the highly prognostic LSC17 signature score recently developed in adults may also be of clinical interest in childhood AML. Here, we demonstrated prognostic relevance of the LSC17 score in pediatric non-core-binding factor AML using Nanostring technology (ELAM02) and RNA-seq data from the NCI (TARGET-AML). AML were stratified by LSC17 quartile groups (lowest 25%, intermediate 50% and highest 25%) and children with low LSC17 score had significantly better event-free survival (EFS: HR = 3.35 (95%CI = 1.64-6.82), P < 0.001) and overall survival (OS: HR = 3.51 (95%CI = 1.38-8.92), P = 0.008) compared with patients with high LSC17 scores. More importantly, the high LSC17 score was an independent negative EFS and OS prognosticator determined by multivariate Cox model analysis (EFS: HR = 3.42 (95% CI = 1.63-7.16), P = 0.001; OS HR = 3.02 (95%CI = 1.16-7.85), P = 0.026). In conclusion, we have demonstrated the broad applicability of the LSC17 score in the clinical management of AML by extending its prognostic relevance to pediatric AML.


Subject(s)
Biomarkers, Tumor/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/pathology , Transcriptome , Adolescent , Case-Control Studies , Child , Child, Preschool , Female , Follow-Up Studies , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Infant , Infant, Newborn , Leukemia, Myeloid, Acute/classification , Male , Neoplastic Stem Cells/metabolism , Prognosis , Survival Rate
18.
Haematologica ; 93(4): 543-50, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18287131

ABSTRACT

BACKGROUND: Genomic gains and losses play a crucial role in the development of diffuse large B-cell lymphomas. High resolution array comparative genomic hybridization provides a comprehensive view of these genomic imbalances but is not routinely applicable. We developed a polymerase chain reaction assay to provide information regarding gains or losses of relevant genes and prognosis in diffuse large B-cell lymphomas. DESIGN AND METHODS: Two polymerase chain reaction assays (multiplex polymerase chain reaction of short fluorescent fragments, QMPSF) were designed to detect gains or losses of c-REL, BCL6, SIM1, PTPRK, MYC, CDKN2A, MDM2, CDKN1B, TP53 and BCL2. Array comparative genomic hybridization was simultaneously performed to evaluate the sensitivity and predictive value of the QMPSF assay. The biological and clinical relevance of this assay were assessed. RESULTS: The predictive value of the QMPSF assay for detecting abnormal DNA copy numbers ranged between 88-97%, giving an overall concordance rate of 92% with comparative genomic hybridization results. In 77 cases of diffuse large B-cell lymphomas, gains of MYC, CDKN1B, c-REL and BCL2 were detected in 12%, 40%, 27% and 29%, respectively. TP53 and CDKN2A deletions were observed in 22% and 36% respectively. BCL2 and CDKN2A allelic status correlated with protein expression. TP53 mutations were associated with allelic deletions in 45% of cases. The prognostic value of a single QMPSF assay including TP53, MYC, CDKN2A, SIM1 and CDKN1B was predictive of the outcome independently of the germinal center B-cell like/non-germinal center B-cell like subtype or the International Prognostic Index. CONCLUSIONS: QMPSF is a reliable and flexible method for detecting somatic quantitative genetic alterations in diffuse large B-cell lymphomas and could be integrated in future prognostic predictive models.


Subject(s)
Gene Dosage , Lymphoma, Large B-Cell, Diffuse/genetics , Neoplasm Proteins/genetics , Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Murine-Derived , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Chromosome Aberrations , Combined Modality Therapy , Female , Gene Amplification , Gene Deletion , Genes, Tumor Suppressor , Hematopoietic Stem Cell Transplantation , Humans , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/mortality , Lymphoma, Large B-Cell, Diffuse/surgery , Male , Middle Aged , Nucleic Acid Hybridization , Prognosis , Proto-Oncogenes , Rituximab , Treatment Outcome
20.
Immunol Lett ; 192: 27-34, 2017 12.
Article in English | MEDLINE | ID: mdl-29030252

ABSTRACT

BACKGROUND: In acute myeloid leukaemia (AML)-affected patients, the presence of heterogeneous sub-clones at diagnosis has been shown to be responsible for minimal residual disease and relapses. The role played by the immune system in this leukaemic sub-clonal hierarchy and maintenance remains unknown. As leukaemic sub-clone immunogenicity could not be evaluated in human AML xenograft models, we assessed the sub-clonal diversity of the murine C1498 AML cell line and the immunogenicity of its sub-clones in immune-competent syngeneic mice. METHODOLOGY: The murine C1498 cell line was cultured in vitro and sub-clonal cells were generated after limiting dilution. The genomic profiles of 6 different sub-clones were analysed by comparative genomic hybridization arrays (CGH). The sub-clones were then injected into immune-deficient and - competent syngeneic mice. The immunogenicities of the sub-clones was evaluated through 1) assessment of mouse survival, 2) determination of leukaemic cell infiltration into organs by flow cytometry and the expression of a fluorescent reporter gene, 3) assessment of the CTL response ex vivo and 4) detection of residual leukaemic cells in the organs via amplification of the genomic reporter gene by real-time PCR (qPCR). RESULTS: Genomic analyses revealed heterogeneity among the parental cell line and its derived sub-clones. When injected individually into immune-deficient mice, all sub-clones induced cases of AML with different kinetics. However, when administered into immune-competent animals, some sub-clones triggered AML in which no mice survived, whereas others elicited reduced lethality rates. The AML-surviving mice presented efficient anti-leukaemia CTL activity ex vivo and eliminated the leukaemic cells in vivo. CONCLUSION: We showed that C1498 cell sub-clones presented genomic heterogeneity and differential immunogenicity resulting either in immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients with AML.


Subject(s)
Antigens, Neoplasm/immunology , Immunotherapy/methods , Leukemia, Myeloid, Acute/genetics , Animals , Antigens, Neoplasm/genetics , Biodiversity , Cell Line, Tumor , Clone Cells , Cytotoxicity, Immunologic , Female , Humans , Immunologic Surveillance , Leukemia, Myeloid, Acute/immunology , Mice , Mice, Inbred C57BL , Mice, SCID , Polymorphism, Genetic
SELECTION OF CITATIONS
SEARCH DETAIL