Jonathan Bond

Toward a NOTCH1/FBXW7/RAS/PTEN–Based Oncogenetic Risk Classification of Adult T-Cell Acute Lymphoblastic Leukemia: A Group for Research in Adult Acute Lymphoblastic Leukemia Study

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.

RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement

Within the human TCR-α/δ locus, ordered rearrangements requires RUNX1, which binds to the Dδ2-23RSS and interacts with RAG1 to enhance RAG1 deposition at this site. Absence of this RUNX1 binding site in the homologous murine Dδ1-23RSS offers an explanation for the lack of ordered TCR-δ gene assembly in mice.

Early Response-Based Therapy Stratification Improves Survival in Adult Early Thymic Precursor Acute Lymphoblastic Leukemia: A Group for Research on Adult Acute Lymphoblastic Leukemia Study.

Purpose Early thymic precursor (ETP) acute lymphoblastic leukemia (ALL) is an immunophenotypically defined subgroup of T-cell ALL (T-ALL) associated with high rates of intrinsic treatment resistance. Studies in children have shown that the negative prognostic impact of chemotherapy resistance is abrogated by the implementation of early response-based intensification strategies. Comparable data in adults are lacking. Patients and Methods We performed comprehensive clinicobiologic, genetic, and survival analyses of a large cohort of 213 adult patients with T-ALL, including 47 patients with ETP-ALL, treated in the GRAALL (Group for Research on Adult Acute Lymphoblastic Leukemia) -2003 and -2005 studies. Results Targeted next-generation sequencing revealed that the genotype of immunophenotypically defined adult T-ALL is similar to the pediatric equivalent, with high rates of mutations in factors involved in cytokine receptor and RAS signaling (62.2%), hematopoietic development (29.7%), and chemical modification of histones (48.6%). In contrast to pediatric cases, mutations in DNA methylation factor genes were also common (32.4%). We found that despite expected high levels of early bone marrow chemotherapy resistance (87%), the overall prognosis for adults with ETP-ALL treated using the GRAALL protocols was not inferior to that of the non-ETP-ALL group (5-year overall survival: ETP, 59.6%; 95% CI, 44.2% to 72.0% v non-ETP, 66.5%; 95% CI, 58.7% to 73.2%; P = 0.33) and that allogeneic stem-cell transplantation had a beneficial effect in a large proportion of patients with ETP-ALL. Conclusion Our results suggest that the use of response-based risk stratification and therapy intensification abrogates the poor prognosis of adult ETP-ALL.

Cryptic XPO1-MLLT10 translocation is associated with HOXA locus deregulation in T-ALL

To the editor: Biological subclasses of T-cell acute lymphoblastic leukemia (T-ALL) can be defined by recurrent gene expression patterns, which typically segregate with specific chromosomal anomalies. The HOXA+ subgroup is characterized by deregulated homeobox A ( HOXA ) gene expression and is

An early thymic precursor phenotype predicts outcome exclusively in HOXA-overexpressing adult T-cell acute lymphoblastic leukemia: a group for research in adult acute lymphoblastic leukemia study

Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acute lymphoblastic leukemias, but it is unclear whether these constitute a homogeneous clinical entity, and the biological consequences of HOXA overexpression have not been systematically examined. We characterized the biology and outcome of 55 HOXA-positive cases among 209 patients with adult T-cell acute lymphoblastic leukemia uniformly treated during the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. HOXA-positive patients had markedly higher rates of an early thymic precursor-like immunophenotype (40.8% versus 14.5%, P=0.0004), chemoresistance (59.3% versus 40.8%, P=0.026) and positivity for minimal residual disease (48.5% versus 23.5%, P=0.01) than the HOXA-negative group. These differences were due to particularly high frequencies of chemoresistant early thymic precursor-like acute lymphoblastic leukemia in HOXA-positive cases harboring fusion oncoproteins that transactivate HOXA. Strikingly, the presence of an early thymic precursor-like immunophenotype was associated with marked outcome differences within the HOXA-positive group (5-year overall survival 31.2% in HOXA-positive early thymic precursor versus 66.7% in HOXA-positive non-early thymic precursor, P=0.03), but not in HOXA-negative cases (5-year overall survival 74.2% in HOXA-negative early thymic precursor versus 57.2% in HOXA-negative non-early thymic precursor, P=0.44). Multivariate analysis further revealed that HOXA positivity independently affected event-free survival (P=0.053) and relapse risk (P=0.039) of chemoresistant T-cell acute lymphoblastic leukemia. These results show that the underlying mechanism of HOXA deregulation dictates the clinico-biological phenotype, and that the negative prognosis of early thymic precursor acute lymphoblastic leukemia is exclusive to HOXA-positive patients, suggesting that early treatment intensification is currently suboptimal for therapeutic rescue of HOXA-positive chemoresistant adult early thymic precursor acute lymphoblastic leukemia. Trial Registration: The GRAALL-2003 and -2005 studies were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively.

Direct interaction of Ikaros and Foxp1 modulates expression of the G protein-coupled receptor G2A in B-lymphocytes and acute lymphoblastic leukemia

Ikaros and Foxp1 are transcription factors that play key roles in normal lymphopoiesis and lymphoid malignancies. We describe a novel physical and functional interaction between the proteins, which requires the central zinc finger domain of Ikaros. The Ikaros-Foxp1 interaction is abolished by deletion of this region, which corresponds to the IK6 isoform that is commonly associated with high-risk acute lymphoblastic leukemia (ALL). We also identify the Gpr132 gene, which encodes the orphan G protein-coupled receptor G2A, as a novel target for Foxp1. Increased expression of Foxp1 enhanced Gpr132 transcription and caused cell cycle changes, including G2 arrest. Co-expression of wild-type Ikaros, but not IK6, displaced Foxp1 binding from the Gpr132 gene, reversed the increase in Gpr132 expression and inhibited G2 arrest. Analysis of primary ALL samples revealed a significant increase in GPR132 expression in IKZF1-deleted BCR-ABL negative patients, suggesting that levels of wild-type Ikaros may influence the regulation of G2A in B-ALL. Our results reveal a novel effect of Ikaros haploinsufficiency on Foxp1 functioning, and identify G2A as a potential modulator of the cell cycle in Ikaros-deleted B-ALL.

Novel Intergenically Spliced Chimera, NFATC3-PLA2G15, Is Associated with Aggressive T-ALL Biology and Outcome

Leukemias are frequently characterized by the expression of oncogenic fusion chimeras that normally arise due to chromosomal rearrangements. Intergenically spliced chimeric RNAs (ISC) are transcribed in the absence of structural genomic changes, and aberrant ISC expression is now recognized as a potential driver of cancer. To better understand these potential oncogenic drivers, high-throughput RNA sequencing was performed on T-acute lymphoblastic leukemia (T-ALL) patient specimens (n = 24), and candidate T-ALL–related ISCs were identified (n = 55; a median of 4/patient). In-depth characterization of the NFATC3-PLA2G15 chimera, which was variably expressed in primary T-ALL, was performed. Functional assessment revealed that the fusion had lower activity than wild-type NFATC3 in vitro, and T-ALLs with elevated NFATC3-PLA2G15 levels had reduced transcription of canonical NFAT pathway genes in vivo. Strikingly, high expression of the NFATC3-PLA2G15 chimera correlated with aggressive disease biology in murine patient-derived T-ALL xenografts, and poor prognosis in human T-ALL patients. Mol Cancer Res; 16(3); 470–5. ©2018 AACR.

Polycomb repressive complex 2 haploinsufficiency identifies a high-risk subgroup of pediatric acute myeloid leukemia

Human polycomb proteins exert their epigenetic functions as part of nuclear multimeric complexes, of which Polycomb Repressive Complexes PRC1 and PRC2 are the best characterized [1]. A variety of alterations in Polycomb protein level and function have been described in hematological malignancy. Reports of PRC1 component changes are essentially limited to overexpression, as has been seen in acute myeloid leukemia (AML) and myelodysplastic syndrome [2, 3]. Described PRC2 alterations are more diverse. On the one hand, the occurrence of EZH2 gain-of-function mutations suggests that PRC2 is oncogenic in non-Hodgkin’s lymphoma [4]. In contrast, mutations that cause reduced PRC2 function are commonly found in the early thymic precursor (ETP) subgroup of T-acute lymphoblastic leukemia [5, 6], and have also been reported in myeloid neoplasia [7, 8]. These mutations have not previously been reported to correlate with prognosis, and the biological consequences of altered Polycomb function in most hematological malignancies remain unclear. In order to characterize the prevalence and clinicobiology of Polycomb gene alterations in pediatric AML, we performed targeted genetic analysis of leukemic DNA extracted from 222 patients treated during the French national ELAM02 study (see Supplementary Files “ELAM02 Key Trial Information” and “ELAM02 Full Protocol French Version”). Co-first authors: Jonathan Bond and Elise Labis.

Early thymic precursor-like lymphomatous presentation of the ETV6-NCOA2 translocation

A 5 year-old, previously healthy boy presented with fever and lymph node enlargement. Clinical examination revealed hepatosplenomegaly, cervical, axillary and inguinal adenopathy and enlarged non-exudative tonsils. He had significant Epstein–Barr virus (EBV) viraemia at 11900 copies/ml. EBV serology was positive, with anti-viral capsid antigen (VCA) IgG levels of >700 u/ml, while anti-VCA IgM were negative, consistent with subacute or recent infection. There was mild pancytopaenia, with Hb of 109 g/l, whit blood cell count of 3 86 9 10/l and a platelet count of 220 9 10/l. Serum lactate dehydrogenase was elevated at 860 iu/l (normal range 120–300 iu/l), but all other biochemical investigations were normal apart from minimal transaminitis (aspartate transaminase 66 iu/l, alanine transaminase 83 iu/l). The atypical clinical presentation and discovery of extensive large cervical, intra-thoracic and intra-abdominal adenopathy by ultrasound, computed tomography and Ffluorodeoxyglucose positron emission tomography (Fig 1A) prompted further investigation. Immunohistological analysis of an inguinal lymph node (Fig 1B) showed replacement of the normal architecture with an infiltrate of medium to large T lymphoid cells with CD45, CD5, CD56 and weak CD3 expression. Staining for CD4, CD8, CD1a, CD30, CD20 and EBV/LMP1 was negative. Immunophenotypic flow cytometric analysis of the nodal aspirate confirmed the immature T-cell population (cCD3+, CD7+, CD2-, sCD3-, CD5 low, CD1a-, CD4-, CD8-), which co-expressed haematopoietic stem cell (HSC) and myeloid antigens (CD34, HLA-DR, CD38, CD123 and CD33). A minority of cells were positive for TdT. Biallelic incomplete TRDD2TRDD3 rearrangements were identified by polymerase chain reaction (PCR), but no clonal rearrangements of TRG, TRB or IGH were detected (Fig 1C). Bone marrow (BM) aspiration revealed 3% malignant infiltration with phenotypically identical immature cells, consistent with a diagnosis of Stage IV Tlymphoblastic lymphoma (T-LBL). Karyotypic analyses of lymph node and BM samples identified a t(5;11)(p1?5;q22) rearrangement involving the ATM gene (data not shown), and a t(8;12)(q13 3;p13) translocation (Fig 1D, upper panel). These anomalies were present in the same clone in 17/20 examined metaphases. The latter translocation prompted us to test for the ETV6-NCOA2 fusion that has previously been reported in T-acute lymphoblastic leukaemia (ALL) (Strehl et al, 2008; Homminga et al, 2011; Zhou et al, 2012). Interphase fluorescent in situ hybridization (FISH) of BM tissue revealed co-localization of the ETV6 and NCOA2 genes in 80% of cells (Fig 1D, lower panel), and expression of the fusion transcript was confirmed by reverse transcription-PCR and direct sequencing (Fig 1E). Treatment was commenced according to the Euro-LB02 LBL protocol, with achievement of complete remission and post-induction negativity (<10 ) for minimal residual disease by both flow cytometry and ETV6-NCOA2 transcript quantification. The patient has maintained an excellent clinical response and, at his last assessment, had remained in molecular remission for 8 months since the end of maintenance treatment, 3 years after diagnosis. To our knowledge, the ETV6-NCOA2 rearrangement has been reported in 8 published cases of T-ALL (Strehl et al, 2008; Homminga et al, 2011; Zhou et al, 2012), but never in association with LBL. The involved exons in this case (Fig 1E) were translocated in only 1/6 cases where breakpoints were analysed (Strehl et al, 2008). We performed PCR screening for the translocation in a further 91 cases of T-LBL (57 adults and 34 children), using primers that are predicted to identify all published ETV6-NCOA2 fusions, but no additional cases were identified (data not shown). Translocations proximate to the ETV6 and NCOA2 loci, i.e. t(8;12)(q13;p13), have additionally been reported in BALL (Pui et al, 1987), acute myeloid leukaemia (Yamamoto et al, 2002) and T-ALL (Schneider et al, 2000), albeit in the absence of molecular confirmation of ETV6-NCOA2 fusion. In the single reported series where immunophenotype was reported in detail, co-occurrence of lymphoid and myeloid antigens was common, and frequently fulfilled the diagnostic criteria for biphenotypic leukaemia (Strehl et al, 2008). Similarly, the case described here demonstrated weak or absent expression of several T-cell markers (CD2, CD5, CD1a, CD4, CD8) and positivity for HSC and myeloid antigens (CD34, HLA-DR, CD33), which, in a leukaemic context, would fulfil the immunophenotypic criteria for early thymic precursor (ETP) ALL. Taken together, it is likely that the translocation occurs preferentially in an early haematopoietic progenitor in which lineage commitment has not yet taken place. The presence of a mediastinal mass in this case is compatible with malignant transformation of an immature thymic precursor, although BM positivity for ETV6-NCOA2+ cells may indicate that the translocation arose in a multipotent marrow progenitor that disseminated and proliferated rapidly in secondary lymphoid organs.

CBF beta-SMMHC regulates ribosomal gene transcription and alters ribosome biogenesis

The core-binding factor (CBF) complex is a heterodimeric transcription factor comprising a CBFβ subunit and a variable DNA-binding RUNX subunit, usually RUNX1 in hematopoietic cells. Aside from its critical hematopoietic functions, CBF regulates the expression of ribosomal protein genes and ribosomal RNA (rRNA) in a cell context-dependent manner.1, 2, 3 Intriguingly, this function may have implications for the pathogenesis of acute myeloid leukemia (AML), as reduced ribosome biogenesis in RUNX1-deficient hematopoietic stem cells has recently been proposed to confer a survival advantage that favors outgrowth of preleukemic RUNX1-deficient clones.3 Furthermore, AML-associated fusion proteins that arise from translocations of CBF subunit genes have been shown to occupy nucleolar organizing regions at mitotic chromosomes,4, 5 suggesting that ribosomal homeostasis might be altered in CBF-AML.