Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has pre... more Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with v...
viable cells, distinct hematopoietic populations were sorted using FACS Aria sorter in an MRD-neg... more viable cells, distinct hematopoietic populations were sorted using FACS Aria sorter in an MRD-negative follow-up sample and analyzed for the presence of the TP53 mt using allele-specific digital droplet PCR (ddPCR, sensitivity: 1,5x10-3-1x10-2). MRD was monitored using real-time quantitative PCR of clonal immune gene rearrangements (sensitivity 1x10-5) and multiparameter flow cytometry. Results: In our series of 43 TP53 mt ALL patients (B-ALL 90.7%, TALL 9.3%) MRD analysis revealed quantifiable IG/TR MRD persistence in 7 patients (16.3%), complete MRD remission in 30 patients (69.8%), and MRD persistence at not quantifiable levels in 6 patients (14.0%). In 9 of the MRD-negative patients (20.9%) the TP53-mutation was detected at levels between 2.0 and 17.4% (median 8.1%), pointing to the presence of TP53 mt in a pre-leukemic compartment. In 5 of 7 MRD-persisters also TP53 mutations were detected. In the 2 remaining patients MRD-level was below the TP53-assay sensitivity. ddPCR analysis of the sorted, progenitor and mature hematopoietic populations revealed the presence of TP53 mt in early hematopoietic cells including multipotent progenitors (MPP: CD19-34+38-), common myeloid/lymphoid progenitors (CD19-34+38+13/33-) and early myeloid progenitors (CD34+19-38+13/33+) but also in differentiated cells as granulocytes (SSC high CD13/33 dim) and monocytes (CD19-34-3-13/33 high). TP53 mt was generally absent in the mature T cells but present in the B-cell precursors defined as CD19+10+20-34+ cells (Figure 1); altogether this data point to the pre-leukemic origin of TP53 mt. Interestingly, one patient who relapsed after two years with a fully unrelated IG/TR rearrangement still harbored the TP53 mt from Dx sample, pointing to the involvement of the TP53 mt in the ALL reoccurrence. The detailed analysis of other relapse-associated mutations is currently ongoing. The correlation analysis of clinical outcome with the observed TP53 mutation pattern will also be performed. Summary/Conclusion: Hereby, we have shown that in a considerable number of adult ALL patients a TP53 mutation is present in pre-leukemic cells, causing clonal expansions in different hematopoietic compartments, and thus persisting in MRD negative follow-up samples. Considering TP53 somatic mutations as an MRD-marker requires careful interpretation.
Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has pre... more Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL, and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: i) typical T, B, or Myeloid without or; ii) with a transitional component to another lineage; iii) atypical; or iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and dat...
T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precur... more T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precursors arrested at specific differentiation stages, including an ‘early-cortical’ thymic maturation arrest characterized by expression of cytoplasmic TCRβ but no surface T-cell receptor (TCR) and frequent ectopic expression of the TLX1/3 NK-like homeotic proteins (NKL). We designed a TCRα VJC PCR to identify clonal TCRα rearrangements in 32% of 127 T-ALLs, including 0/52 immature/TCRγδ lineage cases and 41/75 (55%) TCRαβ lineage cases. Amongst the latter, TCRα rearrangements were not identified in 30/54 (56%) of IMβ/pre-αβ early-cortical T-ALLs, of which the majority (21/30) expressed TLX1/3. We reasoned that the remaining T-ALLs might express other NKL proteins, so compared transcript levels of 46 NKL in T-ALL and normal thymic subpopulations. Ectopic overexpression of 10 NKL genes, of which six are unreported in T-ALL (NKX2-3, BARHL1, BARX2, EMX2, LBX2 and MSX2), was detectable in 17/104 (16%) T-ALLs. Virtually all NKL overexpressing T-ALLs were TCRα unrearranged and ectopic NKL transcript expression strongly repressed Eα activity, suggesting that ectopic NKL expression is the major determinant in early-cortical thymic T-ALL maturation arrest. This immunogenetic T-ALL subtype, defined by TCRβ VDJ but no TCRα VJ rearrangement, is associated with a favorable outcome in GRAALL-treated adult T-ALLs.
V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rende... more V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus, Dδ2-Dδ3 rearrangements occur at a very immature thymic, CD34+/CD1a−/CD7+dim stage, before Dδ2(Dδ3)-Jδ1 rearrangements. These strictly ordered rearrangements are regulated by mechanisms acting beyond chromatin accessibility. Importantly, direct Dδ2-Jδ1 rearrangements are prohibited by a B12/23 restriction and ordered human TCR-δ gene assembly requires RUNX1 protein, which binds to the Dδ2-23RSS, interacts with RAG1, and enhances RAG1 deposition at this site. This RUNX1-mediated V(D)J recombinase targeting imposes the use of two Dδ gene segments in human TCR-δ chains. Absence of this RUNX1 binding site in the homologous mouse Dδ1-23RSS provides a molecular explanation for the lack of ordered TCR-δ gene assembly in mice and may underlie differences in early lymph...
Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic h... more Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) and are generally believed to result from illegitimate V(D)J recombination events. However, molecular characterization and evaluation of the extent of recombinase involvement at the TCR-oncogene junction has not been fully evaluated. In the present study, screening for TCRβ and TCRα/δ translocations by FISH and ligation-mediated PCR in 280 T-ALLs allowed the identification of 4 previously unreported TCR-translocated oncogene partners: GNAG, LEF1, NKX2-4, and IL2RB. Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRβ (which are exclusively enhancer driven) and TCRα/δ (which use an enhancer-independent cryptic internal promoter) transl...
T-cell acute lymphoblastic leukemia 1 (TAL1), also known as stem cell leukemia (SCL), plays impor... more T-cell acute lymphoblastic leukemia 1 (TAL1), also known as stem cell leukemia (SCL), plays important roles in differentiation of hematopoietic and endothelial cells and is deregulated in a high percentage of T-cell acute lymphoblastic leukemia (T-ALL). In this report we show that the intracellular concentration of TAL1 is regulated by transforming growth factor β (TGF-β), which triggers its polyubiquitylation and degradation by the proteasome. This effect is mediated by AKT1, which phosphorylates TAL1 at threonine 90. Immunoprecipitation experiments showed that this event increases association of TAL1 with the E3 ubiquitin ligase CHIP. The E47 heterodimerization partner of TAL1 hinders this association. Our observations indicate that activation of the TGF-β and phosphatidylinositol 3-kinase/AKT pathways might reverse overexpression of TAL1 in leukemic cells by inducing proteolysis of this important oncogene.
Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acut... more Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acute lymphoblastic leukemia, but it is unclear if this constitutes a homogeneous clinical entity, and the biological consequences of HOXA overexpression have not been systematically examined. We characterized the biology and outcome of 55 HOXA positive cases among 209 adult T-cell acute lymphoblastic leukemia patients uniformly treated during the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. HOXA positive patients had markedly higher rates of an early thymic precursor-like immunophenotype (40.8% v 14.5%, p = 0.0004), chemoresistance (59.3% v 40.8%, p = 0.026) and positive minimal residual disease (48.5% v 23.5%, p = 0.01) than the HOXA negative group. These differences were due to particularly high frequencies of chemoresistant early thymic precursor-like acute lymphoblastic leukemia in HOXA positive cases harboring fusion onco-proteins that trans...
Introduction La fievre mediterraneenne familiale (FMF) est la maladie auto inflammatoire la plus ... more Introduction La fievre mediterraneenne familiale (FMF) est la maladie auto inflammatoire la plus frequente ; elle est associee a des mutations du gene MEFV. Aucun travail specifique n’a ete publie a ce jour sur les cytopenies inexpliquees ou les monocytoses chroniques chez les patients adultes atteints de FMF sur une large cohorte. Plusieurs etudes ont porte sur les parametres hematologiques des enfants et des adultes atteints de FMF. En dehors des crises inflammatoires, les parametres hematologiques significativement differents par rapport aux temoins sont l’hyperleucocytose, en particulier le taux de neutrophiles. Dans ces etudes, les cytopenies ainsi que la monocytose chronique n’ont pas ete observees et leur presence impose des investigations specifiques. Le but de ce travail etait de rechercher des causes de cytopenies ou de monocytose inexpliquees chez les patients avec une FMF presentant une mutation homozygote M694 V du gene MEFV. Patients et methodes A partir des patients a...
Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has pre... more Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with v...
viable cells, distinct hematopoietic populations were sorted using FACS Aria sorter in an MRD-neg... more viable cells, distinct hematopoietic populations were sorted using FACS Aria sorter in an MRD-negative follow-up sample and analyzed for the presence of the TP53 mt using allele-specific digital droplet PCR (ddPCR, sensitivity: 1,5x10-3-1x10-2). MRD was monitored using real-time quantitative PCR of clonal immune gene rearrangements (sensitivity 1x10-5) and multiparameter flow cytometry. Results: In our series of 43 TP53 mt ALL patients (B-ALL 90.7%, TALL 9.3%) MRD analysis revealed quantifiable IG/TR MRD persistence in 7 patients (16.3%), complete MRD remission in 30 patients (69.8%), and MRD persistence at not quantifiable levels in 6 patients (14.0%). In 9 of the MRD-negative patients (20.9%) the TP53-mutation was detected at levels between 2.0 and 17.4% (median 8.1%), pointing to the presence of TP53 mt in a pre-leukemic compartment. In 5 of 7 MRD-persisters also TP53 mutations were detected. In the 2 remaining patients MRD-level was below the TP53-assay sensitivity. ddPCR analysis of the sorted, progenitor and mature hematopoietic populations revealed the presence of TP53 mt in early hematopoietic cells including multipotent progenitors (MPP: CD19-34+38-), common myeloid/lymphoid progenitors (CD19-34+38+13/33-) and early myeloid progenitors (CD34+19-38+13/33+) but also in differentiated cells as granulocytes (SSC high CD13/33 dim) and monocytes (CD19-34-3-13/33 high). TP53 mt was generally absent in the mature T cells but present in the B-cell precursors defined as CD19+10+20-34+ cells (Figure 1); altogether this data point to the pre-leukemic origin of TP53 mt. Interestingly, one patient who relapsed after two years with a fully unrelated IG/TR rearrangement still harbored the TP53 mt from Dx sample, pointing to the involvement of the TP53 mt in the ALL reoccurrence. The detailed analysis of other relapse-associated mutations is currently ongoing. The correlation analysis of clinical outcome with the observed TP53 mutation pattern will also be performed. Summary/Conclusion: Hereby, we have shown that in a considerable number of adult ALL patients a TP53 mutation is present in pre-leukemic cells, causing clonal expansions in different hematopoietic compartments, and thus persisting in MRD negative follow-up samples. Considering TP53 somatic mutations as an MRD-marker requires careful interpretation.
Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has pre... more Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL, and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: i) typical T, B, or Myeloid without or; ii) with a transitional component to another lineage; iii) atypical; or iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and dat...
T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precur... more T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precursors arrested at specific differentiation stages, including an ‘early-cortical’ thymic maturation arrest characterized by expression of cytoplasmic TCRβ but no surface T-cell receptor (TCR) and frequent ectopic expression of the TLX1/3 NK-like homeotic proteins (NKL). We designed a TCRα VJC PCR to identify clonal TCRα rearrangements in 32% of 127 T-ALLs, including 0/52 immature/TCRγδ lineage cases and 41/75 (55%) TCRαβ lineage cases. Amongst the latter, TCRα rearrangements were not identified in 30/54 (56%) of IMβ/pre-αβ early-cortical T-ALLs, of which the majority (21/30) expressed TLX1/3. We reasoned that the remaining T-ALLs might express other NKL proteins, so compared transcript levels of 46 NKL in T-ALL and normal thymic subpopulations. Ectopic overexpression of 10 NKL genes, of which six are unreported in T-ALL (NKX2-3, BARHL1, BARX2, EMX2, LBX2 and MSX2), was detectable in 17/104 (16%) T-ALLs. Virtually all NKL overexpressing T-ALLs were TCRα unrearranged and ectopic NKL transcript expression strongly repressed Eα activity, suggesting that ectopic NKL expression is the major determinant in early-cortical thymic T-ALL maturation arrest. This immunogenetic T-ALL subtype, defined by TCRβ VDJ but no TCRα VJ rearrangement, is associated with a favorable outcome in GRAALL-treated adult T-ALLs.
V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rende... more V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus, Dδ2-Dδ3 rearrangements occur at a very immature thymic, CD34+/CD1a−/CD7+dim stage, before Dδ2(Dδ3)-Jδ1 rearrangements. These strictly ordered rearrangements are regulated by mechanisms acting beyond chromatin accessibility. Importantly, direct Dδ2-Jδ1 rearrangements are prohibited by a B12/23 restriction and ordered human TCR-δ gene assembly requires RUNX1 protein, which binds to the Dδ2-23RSS, interacts with RAG1, and enhances RAG1 deposition at this site. This RUNX1-mediated V(D)J recombinase targeting imposes the use of two Dδ gene segments in human TCR-δ chains. Absence of this RUNX1 binding site in the homologous mouse Dδ1-23RSS provides a molecular explanation for the lack of ordered TCR-δ gene assembly in mice and may underlie differences in early lymph...
Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic h... more Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) and are generally believed to result from illegitimate V(D)J recombination events. However, molecular characterization and evaluation of the extent of recombinase involvement at the TCR-oncogene junction has not been fully evaluated. In the present study, screening for TCRβ and TCRα/δ translocations by FISH and ligation-mediated PCR in 280 T-ALLs allowed the identification of 4 previously unreported TCR-translocated oncogene partners: GNAG, LEF1, NKX2-4, and IL2RB. Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRβ (which are exclusively enhancer driven) and TCRα/δ (which use an enhancer-independent cryptic internal promoter) transl...
T-cell acute lymphoblastic leukemia 1 (TAL1), also known as stem cell leukemia (SCL), plays impor... more T-cell acute lymphoblastic leukemia 1 (TAL1), also known as stem cell leukemia (SCL), plays important roles in differentiation of hematopoietic and endothelial cells and is deregulated in a high percentage of T-cell acute lymphoblastic leukemia (T-ALL). In this report we show that the intracellular concentration of TAL1 is regulated by transforming growth factor β (TGF-β), which triggers its polyubiquitylation and degradation by the proteasome. This effect is mediated by AKT1, which phosphorylates TAL1 at threonine 90. Immunoprecipitation experiments showed that this event increases association of TAL1 with the E3 ubiquitin ligase CHIP. The E47 heterodimerization partner of TAL1 hinders this association. Our observations indicate that activation of the TGF-β and phosphatidylinositol 3-kinase/AKT pathways might reverse overexpression of TAL1 in leukemic cells by inducing proteolysis of this important oncogene.
Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acut... more Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acute lymphoblastic leukemia, but it is unclear if this constitutes a homogeneous clinical entity, and the biological consequences of HOXA overexpression have not been systematically examined. We characterized the biology and outcome of 55 HOXA positive cases among 209 adult T-cell acute lymphoblastic leukemia patients uniformly treated during the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. HOXA positive patients had markedly higher rates of an early thymic precursor-like immunophenotype (40.8% v 14.5%, p = 0.0004), chemoresistance (59.3% v 40.8%, p = 0.026) and positive minimal residual disease (48.5% v 23.5%, p = 0.01) than the HOXA negative group. These differences were due to particularly high frequencies of chemoresistant early thymic precursor-like acute lymphoblastic leukemia in HOXA positive cases harboring fusion onco-proteins that trans...
Introduction La fievre mediterraneenne familiale (FMF) est la maladie auto inflammatoire la plus ... more Introduction La fievre mediterraneenne familiale (FMF) est la maladie auto inflammatoire la plus frequente ; elle est associee a des mutations du gene MEFV. Aucun travail specifique n’a ete publie a ce jour sur les cytopenies inexpliquees ou les monocytoses chroniques chez les patients adultes atteints de FMF sur une large cohorte. Plusieurs etudes ont porte sur les parametres hematologiques des enfants et des adultes atteints de FMF. En dehors des crises inflammatoires, les parametres hematologiques significativement differents par rapport aux temoins sont l’hyperleucocytose, en particulier le taux de neutrophiles. Dans ces etudes, les cytopenies ainsi que la monocytose chronique n’ont pas ete observees et leur presence impose des investigations specifiques. Le but de ce travail etait de rechercher des causes de cytopenies ou de monocytose inexpliquees chez les patients avec une FMF presentant une mutation homozygote M694 V du gene MEFV. Patients et methodes A partir des patients a...
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