B-lineage Acute Lymphoblastic Leukemia Research Articles

Clinical Significance of Occult Central Nervous System Disease in Adult ACUTE Lymphoblastic Leukemia. a Multicenter,Report from the Campus ALL/Gimema Network

Introduction. In a modern context of improved management of acute lymphoblastic leukemia (ALL), central nervous system (CNS) involvement at diagnosis remains an obstacle towards long-term cure. We have previously reported that flow cytometry (FCM) is better than conventional cytology (CC) in demonstrating the presence of leukemic cells in patients' (pts) cerebrospinal fluid (CSF), especially in samples with low cell counts. In the framework of the national Campus ALL program aimed at improving the management of adult ALL patients enrolled in the GIMEMA protocols, we retrospectively evaluated the incidence of occult CNS positivity and its impact on outcome in 221 adult pts with newly diagnosed ALL from 11 centers.Methods. Ninety-four patients (42%) were females and 127 (58%) males, with a median age of 44 years (range 17-80), a median white blood cell (WBC) count of 10.6x109/L (range 0.1-457). One hundred and seventy pts (77%) had B-lineage ALL. Cytogenetic/genetic data were available in 167 (75%) pts: 58 (35%) had a BCR/ABL rearrangement, 14 (8%) a complex karyotype and 11 (6%) a MLL rearrangement. Pts were treated according to the GIMEMA/NILG ALL protocols or with the Hyper-CVAD program. Ninety-eight pts underwent an allogeneic stem cell transplant (ASCT). Median follow up was 26.8 months (range 1-136.6). All CSF samples were evaluated both by CC and FCM. The presence of ≥10 clonally restricted or phenotypically abnormal events was regarded as a FCM positivity. Based on the results of CSF examination, three different categories were recognized: manifest CNS+ (CC+FCM+), occult CNS+ (CC-FCM+) and CNS- (CC-FCM-).Results. Overall, 16 (7%) pts had manifest CNS+, 39 (17%) occult CNS+ and 166 (75%) were CNS-Median age, WBC count, B/T lineage, cytogenetic/genetic features did not differ significantly between the three categories. A complete remission (CR) was achieved in 178 (80%) pts, 9 (4%) died early in induction and 104/178 (58%) experienced a relapse. The frequency of CR rate did not vary significantly across the three identified categories. In univariate analysis, the CNS status correlated significantly with the incidence of relapse (p=.004) and with censor (<.00002). The four-year overall survival (OS) for manifest CNS+, occult CNS+ and CNS- pts was 6%, 17% and 46%, respectively (p=.0007) (Figure). No difference in terms of OS was observed between manifestand occult CNS+ pts (p=.30). In multivariate analysis a baseline status of manifestor occult CNS+ (RR: 1.98, 95%CI 1.47-2.43, p=0.00001) and age>45 years (RR:1.45, 95% CI 1.47-2.43, p=0.04)were independently associated with a lower OS.Conclusions. Our large, multicenter CAMPUS ALL study showed that i) in ALL adult pts, FCM allows to detect occult CNS disease, even in conditions of low spinal fluid leukemic count; ii) the presence of occult CNS disease is associated with an unfavorable outcome. Further prospective studies on larger series are needed to confirm these data. DisclosuresDel Principe:Gilead: Membership on an entity's Board of Directors or advisory committees. Fracchiolla:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees. Foà:NOVARTIS: Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; GILEAD: Speakers Bureau; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; INCYTE: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY BOARD, Speakers Bureau.

Digital-Droplet PCR, an Accurate Method for IG/TR PCR-MRD Stratification in Childhood Acute Lymphoblastic Leukemia

Digital-Droplet PCR, an Accurate Method for IG/TR PCR-MRD Stratification in Childhood Acute Lymphoblastic Leukemia

Gene Aberrations in Adult Patients with Ph-Negative ALL in the Czech Republic

IntroductionPhiladelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an identified high-risk B-lineage ALL subtype, accounting for approximately 20% of adult patients with B-ALL. Ph-like ALL is characterized by poor outcome, very high risk of disease relapse and poor overall survival (OS). The leukemic cell gene expression profile of Ph-like ALL is similar to that of Ph-positive ALL. However, instead of BCR-ABL1, such patients harbor a highly diverse range of genetic alterations. The gene aberrations detected in Ph-like ALL patients are associated with JAK/STAT, Ras, Ikaros and ABL signaling pathways. These aberrations can be subdivided into five distinct subgroups based on the type of cytokine receptor or kinase fusion present: 1) rearrangements of CRLF2; 2) ABL-class gene rearrangements; 3) JAK2 and EPOR rearrangements; 4) sequence mutations or deletions activating JAK-STAT or MAPK signaling pathways and 5) other rare kinase alterations. The study aimed to define the frequency of high-risk genetic aberrations associated with Ph-like subtype in adults with Ph-negative ALL from the Czech Republic. The genetic basis in adult patients with Ph-like ALL has not been studied to date.Patients and MethodsThe molecular genetic analysis was performed in 74 Ph-negative ALL adult patients from routine hematologic practice. DNA and RNA were isolated from patient´s peripheral blood or bone marrow. The mutational analysis was focused on the detection of the P2RY8-CRLF2 fusion gene (real-time PCR) and the “hot spot” region of the JAK2 gene (PCR/Sanger sequencing) in patient´s cohort. Minimal residual disease (MRD) value was also assessed in all patients prior to the first consolidation cycle (week 11) by real-time PCR. The detection of copy number variations (MLPA method) and targeted sequencing (NGS) was performed in a selected set of patients.ResultsThe patient cohort of Ph-negative ALL consisted of 46.6% young adults (age 15 to 39 years), 35.6% adults (age 40 to 59 years), and 17.8% older adults (age 60 to 75 years). Among all patients, increasing age was associated with an inferior outcome of the disease. The difference in OS was significant for young adults compared with adults and older adults (median: 126.7, 23.5 and 22.6, respectively; P=0.0087). The above-mentioned gene aberrations were identified in 36.5% of patients with Ph-negative ALL. The 3-year OS for patients with and without gene aberrations was 36% and 49%. Copy number alterations were detected in almost a third of patient cohort in several genes (IKZF1, PAX5, BTG, ETV6, EBF1, CDKN2A/2B, and SHOX). Deletions in IKZF1 were the most frequent aberration (17.8%). Within the patient cohort, the presence of IKZF1 deletions did not influence the OS significantly. The presence of P2RY8-CRLF2 fusion gene was identified in 16.4% patients with markedly inferior OS in comparison to a patient group without detected P2RY8-CRLF2 (median: 33.2 and 126.7). Sequence mutations of TP53, IL7R, and JAK2 genes occurred in 14.3%, 2.9%, and 1.6% patients, respectively. All patients with the detected sequence variant were carriers of at least one other gene aberration. Achieving MRD negativity in patients with Ph-like aberrations tended to more favorable OS in comparison to those MRD positive, but this trend was not statistically significant enough.ConclusionsOur findings demonstrate the representation of selected gene aberrations in Ph-negative ALL adults patients. Also they have led to the introduction of molecular genetic diagnostics of Ph-like ALL to routine hematological practice in the Czech Republic. MRD negativity and younger age are the most important prognostic factors in this subgroup of patients according to our analysis.This study was supported by TA CR (TE02000058), MH CZ - DRO (FNBr, 65269705) and CEITEC 2020 (LQ1601), and MEDGENET 2020 (692298). DisclosuresFolber:Affimed: Research Funding. Doubek:Roche: Consultancy, Honoraria; Affimed: Research Funding; AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy.

Excellent Response to Blinatumomab in Children and Young Adults with Refractory B Lineage Acute Lymphoblastic Leukaemia for Persistent MRD or after Debulking Chemotherapy for Higher Disease Burden

Introduction: A recent phase I/II study of blinatumomab in children with relapsed/refractory B cell acute lymphoblastic leukaemia (B-ALL), found 39-51% achieved complete, often MRD negative, remission after two cycles of therapy. Higher responses were observed in patients with less than 50% bone marrow blasts, compared to greater than 50% (55.6% vs 32.7% (95% CI 30.8-78.5 and 20.3-47.1 respectively)1. Furthermore, an adult study showed complete minimal residual disease (MRD) response rates of 78% when blinatumomab was used to treat MRD-positive ALL in haematological remission 2. Hence response rates (and toxicity) to blinatumomab are highly correlated with pre-treatment disease burden. We present preliminary data showing an excellent response to blinatumomab in children and young adults with resistant B-ALL who had persistent MRD, or after debulking chemotherapy to achieve a partial remission.Methods: Eleven patients were identified through a national survey. The mean age of patients was 10 years (range 0.7-22 years, 3 infants and 1 Down syndrome ALL). All patients had B-lineage ALL which was CD19 positive. None had active CNS disease at the point of receiving blinatumomab. Prior to administration of blinatumomab, all patients either had persistent MRD following several courses of intensive chemotherapy or received debulking chemotherapy for heavier marrow infiltrates. Pre-blinatumomab chemotherapy to which the patients had failed to obtain an adequate MRD response included UKALL 2011 Regimens A or C, UKALL R3, Interfant 06 or NOPHO high risk blocks. Patients received 5-15 µg/m2 of blinatumomab for 1-2 cycles prior to definitive therapy.Results: Pre-blinatumomab, all patients except one were in morphological remission with MRD measurable by PCR (0.003-1%), the remaining patient had 9% marrow disease by morphology. After 1-2 cycles of blinatumomab all patients had negative MRD when measured by flow cytometry and/or by PCR, giving a 100% response rate. This was followed by Haemopoietic stem cell transplant (HSCT) in nine patients and the remaining two are awaiting transplant. Further data on patient characteristics, CNS status, relapse and survival outcome are being collected and will be presented at the meeting.Minimal toxicity was observed; of the seven patients in whom toxicity data were available, three had grade 1 CRS, which resolved spontaneously without interruption of therapy or treatment with corticosteroids or Tocilizumab. One patient reported grade 1 neurotoxicity.This preliminary UK experience demonstrates that excellent MRD response is observed with minimal toxicity in children and young adults who receive blinatumomab for persistent MRD or after debulking chemotherapy. This provided a bridge to transplant in patients who would otherwise not have benefited from the procedure because of persistent MRD. We are planning to extend these observations by undertaking a study of this strategy in first high-risk B-ALL relapse.Stackelberg Av, Locatelli F, Zugmaier G, et al. Phase I/Phase II Study of Blinatumomab in Pediatric Patients With Relapsed/Refractory Acute Lymphoblastic Leukemia. Journal of Clinical Oncology. 2016;34(36):4381-4389.Gokbuget N, Dombret H, Bonifacio M, et al. Blinatumomab for minimal residual disease in adults with B-cell precursor acute lymphoblastic leukemia. Blood. 2018;131(14):1522-1531. DisclosuresGhorashian:Celgene: Other: travel support; Novartis: Honoraria. Marks:Novartis: Consultancy; Pfizer: Consultancy; Amgen: Consultancy. Vora:Amgen: Other: Advisory board; Novartis: Other: Advisory board; Jazz: Other: Advisory board; Medac: Other: Advisory board; Pfizer: Other: Advisory board.

Divergent Evolutionary Trajectories of Erk- and Stat5-Activating Lesions in Acute Lymphoblastic Leukemia

Background and significance: Activation of STAT5- and ERK-signaling are segregated to early and later stages of normal B cell development, respectively. While pro-B cells depend on activation of Stat5 downstream of cytokine receptors, pre-B and immature B cells depend on Erk-activation downstream of pre-B cell receptor (pre-BCR). Since Stat5- (cytokine receptor) and Erk- (pre-BCR) signaling antagonize each other, we studied evolutionary trajectories of Stat5- and Erk-activating lesions in B-cell lineage ALL. We tested the hypothesis that an initial activating mutation in one of the two pathways represents a commitment step that makes the tumor clone non-permissive to activating lesions in the other pathway.Results: Studying B-lineage ALL (n=578), we found that STAT5-activating (e.g. BCR-ABL1, JAK2, cytokine receptors) and ERK-activating (NRAS, KRAS, PTPN11, NF1) lesions were mutually exclusive, with only 9 cases (1.6%) carrying lesions in both pathways. Even in the few remaining cases, low allele frequencies of one of the two lesions suggests that they do not co-occur in the same cells. Reverse phase protein array measurements revealed that phosphorylation of ERK and its upstream kinase MEK were inversely correlated with STAT5-phosphorylation (MDACC, 1983-2007; P<0.001). These findings prompted us to study mechanisms of biochemical cross-inhibition between Erk and Stat5 pathways. In support of this notion, we found that inducible NRASG12D activated Erk at the expense of Stat5 phosphorylation. This was due to stabilization and increased activation of the Stat5-phosphatase Ptpn6 (SHP1). Inducible ablation of Ptpn6 elevated phospho-Stat5 levels, while genetic inactivation of Stat5 strongly increased Erk activity. Studying a matched patient-derived pre-B ALL sample at diagnosis and at relapse (acquired KRASG12V mutation) revealed activation of ERK in association with increased BCL6 and decreased Stat5 levels in the KRASG12V relapsed ALL sample. With engagement of BCL6 and the Stat5-inhibitory phosphatase Ptpn6 downstream of Erk signaling, these findings suggest that occupancy of either Erk or Stat5-pathway represents a commitment step that renders cells non-permissive to the respective alternative pathway. To test this hypothesis, we induced B cell transformation with either Erk or Stat5-pathway oncogenes and then subsequently transduced with either empty vectors or vectors carrying the alternative oncogene. Compared to empty vector controls, Erk- and Stat5-driven B-ALL cells did not tolerate transformation with the alternative oncogene. To test the antagonistic relationship between Stat5- and Erk-activating lesions in B-ALL, we transformed Stat5fl/fl B-ALL cells with NRASG12D for Erk-activation and Erk2fl/fl B-ALL with BCR-ABL1 for activation of Stat5. While Erk-driven B-ALL clones were non-permissive to transduction with Stat5-activating oncogenes (BCR-ABL1), Cre-mediated inducible ablation of Stat5 increased competitive fitness and colony formation ability. Likewise, Stat5-driven B-ALL clones were non-permissive to transduction with Erk-activating oncogenes (NRASG12D). However, Cre-mediated inducible ablation of Erk2 increased competitive fitness and colony formation ability.Conclusion: These results support a model of clonal evolution that selects malignant B-ALL clones based on increased polarization in favor of one single dominant pathway of oncogenic signal transduction with concurrent silencing of alternative pathways. This concept suggests that malignant transformation is not only associated with progressively increased signaling strength of the central oncogenic driver but also concomitant silencing of cytokine and growth factor receptors that normal cells would respond to. DisclosuresWhite:BMS: Research Funding; Novartis: Honoraria, Research Funding.

Multiplex CRISPR/Cas9-Based Genome Editing of Mouse Hematopoietic Stem Cells Recapitulates Acute Erythroid Leukemia and Identifies Therapeutic Targets

Introduction. Acute erythroid leukemia (AEL) is a high-risk leukemia subtype of poorly understood genetic basis. In integrated comparative genomic analysis of 159 AEL and 1509 non AEL myeloid tumors, we identified 5 age-related AEL subtypes with distinct genomic features and outcome: adult AEL with bi-allelic alterations in TP53 (31%), frequently co-occurring with alterations in DNMT3A,BCOR, EZH2, RB1, or NFIX; NPM1-mutated (12%); KMT2A-mutated/rearranged (11%), mostly co-mutated with STAG2; pediatric, NUP98-rearranged (5%) and adult, DDX41-mutated (3%). Thirty-eight percent of cases lacked an identifiable exclusive recurrent founding alteration but were enriched in mutations in ASXL1, TET2 and splicing factors.Methods. To explore the roles and cooperativity of the identified alterations in leukemogenesis we used CRISPR-Cas9 genome editing to induce combinations of loss-of-function mutations in 9 recurrently mutated genes in AEL (Tp53, Tet2, Dnmt3a, Asxl1, Ezh2, Stag2, Bcor, Ppm1d, Rb1 and Nfix). Based on patterns of mutation association, we generated 6 pools of lentiviral vectors (Table 1) with different combinations of single guide RNA (sgRNA) to induce multiplex genome editing in Cas9-eGFP-mouse lineage-negative hematopoietic stem cells (HSCs). Transduced cells were transplanted into lethally irradiated congenic mice. Tumors were characterized by morphology, immunophenotyping, and genomic analysis (sequencing of sites of editing, and exome, methylation and transcriptome sequencing). Ex vivo drug screening was performed to test sensitivity to 192 therapeutic agents, including conventional chemotherapeutic agents, and compounds targeting epigenetic regulators, protein kinases and cell cycle checkpoints.Results. In contrast to the uniform representation of guide RNAs observed in HSCs pre-transplant, tumors exhibited enrichment of specific sgRNAs with tumors of specific phenotype. We frequently observed bi-allelic editing of Tp53, Bcor, Tet2, Dnmt3a, Rb1 and Nfix in agreement with the presence of bi-allelic loss in patients. Concomitant editing and inactivation of Tp53/Bcor/Dnmt3a, or Tp53/Bcor/Rb1/Nfix promoted the development of acute erythroid leukemia (GATA1+/RUNX1+/GlyA+/-Ter119+/- and B220/CD19/PAX5/CD3/Mac1/Gr1-). Concomitant editing of Tp53/Bcor/Tet2 promoted the development of B-lineage ALL, and editing of Dnmt3a and Tet2 without Tp53 promoted T-cell ALL. Leukemic clones from primary tumors were serially transplantable across multiple different genetic backgrounds, with the same dominant clone present in all transplanted mice. Notably, mice that did not develop leukemia showed enrichment of different combinations of sgRNAs for Tet2, Dnmt3a and/or Asxl1, genes commonly mutated in clonal hematopoiesis of indeterminate potential, confirming that these mutations are alone not sufficient to induce leukemogenesis. Additional somatic mutations were acquired during clonal expansion of leukemic cells such as alterations of Notch1 and Ikzf1 in T-ALL; Setd2 at the serial passaging of T-ALL; Ptpn11, Kit (D816V), Kras (Q61L) and Lmo7 in the AEL models; and Sf3b3 in the B-ALL model (Fig 1A). Tumors with mutated Tp53 acquired aneuploidy whereas Tet2-mutated cells were genomically stable (Fig. 1B). Unsupervised hierarchical clustering of gene expression profiling identified 3 subgroups (Fig. 1C) associated with distinct genotypes and methylation profiles (Fig. 1D). Tet2-mutated tumors showed increase of hypermethylated sites and co-mutated Bcor/Dnmt3a leukemic cells showed loss of methylation. Eleven tumors representative of key AEL genotypes from the established models were selected to explore drug sensitivity. Response to individual drugs was associated with genotype with co-mutated Tet2/Dnmt3a T-ALL cells sensitive to bromodomain and histone methyltransferase inhibitors; co-mutated Bcor/Tp53/Dnmt3a or Bcor/Rb1 AEL cells to CDK9 inhibitor (LY2857785); Tp53 mutations alone were exclusively sensitive only to PARP inhibition (Talazoparib). In vivo mouse studies are ongoing to confirm ex vivo results.Conclusions: We successfully generated genetically defined models of AEL, demonstrated the role of Tp53 and Bcor mutations in driving the erythroid phenotype, and showed that sensitivity to different classes of compounds is genotype-dependent. These results provide the rational for testing new targeted agents in AEL. DisclosuresMullighan:Abbvie: Research Funding; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Effective Chimeric Antigen Receptor T Cell Therapy for Relapsed and Refractory Acute Lymphoblastic Leukemia in Children Can be Administered Safely Under the Real-Time Monitoring of Th1/Th2 Cytokine Patterns Measured By the Cytometric Bead Array Technology

Background and Objectives: Chimeric antigen receptor modified T (CAR-T) cell therapy has emerged as a promising strategy for the treatment of relapsed and refractory hematological malignancies recently. CD19 chimeric antigen receptor T (CD19CAR-T) cell therapy is a representative case showing promising results in treating relapsed and refractory B-lineage acute lymphoblastic leukemia (r/r B-ALL). However, challenges remain due to the concurrent severe side-effects such as cytokine release syndrome (CRS) or neurological toxicities which may cause death if they are not properly managed. So, how to safely use the CAR-T cell therapy in patients has drawn a lot of concern and attention worldwide. The objective of this study was to investigate the cytokine patterns to early identify and monitor the severe side-effects of cytokine release syndrome (CRS) and neurotoxicity caused by the therapy so that the therapy can be safely administered. Patients and Method: From December 2015 to March 2017, five patients with r/r B-ALL treated with CD19CAR-T cells were enrolled in this study. The Th1/Th2 cytokines including Interleukin (IL)-2, IL-4, IL6, IL-10, Tumor necrosis factor (TNF) and Interferon-gamma (IFN-γ ) in the serum and cerebral spinal fluid (CSF) were quickly measured by cytometric bead array (CBA) technology which allowed the result being available within 5 hours after the samples taken. The test were repeated more than once a day during the therapy. Results: 5 pediatric patients (2 boys and 3 girls) with r/r B-ALL aged 4 months -12 years old were treated by CD19CAR-T cell therapy. Four patients achieved complete remission (80% of CR rate) with minimal residual diseases (MRD) negative detected by flow cytometry within 2 weeks. One patient did not respond to the therapy due to the poor quality of his T cells. Five to 10 times of cytokine measurements were carried out for the five patients. Two types of cytokine patterns were identified: (1) very high levels of IL-6, IFN-γ with moderately elevated IL-10 indicated the CAR-T therapy effective pattern; (2) IL-6 slightly - severely elevated IL-6 level without concurrent elevation of IFN-γ represented a CAR-T cell therapy ineffective pattern but rather indicating a current infection. All the four responding patients presented the CAR-T therapy effective cytokine pattern, while the non-responding patient presented the CAR-T therapy ineffective cytokine pattern. We also analyzed the relationship between the cytokine levels and the severity of the side-effects and found that they were highly correlated. The cytokine levels in the CSF of two patients with seizure were also measured and we found that the elevation of the cytokine pattern in the CSF was paralleled to those in their serum but CNS-leukemia and CAR-T cells in their CSFs were not detected, indicating that the neurotoxicity concurrently presented in these two patients might be caused by the cytokines in the serum leaked into the CSF through the blood brain barrier. Extremely high level of cytokines occurred at 12 hours after CAR-T cell infusion in a patient was identified immediately and treated timely in onn-ICU setting to avoid the fatal consequence. Acute toxicities including fever, hypotension and other neurological toxicities occurred in responding patients within 2 weeks post infusion and managed properly with tocilizumab and/or steroids according to the “real-time” monitoring of this simple 6-Th1/Th2 cytokine patterns at the non-ICU setting. In conclusion, our study demonstrates that CD19CAR-T cell therapy can be administered safely for patients with relapsed and refractory leukemia under the “real-time” monitoring of a simple 6-cytokine pattern. DisclosuresNo relevant conflicts of interest to declare.

Effect of transplant status in CD19-targeted CAR T-cell therapy: a systematic review and meta-analysis.

Chimeric antigen receptor (CAR) T-cell therapy has shown promise for relapsed/refractory malignancies. Many patients have undergone prior hematopoietic stem cell transplant (HSCT), yet effects of transplant status on CAR T-cell therapy efficacy and safety have not been reported. The purpose of the study is to systematically evaluate the likelihood of achieving optimum response, severe cytokine release syndrome (sCRS), and neurotoxicity in the context of CAR T-cell therapy for HSCT-naïve patients versus those with prior HSCT. Trials were identified in ClinicalTrials.gov, Cochrane Library, and PubMed, and through reference pearl growing. Included studies used CD19-directed CAR T-cells for relapsed/refractory B-lineage Acute Lymphoblastic Leukemia and B cell Chronic Lymphocytic Leukemia, enrolled both HSCT-naïve and prior-HSCT patients, and denoted transplant status with outcomes. Six studies were included for optimum response, five for sCRS incidence, and four for neurotoxicity incidence. The pooled odds ratio for optimum response was 1.57 favoring HSCT-naïve patients (95% CI 0.54-4.61), whereas the pooled odds ratios for sCRS and neurotoxicity were 1.41 (95% CI 0.51-3.94) and 1.37 (95% CI 0.28-6.77), respectively, toward HSCT-naïve patients. Odds ratios were non-statistically significant. Overall risk of bias was moderate. While pooled estimates showed an advantage among HSCT-naïve patients for achieving optimum response and increased likelihood for sCRS and neurotoxicity, findings were not statistically significant. Any differences in efficacy and safety of CAR T-cell therapy cannot be verifiably attributed to transplant status, and additional controlled trials with increased sample sizes are needed to determine whether suggestive patterns favoring HSCT-naïve patients are validated.

Keeping PACE with Ph Positive to Ph-Like Detection in B-Lineage Acute Lymphoblastic Leukemia: A Practical and Cost Effective (PACE) Approach in a Resource Constrained Setting.

Philadelphia (Ph)-like or BCR-ABL like acute lymphoblastic leukemia (ALL) is defined on the basis of a gene expression profile that is similar to Ph-positive ALL. It comprises a wide spectrum of genetic lesions affecting primarily the cytokine receptor and/or kinase signalling genes. It accounts for approximately 10-15% of pediatric ALL, and is more common in patients who are high-risk according to the National Cancer Institute criteria. Presence of Ph-like mutations is an independent predictor of poor outcome. However, there is vast potential to utilize targeted therapy to improve survival in this group. The sizeable range of genetic lesions varying from translocations, fusions, point mutations and deletions make the diagnosis challenging. Hence, a practical and cost effective approach is required to enable identification in resource constrained settings. Patients with recurrent cytogenetic abnormalities such as ETV6-RUNX1, high hyperdiploidy, TCF3-PBX1, BCR-ABL1 and KMT2A (MLL) rearrangement need not be tested, as these are mutually exclusive with BCR-ABL like mutations. Detection of CRLF2 overexpression, which is the commonest abnormality, is employed as the first step. In patients lacking overexpression, testing for tyrosine kinase fusions can be performed. However, the goal should be to employ a combination of molecular diagnostic techniques such as reverse transcriptase polymerase chain reaction (PCR), real time quantitative PCR, fluorescence in situ hybridization and Sanger sequencing to detect genetic lesions that are amenable to targeted therapy.

Abstract 5469: RAS and STAT5 pathway lesions are mutually exclusive in B-cell malignancies through mechanisms of biochemical cross-inhibition

Abstract Activation of STAT5- and RAS-signaling are segregated to early and later stages of normal B cell development, respectively. Studying B-lineage acute lymphoblastic leukemia (ALL; n=578), we found that STAT5 (e.g. BCR-ABL1, JAK2, cytokine receptors) and RAS (NRAS, KRAS, PTPN11, NF1) lesions were mutually exclusive, with only 9 cases (1.6%) carrying lesions in both pathways. Reverse phase protein array measurements revealed that phosphorylation of MEK and ERK1/2 were inversely correlated with STAT5-phosphorylation (MDACC, 1983-2007; P&amp;lt;0.001). These findings prompted us to study mechanisms of cross-inhibition between RAS and STAT5 pathways. Inducible NRASG12D activated ERK at the expense of STAT5 phosphorylation. This was due to stabilization and increased activation of the STAT5-phosphatase PTPN6 (SHP1). Inducible ablation of Ptpn6 elevated phospho-STAT5 levels, while genetic inactivation of Stat5 strongly increased ERK activity. Constitutively active STAT5 suppressed phosphorylation of ERK. Interestingly, STAT5 negatively regulated BCL6, which marks the transition from cytokine receptor-dependent pro-B cells (Stat5+) to pre-BCR dependent stages of development (ERK+). While oncogenic RAS suppressed STAT5, we also found that induction of RAS induced BCL6 expression at both the mRNA and protein levels. Increases in BCL6 expression in response to NRASG12D were abrogated upon treatment with a MEK kinase inhibitor or activation of STAT5. Studying a matched patient-derived pre-B ALL sample at diagnosis and at relapse (acquired KRASG12V mutation) revealed activation of ERK in association with increased BCL6 and decreased STAT5 levels in the KRASG12V relapsed ALL sample. With engagement of BCL6 and the STAT5-inhibitory phosphatase PTPN6 downstream of RAS and ERK signaling, these findings suggest that occupancy of either RAS or STAT5-pathway represents a commitment step that renders cells non-permissive to the respective alternative pathway. To test this hypothesis, we induced B cell transformation with either RAS or STAT5-pathway oncogenes and then subsequently transduced with either empty vectors (EV) or vectors carrying the alternative oncogene. While EVs were easily transduced, RAS- and STAT5-transformed B cells did not tolerate the alternative oncogene. Reflecting early (STAT5) and later (RAS) stages of B cell development, oncogenic activation of these pathways occurs in a mutually exclusive way, owing to biochemical cross-inhibition between STAT5 and RAS. Citation Format: Lai N. Chan, Seyedmehdi Shojaee, Christian Hurtz, Rebecca Caeser, Gang Xiao, Huimin Geng, Steven Kornblau, Markus Muschen. RAS and STAT5 pathway lesions are mutually exclusive in B-cell malignancies through mechanisms of biochemical cross-inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5469.

Feasibility of Outpatient High-Dose Methotrexate Infusions in Pediatric Patients With B-Lineage Acute Lymphoblastic Leukemia

High-dose methotrexate (MTX) given in four hospitalizations during interim maintenance for high-risk pediatric B-lineage acute lymphocytic leukemia significantly improves survival but increases resource utilization. Children remain hospitalized for intravenous hydration and blood or urine monitoring until MTX clearance parameters are reached. Improved supportive care, extended infusion center hours, and pediatric home health expertise afford alternatives to prolonged hospital admissions, potentially offering quality, cost-effective approaches that positively impact the delivery of care.

Rapid identification of BCR/ABL1-like acute lymphoblastic leukaemia patients using a predictive statistical model based on quantitative real time-polymerase chain reaction: clinical, prognostic and therapeutic implications.

BCR/ABL1-like acute lymphoblastic leukaemia (ALL) is a subgroup of B-lineage acute lymphoblastic leukaemia that occurs within cases without recurrent molecular rearrangements. Gene expression profiling (GEP) can identify these cases but it is expensive and not widely available. Using GEP, we identified 10 genes specifically overexpressed by BCR/ABL1-like ALL cases and used their expression values - assessed by quantitative real time-polymerase chain reaction (Q-RT-PCR) in 26 BCR/ABL1-like and 26 non-BCR/ABL1-like cases to build a statistical "BCR/ABL1-like predictor", for the identification of BCR/ABL1-like cases. By screening 142 B-lineage ALL patients with the "BCR/ABL1-like predictor", we identified 28/142 BCR/ABL1-like patients (19·7%). Overall, BCR/ABL1-like cases were enriched in JAK/STAT mutations (P<0·001), IKZF1 deletions (P<0·001) and rearrangements involving cytokine receptors and tyrosine kinases (P=0·001), thus corroborating the validity of the prediction. Clinically, the BCR/ABL1-like cases identified by the BCR/ABL1-like predictor achieved a lower rate of complete remission (P=0·014) and a worse event-free survival (P=0·0009) compared to non-BCR/ABL1-like ALL. Consistently, primary cells from BCR/ABL1-like cases responded invitro to ponatinib. We propose a simple tool based on Q-RT-PCR and a statistical model that is capable of easily, quickly and reliably identifying BCR/ABL1-like ALL cases at diagnosis.

Targeting the phosphatidylinositol 3-kinase/Akt/mechanistic target of rapamycin signaling pathway in B-lineage acute lymphoblastic leukemia: An update.

Despite considerable progress in treatment protocols, B-lineage acute lymphoblastic leukemia (B-ALL) displays a poor prognosis in about 15-20% of pediatric cases and about 60% of adult patients. In addition, life-long irreversible late effects from chemo- and radiation therapy, including secondary malignancies, are a growing problem for leukemia survivors. Targeted therapy holds promising perspectives for cancer treatment as it may be more effective and have fewer side effects than conventional therapies. The phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway is a key regulatory cascade which controls proliferation, survival and drug-resistance of cancer cells, and it is frequently upregulated in the different subtypes of B-ALL, where it plays important roles in the pathophysiology, maintenance and progression of the disease. Moreover, activation of this signaling cascade portends a poorer prognosis in both pediatric and adult B-ALL patients. Promising preclinical data on PI3K/Akt/mTOR inhibitors have documented their anticancer activity in B-ALL and some of these novel drugs have entered clinical trials as they could lead to a longer event-free survival and reduce therapy-associated toxicity for patients with B-ALL. This review highlights the current status of PI3K/Akt/mTOR inhibitors in B-ALL, with an emphasis on emerging evidence of the superior efficacy of synergistic combinations involving the use of traditional chemotherapeutics or other novel, targeted agents.

Prognostic impact of kinase-activating fusions and IKZF1 deletions in pediatric high-risk B-lineage acute lymphoblastic leukemia

AbstractRecurrent chromosomal rearrangements carry prognostic significance in pediatric B-lineage acute lymphoblastic leukemia (B-ALL). Recent genome-wide analyses identified a high-risk B-ALL subtype characterized by a diverse spectrum of genetic alterations activating kinases and cytokine receptor genes. This subtype is associated with a poor prognosis when treated with conventional chemotherapy but has demonstrated sensitivity to the relevant tyrosine kinase inhibitors. We sought to determine the frequency of kinase-activating fusions among National Cancer Institute (NCI) high-risk, Ph-negative, B-ALL patients enrolled on Dana-Farber Cancer Institute ALL Consortium Protocol 05-001 and to describe their associated clinical characteristics and outcomes. Among the 105 patients screened, 16 (15%) harbored an ABL-class fusion (ETV6-ABL1: n = 1; FOXP1-ABL1: n = 1; SFPQ-ABL1: n = 1; ZC3HAV1-ABL2: n = 1) or a fusion activating the JAK-STAT pathway (P2RY8-CRLF2: n = 8; PAX5-JAK2: n = 4). Sixty-nine percent of patients with an identified fusion had a concomitant IKZF1 deletion (n = 11). In univariate analysis, fusion-positivity and IKZF1 deletion were each associated with inferior event-free survival; IKZF1 deletion retained statistical significance in multivariable analysis (hazard ratio, 2.64; P = .019). Our findings support therapy intensification for IKZF1-altered patients, irrespective of the presence of a kinase-activating fusion.

No prognostic significance of immunophenotypic changes at the end of remission induction therapy in children with B-lineage acute lymphoblastic leukemia

Detection of aberrant antigen expression in acute lymphoblastic leukemia (ALL) by flow cytometric is proposed for the quantification of minimal residual disease (MRD). There are few studies that investigate the stability of the antigen expression in children with B lineage ALL at the end of remission induction therapy and determine its prognostic impact. Between 2010 and 2015, 691 bone marrow specimens of childhood ALL were sent at diagnosis for immunophenotypic characterization, and follow-up samples for MRD were analyzed on day 33. Of these, 155 patients with MRD more than or equal to 0.01% were eligible for the study. Immunophenotypic studies were performed by multiparametric flow cytometry using four-colour monoclonal antibody combinations. Overall, 86 of 155 (55.5%) cases showed phenotype shifts at least one marker. CD19 was the most stable markers. By contrast, CD20 was significantly different between diagnosis and day 33 in nearly one third of the cases. Shifts of antigen expression was not significantly associated with EFS, RFS or OS (P > 0.05). Multivariate analysis showed that WBC and BCR-ABL have independent prognostic value in childhood ALL. Changes in antigen expressions were commonly occurred at the end of induction and not associated with prognostic value in patients whose MRD were positive on day 33.

C-Myc regulates the CDK1/cyclin B1 dependent‑G2/M cell cycle progression by histone H4 acetylation in Raji cells.

Overexpression of c-Myc is involved in the tumorigenesis of B-lineage acute lymphoblastic leukemia (B-ALL), but the mechanism is not well understood. In the present study, a c-Myc-knockdown model (Raji-KD) was established using Raji cells, and it was indicated that c-Myc regulates the expression of genes associated with cell cycle progression in G2/M-phase, cyclin D kinase (CDK)1 and cyclin B1, by modulating 60 kDa Tat-interactive protein (TIP60)/males absent on the first (MOF)-mediated histone H4 acetylation (AcH4), which was then completely restored by re-introduction of the c-Myc gene into the Raji-KD cells. The expression of CDK1 and cyclin B1 was markedly suppressed in Raji-KD cells, resulting in G2/M arrest. In comparison to Raji cells, the proliferation of Raji-KD cells was significantly reduced, and it was recovered via re-introduction of the c-Myc gene. In the tumorigenesis assays, the loss of c-Myc expression significantly suppressed Raji cell-derived lymphoblastic tumor formation. Although c-Myc also promotes Raji cell apoptosis via the caspase-3-associated pathway, CDK1/cyclin B1-dependent-G2/M cell cycle progression remains the major driving force of c-Myc-controlled tumorigenesis. The present results suggested that c-Myc regulates cyclin B1- and CDK1-dependent G2/M cell cycle progression by TIP60/MOF-mediated AcH4 in Raji cells.

Cyclin dependent kinase inhibitor 2A/B gene deletions are markers of poor prognosis in Indian children with acute lymphoblastic leukemia.

Cyclin dependent kinase inhibitor 2A/B (CDKN2A/B) genes are implicated in many malignancies including acute lymphoblastic leukemia (ALL). These tumor suppressor genes, with a key regulatory role in cell cycle are located on chromosome 9p21.3. Previous studies involving CDKN2A/B gene deletions have shown mixed associations with survival outcome in childhood ALL. Hundred and four newly diagnosed children with ALL (1-14 years) were enrolled in this study. Genomic DNA from pretreatment bone marrow/peripheral blood samples of these children was investigated for copy number alterations in CDKN2A/B genes using multiplex ligation dependent probe amplification assay. Immunophenotype subtyping and cytogenetic and molecular analysis of ALL was performed at start of induction chemotherapy in all children. Children were monitored for response to prednisolone (Day 8), complete morphological remission, and minimal residual disease at the end of induction. The minimum postinduction follow-up period was 6 months. CDKN2A/B deletions were seen in 19.8% (18/91) of B lineage acute lymphoblastic leukemia (B-ALL) and 38.5% (5/13) of T lineage acute lymphoblastic leukemia (T-ALL). Monoallelic CDKN2A/B deletions were found in 61.1% of total deletions in B-ALL while all the children with T-ALL harbored biallelic deletions. The prevalence of CDKN2A/B gene deletions was found to be significantly higher in older children (P=0.002), in those with higher leukocyte count (P=0.037), and in National Cancer Institute high risk group patients (P=0.001) in the B-ALL subgroup. Hazard ratio was significantly high for children with CDKN2A/B deletions in total cohort (P=0.004). Children with CDKN2A/B deletion had significantly lesser event free survival (P=0.03). CDKN2A/B deletions were significantly more prevalent in T-ALL subgroup and were found to have higher hazard ratio and lesser event free survival in total cohort in our study.

HETEROGENEITY OF CHILDHOOD B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (EGIL SUBTYPE BIV)

Introduction. Surface immunoglobulin expression is a main immunophenotypic criteria of mature subtype of B-lineage acute lymphoblastic leukemia. Although the majority of such cases represents Burkitt leukemia/lymphoma, it was shown for several times that membrane IgM could be detected in the absence of other mature lymphomas signs. The aim of the study was to evaluate heterogeneity of childhood acute lymphoblastic leukemia (ALL) with surface IgM expression and to assess correspondence of BIV EGIL ALL subtype with Burkitt lymphoma (BL) bone marrow dissemination. Materials and methods . Immunophenotypic, cytomorfologic and genetic data of 54 BIV-ALL cases were analyzed. Results . Among the studied patients 39 had BL, while others belonged to B-cell precursor ALL (BCP-ALL). All BL patients and none of BCP-ALL patients carried C-MYC rearrangement while in BCP-ALL group in 8 cases and in any BL cases KMT2A rearrangements were found. None of BCP-ALL children had L3 morphology according to FAB classification. Conclusions. B-lineage ALL with surface IgM expression is rather heterogeneous group of cases including typical BL and rare cases of BCP-ALL even with KMT2A-rearrangements. Combination of all available diagnostic technologies will allow precise split of these two different disease and select the appropriate treatment scheme.

Flow cytometric false myeloperoxidase-positive childhood B-lineage acute lymphoblastic leukemia.

Flow cytometric intracellular myeloperoxidase (MPO) staining of leukemic blasts is a useful tool in diagnosis of leukemia subtype. Interpretation of high MPO-positivity can be a diagnostic challenge in B-lineage acute lymphoblastic leukemia (B-ALL). While very few such cases have been reported, high MPO positive B-ALL cases without additional myeloid antigen positivity are suspect and require further investigation. Three pediatric cases of B-ALL with strong MPO staining (clone 8E6; Invitrogen) at diagnosis and three others with negative MPO staining were studied by flow cytometry and immunohistochemistry. In-vitro drug cytotoxicity, oxidative stress generation, and immunophenotyping using other MPO clones were performed to further investigate MPO presence. Expectedly, normal myeloid cells in all six samples were positive and mature lymphocytes negative for MPO staining. However, MPO monoclonal antibody (mAb) obtained from clones other than Invitrogen and other myeloid-specific mAbs gave negative results suggesting false positivity of the initial MPO staining. Immunohistochemistry for MPO was also negative on all six cases tested. Furthermore, in-vitro vincristine cytotoxicity was greater in leukemic cells from MPO false-positive cases compared with MPO-negative B-ALL samples, demonstrating indirect lack of MPO activity. Moreover, drug treatment did not lead to generation of reactive oxidative species, also reflective of lack of significant MPO presence. The cause of false-positive MPO staining remains unknown in these three cases; a cross reactivity could be the culprit. Caution should be given to similar phenomena and detailed investigation may contribute to the understanding of altered protein expression in such outlier cases. © 2017 International Clinical Cytometry Society.

Rituximab in the management of acute lymphoblastic leukemia

ABSTRACTIntroduction: The anti-CD20 chimeric monoclonal antibody rituximab has revolutionized the treatment of B-cell malignancies, significantly improving patient clinical outcome. Recently, some single-group studies have suggested that adding rituximab to chemotherapy can improve the outcome of CD20-positive B-cell acute lymphoblastic leukemia (ALL) patients.Areas covered: An overview of the current insights of rituximab in adult ALL patients is presented here. In particular, we focused on results of multicenter randomized phase III trial (GRAALL-2005 – Group for Research on Adult Acute Lymphoblastic Leukemia) that evaluated the benefit of associating rituximab to chemotherapy in Ph-negative, B-lineage ALL expressing the CD20 antigen.Expert opinion: Data from clinical trials confirm that rituximab enhances the efficacy of chemotherapy without additive toxicity in ALL. However, results of GRAAL 2005 study represent only a modest incremental improvement in the treatment of ALL. Other promising compounds as single agent or in combination with chemotherapy are currently in different stages of clinical development. The GRAALL 2005 study sets the stage for other prospective studies which will further elucidate the role of monoclonal antibodies in the management of ALL.