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Low hypodiploidy defines a rare subtype of B-cell acute lymphoblastic leukemia (B-ALL) with a dismal outcome. To investigate the genomic basis of low-hypodiploid ALL (LH-ALL) in adults, we analyzed copy-number aberrations, loss of heterozygosity, mutations, and cytogenetics data in a prospective cohort of Philadelphia (Ph)-negative B-ALL patients (n = 591, ages 18–84 years), allowing us to identify 80 LH-ALL cases (14%). Genomic analysis was critical for evidencing low hypodiploidy in many cases missed by cytogenetics. The proportion of LH-ALL within Ph-negative B-ALL dramatically increased with age, from 3% in the youngest patients (under 40 years old) to 32% in the oldest (over 55 years old). Somatic TP53 biallelic inactivation was the hallmark of adult LH-ALL, present in virtually all cases (98%). Strikingly, we detected TP53 mutations in posttreatment remission samples in 34% of patients. Single-cell proteogenomics of diagnosis and remission bone marrow samples evidenced a preleukemic, multilineage, TP53-mutant clone, reminiscent of age-related clonal hematopoiesis.Significance:We show that low-hypodiploid ALL is a frequent entity within B-ALL in older adults, relying on somatic TP53 biallelic alteration. Our study unveils a link between aging and low-hypodiploid ALL, with TP53-mutant clonal hematopoiesis representing a preleukemic reservoir that can give rise to aneuploidy and B-ALL.See related commentary by Saiki and Ogawa, p. 102.This article is highlighted in the In This Issue feature, p. 101

T-cell acute lymphoblastic leukemias (T-ALL) represent 15% of pediatric and 25% of adult ALL. Since they have a particularly poor outcome in relapsed/refractory cases, identifying prognosis factors at diagnosis is crucial to adapting treatment for high-risk patients. Unlike acute myeloid leukemia and BCP ALL, chromosomal rearrangements leading to chimeric fusion-proteins with strong prognosis impact are sparsely reported in T-ALL. To address this issue an RT-MPLA assay was applied to a consecutive series of 522 adult and pediatric T-ALLs and identified a fusion transcript in 20% of cases. PICALM-MLLT10 (4%, n = 23), NUP214-ABL1 (3%, n = 19) and SET-NUP214 (3%, n = 18) were the most frequent. The clinico-biological characteristics linked to fusion transcripts in a subset of 235 patients (138 adults in the GRAALL2003/05 trials and 97 children from the FRALLE2000 trial) were analyzed to identify their prognosis impact. Patients with HOXA trans-deregulated T-ALLs with MLLT10, KMT2A and SET fusion transcripts (17%, 39/235) had a worse prognosis with a 5-year EFS of 35.7% vs 63.7% (HR = 1.63; p = 0.04) and a trend for a higher cumulative incidence of relapse (5-year CIR = 45.7% vs 25.2%, HR = 1.6; p = 0.11). Fusion transcripts status in T-ALL can be robustly identified by RT-MLPA, facilitating risk adapted treatment strategies for high-risk patients.

IDH1 and IDH2 mutations (IDH1/2Mut) are recognized as recurrent genetic alterations in acute myeloid leukemia (AML) and associated with both clinical impact and therapeutic opportunity due to the recent development of specific IDH1/2Mut inhibitors. In T-cell acute lymphoblastic leukemia (T-ALL), their incidence and prognostic implications remain poorly reported. Our targeted next-generation sequencing approach allowed comprehensive assessment of genotype across the entire IDH1 and IDH2 locus in 1085 consecutive unselected and newly diagnosed patients with T-ALL and identified 4% of, virtually exclusive (47 of 49 patients), IDH1/2Mut. Mutational patterns of IDH1/2Mut in T-ALL present some specific features compared to AML. Whereas IDH2R140Q mutation was frequent in T-ALL (25 of 51 mutations), the IDH2R172 AML hotspot was absent. IDH2 mutations were associated with older age, an immature phenotype, more frequent RAS gain-of-function mutations and epigenetic regulator loss-of-function alterations (DNMT3A and TET2). IDH2 mutations, contrary to IDH1 mutations, appeared to be an independent prognostic factor in multivariate analysis with the NOTCH1/FBXW7/RAS/PTEN classifier. IDH2Mut were significantly associated with a high cumulative incidence of relapse and very dismal outcome, suggesting that IDH2-mutated T-ALL cases should be identified at diagnosis in order to benefit from therapeutic intensification and/or specific IDH2 inhibitors.

TO THE EDITOR: T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are aggressive neoplasms that result from the proliferation of T-lymphoid progenitors blocked at thymic stages of differentiation. They account for 15% and 25% of pediatric and adult ALLs, respectively. T-ALL/T-LBL are associated with a wide range of acquired genetic abnormalities that contribute to developmental arrest and abnormal proliferation. Although intensive treatment protocols have markedly improved the outcomes of children with T-ALL, cure rates remain below 60% for adults and 85% for children. The prognosis is particularly poor in relapsing patients, highlighting an urgent need for risk stratification factors at diagnosis.[...]

TO THE EDITOR: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a rare aggressive malignancy in adults. BCP-ALL is frequently characterized by recurrent chromosomal translocations that deregulate proto-oncogenes or result in fusion genes encoding chimeric oncoproteins.[1][1] Gene

ETV6 is a target of recurrent aberrations in sporadic and familial acute lymphoblastic leukemia (ALL). Here, we report on a new pedigree with a germline ETV6 mutation in which the index patient and his father developed high hyperdiploid (HeH) ALL and polycythemia vera at age 13 and 51, respectively. The index patient achieved durable complete remission without transplantation but had persistent moderate thrombocytopenia without bleeding tendency. To determine the prevalence of ETV6 alterations in HeH-ALL, we screened 81 unrelated subjects with HeH-ALL by single nucleotide polymorphism array and high-throughput sequencing for the ETV6 gene. Overall, ETV6 microdeletions and mutations were identified in 9% of cases, all of which were somatic and considered as secondary events. Apart from the index patient, no germline ETV6 aberration was identified. Finally, we reviewed the literature for ETV6 germline aberrations and predispositions to ALL.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) is a phosphatase known to be a tumor suppressor gene in T-cell acute lymphoblastic leukemia (T-ALL). Because the full clinicobiologic characteristics of PTPN2 loss remain poorly reported, we aimed to provide a comprehensive analysis of PTPN2 deletions within a cohort of 430 patients, including 216 adults and 214 children treated according to the GRAALL03/05 (#NCT00222027 and #NCT00327678) and the FRALLE2000 protocols, respectively. We used multiplex ligation-dependent probe amplification to identify an 8% incidence of PTPN2 deletion, which was comparable in adult (9%) and pediatric (6%) populations. PTPN2 deletions were significantly associated with an αβ lineage and TLX1 deregulation. Analysis of the mutational genotype of adult T-ALL revealed a positive correlation between PTPN2 deletions and gain-of-function alterations in the IL7R/JAK-STAT signaling pathway as well as PHF6 and WT1 mutations. Of note, PTPN2 and PTEN (phosphatase and tensin homolog) deletions were mutually exclusive. Regarding treatment response, PTPN2-deleted T-ALLs were associated with a higher glucocorticoid response and a trend for improved survival in children, but not in adults, with a 5-year cumulative incidence of relapse of 8% for PTPN2-deleted pediatric cases vs 26% (P = .177).

Risk stratification in childhood T-cell acute lymphoblastic leukemia (T-ALL) is mainly based on minimal residual disease (MRD) quantification. Whether oncogenetic mutation profiles can improve the discrimination of MRD-defined risk categories was unknown. Two hundred and twenty FRALLE2000T-treated patients were tested retrospectively for NOTCH1/FBXW7/RAS and PTEN alterations. Patients with NOTCH1/FBXW7 (N/F) mutations and RAS/PTEN (R/P) germ line (GL) were classified as oncogenetic low risk (gLoR; n = 111), whereas those with N/F GL and R/P GL mutations or N/F and R/P mutations were classified as high risk (gHiR; n = 109). Day 35 MRD status was available for 191 patients. Five-year cumulative incidence of relapse (CIR) and disease-free survival were 36% and 60% for gHiR patients and 11% and 89% for gLoR patients, respectively. Importantly, among the 60% of patients with MRD

Differential impact of drugs on the outcome of ETV6-RUNX1 positive childhood B-cell precursor acute lymphoblastic leukaemia: results of the EORTC CLG 58881 and 58951 trials

Objectives Until recently, diagnostic laboratories that wanted to report on the international scale had limited options: they had to align their BCR–ABL1 quantification methods through a sample exchange with a reference laboratory to derive a conversion factor. However, commercial methods calibrated on the World Health Organization genetic reference panel are now available. We report results from a study designed to assess the comparability of the two alignment strategies. Design and methods Sixty follow-up samples from chronic myeloid leukemia patients were included. Two commercial methods calibrated on the genetic reference panel were compared to two conversion factor methods routinely used at Saint-Louis Hospital, Paris, and at Lille University Hospital. Results were matched against concordance criteria (i.e., obtaining at least two of the three following landmarks: 50, 75 and 90% of the patient samples within a 2-fold, 3-fold and 5-fold range, respectively). Results Out of the 60 samples, more than 32 were available for comparison. Compared to the conversion factor method, the two commercial methods were within a 2-fold, 3-fold and 5-fold range for 53 and 59%, 89 and 88%, 100 and 97%, respectively of the samples analyzed at Saint-Louis. At Lille, results were 45 and 85%, 76 and 97%, 100 and 100%, respectively. Agreements between methods were observed in the four comparisons performed. Conclusion Our data show that the two commercial methods selected are concordant with the conversion factor methods. This study brings the proof of principle that alignment on the international scale using the genetic reference panel is compatible with the patient sample exchange procedure. We believe that these results are particularly important for diagnostic laboratories wishing to adopt commercial methods.

Mutation of the MYD88 gene has recently been identified in activated B-cell-like diffuse cell lymphoma and enhanced Janus kinase/signal transducer and activator of transcription (JAK-STAT) and nuclear factor κB (NF-κB) signaling pathways. A whole exome-sequencing study of Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. The genetic background is not fully deciphered in WM, although the role of NF-κB and JAK-STAT has been demonstrated. We analyzed MYD88 mutation in exon 5 and characterized the clinical significance of this genetic alteration in 67 WM patients. Clinical features; immunophenotypic markers; and conventional cytogenetic, fluorescence in situ hybridization, and single nucleotide polymorphism array data were analyzed. MYD88 L265P mutation was acquired in 79% of patients. Overall, we have identified alteration of the MYD88 locus in 91% of WM patients, including 12% with gain on chromosome 3 at the 3p22 locus that included the MYD88 gene. Patients with absence of MYD88 mutation were WM characterized with a female predominance, a splenomegaly, gain of chromosome 3, and CD27 expression. Importantly, inhibition of MYD88 signaling induced cytotoxicity and inhibited cell growth of cell lines issued from patients with WM. In conclusion, these results confirm a high frequency of MYD88 L265P mutation in WM. The discovery of MYD88 L265P mutation may contribute to a better understanding of the physiopathogeny of WM.

International audience; Minimal residual disease (MRD) is known to be an independent prognostic factor in patients with acute lymphoblastic leukemia (ALL). High-throughput sequencing (HTS) is currently used in routine practice for the diagnosis and follow-up of patients with hematological neoplasms. In this retrospective study, we examined the role of immunoglobulin/T-cell receptor-based MRD in patients with ALL by HTS analysis of immunoglobulin H and/or T-cell receptor gamma chain loci in bone marrow samples from 11 patients with ALL, at diagnosis and during follow-up. We assessed the clinical feasibility of using combined HTS and bioinformatics analysis with interactive visualization using Vidjil software. We discuss the advantages and drawbacks of HTS for monitoring MRD. HTS gives a more complete insight of the leukemic population than conventional real-time quantitative PCR (qPCR), and allows identification of new emerging clones at each time point of the monitoring. Thus, HTS monitoring of Ig/TCR based MRD is expected to improve the management of patients with ALL.

Age-related EBV-associated lymphoproliferative disorders form a new clinicopathological group. Until recently, this group was associated with diffuse large B-cell lymphoma (DLBCL), characterised by an aggressive clinical presentation and a poor prognosis. Recent findings in Western Caucasian patients, however, suggest that this entity covers a wide spectrum of diseases, ranging from reactive follicular hyperplasia (HR) to DLBCL. We report the case of an 85-year-old Caucasian man showing lymphadenopathy and numerous hypodense lesions of the liver. Examination of a lymph node revealed follicular hyperplasia with EBV expression confined to germinal centres. The patient was treated with Rituximab and subsequently, the lesions of the liver were explored. They showed extensive necrosis and a polymorphic large B-cell population with strong EBV expression. This is the first report to describe age-related EBV-associated follicular hyperplasia at one site coexisting with DLBCL at another. This case warrants undertaking further investigations each time a diagnosis of age-related EBV-HR is associated with extranodal lesions.

In a case with secondary myelofibrosis occurring after essential thrombocythemia, cytogenetic analysis revealed an isolated translocation t(X;17)(q27;q22) in all cells. We found that a bacterial artificial chromosome (BAC) encompassing the breakpoint on chromosome 17 long arm contained only one gene, NOG. We therefore investigated the occurrence of this rare breakpoint in myeloproliferative disorders (MPDs). We identified three more patients with a 17q abnormality in MPDs: myelofibrosis with myeloid metaplasia (MMM); chronic myeloid leukemia positive for t(9;22)(q34;q11) with additional t(4;17)(p15;q22) at diagnosis; and myelofibrosis complicating polycythemia vera. All three cases exhibited a split of BACs containing NOG. The protein encoded by NOG, noggin, acts as an antagonist to bone morphogenetic secreted protein 2 and 4 (BMP2 and BMP4). A comparative analysis of gene expression on Agilent 22K oligonucleotide microarrays in purified CD34+ cells from the blood of MMM patients showed significant downregulation of BMPR2, BMPR1B, BMP2, and BMP8; upregulation of BMP3 and BMP10; and a trend to lower expression of NOG. Thus, given that expression and release of BMPs are important in the induction of osteosclerosis and angiogenic activity, the observed BMP deregulations could be triggered by potential NOG genetic alterations in the four cases here described, and may contribute to the myelofibrotic process characterized by bone marrow stromal reaction including collagen fibrosis, osteosclerosis, and angiogenesis.

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P

International audience; 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.

International audience; Relapse after transplantation is a major cause of treatment failure in paediatric acute lymphoblastic leukaemia (ALL). Here, we report the findings of a prospective national study designed to investigate the feasibility of immune intervention in children in first or subsequent remission following myeloablative conditioning. This study included 133 children who received a transplant for ALL between 2005 and 2008. Minimal Residual Disease (MRD) based on T cell receptor/immunoglobulin gene rearrangements was measured on days -30, 30, 90 and 150 post-transplantation. Ciclosporin treatment was rapidly discontinued and donor lymphocyte infusions (DLI) were programmed for patients with a pre- or post-transplant MRD status ≥10(-3) . Only nine patients received DLI. Pre- and post-transplant MRD status, and the duration of ciclosporin were independently associated with 5-year overall survival (OS), which was 62·07% for the whole cohort. OS was substantially higher in patients cleared of MRD than in those with persistent MRD (52·3% vs. 14·3%, respectively). Only pre-transplant MRD status (Hazard Ratio 2·57, P = 0·04) and duration of ciclosporin treatment (P < 0·001) were independently associated with relapse. The kinetics of chimerism were not useful for predicting relapse, whereas MRD monitoring up to 90 d post-transplantation was a valuable prognostic tool to guide therapeutic intervention.

Thymomas are low-grade epithelial cancers of the thymus whose prevalence varies between 0.1/100,000 and 0.4/100,000. Familial occurrence of thymoma is very rare. We studied a family bearing the constitutional chromosome translocation t(14;20)(q24;p12), 3 of whose members had a thymoma. In this family, among 27 patients, 11 had the translocation: 3 had thymoma and 4 others had 5 different autoimmune diseases: type 1 diabetes mellitus, Graves' disease, pernicious anemia, primitive Sjögren disease, and autoimmune pancytopenia. FISH studies allowed us to be more specific about the translocation breakpoints. The 14q24 breakpoint was in intron 5 of RAD51L1, and the 20p12 breakpoint was 100 kb telomeric to BMP2. RAD51L1 is a tumor-suppressor gene belonging to the RAD51 family, already implicated in many tumors (uterine leiomyomas, pseudo-Meigs syndromes, pulmonary chondroid hamartomas) and involved in recombinational repair of DNA double-strand breaks. BMP2 belongs to the TGFbeta superfamily, and the BMP2-BMP4 genes are involved in thymocyte differentiation by blocking progression from CD4-CD8- to CD4+CD8+ while maintaining a sufficient pool of immature precursors. Dysregulation of RAD51L1 and/or BMP2 may explain this familial occurrence of thymomas and autoimmune diseases. Using QRT-PCR, we studied the expression of BMP2 in 20 sporadic thymomas and found various levels of expression that may be associated with autoimmune diseases.

Despite the favorable prognosis of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22) translocation, relapses still occur in about 30% of the cases but no initial factors can strongly predict the risk of relapse. Several recent studies suggest that monitoring minimal residual disease (MRD) may identify patients at risk of relapse. We prospectively monitored AML1-ETO rearrangement by real-time quantitative PCR (RQ-PCR) in 21 patients uniformly treated in our center. Blood (PB) and bone marrow (BM) samples were collected during and after therapy. At diagnosis, levels of AML1-ETO transcript showed large variations and there was a trend for a higher relapse rate in patients with high pretreatment expression levels (P=0.065). After induction therapy, absolute transcript levels (below 10(-3), compared to Kasumi cell line), or a greater than 3 log decrease by comparison to diagnosis levels, were significant predictors of the absence of relapse (P=0.02 and P=0.02, respectively). MRD levels after consolidation therapy were also significant indicators of relapse (P=10(-5)). Comparison of BM and PB samples showed similar sensitivity for detecting AML1-ETO transcript. In conclusion, RQ-PCR appears to be an early predictive factor of the relapse risk in AML with t(8;21). PB samples can be used adequately to evaluate the level of MRD by this technique.

Targeting the tyrosine kinase activity ofBCR-ABL represents a very promising therapeutic strategy in chronic myeloid leukemia (CML). Despite strong efficacy of the tyrosine kinase inhibitor STI571, resistance has been observed in a significant proportion of patients in advanced CML stage or in Ph-positive acute lymphoid leukemia (ALL). We investigated in this study the mechanism of resistance to STI571 through point mutations in the tyrosine kinase domain and/or BCR-ABL gene amplification in 24 patients (16 in chronic phase and 8 in accelerated phase of the disease) who obtained no cytogenetic response to STI571 treatment. Screening for the already-described Thr315Ile point mutation in the ABL domain using a reverse transcription polymerase chain reaction restriction fragment length polymorphism (RT-PCR-RFLP) technique, 3 patients showed a proportion of mutated transcript at the time of resistance. The same technique failed to detect mutation at diagnosis, but a specific allele-specific oligonucleotide (ASO)–PCR on DNA for the Thr315Ile mutation and, after sequencing, for 2 newly described Phe311Leu and Met351Thr substitutions, showed the presence of rare mutated cells prior to STI571 therapy. Furthermore, the increased proportion of mutated cells during treatment detected by ASO-PCR strongly suggested clonal selection by the functional inhibiting effect of these mutations. Finally, no BCR-ABL gene amplification was detected by fluorescent in situ hybridization (FISH) in the 24 STI571-resistant patients. Our data support that in CML patients treated with STI571, ABL mutations are not restricted to the accelerated phase of the disease and that, at least in some cases, mutations seem to occur prior to STI571 therapy, probably as second mutational events during the course of CML.

The CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome is characterized by hypereosinophilia and clonal circulating CD3(-)CD4(+) T cells. Peripheral T-cell lymphoma has been described during this disease course, and we observed in our cohort of 23 patients 2 cases of angio-immunoblastic T-cell lymphoma. We focus here on histopathological (n=12 patients) and immunophenotypic (n=15) characteristics of CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome. Atypical CD4(+) T cells lymphoid infiltrates were found in 10 of 12 CD3(-)CD4(+) L-HES patients, in lymph nodes (n=4 of 4 patients), in skin (n=9 of 9) and other extra-nodal tissues (gut, lacrymal gland, synovium). Lymph nodes displayed infiltrates limited to the interfollicular areas or even an effacement of nodal architecture, associated with proliferation of arborizing high endothelial venules and increased follicular dendritic cell meshwork. Analysis of 2 fresh skin samples confirmed the presence of CD3(-)CD4(+) T cells. Clonal T cells were detected in at least one tissue in 8 patients, including lymph nodes (n=4 of 4): the same clonal T cells were detected in blood and in at least one biopsy, with a maximum delay of 23 years between samples. In the majority of cases, circulating CD3(-)CD4(+) T cells were CD2(hi) (n=9 of 14), CD5(hi) (n=12 of 14), and CD7(-)(n=4 of 14) or CD7(low) (n=10 of 14). Angio-immunoblastic T-cell lymphoma can also present with CD3(-)CD4(+) T cells; despite other common histopathological and immunophenotypic features, CD10 expression and follicular helper T-cell markers were not detected in lymphoid variant of hypereosinophilic syndrome patients, except in both patients who developed angio-immunoblastic T-cell lymphoma, and only at T-cell lymphoma diagnosis. Taken together, persistence of tissular clonal T cells and histopathological features define CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome as a peripheral indolent clonal T-cell lymphoproliferative disorder, which should not be confused with angio-immunoblastic T-cell lymphoma.

With intensified pediatric-like therapy and genetic disease dissection, the field of adult acute lymphoblastic leukemia (ALL) has evolved recently. In this new context, we aimed to reassess the value of conventional risk factors with regard to new genetic alterations and early response to therapy, as assessed by immunoglobulin/T-cell receptor minimal residual disease (MRD) levels. The study was performed in 423 younger adults with Philadelphia chromosome-negative ALL in first remission (265 B-cell precursor [BCP] and 158 T-cell ALL), with cumulative incidence of relapse (CIR) as the primary end point. In addition to conventional risk factors, the most frequent currently available genetic alterations were included in the analysis. A higher specific hazard of relapse was independently associated with postinduction MRD level ≥10(-4) and unfavorable genetic characteristics (ie, MLL gene rearrangement or focal IKZF1 gene deletion in BCP-ALL and no NOTCH1/FBXW7 mutation and/or N/K-RAS mutation and/or PTEN gene alteration in T-cell ALL). These 2 factors allowed definition of a new risk classification that is strongly associated with higher CIR and shorter relapse-free and overall survival. These results indicate that genetic abnormalities are important predictors of outcome in adult ALL not fully recapitulated by early response to therapy. Patients included in this study were treated in the multicenter GRAALL-2003 and GRAALL-2005 trials. Both trials were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively.

International audience; Minimal residual disease (MRD) is a major predictive factor of the cure rate of acute lymphoblastic leukaemia (ALL). Haematopoietic cell transplantation is a treatment option for patients at high risk of relapse. Between 2005 and 2008, we conducted a prospective study evaluating the feasibility and efficacy of the reduction of immunosuppressive medication shortly after a non-ex vivo T depleted myeloablative transplantation. Immunoglobulin (Ig)H/T-cell receptor MRD 30 d before transplant could be obtained in 122 of the 133 cases of high-risk paediatric ALL enrolled. There were no significant demographic differences except remission status (first or second complete remission) between the 95 children with MRD \textless10(-3) and the 27 with MRD ≥10(-3) . Multivariate analysis identified sex match and MRD as being significantly associated with 5-year survival. MRD ≥10(-3) compromised the 5-year cumulative incidence of relapse (43*6 vs. 16*7%). Complete remission status and stem cell source did not modify the relationship between MRD and prognosis. Thus, pre-transplant MRD is still a major predictor of outcome for ALL. The MRD-guided strategy resulted in survival for 72*3% of patients with MRD\textless10(-3) and 40*4% of those with MRD ≥10(-3).

International audience

Systemic EBV+ T-cell lymphoproliferative disease of childhood is a recent entity described in the 2008 World Health Organisation tumours of haematopoietic system and lymphoid tissues as a clonal T-cell EBV+ systemic proliferation. It occurs after acute or chronic active EBV infection. We report the case of a caucasian, immunocompetent 12-year-old girl, with no particular history, who presented with hemophagocytic lymphohistiocytosis in the aftermath of an infectious mononucleosis. Main symptoms were multiple organ failure, hepatosplenomegaly and pancytopenia. Histopathology of peripheral lymph node and bone marrow revealed a T-cell, CD8+, EBV+ lymphoproliferation. An elevated viral load was detected in blood by PCR. The patient died within 3 weeks. Since most of the cases have been reported in Asia and South America, few cases still have been described in Europe. Unlike B-cell lymphoproliferation in immunocompromised individuals, T-cell EBV+ lymphoproliferation occurs in immunocompetent patients and seems to be the consequence of a proliferative disorder of EBV-infected T-cells, attributed to a cytotoxic T-cell response deficiency. These T-cell proliferations are more frequently immunoreactive for CD8 than CD4. A key feature of the diagnosis might be EBV viral load.

The core-binding factor (CBF) complex is a heterodimeric transcription factor composed of 2 subunits, CBFalpha and CBFbeta, that play a major role in hematopoiesis. Both members of the CBF complex are frequently altered in acute myeloid leukemia (AML) by translocation, most commonly t(8;21), t(12;21), and t(3;21) for CBFalpha, located in 21q22, and inv(16)(p13;q22) for CBFbeta, located on 16q22. Recently, a new mechanism of alteration of CBFalpha, by point mutation, has been reported in myeloid malignancies, particularly in M0 AML. In the present study, we found no point mutation of the CBFbeta gene in 30 myelodysplastic syndromes and 100 AMLs, suggesting a limited role, if any, of CBFbeta point mutations in those disorders.

International audience; To ascertain genomic alterations associated with Imatinib resistance in chronic myeloid leukaemia, we performed high resolution genomic analysis of CD34(+) cells from 25 Imatinib (IM) resistant and 11 responders CML patients. Using patients' T-cells as reference, we found significant association between number of acquired cryptic copy number alterations (CNA) and disease phase (p=0.036) or loss of IM response for patients diagnosed in chronic phase (CP) (p=0.04). Recurrent cryptic losses were identified on chromosomes 7, 12 and 13. On chromosome 7, recurrent deletions of the IKZF1 locus were detected, for the first time, in 4 patients in CP.

Adult and child B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) differ in terms of incidence and prognosis. These disparities are mainly due to the molecular abnormalities associated with these two clinical entities. A genome-wide analysis using oligo SNP arrays recently demonstrated that PAX5 (paired-box domain 5) is the main target of somatic mutations in childhood BCP-ALL being altered in 38.9% of the cases. We report here the most extensive analysis of alterations of PAX5 coding sequence in 117 adult BCP-ALL patients in the unique clinical protocol GRAALL-2003/GRAAPH-2003. Our study demonstrates that PAX5 is mutated in 34% of adult BCP-ALL, mutations being partial or complete deletion, partial or complete amplification, point mutation or fusion gene. PAX5 alterations are heterogeneous consisting in complete loss in 17%, focal deletions in 10%, point mutations in 7% and translocations in 1% of the cases. PAX5 complete loss and PAX5 point mutations differ. PAX5 complete loss seems to be a secondary event and is significantly associated with BCR-ABL1 or TCF3-PBX1 fusion genes and a lower white blood cell count.

In a series of 153 children with T-cell malignancies enrolled in 2 consecutive European Organization for Research and Treatment of Cancer (EORTC) trials, we assessed the HOX11L2 expression and/or the presence of a t(5;14)(q35;q32). Additionally, in 138 of these patients, HOX11 expression and SIL-TAL rearrangement were also assessed. These alterations were mutually exclusive, and their frequency was 23% (n = 35), 7% (n = 10), and 12% (n = 17), respectively. HOX11L2/t(5;14) positivity was more frequent in acute lymphoblastic leukemia (ALL) with cortical T immunophenotype and in children aged between 6 and 9 years. In contrast with previously reported data, patients positive and negative for HOX11L2/t(5;14) were comparable with regard to clinical outcome as well as to the response to a 7-day prephase treatment or to residual disease at completion of induction therapy. The 3-year event-free survival (EFS) rate (+/- SE percentage) for patients positive and negative for HOX11L2/t(5;14) was 75.5% (+/- 8.1%) and 68.3% (+/- 5.0%), respectively; the hazard ratio was 0.84 (95% confidence interval, 0.40-1.80). Patients with HOX11-high expression and those with SIL-TAL fusion had low levels of residual disease at the end of induction and a favorable prognosis: the 3-year EFS rate was 83.3% (+/- 8.5%) and 75.3% (+/- 12.6%), respectively. The results obtained in HOX11L2/t(5;14) patients in this study do not confirm the unfavorable prognosis reported in previous studies.

Cytogenetic analysis of mantle cell lymphoma (MCL), characterized by the presence of t(11;14)(q13;q32) translocation, is often difficult because of the low proliferating rate of MCL cells and the presence of normal cells in bone marrow which may interfere with growth of MCL cells. We describe herein a TPA (12-O-tetradecanoylphorbol 13-acetate) stimulated culture to improve detection of t(11;14)(q13;q32) in 20 MCL patients regardless of the samples used.

FLT3 internal tandem duplications (FLT3-ITDs) are present in nearly 25% of patients with AML and have been associated with poor response to conventional therapy and poor outcome. We retrospectively evaluated the effect of reinforced courses of chemotherapy on the prognostic value of FLT3-ITDs in 159 AML patients prospectively enrolled in the ALFA-9000 trial, which randomly compared three reinforced induction regimens (standard 3+7 including high-dose daunorubicin, double induction, and timed-sequential therapy). FLT3-ITD was present in 40/159 (25%) blast samples and associated with high WBC (P = 0.002) and cytogenetics (P < 0.001) with a higher incidence (35%) in patients with a normal karyotype. There was no difference in CR rate between FLT3-wt and FLT3-ITD patients (80% vs 78%). Relapse-free survival (RFS) was similar in both groups (5-year RFS, 33% vs 32%; P = 0.41), even after adjustment for age, sex, WBC, cytogenetics, and treatment arm. A trend to a worse survival was observed in the FLT3-ITD group (estimated 5-year OS, 23% vs 37%; P = 0.09), mainly in patients with a normal karyotype. This was associated with a dramatic outcome in relapsing FLT3-ITD patients (estimated 3-year post-relapse survival, 0% vs 27%; P = 0.04). These results suggest that the bad prognosis associated with FLT3-ITDs in AML might be partly overcome using reinforced chemotherapy. Early detection of FLT3 mutations might thus be useful to intensify induction as well as post-remission therapy in FLT3-ITD patients.

Increased apoptosis of myeloid precursors appears to contribute to the pathophysiology of cytopenias in myelodysplastic syndromes (MDS). Fas /APO-1(CD95) is a cell surface protein inducing an apoptotic signal after its binding to Fas ligand or to a functional anti-Fas antibody. Here we studied Fas expression by immunocytochemistry on marrow slides from 30 cases of MDS. Increased Fas expression in erythroblasts and/or immature granulocytes, compared to controls, was seen in 12 (40%) of the cases. In addition, in 16 of the 18 cases withor = 5% marrow blasts, a variable proportion of blasts expressed Fas. Increased apoptosis was found by morphological analysis and/or TUNEL technique in marrow cells from 8 of the 26 cases analyzed (31%) The ability of Fas antigen to trigger apoptosis was studied after addition of a functional anti Fas antibody in 5 of the patients with Fas overexpression. Addition of this antibody, however, only lead to mild increase of apoptosis in immature granulocytes (but not other myeloid cells) in 2 of the 5 cases. Thus, increased Fas expression is seen in myeloid and/or blast cells in the majority of MDS cases. However, the relationship between this finding and increased apoptosis in MDS still remains to be established.

The major vault lung resistance protein LRP is a cytoplasmic protein involved in drug resistance, especially in acute myeloid leukemia. We looked for LRP overexpression, using immunocytochemistry with LRP 56 monoclonal antibody, on marrow slides from 41 cases of myelodysplastic syndromes (MDS). LRP overexpression (LRP+) was defined by expression of LRP 56 in at least 20% of marrow blasts. LRP overexpression was seen in 19 (46%) cases. Concordant results between LRP overexpression and P-glycoprotein (PGP) expression were seen in 66% of the cases (p = 0.03), and discordant results (LRP+ and PGP-, or LRP- and PGP+) in 33% of the cases. No correlation was seen between LRP overexpression and FAB type, karyotype, CD34, p53 expression and bcl2 overexpression in blasts. Furthermore, in the 18 cases treated with anthracycline-AraC intensive chemotherapy and the 7 cases treated with low dose AraC, the response rate was not significantly different in LRP+ and LRP- patients. Survival was also similar in LRP+ and LRP- patients. In conclusion, LRP overexpression is probably more frequent in MDS than in de novo AML and, as in AML, is only partially correlated with PGP expression. In our experience, however, LRP was not a prognostic factor for response to chemotherapy and survival in MDS.

We looked for increased apoptosis in fresh bone marrow aspirates in 40 cases of myelodysplastic syndrome (MDS), by detection of DNA fragmentation using TdT incorporation of nucleotides on 3′ ends of DNA (TUNEL technique). No DNA laddering was seen. In six cases (15%) the TUNEL technique showed a moderate increase in the percentage of apoptotic cells (2.5–5% in comparison with

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. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-409) contains supplementary material, which is available to authorized users.