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Supplementary Table 1 from T Cell Receptor Genotyping and HOXA/TLX1 Expression Define Three T Lymphoblastic Lymphoma Subsets which Might Affect Clinical Outcome

Supplementary Figure 2 from T Cell Receptor Genotyping and HOXA/TLX1 Expression Define Three T Lymphoblastic Lymphoma Subsets which Might Affect Clinical Outcome

Purpose: T lymphoblastic lymphomas (T-LBL) are rare disorders of immature T cells which predominantly involve the mediastinum. Their oncogenic pathways and prognostic variables are not clear.Experimental Design: We undertook a retrospective study of 41 cytoplasmic CD3+ T-LBL (nine cases aged Results: Application of a TCR-based immunogenetic classification allowed the identification of three subcategories: 11 immature IM0/D-LBL showed no TCR or only incomplete TCRD DJ rearrangement and corresponded to cytoplasmic CD3+ precursors of uncertain lineage. Sixteen mature TCRDdel-LBL showed biallelic TCRD deletion and both TCRG and TCRB rearrangement, consistent with TCRαβ lineage restriction. Fourteen intermediate LBL (Int-LBL) showed complete TCRD VDJ and TCRG VJ rearrangement, with TCRB VDJ rearrangement in the majority. All Int-LBL expressed HOX11/TLX1 or HOXA9 transcripts and a proportion of the latter were associated with CALM-AF10 or NUP214-ABL fusion transcripts. IM0/D-LBL were restricted to adults with extrathymic disease and bone marrow involvement, whereas Int-LBL and TCRDdel-LBL were found in children and adults with predominantly thymic disease. In adults, the Int-LBL subgroup was associated with a significantly superior clinical outcome. This subgroup can be identified either by TCR immunogenotyping or HOXA9/TLX1 transcript quantification.Conclusion: Application of this molecular classification will allow the prospective evaluation of prognostic effects within pediatric and adult protocols.

Supplementary Figure 1 from T Cell Receptor Genotyping and HOXA/TLX1 Expression Define Three T Lymphoblastic Lymphoma Subsets which Might Affect Clinical Outcome

Supplemental Digital Content is available in the text, T-cell Receptor Gamma (TRG) rearrangements are commonly used to detect clonal lymphoproliferations in hematopathology, since they are rearranged in virtually all T lymphocytes and have a relatively limited recombinatorial repertoire, which reduces the risk of false negative results, at the cost of potential false positivity. We developed an initial one-tube, 2-fluorochrome EuroClonality TRG PCR multiplex (TRG-1T-2F) which was compared to the original 2-tube, 2-fluorochrome EuroClonality/BIOMED-2 TRG PCR (TRG-2T-2F) and a commercial Invivoscribe one-tube, one-fluorochrome kit (IVS-1T-1F) on a series of 239 samples, including both T-cell malignancies and reactive cases. This initial assay yielded discrepant results between the 10 participating EuroClonality laboratories when using 2 fluorochromes, leading to adoption of a final single color EuroClonality strategy (TRG-1T-1F). Compared to TRG-2T-2F, both TRG-1T-1F and IVS-1T-1F demonstrated easier interpretation and a lower risk of false positive from minor peaks in dispersed repertoires. Both generate smaller fragments and as such are likely to be better adapted to analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples. Their differential performance was mainly explained by (i) superposition of biallelic rearrangements with IVS-1T-1F, due to more extensive overlapping of the repertoires and (ii) intentional omission of the TRGJP primer in TRG-1T-1F, in order to avoid the potential risk of confusion of consensus TRG V9-JP normal rearrangements with a pathological clone.

Although purine analogues have significantly improved the outcome of hairy cell leukaemia (HCL) patients, 30-40% relapse, illustrating the need for minimal residual disease (MRD) markers that can aid personalized therapeutic management. Diagnostic samples from 34 HCL patients were used to design an 8-colour flow cytometry (8-FC) tube for blood MRD (B/RD) analysis (188 samples) which was compared to quantitative IGH polymerase chain reaction (Q-PCR) on 83 samples and to qualitative consensus IGH PCR clonality analysis on 165 samples. Despite heterogeneous HCL phenotypes at diagnosis, discrimination from normal B lymphocytes was possible in all cases using a single 8-FC tube, with a robust sensitivity of detection of 10(-4) , comparable to Q-PCR at this level, but preferable in terms of informativeness, simplicity and cost. B/RD assessment of 15 patients achieving haematological complete remission after purine analogues was predictive of a clinically significant relapse risk: with a median follow-up of 95 months; only one of the nine patients with reproducible 8-FC B/RD levels below 10(-4) (B/RD(neg) ) relapsed, compared to 5/6 in the B/RD(pos) group (P = 0.003). These data demonstrate the clinical interest of a robust 8-FC HCL B/RD strategy that could become a surrogate biomarker for therapeutic stratification and new drug assessment, which should be evaluated prospectively.

Purpose Multicentric Castleman disease (MCD) is a distinct lymphoproliferative disorder characterized by inflammatory symptoms, lymphadenopathy, splenomegaly, and cytopenia. Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8), is the cause of virtually all cases of MCD occurring in patients with HIV infection. MCD lesions characteristically contain HHV-8-infected polyclonal IgMλ plasmablasts. A high frequency of HHV-8-related non-Hodgkin lymphoma has been reported in these patients. Patients and methods We now report on three patients who presented with severe symptoms of MCD, extreme splenomegaly, and rapid expansion of B-cell lymphocytosis (44–81%) attributable to circulating HHV-8 positive plasmablasts. Results The circulating plasmablastic cells shared the phenotype (IgMλ, CD19+, CD20− CD138−) of HHV-8-infected cells from MCD lesions, mimicking the leukemic phase of large B-cell lymphoma occurring in HHV-8-related MCD. These patients displayed a very high HHV-8 viral load in blood (>7 logs HHV-8 DNA copies/ml) and high levels of serum vIL-6, the viral homolog of human interleukin 6. Serum IL-6 and IL-10 were also abnormally elevated. HHV-8-infected cells were demonstrated by immunoglobulin gene rearrangement analysis, to be polyclonal and likely represent an expansion of HHV-8-infected cells similar to those found in MCD lesions. Conclusion Thus, the spectrum of HHV-8-related plasmablastic lymphoproliferative disorders in patients with HIV infection is expanded to include HHV-8+ polyclonal IgMλ B-cell lymphocytosis. At onset, this lymphoproliferative disorder may mimic plasmablastic leukemia/lymphoma. Recognizing this unusual complication may have important implications in treatment decision avoiding unnecessary toxicity to the patients.

International audience; Treatment with rituximab has improved the outcome for patients with non-Hodgkin's lymphoma. Patients with B-lineage acute lymphoblastic leukemia (ALL) may also have the CD20 antigen, which is targeted by rituximab. Although single-group studies suggest that adding rituximab to chemotherapy could improve the outcome in such patients, this hypothesis has not been tested in a randomized trial. We randomly assigned adults (18 to 59 years of age) with CD20-positive, Philadelphia chromosome (Ph)-negative ALL to receive chemotherapy with or without rituximab, with event-free survival as the primary end point. Rituximab was given during all treatment phases, for a total of 16 to 18 infusions. From May 2006 through April 2014, a total of 209 patients were enrolled: 105 in the rituximab group and 104 in the control group. After a median follow-up of 30 months, event-free survival was longer in the rituximab group than in the control group (hazard ratio, 0.66; 95% confidence interval [CI], 0.45 to 0.98; P=0.04); the estimated 2-year event-free survival rates were 65% (95% CI, 56 to 75) and 52% (95% CI, 43 to 63), respectively. Treatment with rituximab remained associated with longer event-free survival in a multivariate analysis. The overall incidence rate of severe adverse events did not differ significantly between the two groups, but fewer allergic reactions to asparaginase were observed in the rituximab group. Adding rituximab to the ALL chemotherapy protocol improved the outcome for younger adults with CD20-positive, Ph-negative ALL. (Funded by the Regional Clinical Research Office, Paris, and others; ClinicalTrials.gov number, NCT00327678 .)

International audience; Deletion of the Ikaros (IKZF1) gene is an oncogenic lesion frequently associated with BCR-ABL1-positive acute lymphoblastic leukemias. It is also found in a fraction of BCR-ABL1-negative B-cell precursor acute lymphoblastic leukemias, and early studies showed it was associated with a higher risk of relapse. Therefore, screening tools are needed for evaluation in treatment protocols and possible inclusion in risk stratification. Besides monosomy 7 and large 7p abnormalities encompassing IKZF1, most IKZF1 alterations are short, intragenic deletions. Based on cohorts of patients, we mapped the microdeletion breakpoints and developed a breakpoint-specific fluorescent multiplex polymerase chain reaction that allows detection of recurrent intragenic deletions. This sensitive test could also detect IKZF1 subclonal deletions, whose prognostic significance should be evaluated. Moreover, we show that consensus breakpoint sequences can be used as clonal markers to monitor minimal residual disease. This paper could be useful for translational studies and in clinical management of BCP-ALL.

Slow T-cell reconstitution is a major clinical concern after transplantation of cord blood (CB)-derived hematopoietic stem cells. Adoptive transfer of in vitro-generated T-cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T-cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Culture of human CD34+ CB cells in this new DL-4 system enabled the in vitro generation of large amounts of T-cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc−/− (NSG) mice, DL-4 primed T-cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αβ T cells that subsequently left the thymus and accelerated T-cell reconstitution. T-cell reconstitution was even faster and more robust when ex vivo-manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro-generated human T-cell progenitors to accelerate T-cell reconstitution and also introduces a feeder-cell-free culture technique with the potential for rapid, safe transfer to a clinical setting.

An important proof of principle has been achieved with the development of an in vitro T-cell differentiation assay based on the coculture of hematopoietic progenitors with the OP9-Delta1 stromal cell line. The original murine T cell differentiation assay has since been adapted for human T-cell differentiation, however with lower efficiency. The choice of both medium and cytokines is crucial in this assay, therefore our work has been focused on these two factors. The use of freshly reconstituted medium, the optimization of interleukine-7 (IL-7) concentration, and the addition of stem cell factor (SCF) have allowed to improve the proliferation of progenitors and T-cell precursors as well as the yield of double positive CD4+CD8+ T cells, and mature γδ and αβ T cells. These optimizations make the OP9-Delta1 system sensitive enough to perform both quantitative and qualitative assays with various type of progenitors, including those transduced by a retroviral vector. The improved OP9-Delta1 assay therefore constitutes an extremely useful test for basic research purposes and for translational medicine.

There is a causal association between Helicobacter pylori (Hp) gastric infection and the development of gastric MALT lymphoma. In contrast, the link between Hp gastric infection and the development of extragastric lymphoma has not been thoroughly investigated. We, therefore, studied the prevalence of gastric Hp infection at initial diagnosis of ophthalmologic and nonophthalmologic extragastric lymphoma patients. Three cohorts of patients were studied: a first one of 83 patients with OAL, a second one of 101 patients with extraophthalmologic extragastric lymphoma, and a third one of 156 control individuals (control) without malignant lymphoma. Gastric Hp infection was investigated by histopathological analysis and Hp-specific PCR assay on gastric biopsy tissue samples. We found gastric Hp infection in 37 OAL patients (45%), in 25 extraophthalmologic extragastric lymphoma cases (25%), and in 18 controls individuals (12%) (P < 0.0001 OAL/C and P < 0.01 OAL/extra-OAL cases). Gastritis was found in 51% and 9% of Hp-positive and Hp-negative lymphoma patients, respectively (P < 10−4). Gastric Hp infection only correlated with MALT/LPL lymphoma (P = 0.03). There is a significant association between gastric Hp infection and MALT/LPL OAL. This suggests a novel mechanism of indirect infection-associated lymphomagenesis whereby chronic local antigen stimulation would lead to the emergence of ectopic B-cell lymphoma. © 2010 Wiley-Liss, Inc. Am. J. Hematol.

Purpose: T lymphoblastic lymphomas (T-LBL) are rare disorders of immature T cells which predominantly involve the mediastinum. Their oncogenic pathways and prognostic variables are not clear. Experimental Design: We undertook a retrospective study of 41 cytoplasmic CD3+ T-LBL (nine cases aged Results: Application of a TCR-based immunogenetic classification allowed the identification of three subcategories: 11 immature IM0/D-LBL showed no TCR or only incomplete TCRD DJ rearrangement and corresponded to cytoplasmic CD3+ precursors of uncertain lineage. Sixteen mature TCRDdel-LBL showed biallelic TCRD deletion and both TCRG and TCRB rearrangement, consistent with TCRαβ lineage restriction. Fourteen intermediate LBL (Int-LBL) showed complete TCRD VDJ and TCRG VJ rearrangement, with TCRB VDJ rearrangement in the majority. All Int-LBL expressed HOX11/TLX1 or HOXA9 transcripts and a proportion of the latter were associated with CALM-AF10 or NUP214-ABL fusion transcripts. IM0/D-LBL were restricted to adults with extrathymic disease and bone marrow involvement, whereas Int-LBL and TCRDdel-LBL were found in children and adults with predominantly thymic disease. In adults, the Int-LBL subgroup was associated with a significantly superior clinical outcome. This subgroup can be identified either by TCR immunogenotyping or HOXA9/TLX1 transcript quantification. Conclusion: Application of this molecular classification will allow the prospective evaluation of prognostic effects within pediatric and adult protocols.

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34(+)CD10(+) progenitor population and which is distinct from B cell-committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin(-)CD34(+)CD10(+)CD24(-) progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor alpha, and CD3epsilon. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus.

Contains fulltext : 51510.pdf (Publisher’s version ) (Closed access) Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease. 7 p.

Contains fulltext : 52315.pdf (Publisher’s version ) (Closed access) Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Acute promyelocytic leukemia (APL) is the most differentiated form of acute myeloid leukemia (AML) and has generally been considered to result from transformation of a committed myeloid progenitor. Paradoxically, APL has long been known to express the T-cell lymphoid marker, CD2. We searched for other parameters indicative of T-cell lymphoid specification in a cohort of 36 APL cases, revealing a frequent but asynchronous T-cell lymphoid program most marked in the hypogranular variant (M3v) subtype, with expression of PTCRA, sterile TCRA, and TCRG transcripts and TCRG rearrangement in association with sporadic cytoplasmic expression of CD3 or TdT proteins. Gene-expression profiling identified differentially expressed transcription factors that have been implicated in lymphopoiesis. These data carry implications for the hematopoietic progenitor targeted by the PML-RARA oncoprotein in APL and are suggestive of a different cellular origin for classic hypergranular (M3) and variant forms of the disease. They are also consistent with the existence and subsequent transformation of progenitor populations with lymphoid/myeloid potential.

HOXA cluster deregulation in T-ALL associated with both a TCRD-HOXA and a CALM-AF10 chromosomal translocation

Postnatal thymic involution occurs progressively throughout the first 3 decades of life. It predominantly affects T-cell receptor (TCR) alphabeta-lineage precursors, with a consequent proportional increase in multipotent thymic precursors. We show that T-acute lymphoblastic leukemias (T-ALLs) demonstrate a similar shift with age from predominantly TCR expressing to an immature (IM0/delta/gamma) stage of maturation arrest. Half demonstrate HOX11, HOX11L2, SIL-TAL1, or CALM-AF10 deregulation, with each being associated with a specific, age-independent stage of maturation arrest. HOX11 and SIL-TAL represent alphabeta-lineage oncogenes, whereas HOX11L2 expression identifies an intermediate alphabeta/gammadelta-lineage stage of maturation arrest. In keeping with preferential alphabeta-lineage involution, the incidence of SIL-TAL1 and HOX11L2 deregulation decreased with age. In contrast, HOX11 deregulation became more frequent, suggesting longer latency. TAL1/LMO1 deregulation is more frequent in alphabeta-lineage T-ALL, when it is predominantly due to SIL-TAL1 rearrangements in children but to currently unknown mechanisms in adolescents and adults. LMO2 was more frequently coexpressed with LYL1, predominantly in IM0/delta/gamma adult cases, than with TAL1. These age-related changes in phenotype and oncogenic pathways probably reflect progressive changes in the thymic population at risk of malignant transformation.

cCD79a and IgH VDJ/DJ rearrangements are considered to be relatively specific for B lymphoid precursors. We looked for both in cCD3+, CD7+, CD19- T-ALLs classified by TCR status into alphabeta or gammadelta/immature (IM) lineages, with individualization of HOX11L2+ T-ALLs since they represent an intermediate alphabeta/gammadelta category. cCD79a was expressed at low levels in 47% of T-ALL and was most frequent in IMgamma T-ALLs. IgH rearrangements were common in gammadelta/IM (45%) and HOX11L2+ (35%) T-ALLs compared to HOX11L2-negative cases (3%; P0.001). CD127 (IL7Ralpha) expression was also more common in the gammadelta/IM lineage but its expression was virtually mutually exclusive of IgH rearrangement. Low-level cCD79a expression alone should therefore not be interpreted as evidence of B lineage affiliation in immature leukemias. gammadelta/IM lineage T-ALLs potentially include two distinct categories: predominantly IgH+, cCD79a+, CD127- cases which retain gammadelta and B lymphoid potential and IgH-, cCD79a-, CD127+ cases with restricted T lineage potential.

MLL rearrangements in acute myeloid leukemia (AML) include translocations and intragenic abnormalities such as internal duplication and breakage induced by topoisomerase II inhibitors. In adult AML, FLT3 internal tandem duplications (ITDs) are more common in cases with MLL intragenic abnormalities (33%) than those with MLL translocation (8%). Mutation/deletion involving FLT3 D835 are found in more than 20% of cases with MLL intragenic abnormalities compared with 10% of AML with MLL translocation and 5% of adult AML with normal MLL status. Real-time quantification of FLT3 in 141 cases of AML showed that all cases with FLT3 D835 express high level transcripts, whereas FLT3-ITD AML can be divided into cases with high-level FLT3 expression, which belong essentially to the monocytic lineage, and those with relatively low-level expression, which predominantly demonstrate PML-RARA and DEK-CAN. FLT3 abnormalities in CBF leukemias with AML1-ETO or CBFβ-MYH11 were virtually restricted to cases with variant CBFβ-MYH11 fusion transcripts and/or atypical morphology. These data suggest that the FLT3 and MLL loci demonstrate similar susceptibility to agents that modify chromatin configuration, including topoisomerase II inhibitors and abnormalities involving PML and DEK, with consequent errors in DNA repair. Variant CBFβ-MYH11 fusions and bcr3 PML-RARA may also be initiated by similar mechanisms.

Adult Acute Lymphoblastic Leukemia (ALL) therapies have been improved by pediatric-like approaches. However, treatment failures and relapses are common and new markers are needed to identify patients with poor prognosis in prospective trials. The p16(INK4A)/CDK4-6/pRb pathway and telomerase activity, which are implicated in cell activation and aging, were analyzed to identify new prognostic markers. Proteins of the p16(INK4A)/CDK4-6/pRb pathway and telomerase activity were analyzed in 123 adult B-cell precursor (BCP) ALL cases included in the GRAALL/GRAAPH trials. We found a significantly increased expression of p16(INK4A) in BCP-ALLs with MLL rearrangement. Telomerase activity was significantly lower in Philadelphia chromosome-negative/IKAROS-deleted (BCR-ABL1(-)/IKAROS(del)) cases compared to Philadelphia chromosome-positive (BCR-ABL1+) BCP-ALLs. In BCR-ABL1+ ALLs, high CDK4 expression, phosphorylated pRb (p-pRb) and telomerase activity were significantly associated with a shorter disease-free survival (DFS) and event-free survival (EFS). Enhanced p16(INK4A) expression was only related to a significantly shorter DFS. In vitro analyses of normal stimulated lymphocytes after short- and long-term cultures demonstrated that the observed protein variations of poor prognosis in BCR-ABL1+ ALLs may be related to cell activation but not to cell aging. For these patients, our findings argue for the development of therapeutic strategies including the addition of new lymphocyte activation inhibitors to current treatments.

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.

B-cell precursor acute lymphoblastic leukemias (BCP-ALLs) are increasingly treated on risk-adapted protocols based on presenting clinical and biological features. Residual molecular positivity of clonal immunoglobulin (IG) and T-cell receptor (TCR) rearrangements allows detection of patients at an increased risk of relapse. If these rearrangements are to be used for universal follow-up, it is important to determine the extent to which they are informative in different BCP-ALL subsets. We show thatIGH V-D-J rearrangements occur in 89% of 163 BCP-ALL, with no significant variation according to age or genotype (BCR-ABL, TEL-AML1, MLL-AF4, and E2A-PBX1). In contrast,TCRG rearrangements, which occur in 60% of patients overall, are frequent in BCR-ABL and TEL-AML1, are less so in MLL-AF4, and are virtually absent in infants aged predominantly from 1 to 2 years and in E2A-PBX1 ALLs. Incidence of the predominant TCRD Vδ2-Dδ3 rearrangement decreases with age but is independent of genotype. These differences are not due to differential recombination activating gene activity, nor can they be explained adequately by stage of maturation arrest. Analysis of MLL-AF4 BCP-ALL is in keeping with transformation of a precursor at an early stage of ontogenic development, despite the adult onset of the cases analyzed. We postulate that the complete absence of TCRG rearrangement in E2A-PBX1 cases may result from deregulated E2A function. These data also have practical consequences for the use ofTCR clonality for the molecular follow-up of BCP-ALL.

Detection of clonal T cell receptor γ (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10−3 at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V–J rearrangements from childhood cases. We describe rapid, multifluorescent Vγ and Jγ PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of Vγ and Jγ segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10−2–10−3 level. We demonstrate preferential V–J combinations but no difference in V–J usage between children and adults, nor between SIL-TALI-negative and -positive cases. A combination of fluorescent multiplex and Vγ–Jγ-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.

To the editor: The CCAAT/enhancer binding protein α (C/EBPA) is a stage-specific transcription factor that controls proliferation and differentiation toward a myeloid and against a T-lymphoid fate.[1][1] Nonconditional targeted disruption of C/EBPA leads to a selective early block to granulocyte

Pure red cell aplasia (PRCA) is defined by an isolated severe anemia, contrasting with a normal white blood cell and platelet counts, severe reticulocytopenia and a selective absence of erythroid progenitors in the bone marrow smears.[1][1] Although an immune origin is involved in the majority of

Activation of tyrosine kinase genes is a frequent event in human hematologic malignancies. Because gene activation could be associated with gene dysregulation, we attempted to screen for activating gene mutation based on high-level gene expression. We focused our study on the Janus kinase 2 (JAK2) gene in 90 cases of acute leukemia. This strategy led to the identification of a novel JAK2-acquired mutation in a patient with Down syndrome (DS) with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This mutation involves a 5–amino acid deletion within the JH2 pseudokinase domain (JAK2ΔIREED). Expression of JAK2ΔIREED in Ba/F3 cells induced constitutive activation of the JAK-STAT pathway and growth factor–independent cell proliferation. These results highlight the JAK2 pseudokinase domain as an oncogenic hot spot and indicate that activation of the JAK-STAT pathway may contribute to lymphoid malignancies and hematologic disorders observed in children with DS.

Purpose The Group for Research in Adult Acute Lymphoblastic Leukemia (GRAALL) recently reported a significantly better outcome in T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 (N/F) mutations compared with unmutated T-ALL. Despite this, one third of patients with N/F-mutated T-ALL experienced relapse. Patients and Methods In a series of 212 adult T-ALLs included in the multicenter randomized GRAALL-2003 and -2005 trials, we searched for additional N/K-RAS mutations and PTEN defects (mutations and gene deletion). Results N/F mutations were identified in 143 (67%) of 212 patients, and lack of N/F mutation was confirmed to be associated with a poor prognosis. K-RAS, N-RAS, and PTEN mutations/deletions were identified in three (1.6%) of 191, 17 (8.9%) of 191, and 21 (12%) of 175 patients, respectively. The favorable prognostic significance of N/F mutations was restricted to patients without RAS/PTEN abnormalities. These observations led us to propose a new T-ALL oncogenetic classifier defining low-risk patients as those with N/F mutation but no RAS/PTEN mutation (97 of 189 patients; 51%) and all other patients (49%; including 13% with N/F and RAS/PTEN mutations) as high-risk patients. In multivariable analysis, this oncogenetic classifier remained the only significant prognostic covariate (event-free survival: hazard ratio [HR], 3.2; 95% CI, 1.9 to 5.15; P < .001; and overall survival: HR, 3.2; 95% CI, 1.9 to 5.6; P < .001). Conclusion These data demonstrate that the presence of N/F mutations in the absence of RAS or PTEN abnormalities predicts good outcome in almost 50% of adult T-ALL. Conversely, the absence of N/F or presence of RAS/PTEN alterations identifies the remaining cohort of patients with poor prognosis.

The advent of tyrosine kinase inhibitors (TKI) has profoundly changed the current therapeutic approach in some hematologic malignancies, including BCR-ABL1 -positive acute lymphoblastic leukemia (ALL). Recently, next-generation genomic methods have uncovered various alterations in ALL that lead to

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (

Contains fulltext : 107785.pdf (Publisher’s version ) (Open Access) PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

We examined 807 productive IGHV-IGHD-IGHJ gene rearrangements from mantle cell lymphoma (MCL) cases, by far the largest series to date. The IGHV gene repertoire was remarkably biased, with IGHV3-21, IGHV4-34, IGHV1-8, and IGHV3-23 accounting for 46.3% of the cohort. Eighty-four of 807 (10.4%) cases, mainly using the IGHV3-21 and IGHV4-34 genes, were found to bear stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences and were placed in 38 clusters. Notably, the MCL stereotypes were distinct from those reported for chronic lymphocytic leukemia. Based on somatic hypermutation (SHM) status, 238/807 sequences (29.5%) carried IGHV genes with 100% germ line identity; the remainder (569/807; 70.5%) exhibited different SHM impact, ranging from minimal (in most cases) to pronounced. Shared replacement mutations across the IGHV gene were identified for certain subgroups, especially those using IGHV3-21, IGHV1-8, and IGHV3-23. Comparison with other entities, in particular CLL, revealed that several of these mutations were "MCL-biased." In conclusion, MCL is characterized by a highly restricted immunoglobulin gene repertoire with stereotyped VH CDR3s and very precise SHM targeting, strongly implying a role for antigen-driven selection of the clonogenic progenitors. Hence, an antigen-driven origin of MCL could be envisaged, at least for subsets of cases. (Blood. 2011; 118(11):3088-3095) We examined 807 productive IGHV-IGHD-IGHJ gene rearrangements from mantle cell lymphoma (MCL) cases, by far the largest series to date. The IGHV gene repertoire was remarkably biased, with IGHV3-21, IGHV4-34, IGHV1-8, and IGHV3-23 accounting for 46.3% of the cohort. Eighty-four of 807 (10.4%) cases, mainly using the IGHV3-21 and IGHV4-34 genes, were found to bear stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences and were placed in 38 clusters. Notably, the MCL stereotypes were distinct from those reported for chronic lymphocytic leukemia. Based on somatic hypermutation (SHM) status, 238/807 sequences (29.5%) carried IGHV genes with 100% germ line identity; the remainder (569/807; 70.5%) exhibited different SHM impact, ranging from minimal (in most cases) to pronounced. Shared replacement mutations across the IGHV gene were identified for certain subgroups, especially those using IGHV3-21, IGHV1-8, and IGHV3-23. Comparison with other entities, in particular CLL, revealed that several of these mutations were "MCL-biased." In conclusion, MCL is characterized by a highly restricted immunoglobulin gene repertoire with stereotyped VH CDR3s and very precise SHM targeting, strongly implying a role for antigen-driven selection of the clonogenic progenitors. Hence, an antigen-driven origin of MCL could be envisaged, at least for subsets of cases. (Blood. 2011; 118(11):3088-3095)

SummaryThe relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.

Abstract 91 In normal and leukemic hemopoiesis, stem cells differentiate through intermediate progenitors into terminal cells. In human Acute Myeloid Leukemia (AML), there is uncertainty about: (i) whether there is more than one leukemic stem cell (LSC) population in any one individual patient; (ii) how homogeneous AML LSCs populations are at a molecular and cellular level and (iii) the relationship between AML LSCs and normal stem/progenitor populations. Answers to these questions will clarify the molecular pathways important in the stepwise transformation of normal HSCs/progenitors. We have studied 82 primary human CD34+ AML samples (spanning a range of FAB subtypes, cytogenetic categories and FLT3 and NPM1 mutation states) and 8 age-matched control marrow samples. In ∼80% of AML cases, two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. One population is CD34+CD38-CD90-CD45RA+ (CD38-CD45RA+) and the other CD34+CD38+CD110-CD45RA+ (GMP-like). Both populations from 7/8 patients have leukemic stem cell (LSC) activity in primary and secondary xenograft assays with no LSC activity in CD34- compartment. The two CD34+ LSC populations are hierarchically ordered, with CD38-CD45RA+ LSC giving rise to CD38+CD45RA+ LSC in vivo and in vitro. Limit dilution analysis shows that CD38-CD45RA+LSCs are more potent by 8–10 fold. From 18 patients, we isolated both CD38-CD45RA+ and GMP-like LSC populations. Global mRNA expression profiles of FACS-sorted CD38-CD45RA+ and GMP-like populations from the same patient allowed comparison of the two populations within each patient (negating the effect of genetic/epigenetic changes between patients). Using a paired t-test, 748 genes were differentially expressed between CD38-CD45RA+ and GMP-like LSCs and separated the two populations in most patients in 3D PCA. This was confirmed by independent quantitative measures of difference in gene expression using a non-parametric rank product analysis with a false discovery rate of 0.01. Thus, the two AML LSC populations are molecularly distinct. We then compared LSC profiles with those from 4 different adult marrow normal stem/progenitor cells to identify the normal stem/progenitor cell populations which the two AML LSC populations are most similar to at a molecular level. We first obtained a 2626 gene set by ANOVA, that maximally distinguished normal stem and progenitor populations. Next, the expression profiles of 22 CD38-CD45RA+ and 21 GMP-like AML LSC populations were distributed by 3D PCA using this ANOVA gene set. This showed that AML LSCs were most closely related to their normal counterpart progenitor population and not normal HSC. This data was confirmed quantitatively by a classifier analysis and hierarchical clustering. Taken together, the two LSC populations are hierarchically ordered, molecularly distinct and their gene expression profiles do not map most closely to normal HSCs but rather to their counterpart normal progenitor populations. Finally, as global expression profiles of CD38-CD45RA+ AML LSC resemble normal CD38-CD45RA+ cells, we defined the functional potential of these normal cells. This had not been previously determined. Using colony and limiting dilution liquid culture assays, we showed that single normal CD38-CD45RA+ cells have granulocyte and macrophage (GM), lymphoid (T and B cell) but not megakaryocyte-erythroid (MK-E) potential. Furthermore, gene expression studies on 10 cells showed that CD38-CD45RA+ cells express lymphoid and GM but not Mk-E genes. Taken together, normal CD38-CD45RA+ cells are most similar to mouse lymphoid primed multi-potential progenitor cells (LMPP) cells and distinct from the recently identified human Macrophage Lymphoid progenitor (MLP) population. In summary, for the first time, we show the co-existence of LMPP-like and GMP-like LSCs in CD34+ AML. Thus, CD34+ AML is a progenitor disease where LSCs have acquired abnormal self-renewal potential (Figure 1). Going forward, this work provides a platform for determining pathological LSCs self-renewal and tracking LSCs post treatment, both of which will impact on leukemia biology and therapy. Disclosures: No relevant conflicts of interest to declare.

Expression of cell–cell interacting genes distinguishes HLXB9/TEL from MLL-positive childhood acute myeloid leukemia

The prognostic impact of minimal residual disease (MRD) was analyzed in 259 patients with mantle cell lymphoma (MCL) treated within 2 randomized trials of the European MCL Network (MCL Younger and MCL Elderly trial). After rituximab-based induction treatment, 106 of 190 evaluable patients (56%) achieved a molecular remission (MR) based on blood and/or bone marrow (BM) analysis. MR resulted in a significantly improved response duration (RD; 87% vs 61% patients in remission at 2 years, P = .004) and emerged to be an independent prognostic factor for RD (hazard ratio = 0.4, 95% confidence interval, 0.1-0.9, P = .028). MR was highly predictive for prolonged RD independent of clinical response (complete response [CR], complete response unconfirmed [CRu], partial response [PR]; RD at 2 years: 94% in BM MRD-negative CR/CRu and 100% in BM MRD-negative PR, compared with 71% in BM MRD-positive CR/CRu and 51% in BM MRD-positive PR, P = .002). Sustained MR during the postinduction period was predictive for outcome in MCL Younger after autologous stem cell transplantation (ASCT; RD at 2 years 100% vs 65%, P = .001) and during maintenance in MCL Elderly (RD at 2 years: 76% vs 36%, P = .015). ASCT increased the proportion of patients in MR from 55% before high-dose therapy to 72% thereafter. Sequential MRD monitoring is a powerful predictor for treatment outcome in MCL. These trials are registered at www.clinicaltrials.gov as #NCT00209222 and #NCT00209209.

Purpose Retrospective comparisons have suggested that adolescents or teenagers with acute lymphoblastic leukemia (ALL) benefit from pediatric rather than adult chemotherapy regimens. Thus, the aim of the present phase II study was to test a pediatric-inspired treatment, including intensified doses of nonmyelotoxic drugs, such as prednisone, vincristine, or l-asparaginase, in adult patients with ALL up to the age of 60 years. Patients and Methods Between 2003 and 2005, 225 adult patients (median age, 31 years; range, 15 to 60 years) with Philadelphia chromosome–negative ALL were enrolled onto the Group for Research on Adult Acute Lymphoblastic Leukemia 2003 protocol, which included several pediatric options. Some adult options, such as allogeneic stem-cell transplantation for patients with high-risk ALL, were nevertheless retained. Results were retrospectively compared with the historical France-Belgium Group for Lymphoblastic Acute Leukemia in Adults 94 (LALA-94) trial experience in 712 patients age 15 to 55 years. Results Complete remission rate was 93.5%. At 42 months, event-free survival (EFS) and overall survival (OS) rates were 55% (95% CI, 48% to 52%) and 60% (95% CI, 53% to 66%), respectively. Age remained an important bad prognostic factor, with 45 years of age as best cutoff. In older versus younger patients, there was a higher cumulative incidence of chemotherapy-related deaths (23% v 5%, respectively; P < .001) and deaths in first CR (22% v 5%, respectively; P < .001), whereas the incidence of relapse remained stable (30% v 32%, respectively). Complete remission rate (P = .02), EFS (P < .001), and OS (P < .001) compared favorably with the previous LALA-94 experience. Conclusion These results suggest that pediatric-inspired therapy markedly improves the outcome of adult patients with ALL, at least until the age of 45 years.

The majority of haematological cancers involve the lymphoid system. They include acute lymphoblastic leukemias (ALL), which are arrested at variable stages of development and present with blood and bone marrow involvement and chronic leukemias, lymphomas and myelomas, which present with infiltration of a large variety of hematopoietic and non hematopoietic tissues by mature lymphoid cells which express a surface antigen receptor. The majority involve the B-cell lineage and the vast majority have undergone clonal rearrangement of their Ig and/or TCR rearrangements. Analysis of Ig/TCR genomic V(D)J repertoires by PCR based lymphoid clonality analysis within a diagnostic setting allows distinction of clonal from reactive lymphoproliferative disorders, clonal tracking for evidence of tumor dissemination and follow-up, identification of a lymphoid origin in undiagnosed tumors and evaluation of clonal evolution. Ig/TCR VDJ errors are also at the origin of recombinase mediated deregulated expression of a variety of proto-oncogenes in ALL, whereas in lymphoma it is increasingly clear that IgH containing translocations result from abnormalities other than VDJ errors (somatic hypermutation and/or isotype switching). In addition to this mechanistic contribution to lymphoid oncogenesis, it is possible that failure to successfully complete expression of an appropriate Ig or TCR may lead to maturation arrest in a lymphoid precursor, which may in itself contribute to altered tissue homeostasis, particularly if the arrest occurs at a stage of cellular expansion.

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.

We conducted a study of 152 HIV-1-seropositive individuals in order to evaluate the possible correlations between the isolation of HIV from peripheral blood mononuclear cells or from plasma and CD4 cell counts. HIV was isolated from only 36% of plasma samples, and the isolation rate was closely related to CD4 cell counts, increasing gradually from 0% in subjects with greater than 800 x 10(6)/l CD4 cells to 88% in those with less than 100 x 10(6)/l CD4 cells. In contrast, HIV was isolated from 92% of cell samples (99% in subjects with less than 900 x 10(6)/l CD4 cells, 46% in those with CD4 counts greater than or equal to 900 x 10(6)/l). Since most cell samples were positive, a scoring method was designed to quantify the cellular viral load. The results obtained demonstrated that the cellular viral load was closely related to CD4 counts. We also found that the cellular viral load was higher in subjects with either positive plasma isolation or positive p24 antigenaemia. The measurement of the cellular viral load by this scoring method appears to be useful for the management of HIV-seropositive individuals and for the evaluation of therapeutic trials.

Many somatic genetic abnormalities have been identified in T-cell acute lymphoblastic leukemia (T-ALL) but each individual abnormality accounts for a small proportion of cases; therapeutic stratification consequently still relies on classical clinical markers. NOTCH1 and/or FBXW7 mutations both lead to activation of the NOTCH1 pathway and are among the most frequent mutations in T-ALL. We screened 141 adult diagnostic T-ALL samples from patients treated on either the Lymphoblastic Acute Leukemia in Adults (LALA)-94 (n = 87) or the GRAALL-2003 (n = 54) trials. In 88 cases (62%) there were demonstrated NOTCH1 mutations (42% heterodimerization [HD], 10% HD+proline glutamate serine threonine [PEST], 6% PEST, 2% juxtamembrane mutations, 2% transactivation domain [TAD]) and 34 cases (24%) had FBXW7 mutations (21 cases had both NOTCH1 and FBXW7 mutations); 40 cases (28%) were wild type for both. There was no significant correlation between NOTCH1 and/or FBXW7 mutations and clinico-biologic features. Median event-free survival (EFS) and overall survival (OS) were 36 versus 17 months (P = .01) and not reached versus 32 months (P = .004) in patients with NOTCH1 and/or FBXW7 mutations versus other patients, respectively. Multivariate analysis showed that the presence of NOTCH1/FBXW7 mutations was an independent good prognostic factor for EFS and OS (P = .02 and P = .01, respectively). These data demonstrate that NOTCH1 pathway activation by either NOTCH1 or FBXW7 mutation identifies a large group of patients with a favorable outcome that could justify individual therapeutic stratification for T-ALL.

Previously, several individuals with X-linked SCID (SCID-X1) were treated by gene therapy to restore the missing IL-2 receptor gamma (IL2RG) gene to CD34+ BM precursor cells using gammaretroviral vectors. While 9 of 10 patients were successfully treated, 4 of the 9 developed T cell leukemia 31-68 months after gene therapy. In 2 of these cases, blast cells contained activating vector insertions near the LIM domain-only 2 (LMO2) proto-oncogene. Here, we report data on the 2 most recent adverse events, which occurred in patients 7 and 10. In patient 10, blast cells contained an integrated vector near LMO2 and a second integrated vector near the proto-oncogene BMI1. In patient 7, blast cells contained an integrated vector near a third proto-oncogene,CCND2. Additional genetic abnormalities in the patients' blast cells included chromosomal translocations, gain-of-function mutations activating NOTCH1, and copy number changes, including deletion of tumor suppressor gene CDKN2A, 6q interstitial losses, and SIL-TAL1 rearrangement. These findings functionally specify a genetic network that controls growth in T cell progenitors. Chemotherapy led to sustained remission in 3 of the 4 cases of T cell leukemia, but failed in the fourth. Successful chemotherapy was associated with restoration of polyclonal transduced T cell populations. As a result, the treated patients continued to benefit from therapeutic gene transfer.

International audience; Severe combined immunodeficiency (SCID) caused by mutation of the recombination-activating gene 1 (RAG1) or Artemis gene lead to the absence of B- and T-cell differentiation. The only curative treatment is allogeneic bone marrow (BM) transplantation, which displays a high survival rate when an HLA compatible donor is available but has a poorer prognosis when the donor is partially compatible. Consequently, gene therapy may be a promising alternative strategy for these diseases. Here, we report that lentiviral gene-corrected BM CD34(+) cells (isolated from Artemis- or RAG1-deficient patients) sustain human B-cell differentiation following injection into non-obese diabetic/SCID (NOD-SCID) mice previously infused with anti-interleukin-2 receptor beta chain monoclonal antibody. In most of the mice BM, engrafted with Artemis-transduced cells, human B-cell differentiation occurred until the mature stage. The B cells were functional as human immunoglobulin M (IgM) was present in the serum. Following injection with RAG1-transduced cells, human engraftment occurred in vivo but B-cell differentiation until the mature stage was less frequent. However, when it occurred, it was always associated with human IgM production. This overall approach represents a useful tool for evaluating gene transfer efficiency in human SCID forms affecting B-cell development (such as Artemis deficiency) and for testing new vectors for improving in vivo RAG1 complementation.