T-lineage Acute Lymphoblastic Leukemia Research Articles

Genetic profile of T-cell acute lymphoblastic leukemias with MYC translocations

AbstractMYC translocations represent a genetic subtype of T-lineage acute lymphoblastic leukemia (T-ALL), which occurs at an incidence of ∼6%, assessed within a cohort of 196 T-ALL patients (64 adults and 132 children). The translocations were of 2 types; those rearranged with the T-cell receptor loci and those with other partners. MYC translocations were significantly associated with the TAL/LMO subtype of T-ALL (P = .018) and trisomies 6 (P < .001) and 7 (P < .001). Within the TAL/LMO subtype, gene expression profiling identified 148 differentially expressed genes between patients with and without MYC translocations; specifically, 77 were upregulated and 71 downregulated in those with MYC translocations. The poor prognostic marker, CD44, was among the upregulated genes. MYC translocations occurred as secondary abnormalities, present in subclones in one-half of the cases. Longitudinal studies indicated an association with induction failure and relapse.

Impact of TCF3 Rearrangement on CNS Relapse in Egyptian Pediatric Acute Lymphoblastic Leukemia

Background: TCF3 rearrangement mostly t(1;19) (q23;p13)/ TCF3-PBX1 gene is associated with favorable outcome in acute lymphoblastic leukemia (ALL) upon treatment with intensification protocols; however, it is associated with higher incidence of central nervous system (CNS) relapse which may affect outcome of patients. Objectives: We aimed to assess TCF3 rearrangement in newly diagnosed pediatric ALL patients in relation to clinical and laboratory parameters, CNS relapse, and clinical outcome. Patients and Methods: Eighty newly diagnosed pediatric ALL patients following at Pediatric Hematology Oncology Clinic, Ain Shams University Hospitals were included in this study. Their ages ranged from 0.75 to 16 years. Seventy six (95%) patients had B-lineage ALL and four (5%) had T-lineage ALL. Data recorded included; age, sex, extramedullary manifestations, CNS, and testes infiltrations, risk stratification, response to treatment, and CBC and BM findings. TCF3 rearrangement was assessed by FISH technique using dual color break-apart probe. Results: TCF3 rearrangement [t(1;19) (q23;p13)] was detected in 16 (20%) out of the 80 studied patients, and it was significantly associated with splenomegaly, lymphadenopathy, CNS infiltration at presentation, high total leucocytic count, low platelet count, high-risk group, and isolated CNS relapse. These results identify a group of high-risk ALL patients with high incidence of CNS relapse and poor response to standard therapeutic regimen. Conclusion: Analysis of TCF3 rearrangement [t(1;19) (q23;p13)] at diagnosis may provide a valuable target for modified and intensified CNS-directed chemotherapeutic protocol aiming to improve the patients’ outcome.

Cranial radiation for pediatric T-lineage acute lymphoblastic leukemia: a systematic review and meta-analysis.

There are heterogeneous approaches to cranial radiation therapy (CRT) for T-lineage acute lymphoblastic leukemia (T-ALL). We performed a systematic review of studies that specified a radiation strategy and reported survival for pediatric T-ALL. Our analysis included 62 publications reporting 78 treatment groups (patient n = 5844). The average event-free survival (EFS) was higher by 6% per 5 years (P < 0.001). Adjusting for year, EFS differed by radiation strategy. Compared to the reference group (CRT for all) which had a year-adjusted EFS of 65% (95% confidence interval, CI: 61-69%) the adjusted EFS was significantly worse (rate difference (RD) = -9%, 95% CI: -15 to -2%) among studies that used a risk-directed approach to CRT (P = 0.004). The adjusted EFS for the other strategies were not significantly different compared to the reference group: CRT for central nervous system positive patients only (RD = -3%, 95% CI: -14 to 7%, P = 0.49); CRT omitted for all patients (RD = 5%, 95% CI: -4 to 15%, P = 0.33). CRT may not be necessary with current chemotherapy for T-ALL. These findings, however, are susceptible to bias and caution should be applied in drawing conclusions on the comparative effectiveness of alternative CRT strategies.

TP53 mutations occur in 15.7% of ALL and are associated with MYC-rearrangement, low hypodiploidy, and a poor prognosis

AbstractTP53 is the most extensively studied gene in cancer. However, data on frequency and the prognostic impact of TP53 mutations in acute lymphoblastic leukemia (ALL) remain scarce. Thus, we aimed at identifying the mutation frequency of TP53, its association with cytogenetic subgroups, and its impact on survival in a large cohort of 625 patients with ALL. Our data revealed an overall mutation incidence of 15.7%, which increases with age. Correlation with cytogenetic subgroups showed that mutations were most frequent in ALL with low hypodiploidy or MYC-rearrangements. Furthermore, for a large number of patients, both TP53 alleles were altered, either by 2 TP53 mutations (12%) or by a TP53 mutation and a TP53 deletion in the second allele (39%). A high TP53 mutation load was correlated to low hypodiploidy, high hyperdiploidy, and a complex karyotype. Moreover, a higher mutation load was found in B-lineage ALL compared with T-lineage ALL. Similar to other cancers, the median overall survival was significantly shorter in patients with TP53 mutation compared with patients with wild-type TP53. This effect was especially pronounced when both TP53 alleles were affected, either by 2 TP53 mutations or by both a mutation and an accompanying TP53 deletion.

Autologous Hematopoietic Stem Cell Transplantation for High-risk Acute Lymphoblastic Leukemia: non-Randomized Study with a maximum Follow-up of more than 22 Years.

ObjectiveTo evaluate the efficacy and toxicity of autologous hematopoietic stem cell transplantation (AHSCT) for high-risk acute lymphoblastic leukemia (ALL).Material and methodsOverall, 128 high-risk ALL patients at a median age of 26 years (range 18–56 years) at diagnosis received AHSCT between 1991–2008. Induction treatment was anthracycline-based in all patients. Conditioning regimen consisted of CAV (cyclophosphamide, cytarabine, etoposide) in 125 patients whereas 3 subjects received cyclophosphamide and TBI (total body irradiation). Bone marrow was stored for 72 hours in 4°C and re-infused 24 hours after conditioning completion. Bone marrow was a source of stem cells in 119 patients, peripheral blood in 2 and 7 subjects received both bone marrow and peripheral blood.ResultsWith a median follow-up after AHSCT of 1.6 years (range 0.1–22.3 years), the probability of leukemia-free survival (LFS) for the whole group at 10 years was 27% and 23% at 20 years. Transplant-related mortality at 100 days after AHSCT was 3.2%. There was a strong tendency for better LFS for MRD-negative patients if compared with patients who had positive or unknown MRD status at AHSCT (32% vs 23% and 25%, respectively; p=0.06). There was no difference in LFS between B- and T-lineage ALL as well as between patients transplanted in first complete remission (CR1) and CR2. LFS at 10 years for patients with Philadelphia-positive (Ph+) ALL at transplant was 20% and this was comparable with subjects with negative and missing Ph status (26% and 28%; p=0.97).ConclusionsThe results of AHSCT for high-risk ALL remains unsatisfactory with low probability of long-term LFS.

Treatment outcome of childhood standard-risk and median-risk acute lymphoblastic leukemia with CCLG-2008 protocol

To estimate the significance of the adjustment of acute lymphoblastic leukemia (ALL) risk group by monitoring minimal residual disease(MRD). Totally 285 children ALL patients who were diagnosed and systematically treated according to CCLG-2008 in Institute of Hematology and Blood Diseases Hospital, CAMS and PUMC, from April 2008 to August 2011 were prospectively selected. Among these cases, 62.8% (n = 179) were boys and 37.2% (n = 106) were girls and the median age was 5.3(0.5-14.0). The patients who were at high-risk group initially were excluded. The grouping of cases: the patients were divided into two groups according to the dates of initial diagnosis. Group I had 126 patients who were initially diagnosed between April 2008 and December 2009 in whom therapeutic regimen was not adjusted by reassignment of risk group by MRD. Group II had 159 patients who were initially diagnosed between January 2010 and August 2011 whose therapeutic regimen was adjusted by reassignment of risk group by MRD at specific time (33rd day of induction chemotherapy and 12 weeks after the beginning of chemotherapy). MP-FCM Coulter FC-500 was used in the detection of MRD. Among these 285 patients, 94.0% (n = 268) were diagnosed as B-lineage acute lymphoblastic leukemia and 6.0% (n = 17) were T-lineage acute lymphoblastic leukemia. In group I, 61.9% (n = 78) patients belonged to low-risk group, 38.1% (n = 48) median-risk; in group II, before the adjustment, the rates of the low-risk group and median-risk group were 68.6% (n = 109) and 31.4% (n = 50) , respectively, while after the adjustment they were altered to 53.5% (n = 85) and 39.6% (n = 63) , furthermore 6.9% (n = 11) patients went into the high-risk group. Both groups were followed up for 2.5 years after their diagnoses, the disease of 7.4% (n = 21) patients relapsed, and the rates of two groups were 12.7% (n = 16) and 3.1% (n = 5) respectively, P = 0.009. The rate of serious infection (such as sepsis, pulmonary infection) of all these patients was 32.3% (92/285) , there was no significant difference between the two groups [28.6% (36/126) vs.35.2% (56/159) , P = 0.392]. The mortality of all these patients was 6.7% (19/285) , and that of group I was higher than that of group II [10.3% (13/126) vs. 3.8% (6/159) , P = 0.044]. The 2.5 years overall survival (OS), event-free survival (EFS) and disease-free survival (DFS) of group I were all lower than those of group II in Kaplan-Meier survivorship analysis (all P < 0.05). The two groups were followed up for 2.5 years after their diagnoses, after elimination of the confounding influence of sex, age, FAB subtype, WBC count, ratio of blast cells in bone marrow at diagnosed, chromosome karyotype and fusion gene, reassignment of risk group by MRD was used to calculate the OS, EFS and DFS of ALL patients (all P < 0.05). After the adjustment the risk group was more significant in the assessment of prognosis. The reassignment of risk group in low and median risk groups children with acute lymphoblastic leukemia by MRD did not increase the rate of serious infection but could reduce the relapse rate and mortality, and was beneficial to increase the patients' OS, EFS and DFS.

The landscape of somatic mutations in epigenetic regulators across 1,000 paediatric cancer genomes

Studies of paediatric cancers have shown a high frequency of mutation across epigenetic regulators. Here we sequence 633 genes, encoding the majority of known epigenetic regulatory proteins, in over 1,000 paediatric tumours to define the landscape of somatic mutations in epigenetic regulators in paediatric cancer. Our results demonstrate a marked variation in the frequency of gene mutations across 21 different paediatric cancer subtypes, with the highest frequency of mutations detected in high-grade gliomas, T-lineage acute lymphoblastic leukaemia and medulloblastoma, and a paucity of mutations in low-grade glioma and retinoblastoma. The most frequently mutated genes are H3F3A, PHF6, ATRX, KDM6A, SMARCA4, ASXL2, CREBBP, EZH2, MLL2, USP7, ASXL1, NSD2, SETD2, SMC1A and ZMYM3. We identify novel loss-of-function mutations in the ubiquitin-specific processing protease 7 (USP7) in paediatric leukaemia, which result in decreased deubiquitination activity. Collectively, our results help to define the landscape of mutations in epigenetic regulatory genes in paediatric cancer and yield a valuable new database for investigating the role of epigenetic dysregulations in cancer.

Germline variants in IKZF1, ARID5B, and CEBPE as risk factors for adult-onset acute lymphoblastic leukemia: an analysis from the GMALL study group

Germline variants in IKZF1, ARID5B, and CEBPE as risk factors for adult-onset acute lymphoblastic leukemia: an analysis from the GMALL study group

Cure rates of childhood acute lymphoblastic leukemia in Lithuania and the benefit of joining international treatment protocol

BackgroundChildhood acute lymphoblastic leukemia (ALL) represents the largest group of pediatric malignancies with long-term survival rates of more than 80% achieved in developed countries. Epidemiological data and survival rates of childhood ALL in Lithuania were lacking. Therefore, the aim of this study was to analyze the population-based long-term treatment results of childhood ALL in Lithuania during 1992–2012. Materials and methodsData of all 459 children with T-lineage and B-cell precursor ALL treated in Lithuania from 1992 to 2012 were collected and analyzed. Results were compared among four time-periods: 1992–1996 (N=132), 1997–2002 (N=136), 2003–2008 (N=109) and 2009–2012 (N=82). ResultsThe incidence of childhood ALL in Lithuania was 3.2–3.6 cases per 100000 children per year during the study period. Five-year probability of event-free survival increased from 50%±4% in 1992–1996 to 71%±4% in 2003–2008 (P<0.001). Five-year cumulative incidence of relapses reduced from 27%±4.5% in 1992–1996 to 14%±3.6% in 2003–2008 (P=0.042). After introduction of high-dose methotrexate of 5g/m2, cumulative incidence of CNS-involving relapses reduced from 17%±3.9% in 1992–1996 to 1%±1.0% in 2003–2008 (P<0.001). Trend for further improvement in survival was seen in 2009–2012 when Lithuania joined international the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL-2008 treatment protocol. ConclusionsCure rates of childhood ALL in Lithuania are improving steadily and are now approaching those reported by the largest international study groups. The reasons for such a positive effect are both better financial support for treatment of children with cancer in Lithuania and international collaboration with joining international treatment protocol for childhood ALL.

Effective Targeting Of The P53/MDM2 Axis In Preclinical Models Of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

IntroductionWhile the overall cure rate for the most common pediatric cancer, acute lymphoblastic leukemia (ALL) now approaches 90%, infants (<12 months) diagnosed with ALL harboring translocations in the mixed-lineage leukemia oncogene (infant MLL-ALL) experience shorter remission duration and a significantly reduced likelihood of survival (∼50%). Therefore, new treatments that can be incorporated into conventional chemotherapy regimens to extend patient remission and improve survival are urgently required. Mutations in the p53 tumor suppressor are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be of therapeutic benefit. Nutlin cis-imidazole molecules selectively inhibit p53-MDM2 binding, resulting in activation of the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenograft models. MethodsIn vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. P53 protein levels and subcellular distribution were assessed by immunoblotting. Patient-derived xenografts were established from infant MLL-ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice, and their gene expression profiles generated using Illumina Human Ref-12 Expression BeadChips. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the PB. Mice with established disease received vehicle, RG7112 (100 mg/kg daily x 5 p.o.), a combination of vincristine (0.15 mg/kg once i.p.) dexamethasone (5 mg/kg daily x 5 i.p.) and L-asparaginase (1,000 IU/kg daily x 5 i.p.) (VXL), or RG7112 plus VXL. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups from treatment initiation. Therapeutic enhancement was considered to occur when the RG7112/VXL combination significantly extended mouse EFS compared with that of both of the RG7112 and VXL treated groups. ResultsUnsupervised hierarchical clustering of gene expression profiles revealed that the MLL-ALL (n=9), BCP-ALL (n=7) and T-ALL (n=13) xenografts clustered according to leukemia subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA gene family, were significantly upregulated in MLL-ALL xenografts. The specificity of RG7112 was validated by cytotoxicity assays against leukemia cell lines of known p53 status; p53 wild-type cell lines (RS4;11, IC50 1.4 µM; NALM-6, IC50 3.0 µM) were markedly more sensitive than those with mutant p53 (CEM, IC50 >10 µM; JURKAT, IC50 >10 µM). The in vitro sensitivity of BCP-ALL (n=3) and infant MLL-ALL (n=4) xenografts was consistent with wild-type p53 status, with IC50s of 0.11 - 2.2 µM. Exposure of ALL xenograft cells to nutlin-3 (10 µM, 6h) caused marked p53 up-regulation and nuclear translocation. Since we had previously shown that RG7112 administered as a single agent for 14 days induced significant regressions [Complete Responses (CRs) or greater] in 7/7 infant MLL-ALL xenografts in vivo, we assessed its efficacy in a 5-day combination treatment with an induction-type regimen (VXL) against two infant MLL-ALL xenografts (MLL-5 and MLL-14). The RG7112/VXL combination caused a Partial Response in MLL-5 compared with Progressive Disease for both RG7112 and VXL. The efficacy of RG7112/VXL was even more pronounced against MLL-14, causing a Maintained CR compared with CRs for both RG7112 and VXL, which met the criteria for Therapeutic Enhancement (the median EFS of RG7112/VXL-treated mice, 65.0 days, was significantly greater, P< 0.0001, than that of RG7112, 22.2 days, and VXL, 28.5 days). ConclusionsRG7112 induces significant regressions in a high proportion of infant MLL-ALL xenografts and enhances the efficacy of an induction-type regimen. The utility of targeting the p53-MDM2 axis in combination with established drugs for the clinical management of infant MLL-ALL warrants further investigation.This study was supported by NCI NO1CM42216. The authors thank Roche Pharmaceuticals, Inc., for providing RG7112. Disclosures:No relevant conflicts of interest to declare.

Allogeneic Stem Cell Transplantation In Adults With High Risk Acute Lymphoblastic Leukemia (ALL) In 1. Remission (CR1): Promising, But Strongly Influenced By ALL Subtypes

Stem cell transplantation (SCT) has contributed to improved overall survival particularly in high risk adult ALL patients and the indication for SCT is a matter of ongoing discussion. We report the results obtained over a period of 18 years in a large center. Patients received first line treatment according to three consecutive trials of the German Multicenter Study group for Adult ALL (GMALL) and risk stratification was performed accordingly. In the most recent trial patients with high risk and very high risk factors are candidates for allogeneic stem cell transplantation (SCT) in CR1 from matched sibling or unrelated donors. The risk factors are: very high risk (VHR): Philadelphia chromosome positive ALL (Ph+ALL); high risk group (HR) of B-lineage ALL (HR-B): leukocytes > 30/nl at diagnosis and/or delayed CR > 3 weeks (other B) or pro B-ALL and of T-lineage ALL (HR-T): early-T-All and mature T-ALL. In addition standard risk patients were candidates for SCT in case of molecular failure or molecular relapse (SR-Mol). 106 high risk ALL patients in CR1 underwent allogeneic SCT between 1995 and 2012 at our center. Median age was 38 years (17 – 67). 40/106 patients had Ph+ALL (VHR), 57/106 patients were HR patients (HR-B n = 32: pro-B-ALL n = 15, other B n = 17; HR-T n = 25: early T n = 17, mature T n = 4, thymic n = 4). 9/106 patients belonged to the SR-Mol group. Stem cell donors were matched related donors (MRD) in 42 patients and compatible unrelated donors in 64 patients. The conditioning regimen was 12 Gy TBI ± Cyclophosphamide or VP16 in 86 patients, other n = 1, and in patients > 55 years 8 Gy TBI + Fludarabine (n = 5) or Fludarabine + Busulfan (n = 14). ATG as GvHD and rejection prophylaxis was given since 5/2005 in all patients with unrelated SCT and in reduced intensity conditioning regimens (RIC). Results60/106 (57 %) patients with high risk ALL transplanted in CR1 are alive, 46/106 (43 %) patients are dead. The median follow-up of the surviving patients is 64 months (5 – 172). Causes of death were leukemia in 16/106 patients (15%) and transplant-related mortality (TRM) in 30/106 patients (28%), mainly infections ± GvHD (n = 24), secondary malignancies (n = 2) and other (n = 4). In Ph+ALL 19/40 patients (47 %) are alive in CR (CCR), 12/40 are dead due to TRM and 9/40 due to leukemia. In HR-B ALL 23/32 patients (71 %) are in CCR, 8/32 are dead due to TRM and 1/32 due to leukemia. In HR-T ALL 11/25 patients (44 %) are in CCR, 10/25 died due to TRM and 4/25 due to leukemia. In SR-Mol patients 7/9 patients (77 %) are in CCR, 2/9 died due to leukemia. Probability of survival (OS) for all patients at 96 months is 0.48, probability of disease free survival (DFS) at 96 months is 0.47, probability of TRM at 96 months is 0.34. OS was 0.50 for patients with myeloablative conditioning (MAC) (n = 86) and 0.38 for patients with RIC (n = 19), n.s. OS was different for the 4 risk groups: VHR 0.42, HR-B 0.67, HR-T 0.27, SR-Mol 0.74, but not significant (0.095). DFS: VHR 0.42, HR-B 0.68, HR-T 0.24, SR-Mol 0.76 (0.041) and TRM: VHR 0.34, HR-B 0.26, HR-T 0.58, SR-Mol 0. (0.026). In HR-B OS was better for pro-B-ALL than other B-lineage (0.73.vs. 0.66, resp.). OS was lower in early-T-ALL (0.24). In mature T-ALL 3/4 patients are dead, in thymic ALL 1/4 patients. In ConclusionLong term survival and probably cure can be reached in high risk ALL patients after allogeneic stem cell transplantation in CR1. OS in pro-B-ALL and B-lineage-HR-ALL patients is better than in Ph+ALL or in HR-T-ALL due to lower relapse rate and lower TRM. Whether the quality of remission before allogeneic SCT could be improved in Ph+ALL and HR-T-ALL, has to be studied. Allogeneic SCT in SR with molecular failure or relapse show promising data, though in a small patient cohort. For all patients the TRM-rate due to GvHD and infection has to be improved due to better supportive care. Disclosures:No relevant conflicts of interest to declare.

Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) In Adults With Philadelphia Chromosome (Ph)-Negative Acute Lymphoblastic Leukemia (ALL): Results From The Group For Research On Adult ALL (GRAALL)

▪ PurposeThe pediatric-inspired intensified GRAALL protocol yielded a marked improvement in the outcome of adults with Ph-negative acute lymphoblastic leukemia (ALL) (Huguet et al,JCO 2009), raising the issue of the place of allogeneic HSCT in this new context. We report here the results associated with HSCT in first complete remission (CR) in younger adults treated in the GRAALL-2003/2005 trials. Patients and methodsIn these trials, HSCT was offered in first CR to patients aged 15-55 years with ALL at higher risk of relapse. High-risk factors included white blood count >30 x 109/L for B-lineage ALL, central nervous system (CNS) involvement, t(4;11)/MLL anomalies, t(1;19), low hypodiploidy/near triploidy, complex karyotype, early resistance to the steroid prephase (CsR), early resistance to chemotherapy (ChR) as assessed by poor bone marrow blast clearance at day 8, and late CR. Per protocol, HSC donor should be a matched sibling or an unrelated donor (10/10 or eventually 9/10 HLA matched) and conditioning regimen should include single fraction 10-Gy or fractionated 12-Gy total body irradiation (TBI) and high-dose cyclophosphamide. The role of HSCT was evaluated by HSCT versus no-HSCT cohort comparison (Mantel-Byar time-dependent analysis). ResultsAmong the 522 high-risk patients eligible, 283 (54%) actually received HSCT in first CR (185 B-lineage and 98 T-lineage ALL; median age, 31 years). There were no significant differences in baseline characteristics and early response between HSCT and no-HSCT cohorts, except more HSCT patients with t(4;11)/MLL anomalies. Among the 283 HSCT patients, 46 had t(4;11)/MLL anomalies, 13 had CNS disease, 116 had CsR and 176 had ChR ALL. Origin of HSC was 140 sibling and 143 unrelated donors, including 47 unrelated donors with 1 HLA mismatch. The following HSCT protocol violations were observed: a) conditioning did not include TBI in 17 patients; b) 10 patients received reduced-intensity conditioning (RIC); and c) 13 patients received cord blood HSCT. These patients remained in the HSCT cohort for the analysis. With a median post-transplant follow-up of 3.5 years, post-HSCT cumulative incidence of relapse (CIR), non-relapse mortality (NRM) and relapse-free survival (RFS) were estimated respectively at 19% (95% CI, 15-25), 16% (95% CI, 12-21) and 64% (95% CI, 58-70) at 3 years, without any RFS difference between sibling and unrelated donors. A marked trend for a higher NRM was observed in patients aged 45 years or more (26% versus 13% at 3 years; HR, 1.6; p=0.059), while post-transplant CIR was similar in older and younger patients. Mantel-Byar RFS estimations showed no significant difference between both HSCT and no-HSCT cohorts (HR, 0.80; p=0.13), meaning that the risk factors used in these protocols failed to identify patients who could significantly benefit from HSCT in first CR. As minimal residual disease (MRD) had a major prognostic impact, we retrospectively analyzed the effect of HSCT in the 278/522 patients evaluated for 6-week Ig/TCR MRD level (154 HSCT and 124 no-HSCT patients, including 92 and 62 patients with MRD >=10-4, respectively). Mantel-Byar RFS estimations showed that HSCT did not benefit to patients with MRD <10-4 (HR, 1.2; p=0.67) as opposed to those with MRD >=10-4 (HR, 0.6; p=0.02), with a positive HSCT-by-MRD interaction (p=0.04) (Figure 1). [Display omitted] ConclusionIn adult patients with high-risk ALL in first CR treated in the GRAALL-2003/2005 trials, myeloablative allogeneic HSCT from sibling and unrelated donors was associated with a 64% RFS at 3 years. The 26% NRM observed at 3 years in patients aged 45 years or more suggests considering RIC-HSCT in these patients. MRD response appears to be a powerful tool to select patients who might benefit from HSCT. This selection tool will be prospectively evaluated in the next GRAALL-2013 trial.ND and AH contributed equally to the work. Disclosures:No relevant conflicts of interest to declare.

TP53 Mutations Are Present In 15.7% Of ALL, Are Especially Frequent In ALL With MYC-Rearrangement and In ALL With Low Hypodiploid Chromosome Status and Are Associated With Poor Prognosis

BackgroundTP53 is the most frequently mutated gene in cancer. The prognostic impact of TP53 mutations has been demonstrated in CLL, AML, and MDS. However, data on the frequency and prognostic impact of TP53 mutations in ALL is scarce. AimsWe aimed at determining the TP53 mutation frequency, the association with cytogenetic subgroups and age, as well as the impact on survival. Patients and MethodsIn total, a large cohort of 625 patients with ALL was analyzed for TP53 mutations by deep-sequencing allowing to simultaneously quantify the mutation load. In all patients chromosome banding analyses have been performed. In addition, in 341 patients the copy number state of TP53 was determined by FISH. ResultsThe cohort comprised 353 male and 272 female patients, median age was 49.5 years (range: 0.1-91.4 years). The cohort included the following groups: normal karyotype (n=101; 16.2%), t(9;22)(q34;q11) (n=162; 25.9%), MLL-translocations (n=37; 5.9%), MYC-translocations (n=40; 6.4%), t(12;21)(p13;q22) (n=15; 2.4%), low hypodiploidy (<40 chromosomes) (n=24; 3.8%), high hyperdiploidy (51-68 chromosomes) (n=38; 6.1%), complex karyotype (n=69; 11.0%), other cytogenetic abnormalities (n=139; 22.2%). Detailed data on immunophenotyping was available for 408 patients (T-lineage: n=105; B-lineage: n=267, Burkitt: n=36). In the total cohort, the frequency of TP53 mutations was 15.7% (98/625). TP53 mutations were most frequent in ALL with low hypodiploidy (22/24; 91.7%) and MYC-translocated ALL (25/40; 62.5%) and also quite frequent in ALL with complex karyotype (16/69; 23.2%), ALL with normal karyotype (13/101; 17.4%), and in MLL-translocated ALL (6/37; 16.2%). TP53 mutations were rare in t(9;22)(q34;q11) (7/162; 4.3%), high hyperdiploidy (3/38; 6.1%), and other cytogenetic abnormalities (6/139; 4.3%) and absent in ALL with t(12;21)(p13;q22) (0/15; 0%). Furthermore, TP53 mutations were less frequent in T-lineage ALL (8/105; 7.6%) as compared to B-lineage and Burkitt ALL (41/267; 15.4% and 21/36; 58.3%). TP53 mutation frequency increased with age (TP53mut <60 years vs ≥ 60 years 10.8% (45/417) vs 25.5% (53/208), p<0.0001). 25/64 (39.1%) TP53 mutated patients with available TP53 deletion status showed a deletion of the second allele, while in 17/319 (5.3%) TP53wt patients with available TP53 deletion status a TP53 deletion was detected (p<0.001). 11/98 (11.2%) TP53 mutated patients showed two TP53 mutations. Median overall survival (OS) was significantly shorter in TP53mut vs TP53wt patients (18.8 months vs 75.5 months, p<0.0001). OS at 4 years in patients <60 years was 80.1% in TP53wt compared to 56.8% in TP53mut (p=0.012) and 59.3% vs 22.6% in patients ≥ 60 years (p<0.0001). Also within the cytogenetic categories MYC-translocated and complex karyotype TP53 mutations had a significant adverse impact on overall survival. Further, patients with either two TP53 mutations or one TP53 mutation and an accompanying TP53 deletion had a significantly shorter OS as compared to patients with only one altered TP53 allele (median OS 11.5 vs 63.1 months, p=0.009). In contrast, OS in patients with a TP53 deletion without a TP53 mutation did not differ from patients without TP53 alterations. In addition, the TP53 mutation load was investigated by next-generation sequencing and varied between 2% and 98% (median: 41%). Within the subset of patients with TP53 mutation, patients with a mutation load >20% showed a significantly shorter OS as compared to patients with a lower mutation load (median OS 11.5 months vs not reached, p=0.003). Interestingly, OS in patients with a TP53 mutation load ≤ 20% did not differ from TP53wt patients. In multivariate Cox regression analysis including parameters significantly associated with shorter OS in univariate analysis the following factors retained an independent adverse impact on OS: age (<60 years vs ≥ 60 years, HR=2.2; p=0.01), MLL-translocation (HR=2.8; p=0.03), and TP53mut >20% (HR: 3.1, p=0.01). Conclusions1. TP53 is mutated in 15.7% of ALL with the highest frequency in ALL with low hypodiploidy (91.7%) and MYC-translocated ALL (62.5%). 2. The TP53 mutation frequency increases with age. 3. TP53 mutations are associated with short survival independent of age and specific cytogenetic alterations. 4. TP53 mutations had a significant impact on OS only if the mutation load was >20%. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Chromosome 21 Kinase DYRK1A Controls Cell Cycle Exit and Survival During Lymphoid Development and Is a Novel Therapeutic Target In Acute Lymphoblastic Leukemia

The dual specificity tyrosine-regulated kinase 1A (DYRK1A) phosphorylates a growing number of proteins to influence various aspects of cell biology, including survival, apoptosis, proliferation, and neuronal development and function. Encoded on human chromosome 21, the DYRK1A locus resides in the Down syndrome critical region (DSCR) and has been implicated in the pathogenesis of neurocognitive deficits that result from trisomy 21. We recently demonstrated a prominent tumor-promoting role for DYRK1A in acute megakaryoblastic leukemia in children with DS (DS-AMKL). This discovery prompted us to ask whether DYRK1A is important for normal hematopoiesis, and if additional types of leukemia result from enhanced DYRK1A activity.Apart from our study in DS-AMKL, the involvement of DYRK1A in normal and malignant hematopoiesis has not been described. Dyrk1a germline knockout mice die mid-gestation; therefore, we generated conditional knockout (CKO) mice to allow inducible deletion of the kinase in hematopoietic cells with Mx1-Cre. In the bone marrow, CKO mice exhibit significant reductions of the hematopoietic stem cell-enriched LSK and common lymphoid progenitor populations (5-fold and >20-fold reductions vs. controls, respectively). In B cell lineage, CKO mice have 2-fold reductions of small pre-B and immature B cells, and a 5-fold reduction of mature recirculating B cells. In the thymus, CKO mice show dramatic loss of CD4+/CD8+double positive cells (DP, >30-fold fewer at 4 weeks). Furthermore, DYRK1A-deficient cells fail to reconstitute lymphoid cells in competitive bone marrow transplantation experiments and give rise to virtually no pre-B cell colonies in ex vivo assays. In contrast to these lymphoid defects, myeloid development is largely normal, with slightly increased numbers of erythroid precursors, granulocytes, and monocytes.To understand the mechanisms by which DYRK1A controls lymphopoiesis, we performed microwestern blot arrays. This approach allowed us to simultaneously screen >200 antibodies to identify differentially expressed proteins between CKO and control lymphoid cells. We found increased levels of cell cycle regulatory proteins, such as CDK1, suggesting that DYRK1A modulates cell cycle status during lymphoid development. Indeed, cell cycle analysis revealed that there were 20-30% fewer quiescent (G0) cells among CKO small pre-B cells and DP thymocytes, two developmental stages characterized by exit from the cell cycle following a proliferative burst. Among its targets, DYRK1A is known to contribute to DREAM (DP, Rb-like, E2F, and MuvB) complex assembly via phosphorylation of LIN52. The DREAM complex, not previously described in hematopoiesis, promotes quiescence by repressing the transcription of numerous cell cycle-associated genes during G0. RNA sequencing analysis in flow-purified G0 cells confirmed that DREAM complex targets are markedly de-repressed in the affected CKO populations, highlighting the importance of DYRK1A/DREAM complex interaction during lymphoid cell cycle exit.Both B- and T-lineage ALL express high levels of DYRK1A relative to other tumor types. Having observed that DYRK1A is essential for lymphoid cells, we next asked whether targeting DYRK1A with small molecule inhibitors would be an effective therapy for ALL. We found that EHT 1610, a highly selective DYRK1 inhibitor (Leblond B., Casagrande A.-S., Désiré L., Foucourt A., Besson T., DYRK1 inhibitors and uses thereof, WO 2013026806, 2013.02.28), dose-dependently induced apoptosis in B- and T-ALL cell lines and primary human pediatric ALL samples. Thus far, all cell lines and all primary samples tested were exquisitely sensitive to EHT 1610. Moreover, EHT 1610 induced apoptosis of primary ALL cells that were resistant to cytarabine, suggesting that DYRK1A inhibitors may be used in combination with standard ALL therapies for refractory or relapsed cases. Together, our data establish novel essential roles for DYRK1A in both normal and malignant lymphoid development and provide rationale for the design of DYRK1A-targeted ALL therapies. Disclosures:Leblond:Diaxonhit Therapeutics: Employment. Casagrande:Diaxonhit Therapeutics: Employment. Désiré:Diaxonhit Therapeutics: Employment. Crispino:Sanofi: Research Funding.

Diagnosis of a T-lineage acute lymphoblastic leukemia through digitalized cell analysis of the pleural effusion

IntroductionPleural effusion as the first clinical manifestation of acute lymphoblastic leukemia (ALL) is a relatively rare event. An early and accurate diagnosis of this clinical picture is very important for adequate patient management.Case presentationWe report the atypical onset of T-lineage ALL in a 31-year-old man. The patient was admitted to the emergency room due to lung failure; at that moment, the patient’s initial blood count was normal; the chest X-ray radiography showed a massive pleural effusion and a thoracentesis was carried out. Routine investigations performed on the pleural fluid using a new technology system and digitalized cell analysis demonstrated infiltration by immature cells. Therefore, bone marrow aspirate and flow cytometry analyses were performed, leading to the diagnosis of T-lineage ALL. A cord blood transplantation procedure was performed at the first hematological remission following chemotherapy regimens. The patient died of septic shock.ConclusionThe case we reported underlines the usefulness of using automated instruments to identify abnormal lymphoid cells in body fluids.

Implications of 25-Year Follow-Up of White Matter Integrity and Neurocognitive Function of Childhood Leukemia Survivors: A Wake-Up Call

Improved treatment via new drug development, combination therapy, and methods of treatment have resulted in 73% to 80% of children treated for acute lymphoblastic leukemia (ALL) being considered long-term survivors who are cured. This survival rate has not come without a cost, with long-term consequences (late effects) occurring in reproductive capacity, cardiac function, neurocognitive function, endocrine function, pulmonary function, and second malignancy related to treatment. As children survive longer into adulthood, an additional late effect is beginning to be recognized: an onset of diseases associated with aging occurring earlier than in the general population. Though much is known about neurocognitive late effects in children and adolescents during school-age and adolescence, nothing is known about the long-term effects of treatment received during childhood on the survivors of ALL 20 to 30 years after diagnosis. In the article accompanying this editorial, Schuitema et al describe one of the first studies in this area, and the results are consistent with other late-effects studies related to early aging concerns. Ninety-three young adults treated between 1978 and 1990 for childhood ALL with either cranial radiation therapy (CRT) or CNS prophylaxis with chemotherapy were evaluated using magnetic resonance imaging (MRI) and standardized tests of neurocognitive function. Survivors who were treated with CRT had significantly reduced white-matter integrity, as measured by MRI fractional anisotropy (FA), and poorer neurocognitive function (lower intelligence quotient, poorer visuomotor accuracy, stability, and sequential working memory, and poorer work flow during sustained attention). Decreases in FA were observed in chemotherapy-only patients, but those decreases were more moderate than in CRT-treated survivors. Consistent with other reports describing white-matter integrity during childhood and adolescence, younger patient age at the time of CRT and higher dose were associated with worse white-matter integrity. Steep declines in FA in the frontal and parietal white matter were observed related to age of assessment, suggesting an accelerated pattern of aging. To emphasize this point, the authors note anatomic similarities between these long-term ALL survivors and aging patients with cognitive decline or dementia. Accelerated aging of the CNS is a concern of late-effects investigators. Evidence is growing for persistent neurocognitive impairment after both CRT and systemic chemotherapy in survivors of cancers involving treatment of the CNS with CRT, chemotherapy, or combination CRT and chemotherapy. Impairment is most frequently seen in the areas of executive function, processing speed, and short-term or working memory. There is additional risk for increased severity in other areas of cognitive function in children who are younger at time of treatment or who receive higher doses of CRT. For children, school performance is often negatively affected; little is known about daily-function challenges in adult survivors. Treatment of childhood ALL has changed dramatically over the last 30 years, particularly CNS prophylaxis. Before 1985, with the introduction of Pediatric Oncology Group 8602 study, children with B-lineage or T-lineage ALL were typically treated with craniospinal radiation therapy, sometimes alone and sometimes in combination with intrathecal methotrexate. The dose of CRT was typically 24 Gy or 18 Gy. The Pediatric Oncology Group 8602 study eliminated CRT for the majority of children treated for ALL, replacing it with intrathecal methotrexate or triple intrathecal chemotherapy (methotrexate, cytarabine, and hydrocortisone). This strategy reduced some of the neuroendocrine and growth delays that had been associated with CRT, but neurocognitive deficits were still observed 3 or more years after diagnosis. Treatment for ALL since the early 1990s has included a more aggressive use of systemic methotrexate during the consolidation phase of therapy, increasing from 1 gm/m to 5 gms/m. This change, without CRT, has also resulted in reports of increased neurocognitive impairment and computed tomography and MRI evidence of primarily calcification in the CNS. For children with CNS tumors, CRT remains the backbone of therapy after surgical resection. CRT doses are typically much higher than those used in the historical and current treatment of ALL (30 Gy to 65 Gy v 18Gy or less). There have been efforts dating back to the mid-1980s to reduce the dose of CRT, delay its use, or eliminate it from treatment, but most children with high-grade malignant CNS tumors will likely be treated with CRT, either photon-beam or proton-beam. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 27 SEPTEMBER 2

Mutational landscape of adult ETP-ALL

T-lineage acute lymphoblastic leukemia (T-ALL) accounts for approximately 25% of adult acute lymphoblastic leukemia. Improving therapy is a major challenge as outcome in adult T-ALL remains unsatisfactory for the majority of patients [1]. The subgroup of early T-cell precursor (ETP) ALL recently gained great interest [2] due to the fact that this subgroup in pediatric T-ALL patients was clinically characterized by an especially poor outcome and molecularly defined by a distinct gene expression signature and an immature immunophenotype [3]. The gene expression signature displays an expression profile similar to that of normal T cell progenitors that immigrate from the bone marrow to the thymus. In addition to properties of T cell development, gene expression profile and immunophenotype of ETP-ALL also show features of an early myeloid fate. Thus, ETP-ALL might serve as model for a stem cell leukemia at the intersection of the myeloid and lymphoid lineages. The homogeneity of ETP-ALL with respect to gene expression, immunophenotype, and clinical outcome is however not reflected by a common genetic event; in contrast, a comprehensive genomic analysis revealed a highly heterogeneous mutational spectrum of ETP-ALL in pediatric patients [4]. Similarities to myeloid malignancies were found and mutated target genes serving as potentially druggable lesions have recurrently been identified (e.g. cytokine receptors or JAK signalling). In our recent study we further investigated these aspects for ETP-ALL in adult patients [5]. This analysis included whole exome sequencing of five ETP-ALLs and initially resulted in the identification of only two recurrently mutated genes. Interestingly, the affected genes, DNMT3A and FAT3, have not been previously been reported in pediatric ETP-ALL patients. The overall mutational spectrum of adult ETP-ALL also comprised genes that have shown to be mutated in T-ALL (ETV6, NOTCH1, DNM2) as well as in myeloid malignancies (NRAS, JAK1, DNMT3A). The heterogeneity of genetic alterations is not only observed in ETP-ALL, but also in other hematologic malignancies like acute myeloid leukemia [6]. Thus, supposed homogenous disease entities share only to minor degree common genetic lesions. The generation of genomic data opens the opportunity to classify disease entities on an additional level, which may provide the basis for the development of targeted therapy. For the rare subgroup of ETP-ALL, the collected data are obviously not sufficient to establish definite molecular classifiers. But these data can build in the model of the myeloid/lymphoid leukemic intersection and the identification of recurrent mutations might unravel potential targets relevant to stem cell like leukemias. In our work, we were able to identify over 60% of the ETP-ALL patients with a mutation in one of the three genes: FLT3, DNMT3A, and NOTCH1. Thus, based on these findings, molecularly defined leukemic subgroups can potentially be targeted by new treatment approaches altering these pathways: demethylating agents, kinase inhibitors, and gamma secretase inhibitors. In addition to these already recognized alterations, novel recurrent mutations were found in FAT1, FAT3, and MLL2 in a high percentage of adult ETP-ALL patients. These data might increase the number of patients, for whom specific treatment approaches can be explored in the future. For FAT1, the connection to the WNT pathway and possible ways to antagonize the activation has been discussed in a recent work of Morris and colleagues [7]. Another finding of our analysis was the age dependent occurrence of specific mutations. There were several mutations in adult patients that had not been discovered in pediatric patients such as DNMT3A, FAT1, FAT3, and MLL2. Of note, we found DNMT3A mutations in 16% of adult ETP-ALL patients, consistent with recent discoveries of frequent DNMT3A mutations not only in ETP-ALL, but also in T-ALL [8]. We observed a clear age dependency with the occurrence of DNMT3A mutations mainly in older patients. In contrast, other mutations like the histone modifiers EZH2 and SUZ12 were significantly less frequent in adult than in pediatric patients. Together with data from other studies, which show a higher mutational burden in older patients and the occurrence of mutations in epigenetic regulators, e.g. TET2 in elderly healthy persons, this age dependency points to the requirement of age adapted therapies. This does not only include the dose modification of existing therapies and the consideration of comorbidities, but in particular the identification of molecular targets within a different genomic background. Keeping in mind that the majority of our recent data were collected in patients aged 60 years and younger, many more patients over the age of 60 years will have to be studied in detail to unravel genetic alterations in the elderly. In summary, next generation sequencing allowed to explore the mutational spectrum of adult ETP-ALL and led to the identification of novel recurrent mutations and potentially druggable targets. The enormous challenge yet lies ahead: the development of targeted therapies and the implementation of a routine screening for these mutations resulting in consecutive tailored therapy based on the genetic lesions.

Defective K-Ras oncoproteins overcome impaired effector activation to initiate leukemia in vivo

AbstractReversing the aberrant biochemical output of oncogenic Ras proteins is one of the great challenges in cancer therapeutics; however, it is uncertain which Ras effectors are required for tumor initiation and maintenance. To address this question, we expressed oncogenic K-RasD12 proteins with “second site” amino acid substitutions that impair PI3 kinase/Akt or Raf/MEK/ERK activation in bone marrow cells and transplanted them into recipient mice. In spite of attenuated signaling properties, defective K-Ras oncoproteins initiated aggressive clonal T-lineage acute lymphoblastic leukemia (T-ALL). Murine T-ALLs expressing second site mutant proteins restored full oncogenic Ras activity through diverse mechanisms, which included acquiring novel somatic third site KrasD12 mutations and silencing PTEN. T-ALL cell lines lacking PTEN had elevated levels of phosphorylated Akt, a gene expression pattern similar to human early T-cell precursor ALL, and were resistant to the potent and selective MEK inhibitor PD0325901. Our data, which demonstrate strong selective pressure to overcome the defective activation of PI3 kinase/Akt and Raf/MEK/ERK, implicate both Ras effector pathways as drivers of aberrant growth in T-ALL and further suggest that leukemia cells will deploy multiple mechanisms to develop resistance to targeted inhibitors in vivo.

Whole-exome sequencing in adult ETP-ALL reveals a high rate of DNMT3A mutations

AbstractEarly T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a high-risk subgroup of T-lineage ALL characterized by specific stem cell and myeloid features. In adult ETP-ALL, no comprehensive studies on the genetic background have been performed to elucidate molecular lesions of this distinct subgroup. We performed whole-exome sequencing of 5 paired ETP-ALL samples. In addition to mutations in genes known to be involved in leukemogenesis (ETV6, NOTCH1, JAK1, and NF1), we identified novel recurrent mutations in FAT1 (25%), FAT3 (20%), DNM2 (35%), and genes associated with epigenetic regulation (MLL2, BMI1, and DNMT3A). Importantly, we verified the high rate of DNMT3A mutations (16%) in a larger cohort of adult patients with ETP-ALL (10/68). Mutations in epigenetic regulators support clinical trials, including epigenetic-orientated therapies, for this high-risk subgroup. Interestingly, more than 60% of adult patients with ETP-ALL harbor at least a single genetic lesion in DNMT3A, FLT3, or NOTCH1 that may allow use of targeted therapies.

Detection of PICALM-MLLT10 (CALM-AF10) and outcome in children with T-lineage acute lymphoblastic leukemia

Detection of PICALM-MLLT10 ( CALM-AF10 ) and outcome in children with T-lineage acute lymphoblastic leukemia