Positive T-ALL Research Articles

Outcomes of Childhood Noninfant Acute Lymphoblastic Leukemia With 11q23/KMT2A Rearrangements in a Modern Therapy Era: A Retrospective International Study.

We aimed to study prognostic factors and efficacy of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) in first remission of patients with noninfant childhood acute lymphoblastic leukemia (ALL) with 11q23/KMT2A rearrangements treated with chemotherapy regimens between 1995 and 2010. Data were retrospectively retrieved from 629 patients with 11q23/KMT2A-rearranged ALL from 17 members of the Ponte-di-Legno Childhood ALL Working Group. Clinical and biologic characteristics, early response assessed by minimal residual disease at the end of induction (EOI) therapy, and allo-HSCT were analyzed for their impact on outcomes. A specific 11q23/KMT2A translocation partner gene was identified in 84.3% of patients, with the most frequent translocations being t(4;11)(q21;q23) (n = 273; 51.5%), t(11;19)(q23;p13.3) (n = 106; 20.0%), t(9;11)(p21_22;q23) (n = 76; 14.3%), t(6;11)(q27;q23) (n = 20; 3.8%), and t(10;11)(p12;q23) (n = 14; 2.6%); 41 patients (7.7%) had less frequently identified translocation partner genes. Patient characteristics and early response varied among subgroups, indicating large biologic heterogeneity and diversity in therapy sensitivity among 11q23/KMT2A-rearranged ALL. The EOI remission rate was 93.2%, and the 5-year event-free survival (EFS) for the entire cohort was 69.1% ± 1.9%, with a range from 41.7% ± 17.3% for patients with t(9;11)-positive T-ALL (n = 9) and 64.8% ± 3.0% for patients with t(4;11)-positive B-ALL (n = 266) to 91.2% ± 4.9% for patients with t(11;19)-positive T-ALL (n = 34). Low EOI minimal residual disease was associated with favorable EFS, and induction failure was particularly predictive of nonresponse to further therapy and relapse and poor EFS. In addition, EFS was not improved by allo-HSCT compared with chemotherapy only in patients with both t(4;11)-positive B-ALL (n = 64 v 51; P = .10) and 11q23/KMT2A-rearranged T-ALL (n = 16 v 10; P = .69). Compared with historical data, prognosis of patients with noninfant 11q23/KMT2A-rearranged ALL has improved, but allo-HSCT failed to affect outcome. Targeted therapies are needed to reduce relapse and treatment-related mortality rates.

Ectopic expression of a combination of 5 genes detects high risk forms of T-cell acute lymphoblastic leukemia

BackgroundT cell acute lymphoblastic leukemia (T-ALL) defines a group of hematological malignancies with heterogeneous aggressiveness and highly variable outcome, making therapeutic decisions a challenging task. We tried to discover new predictive model for T-ALL before treatment by using a specific pipeline designed to discover aberrantly active gene.ResultsThe expression of 18 genes was significantly associated with shorter survival, including ACTRT2, GOT1L1, SPATA45, TOPAZ1 and ZPBP (5-GEC), which were used as a basis to design a prognostic classifier for T-ALL patients. The molecular characterization of the 5-GEC positive T-ALL unveiled specific characteristics inherent to the most aggressive T leukemic cells, including a drastic shut-down of genes located on the mitochondrial genome and an upregulation of histone genes, the latter characterizing high risk forms in adult patients. These cases fail to respond to the induction treatment, since 5-GEC either predicted positive minimal residual disease (MRD) or a short-term relapse in MRD negative patients.ConclusionOverall, our investigations led to the discovery of a homogenous group of leukemic cells with profound alterations of their biology. It also resulted in an accurate predictive tool that could significantly improve the management of T-ALL patients.

Analysis of the clinical characteristics of 24 cases of hematological malignancies with SET-NUP214 fusion gene

目的探讨SET-NUP214融合基因在血液恶性肿瘤中的表达，分析其相关的临床及生物学特征。方法回顾性分析2012年1月至2018年12月北京大学人民医院诊断的24例SET-NUP214融合基因阳性血液恶性肿瘤患者的临床资料，并采用Kaplan-Meier法进行生存分析。结果24例患者中，急性淋巴细胞白血病（ALL）15例（T-ALL 13例，B-ALL 2例）、急性髓系白血病（AML）7例，T/髓混合急性白血病2例。13例T-ALL患者免疫表型以CD3+CD2−为主要特征，73.3％的ALL患者伴有髓系标志表达，85.7％的AML患者表达CD7。24例患者诱导化疗完全缓解（CR）率91.7％。全部患者均接受异基因造血干细胞移植，中位随访24个月，AML和ALL的3年无复发生存（RFS）率分别为85.7％和33.3％，差异无统计学意义（P＝0.128）。比较13例SET-NUP214阳性与62例SET-NUP214阴性T-ALL患者的疗效，诱导化疗CR率分别为92.3％和93.5％（P＝0.445），诱导化疗4周CR率分别为69.2％和72.6％（P＝0.187），差异均无统计学意义。接受造血干细胞移植后，SET-NUP214阳性T-ALL患者的3年RFS率（38.5％）明显低于SET-NUP214阴性T-ALL患者（66.4％）（P＝0.028）。结论SET-NUP214融合基因主要见于T细胞源性血液肿瘤，伴SET-NUP214融合基因T-ALL预后较差。

The use of 5‐aminolevulinic acid to reduce heat‐stress‐related damages in tall fescue

AbstractHeat stress is a major abiotic stress that damages cool‐season turfgrasses, such as tall fescue (Festuca arundinacea Schreb.), during summer months. Heat stress results in oxidative damage, reductions in photosynthesis, and ultimately can cause plant death. Exogenous applications of 5‐aminolevulinic acid (ALA) have previously been demonstrated to improve abiotic stress tolerance in a number of crops by improving chlorophyll levels and reducing oxidative damage, two major indicators of heat stress in plants. Experiments were conducted in growth chamber conditions to determine the effect of ALA application on heat stress in tall fescue plants and to confirm if ALA could alleviate damage related to inhibited photosynthesis or oxidative damages associated with high temperature. A rate of 100 mg L−1 ALA was determined to be effective in eliciting positive tall fescue responses during heat stress with plants maintaining better visual characteristics. Plants treated with ALA had greater levels of chlorophyll as well as photosynthetic performance under heat stress than plants that were not treated with ALA. Additionally, ALA plants had reduced oxidative damage associated with greater antioxidant activities of the enzymes catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). The use of ALA was demonstrated to be effective in reducing heat stress related damages in tall fescue.

In Vitro Drug Response Profiling in BCP- and T-ALL Primary Samples Adds a Robust Functional Layer Enabling Optimized Guidance of Individualized Therapy in Relapsed and Refractory Pediatric Acute Leukemia Patients

In Vitro Drug Response Profiling in BCP- and T-ALL Primary Samples Adds a Robust Functional Layer Enabling Optimized Guidance of Individualized Therapy in Relapsed and Refractory Pediatric Acute Leukemia Patients

Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Lymphoblastic Leukemia and Karnofsky Performance Status Score Equal or Lower Than 80%. a Study from the Acute Leukemia Working Party (ALWP) of the European Society for Blood and Marrow Transplantation (EBMT)

Recently, an increasing number of patients with acute lymphoblastic leukemia with a poor Karnofsky Performance Status (KPS) score are considered for allogeneic hematopoietic stem cell transplantation (allo-HCT). Nevertheless, there is paucity of available data about outcome of this fragile population. We report here the results of a retrospective study designed to evaluate outcome of ALL patients undergoing allo-HCT with KPS score ≤80%. The analysis included ALL patients aged ≥18 years, undergoing allo-HCT in first remission between 2000 and 2018, with a KPS score of 50% to 80% at the time of transplant. Patients who received manipulated grafts or had incomplete data about cytogenetics were excluded. Conditioning intensity was defined according to EBMT definitions (Aoudjhane M. et al, Leukemia 2005; 19: pp. 2304-2312). A total of 1,010 patients were identified. Median age at transplant was 43 years (18-76 years). Median year of transplant was 2011. The KPS score was =80% in 83% of the patients and <80% in 17% of the patients. Diagnosis was Philadelphia chromosome (Ph) negative B-ALL, Ph positive B-ALL or T-ALL in 34%, 44% and 22% of the patients, respectively. Donor type was MSD or 10/10 UD in 60% and 40% of the patients, respectively. Conditioning was myeloablative (MAC) or reduced-intensity (RIC) in 78% and 22% of the patients, respectively, and it was TBI-based in 79% of the patients. Stem cell source was PBSC in 76% and BM in 24% of the patients, respectively. Anti-thymocyte globulin (ATG) was administered to 21% of the patients receiving MSD and 68% of the patients receiving 10/10 UD as donor type. Cumulative incidence of grade II-IV and III-IV aGVHD was 32% and 9%, respectively. The 2-year cumulative incidence of chronic GVHD (cGvHD) and severe cGVHD was 43% and 18%, respectively. Non relapse mortality (NRM) and relapse incidence (RI) at 2 years were 18% and 28%, respectively. At 2 years, leukemia-free survival (LFS), overall survival (OS) and GVHD-free, relapse-free survival (GRFS) rates were 54%, 64% and 41%, respectively. On multivariate analysis, transplant from 10/10 UD was associated with higher incidence of aGVHD (HR 1.8, p<0.0001) and higher risk of NRM (HR 1.7, p<0.01) as compared to MSD. RIC conditioning was associated with higher risk of relapse (HR 1.2, p=0.02), lower LFS (HR 1.3, p=0.03) and lower GRFS (HR 1.3 p=0.02) as compared to MAC. NRM was not significantly different between MAC and RIC. Factors independently associated with improved OS were younger age at transplant, female sex, more recent year of transplant and Ph+ B ALL phenotype. Administration of ATG was associated with reduced risk of developing grade II-IV aGVHD (HR 0.6, p<0.001), cGVHD (HR 0.5, p<10-4) and severe cGVHD (HR 0.4, p<10-4). In conclusion, allo-HCT is feasible in patients with acute lymphoblastic leukemia in first remission and KPS score ≤80%, with acceptable NRM and survival rates. Transplant from a sibling donor was associated with reduced risk of NRM and aGVHD as compared to matched unrelated donor. Interestingly, despite the poor KPS score of the patients included in the analysis, a MAC protocol was associated with similar NRM, lower relapse and better LFS and GRFS as compared to RIC in the selected population. Finally, administration of ATG was associated with reduced acute and chronic GVHD rates. Disclosures Labopin: Jazz Pharmaceuticals: Honoraria. Socie:Alexion: Consultancy. Kröger:JAZZ: Honoraria; Medac: Honoraria; Neovii: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Riemser: Research Funding; Sanofi-Aventis: Research Funding; Celgene: Honoraria, Research Funding; DKMS: Research Funding. Mohty:Jazz Pharmaceuticals: Honoraria, Research Funding.

Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway or NUP214-ABL1 fusion protein in human Acute Lymphoblastic Leukemia.

Philadelphia chromosome-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) accounts for 25–30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes are markers of poor prognosis and amplification of the NUP214-ABL1 oncogene can be detected mainly in patients with T-ALL. T cell malignancies harboring the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been achieved. The PI3K/Akt/mTOR signaling pathway is often activated in leukemias and plays a crucial role in leukemogenesis.We analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on three NUP214-ABL1 positive T-ALL cell lines that also displayed PI3K/Akt/mTOR activation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis. Four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226, ZSTK474 and Torin-2, showed marked cytotoxic effects on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of these compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL.

Abstract 1145: Zhx2 promotes survival of B-cell acute lymphoblastic leukemia cell lines

Abstract Zhx2, a zinc finger containing transcription factor is frequently overexpressed in B cell malignancies including B cell acute lymphoblastic leukemia (B-ALL), however its role in development of B cell malignancies remains unknown. Previously, dysregulated activity of transcription factors due to mutation e.g. Myc or overexpression e.g. Sox11 has been shown to perturb B cell differentiation leading to malignant phenotypes, resistance to chemotherapeutics and genetic heterogeneity. Zhx2 is required for normal neuronal and erythropoietic differentiation and has been identified as a tumor suppressor in hepatocellular carcinoma. Furthermore, analysis of public databases shows that Zhx2 is highly overexpressed in several cell lines representing B cell malignancies including B cell acute lymphoblastic leukemia (B-ALL) and B cell lymphoma. Consistently, Zhx2 is also overexpressed in patient samples derived from different subtypes of B cell malignancies. Quantification of Zhx2 mRNA expression obtained from human donors showed a marked upregulation during transition from common lymphoid progenitor to Pro B-cell stage suggesting potential role in B cell differentiation. Here we report that Zhx2 is overexpressed in Philadelphia chromosome positive (Ph+) B-ALL and T-ALL cell lines as well as Ph- B-ALL cell line. Zhx2 silencing in BV-173 (Ph+ B-ALL) and CMLT1 (Ph+ T-ALL) showed enhanced apoptosis with low doses of Dasatinib, a dual Src/Abl kinase inhibitor. Furthermore, global gene promoter and gene expression analysis on Zhx2 silenced cells identified putative Zhx2 regulated genes including CREB and YY1. Both of these transcription factors have roles in B cell differentiation, survival, proliferation and are commonly deregulated in B cell malignancies. We propose that Zhx2 regulates a B cell differentiation program and that overexpression of Zhx2 could lead to blocked differentiation of B cell progenitors promoting the malignant phenotype. Therefore, Zhx2 could be exploited as a chemo-therapeutic target in B cell malignancies. Citation Format: Namit Sharma, Huayun Hou, Michael D. Wilson, Catherine Jane McGlade. Zhx2 promotes survival of B-cell acute lymphoblastic leukemia cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1145.

Abstract B21: Genetic characterization and therapeutic targeting of MYC translocated pediatric T-cell acute lymphoblastic leukemia

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive class of hematologic tumors caused by malignant transformation and abnormal proliferation of T-cell progenitors. T-ALL represents 10-15% of pediatric ALL and, despite the improved survival rate during the last decades, 20% of cases still experience relapsed and therapy failure. MYC serves as a pivotal oncogene in the pathogenesis of T-ALL that can be activated through a number of direct and indirect mechanisms. For example, a rare subgroup of primary T-ALL (~1%) is characterized by T cell receptor (TCR) driven translocations that cause massive activation of the MYC oncogene and present an unfavorable prognosis, rapid disease progression and poor response to conventional therapy. Here, we performed detailed molecular genetic characterization of an extensive series of primary T-ALL that present TCR driven MYC translocations and evaluated new therapeutic strategies for this poor prognostic subtype of human leukemia. Molecular genetic characterization of a cohort of 28 pediatric T-ALL patients carrying the t(8;14)(q24;q11) translocation was performed by copy number profiling, gene expression and mutational screening. Xenograft experiments with primary patient material were used to evaluate the therapeutic relevance of JQ1 in the context of MYC translocation positive T-ALL. Copy number analysis of MYC translocated T-ALL cases revealed loss of the previously reported T-ALL tumor suppressor genes PTEN (18%), CDKN2A/B (68%) and LEF1 (7%). Moreover, about 30% of patients harbored genomic deletions that cause aberrant activation of the SIL-TAL1 or LMO2 oncogenes. Interestingly, sequencing analysis revealed no aberrations in NOTCH1 or FBXW7 and a high number (30%) of loss-of-function mutations targeting PTEN. Therefore, TCRAD-MYC translocation positive T-ALL seems to represent a NOTCH1 independent subtype of leukemia that is frequently addicted to activated PI3K/AKT signaling. In line with this notion, the t(8;14)(q24;q11) positive T-ALL cell lines KE-37 and MOLT16 lack NOTCH1/FBXW7 mutations but both present loss of PTEN. Indeed, these tumor lines show aberrant pAKT activation but lack enhanced NOTCH1 activity at the protein level. In accordance with the frequent identification of TAL1 and/or LMO2 rearrangements, genome-wide transcription profiling analysis revealed that TCRAD-MYC positive T-ALL showed a uniform gene expression signature reminiscent of late cortical thymocyte T-ALL that express CD4, CD8 and membrane CD3. Importantly, gene set enrichment analysis revealed a negative enrichment of NOTCH1 target genes in MYC translocated late cortical T-ALLs, including NOTCH3, HES4, DLL4, PTCRA and DTX1. From a therapeutic perspective, in vitro drug sensitivity screening showed that MYC rearranged T-ALL cell lines were amongst the most sensitive to JQ1 treatment (IC50 values: MOLT16, 199nM; KE37, 497nM). MYC mRNA and protein downregulation were confirmed after JQ1 treatment for both the lines. Moreover, in vivo drug treatment experiments using xenografts generated from primary t(8;14)(q24;q11) positive T-ALL patients cells showed the in vivo activity of the JQ1 against this aggressive subtype of human T-ALL, as evaluated by percentage of leukemic blasts in blood or bone marrow and spleen size. Given the high frequency of genetic abnormalities targeting the PI3K/AKT pathway in these NOTCH1 independent human T-ALL, we are currently evaluating the synergistic activity of JQ1/MK-2206 combination therapy using the same in vivo model systems. In this study, we identified TCRAD-MYC rearranged T-cell leukemia as a novel subclass of NOTCH1-independent late cortical T-ALL that frequently depend on aberrant PI3K/AKT signaling. Moreover, we showed that JQ1 might serve as a promising new therapeutic strategy for the treatment of this high-risk subtype of pediatric leukemia. Citation Format: Gloria Milani, Kaat Durinck, Filip Matthijssens, Sofie Peirs, Tim Pieters, Lindy Reunes, Beatrice Lintermans, Niels Vandamme, Tim Lammens, Yunlei Li, Claire Schwab, Susana Raimondi, Barbara De Moerloose, Yves Benoit, Geert Berx, Christine Harrison, Giuseppe Basso, Helene Cavé, Rosemary Sutton, Vahid Asnafi, Charles Mullighan, Jules Meijerink, Mignon Loh, Pieter Van Vlierberghe. Genetic characterization and therapeutic targeting of MYC translocated pediatric T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B21.

Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway and NUP214-ABL1 fusion protein in human acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a neoplasm of precursor cells committed to the B-cell and T-cell lineages involving bone marrow and blood, with a rapid onset and frequent chemotherapy resistance and refractory relapses (1). Philadelphia chromosome-positive (Ph+) ALL accounts for 25–30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes, among which BCR-ABL1 is the most commonly found, are markers of very poor prognosis. Amplification of the NUP214-ABL1 oncogene can be detected only in patients with T-ALL (2). The PI3K/Akt/mTOR signaling pathway is activated in many solid cancers and in leukemias and plays a crucial role in tumorigenesis. Furthermore, the presence of RTKs (Receptor Tyrosine Kinases) by ABL1 fusion proteins may result in activation of the PI3K/Akt/mTOR axis. T cell malignancies bearing the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been found. In this work we analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on two NUP214-ABL1 positive T-ALL cell lines, ALL-SIL and PEER that also displayed Akt hyperactivation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis as deducted by a readout of drug efficacy, four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226 (BGT226), ZSTK474 and Torin-2, showed a relevant cytotoxic efficacy on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of the compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability which was associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL.

Abstract B05: Transcriptional antagonism between the cooperative oncogenes TLX1 and NOTCH1 in T-cell acute lymphoblastic leukemia.

Abstract Introduction: Combined activation of specific oncogenes is a general feature of human cancer and suggests that co-occurrence of particular oncogenic factors provides a selective advantage during cellular transformation. However, the exact molecular mechanisms by which oncoproteins cooperate during malignant transformation often remains elusive. Here, we study the functional relationship between the cooperative oncogenes NOTCH1 and TLX1 in the context of T-cell acute lymphoblastic leukemia to better understand their cooperative mechanism of action during T cell transformation. Methods: In this study, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for the TLX1 homeobox oncoprotein in the T-ALL cell line ALL-SIL and analyzed the transcriptional response before and after TLX1 modulation using microarray based methods. We performed integration of TLX1 ChIPseq data with publically available transcription factor binding profiles in T-ALL and evaluated the immunophenotypic and transcriptional effects of ectopic TLX1 expression in thymus-derived CD34+ T-cell progenitors. Results: Integration of TLX1 ChIPseq data with gene expression profiles after TLX1 knockdown in the TLX1 positive T-ALL cell line ALL-SIL, confirmed the previously established role for TLX1 as transcriptional repressor in T-ALL biology. In line with previous reports (Della-Gatta et al., Nature Medicine, 2012), de novo TLX1 motif discovery identified RUNX1 and ETS1 as important mediators of the global TLX1 transcriptional network. Next, we used TLX1 ChIPseq data to define TLX1 bound super-enhancer including several loci critically involved in T-cell biology (e.g. T-cell receptor loci, RAG2, MYB). Furthermore, Gene set Enrichment Analysis (GSEA) showed that TLX1-defined super-enhancers were significantly affected by JQ1 treatment in ALL-SIL. Integration of our TLX1 ChIP-seq data with publically available ChIP-seq data for ICN1, RUNX1 and ETS1 in T-ALL cells (Wang et al., PNAS, 2013) showed a remarkable genome-wide overlap between the binding sites of these four transcription factors. Integration of these binding patterns with transcriptional read-out revealed an unprecedented transcriptional antagonism between TLX1 and NOTCH1, in which TLX1 suppresses the oncogenic NOTCH1 transcriptional program including IL7R, NOTCH3 and c-MYC. In line with this observation, ectopic TLX1 expression in CD34+ human thymic precursor T-cells broadly interfered with the normal T-cell differentiation program causing differentiation arrest, massive apoptosis and a significant downregulation of NOTCH1 target genes including reduced IL7R-alpha surface expression. Conclusion: In conclusion, our study reveals unexpected transcriptional antagonism between the cooperative oncogenes TLX1 and NOTCH1 in the biology of T-ALL. TLX1 mediated suppression of NOTCH signaling might be critically involved in the pre-leukemic phenotype (reduced thymus size and decreased cellularity) that has been observed in Lck-TLX1 transgenic mice (De Keersmaecker et al., Nature Medicine, 2010). Moreover, our results suggests that full malignant transformation of TLX1-driven leukemias might only be possible through acquisition of secondary NOTCH1 mutations that can overcome the initial TLX1 mediated suppression of NOTCH1 signaling, which is in line with the high frequency of gain-of-function NOTCH1 mutations in TLX1 positive human T-ALL. Citation Format: Kaat Durinck, Wouter Van Loocke, Joni Van der Meulen, Inge Van de Walle, Pieter Rondou, Charles E. De Bock, Bruce Poppe, Jan Cools, Jean Soulier, Tom Taghon, Frank Speleman, Pieter Van Vlierberghe. Transcriptional antagonism between the cooperative oncogenes TLX1 and NOTCH1 in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B05.

Transcriptional Antagonism Between the Cooperative Oncogenes TLX1 and NOTCH1 in T-Cell Acute Lymphoblastic Leukemia

Introduction: Combined activation of specific oncogenes is a general feature of human cancer and suggests that co-occurrence of particular oncogenic factors provides a selective advantage during cellular transformation. However, the exact molecular mechanisms by which oncoproteins cooperate during malignant transformation often remains elusive.Here, we study the functional relationship between the cooperative oncogenes NOTCH1 and TLX1 in the context of T-cell acute lymphoblastic leukemia to better understand their cooperative mechanism of action during T cell transformation.Methods: In this study, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for the TLX1 homeobox oncoprotein in the T-ALL cell line ALL-SIL and analyzed the transcriptional response before and after TLX1 modulation using microarray based methods. We performed integration of TLX1 ChIPseq data with publically available transcription factor binding profiles in T-ALL and evaluated the immunophenotypic and transcriptional effects of ectopic TLX1 expression in thymus-derived CD34+ T-cell progenitors.Results: Integration of TLX1 ChIP-seq data with gene expression profiles after TLX1 knockdown in the TLX1 positive T-ALL cell line ALL-SIL, confirmed the previously established role for TLX1 as transcriptional repressor in T-ALL biology. In line with previous reports (Della-Gatta et al., Nature Medicine, 2012), de novo TLX1 motif discovery identified RUNX1 and ETS1 as important mediators of the global TLX1 transcriptional network.Next, we used TLX1 ChIP-seq data to define TLX1 super-enhancers including several loci critically involved in T-cell biology (e.g. T-cell receptor loci, RAG2, MYB). Furthermore, Gene set Enrichment Analysis (GSEA) showed that TLX1-defined super-enhancers were significantly affected by JQ1 treatment in ALL-SIL.Integration of our TLX1 ChIP-seq data with publically available ChIP-seq data for ICN1, RUNX1 and ETS1 in T-ALL cells (Wang et al., PNAS, 2013) showed a remarkable genome-wide overlap between the binding sites of these four transcription factors. Integration of these binding patterns with transcriptional read-out revealed an unprecedented transcriptional antagonism between TLX1 and NOTCH1, in which TLX1 suppresses the oncogenic NOTCH1 transcriptional program including IL7R, NOTCH3 and c-MYC. In line with this observation, ectopic TLX1 expression in CD34+ human thymic precursor T-cells broadly interfered with the normal T-cell differentiation program causing differentiation arrest, massive apoptosis and a significant downregulation of NOTCH1 target genes including reduced IL7R-alpha surface expression.Conclusion: In conclusion, our study reveals unexpected transcriptional antagonism between the cooperative oncogenes TLX1 and NOTCH1 in the biology of T-ALL. TLX1 mediated suppression of NOTCH signaling might be critically involved in the pre-leukemic phenotype (reduced thymus size and decreased cellularity) that has been observed in Lck-TLX1 transgenic mice (De Keersmaecker et al., Nature Medicine, 2010). Moreover, our results suggests that full malignant transformation of TLX1-driven leukemias might only be possible through acquisition of secondary NOTCH1 mutations that can overcome the initial TLX1 mediated suppression of NOTCH1 signaling, which is in line with the high frequency of gain-of-function NOTCH1 mutations in TLX1 positive human T-ALL. DisclosuresNo relevant conflicts of interest to declare.

FLT3-ITD As a Target for Minimal Residual Disease Monitoring in Early T-Cell Precursor (ETP) Acute Lymphoblastic Leukemia

Background. The 'early T-cell precursor’ (ETP) subtype of T-ALL comprises up to 15% of T-ALL and has been reported to be associated with high risk of relapse. In addition to properties of T cell development, gene expression profile and immunophenotype of ETP-ALL show stem cell and early myeloid features. Consistently, this leukemia subgroup shows lower frequencies of prototypical T-ALL lesions and a higher prevalence of mutations typically associated with AML, including RAS and FLT3 mutations. In particular, FLT3-ITD was identified in up to 35% of adult ETP-ALL but data on its prevalence in pediatric ETP-ALL are lacking.In agreement with its stem-cell signature, ETPs frequently lack Immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, the most used and sensitive targets for MRD monitoring. As a consequence, alternative markers are required to extend the application of molecular MRD to most ETP-ALL patients.Aim. We explored the prevalence of FLT3-ITD mutation in a large series of pediatric ETP-ALL enrolled in two consecutive protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP), and we evaluated the potential use of FLT3-ITD as an alternative DNA marker for MRD monitoring.Methods. Out of 439 T-ALL patients enrolled in Italy into the AIEOP-BFM ALL2000 and AIEOP ALLR2006 consecutive protocols, 145/168 Early-T ALL (TI/II) patients were screened for FLT3-ITD occurrence. Among Early-T patients, 34 were defined as ETP according to immunophenotype, and 31 of them were screened for FLT3-ITD. Twenty-two ETP patients enrolled in Italy into the ongoing AIEOP-BFM ALL2009 were also screened, together with T-ALL cases without IG/TR molecular markers only for the technical validation of the method. PCR screening and RQ-PCR for FLT3-ITD were performed as previously reported (Nakao, Leukemia 1996;10:1911; Beretta, Leukemia 2004;18:1441). Parallel MRD analysis for IG/TR on the same samples, and flow cytometry-MRD were performed by standard procedures. EuroMRD guidelines were applied for performance and interpretation of RQ-PCR.Results. Among ALL2000/R2006 and ALL2009 ETP cases, 4/31 (12.9%) and 3/22 (13.6%) were FLT3-ITD positive, respectively; 5/7 were PPR, and all 7 were stratified as high risk. For ALL2000/R2006 patients, IG/TR MRD monitoring was feasible in 2 cases, and both were MRD-HR; 3/4 cases are alive in CCR, and one died after HSCT. Overall, the FLT3-ITD marker was detected in 12 T-ALL cases; only 4 of them had valuable IG/TR markers, while 8/12 (66%) did not present a suitable IG/TR MRD marker. FLT3-ITD MRD monitoring was performed on 11/12 FLT3-ITD positive T-ALL cases. Mean length of the ITD was 44 nucleotides (nts) (range 24-71), with a mean of 7 randomly inserted nts (range 1-26). Standard curves performed by 10-fold dilutions in DNA from PB Healthy Donor, showed a quantitative range of at least 5.0E-04 in all cases and 1.0E-04 in 5/11. Sensitivity of the assay was at least 1.0E-04 in all tested cases, and 1.0E-05 in 7/11.A comparison between IG/TR and FLT3-ITD was feasible in 3 out of 4 cases (1 is ongoing); all 3 cases were monitored by 2 IG/TR markers. At day15 and day33 of the Induction therapy, when MRD was very high (10-1 to 10-3 range), the IG/TR and FLT3-ITD were fully comparable, with less than 2 times difference. At day78 (after IB Induction block) 1 case was fully negative for both markers, 1 was slightly positive by FLT3-ITD (although not quantifiable and at the limit of the sensitivity) and negative for both IG/TR. The latter case was highly positive for both IG/TR (5.0E-03) but low positive (<1.0E-04) by FLT3-ITD. In this case, the IG/TR and FLT3-ITD were concordant and finally both were negative at subsequent time points.Conclusions. This is the first report on FLT3-ITD prevalence in a consecutive series of children with ETP-ALL, which resulted to be 14.8% (4/31), a value lower than that reported in adult ETP-ALL. The limited number of cases does not allow to draw conclusions on the prognostic impact of FLT3-ITD in ETP-ALL, although 3 out of 4 patients are alive in CCR. Although available in a limited subset, FLT3-ITD can be used as a marker for sensitive molecular MRD monitoring in ETP-ALL, when IG/TR markers are not available (about 2/3 of cases). The results of MRD monitoring in a limited set of cases suggests that ETP patients might respond well to IB Induction therapy. As already known for AML, caution for false negative results is required when only FLT3-ITD is monitored. DisclosuresNo relevant conflicts of interest to declare.

The Sub-Clonal Complexity of STIL-TAL1 T-ALL

IntroductionThe STIL-TAL1 fusion is found in 16% cases of paediatric and adolescent T-ALL, making it one of the most common T-ALL subgroups. Our study considers this leukaemia subtype in the context of a complex ecosystem that is diverse, evolving and subject to selective pressures. We used single cell methods to understand the order of co-operating mutational events and the clonal evolution of mutations in genes that are re-iteratively targeted, such as PTEN.MethodsDiagnostic DNA from five STIL-TAL1 positive T-ALL cases was exome sequenced using Agilent SureSelect Human all Exon kit plus Illumina paired end sequencing. Driver copy number alterations and NOTCH1/PTEN exon 7 mutation status had been identified in a previous study and candidate driver mutations for inclusion in single cell experiments were validated by sequencing or Q-PCR using custom assays. Where more than one mutation was present within the same exon of a candidate driver gene, cloning experiments were carried out to verify the independent mutation sequences. Material from xenograft transplants was available in three of the five cases to assess their clonal heterogeneity in the leukaemia initiating cell compartment. Single cell multiplex Q-PCR was used to examine the single cell genetics of the pre-defined mutation events. Briefly, single cells were sorted and lysed prior to multiplex specific (DNA) target amplification and Q-PCR using the 96.96 dynamic microfluidic array and the BioMark HD (Fluidigm, UK). Copy number assays for the 1p33 deletion and custom assays for the patient specific STIL-TAL1 fusion breakpoints were used to confirm that the 1p33 deletion leading to this gene fusion was a clonal event.ResultsThe only aberrant events common to all five samples were CKDN2A copy number loss and the 1p33 deletion that results in the STIL-TAL1 fusion. Exome sequencing revealed further mutations in known T-ALL drivers including NOTCH1, PTEN and PHF6 as well as candidate driver mutations in FREM2, PIK3CD, RPL14, BMPR1A and CDH18. Both NOTCH1 and PTEN demonstrated re-iterative inactivation and this was investigated in detail for PTEN. Case 1 had multiple PTEN exon 7 mutations and sub-clonal copy number loss. Case 2 had parallel frameshift mutations in PTEN exons 5 and 7. Case 3 contained an exon 8 mutation and multiple PTEN exon 7 mutations. In this case the three most frequent PTEN exon 7 indels were validated and tracked in a single cell multiplex Q-PCR experiment. This revealed a branching sub-clonal genetic architecture (see figure 1) in which all malignant cells at the proposed apex of the branching architecture harboured the STIL-TAL1 fusion and CDKN2A deletion with copy number losses of 4p, 6q and FREM2 and PTEN mutations occurring as sub-clonal events. PTEN indels 2 and 3 were found co-localised in the same sub-clone. Preliminary analysis of the paired mouse xenograft bone marrow did not detect PTEN exon 7 indels 1 – 3 in 84 single cells. However, bulk Sanger Sequencing analysis did identify the PTEN exon 8 mutation in the mouse. Ongoing work is in progress to determine whether single cells of the xenograft carry alternative PTEN exon 7 mutations detected in the diagnostic sample exome data and to characterise in which diagnostic sub-clone the PTEN exon 8 mutation resides.ConclusionsThis study demonstrates how exome sequencing and single cell multiplex Q-PCR can be used as complementary tools to understand the sub-clonal complexity of STIL-TAL1 T-ALL. PTEN inactivation is sub-clonal by single cell analysis, demonstrating the parallel evolution of multiple independent PTEN inactivated sub-clones, highlighting PTEN inactivation as a key event in this T-ALL subgroup. In a wider cohort of 20 patients collected by our group at least 50% had PTEN inactivation as assessed by sequencing of exon 7 and copy number data alone. Results indicate a strong evolutionary pressure selecting for mutational events that result in inactivation of the PTEN-PI3Kinase pathway. These events occur via multiple mechanisms, including copy number loss and truncating mutations, which are not limited to the known T-ALL hotspot in exon 7. Current work is focussing on using a similar approach to examine the clonal evolution of NOTCH1 mutations in STIL-TAL1 T-ALL samples in diagnostic and xenograft samples of cases 4 and 5. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Detection of CALM-AF10 Fusion Transcript Does Not Predict A Poor Prognosis in Children with T-Lineage Acute Lymphoblastic Leukemia Treated with AIEOP ALL 2000 Protocol and Subsequent Modified 2000 Study (R-2006).

Abstract 1578Poster Board I-604 BackgroundT-ALL accounts for about 15% of pediatric ALL and still represents a clinical challenge, because more than 20% of children experience a recurrent disease which has a dismal prognosis. Characterization of molecular alterations with prognostic impact may be useful for an early identification of patients at high risk of failure in whom more intensive treatments, including hematopoietic stem cell transplantation (HSCT) may be considered. CALM-AF10 results from a recurring t(10;11)(p13;q14-21) chromosomal translocation and is the most frequent fusion transcript in both adult and pediatric patients with T-ALL. Its presence has been associated with a poor prognosis (Asnafi V et al. Blood 2003; van Grotel M et al Haematologica 2006). The aim of the present study was i) to define the incidence of CALM-AF10 among homogeneously treated children with T-ALL and ii) to evaluate the outcome of these patients. Materials and MethodsWe studied 201 patients with T-ALL, diagnosed and enrolled between 9/2000 and 12/2007 in the ALL-2000 protocol and the subsequent modified 2000 study (ALL-R-2006) of the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP) which consist of an intensive chemotherapy strategy based on BFM-ALL schedules. Patients were mainly stratified according to prednisone response evaluation (good response: blast count at day 8 less than 1000/mmc) and detection of minimal residual disease (MRD) performed at day 33 (Timepoint 1) and at day 78 (Timepoint 2). When both TPs were negative children were considered to be at Standard Risk (SR); patients with TP1 and/or TP2 positive and TP2 '10-3 were considered to be at Intermediate Risk (MR); children with TP2 positive ≥10-3 belong to the high risk (HR) group. Patients with prednisone poor response and MRD-HR were considered eligible for HSCT from a sibling donor in first complete remission. Event free survival (EFS) and overall survival (OS) estimates with 95% CIs were calculated through the Kaplan-Meier method and differences compared with the log-rank test. RT-PCR reactions for detection of CALM-AF10 were performed as previously reported (Asnafi V et al Blood 2003) ResultsTen patients resulted not eligible and were excluded from analysis. Among the 191 evaluable children with T-ALL, 14 (7,3%) were positive for CALM-AF10. Twelve (85%) of these patients were males. Median age was 8 years (range 2 – 13). Immunophenotyping showed thymic/intermediate features in 6 cases, mature in 5, early in 1, biclonal in 1 and unknown in 1, respectively. Eight cases showed a poor prednisone (PDN) response. Based on a randomized study performed in induction in the frame of the ALL-2000 protocol on the efficacy of PDN vs dexametasone (DXM) 8 children were treated with PDN and 3 with DXM The remaining 3 cases, belonging to the ALL R-2006 protocol, were treated with DXM (n=2) and PDN (n=1). MRD-based stratification allowed the allocation of 3, 8 and 3 patients in the SR, MR and HR, respectively. Four cases relapsed (3 in the central nervous system and 1 in the bone marrow). EFS at 5 years of the 14 CALM-AF10 positive T-ALL children versus the 177 who were negative was 70.1% vs 63.9%, respectively (p-value log-rank=0.61). Small numbers did not allow to fully evaluate the impact of different variables such as initial steroid treatment (PDN vs DXM) or the MRD-based risk-group assignment ConclusionsThis study performed in a vast cohort of children with T-ALL shows that CALM-AF10 is found in about 7% of children with T-ALL and that does not predict a poor outcome when an intensive chemotherapy strategy is employed. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impact of Minimal Residual Disease (MRD) in Children Is Different in B or T Lineage Acute Lymphoblastic Leukemia: Results of Trial AIEOP-BFM ALL 2000.

Slow early response indicates poor prognosis in childhood ALL. We aimed to evaluate if post-induction MRD levels had different prognostic impact in precursor B-cell (pB) or T-cell ALL. From 07/2000 to 06/2006, 4730 pts with ALL were enrolled in trial AIEOP-BFM ALL 2000. MRD levels were centrally measured by real-time quantitative polymerase chain reaction using the identification of clone-specific T-cell receptor and immunoglobulin gene rearrangements. MRD study time-points (TP) were treatment day 33 (TP1, end of induction) and day 78 (TP2, after consolidation). To define MRD negativity, two markers with a sensitivity of at least 10−4 were required. Patients were treated with BFM induction (protocol I-A), consolidation (I-B), extra-compartment/intensified consolidation (HD-MTX in non-high-risk patients, pulses in high-risk patients), reinduction, and maintenance. MRD analysis at one or two time points suceeded in 3707 pts; the immunophenotype was available from 3636 pts. MRD levels and corresponding estimated 5-year event-free survival (5y-pEFS) comparing pB- and T-ALL are shown in Table 1 (3yrs median follow-up). MRD response in T-ALL was slower than in pB-ALL resulting in a higher percentage of pts with high MRD load in T-ALL. In pB-ALL as well as T-ALL, high MRD levels at TP2 were well predictive to identify pts with poor prognosis. For prediction of good prognostic subgroups, TP1 was more appropriate identifying a subgroup with excellent 5y-pEFS of >90% in case of MRD negativity. Specificity of TP1 was poor in T-ALL if the pB-ALL criteria of MRD negativity were applied. If MRD low positive and MRD negative T-ALL pts were combined, the discrimination was as good as in pB-ALL. The optimal choice of MRD evaluation time points depends on biological factors and treatment, and is most relevant for MRD-based risk stratification.Table 1pB-ALLT-ALLn%5y-pEFS % (SE)n%5y-pEFS % (SE)all3177100%82.3 (1.0)459100%77.2 (2.2)MRD TP1 neg139944.192.5 (1.0)7516.494.3 (2.8) 10E-4/−5112235.481.9 (1.7)11625.491.2 (2.8) 10E-339312.466.4 (3.5)11024.175.3 (4.6) ≥10E-22568.153.2 (4.3)15634.159.8 (4.5)MRD TP2 neg246477.687.7 (1.0)22047.991.9 (2.0) 10E-4/−552316.568.9 (2.9)14331.276.6 (3.9) 10E-31073.456.3 (6.5)5812.650.2 (8.1) ≥10E-2822.638.0 (7.3)388.333.2 (8.3)

The Cryptic Chromosomal Deletion, del(11)(p12p13), as a New Activation Mechanism of LMO2 in Pediatric T-Cell Acute Lymphoblastic Leukemia.

To identify new cytogenetic abnormalities associated with leukemogenesis or disease outcome, T-ALL patient samples were analyzed by means of the array-comparative genome hybridization technique (array-CGH). Here, we report the identification of a new recurrent and cryptic deletion on chromosome 11, i.e. the del(11)(p12p13), in about 4% (6/138) of pediatric T-ALL patients. Detailed molecular-cytogenetic analysis revealed that this deletion activates the LMO2 oncogene in 4 out of 6 del(11)(p12p13) positive T-ALL patients, alike patients with an LMO2 translocation (9/138). The LMO2 activation mechanism of this deletion is loss of a negative regulatory region upstream of LMO2, causing activation of the proximal LMO2 promoter. LMO2 rearrangements, including this del(11)(p12p13) and t(11;14)(p13;q11) or t(7;11)(q35;p13), were found in the absence of other recurrent cytogenetic abnormalities involving HOX11L2, HOX11, CALM-AF10, TAL1, MLL or MYC. LMO2 abnormalities, represent about 9% (13/138) of pediatric T-ALL cases, and are more frequent in pediatric T-ALL than appreciated up till now.

HOX11L2 Genotyping Identifies Subset of Adult Thymic T-ALL with Inferior Outcome.

Background: Studies evaluating the prognostic impact of the expression of the oncogenes HOX11 and HOX11L2 in adult T-ALL have been controversial so far.Methods: In the two consecutive German Multicenter ALL (GMALL) therapy trials 5/93 and 6/99, 503 T-ALL pts were recruited between 4/93 and 10/03. Of these, 286 patients with adult T-ALL have been retrospectively investigated for the expression of HOX11 and HOX11L2. This represents the largest cohort of adult T-ALL patients investigated so far. Expression was measured in pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR.Results: The median age was 30 (16–65)yrs, 76% were male, 69% had a mediastinal tumor, 24% WBC > 100.000 and 7% CNS involvement. 61 patients (21%) showed a high and 57 patients (20%) a low HOX11 expression, 165 patients (59%) did not express HOX11. In comparison, 29 patients (10%) expressed HOX11L2 whereas 256 (91%) did not show a HOX11L2 expression. The prevalence of HOX11L2 expression was higher in the youngest age group (median 20 yrs vs. 31 yrs in “negative” pts, p=0,025). CNS involvement was more frequently found in the high HOX11 expression group (p=0,025). High HOX11 expression and HOX11L2 expression were both significantly associated with the thymic immunophenotype (p=0.0077 and 0,031, respectively). There were no significant differences in CR rates between the HOX11- and HOX11L2-expression subgroups. In contrast, HOX11L2 positive patients showed a higher relapse rate (52% vs. 28%; P=0.011). The overall survival (OS) was higher for pts. with high HOX11 expression (OS at 5 years: high 69% vs low 36% vs negative 41%; p=0.007), and disease free survival (DFS) was significantly longer in the high HOX11 expression group (at 5 years high 73% vs low 41% vs negative 47%; p=0.007). For HOX11L2 expression the difference in OS did not reach statistical significance regarding all T-ALL patients (at 5 yrs: positive 13% vs negative 49%; p=0.06). Controlling for the immunophenotype, a significant difference in OS became evident (at 5 yrs: positive 19% vs negative 64%; p=0.01). DFS was significantly shorter in HOX11L2 positive patients (at 5 yrs: positive 24% vs negative 56%; p=0.03). In a multivariate analysis the expression of HOX11 could not be confirmed as independent prognostic factor for OS or DFS (p≥0,05). In contrast, HOX11L2 expression was found to be the only relevant prognostic factor besides the immunophenotype: The hazard ratio of death was 1,8 for HOX11L2 positive T-ALL patients (95% CI 1.1–3,0; P=0,021), and the hazard ratio for a relapse was 2,0 (95% CI 1.1–3,7; P=0.023).Conclusions: HOX11 expression is highly associated with the thymic immunophenoytpe and is not an independent prognostic factor. The expression of HOX11L2 is also predominantly found in thymic T-ALL. Whereas the majority of thymic T-All patients do not express HOX11L2 and have an improved outcome, HOX11L2 expression identifies a small subset of thymic T-ALL patients with a reduced OS and DFS, similarly to early and mature T-ALL. If this can be confirmed prospectively, thymic T-ALL patients need to be treated according to their HOX11L2 expression status as different entities: HOX11L2 positive thymic T-ALL patient requiring a rather aggressive therapeutical management.

Rearrangements of the T cell receptor delta gene in T acute lymphoblastic leukemia cells are distinct from those occurring in B lineage acute lymphoblastic leukemia and preferentially involve one V delta gene segment.

Rearrangement of the TCR-delta gene was studied using J delta, C delta, and V delta probes in 61 cases of acute lymphoblastic leukemia (ALL) and several cases of chronic lymphoid neoplasms to define the specificity and the diversity of rearrangements occurring at the delta locus. TCR-delta rearrangements or deletions were found in all T (33 cases) and B lineage (28 cases) ALL but not in any case of B cell chronic proliferations (13 cases). The restriction patterns of rearrangement were clearly distinct between T and B ALL and use of one V delta probe showed that rearrangement of the V delta IDP2 gene segment which is also productively rearranged in the Peer cell line, occurred frequently in T-ALL but never in B lineage ALL. Studies of WT31 and delta TCS1 antibody reactivity showed that at least 4 of 13 CD3+ T-ALL cases expressed the delta protein. CD4 and/or CD8 Ag expression were observed in some of the gamma delta expressing T-ALL. These data show that particular TCR-delta gene rearrangements occur in neoplastic early B cells and that the combinatorial diversity of TCR-delta rearrangements in T cells is higher than initially expected. In addition this study shows that an important proportion of CD3 positive T-ALL cases express the gamma delta heterodimer.