Adult B-cell Acute Lymphoblastic Leukemia Research Articles

A phase I trial of palbociclib in combination with dexamethasone in relapsed or refractory adult B-cell acute lymphoblastic leukemia (ALL).

TPS7065 Background: c-Myb is a DNA-binding transcription factor that is highly expressed in immature hematopoietic cells. c-Myb and its products are essential in regulating normal hematopoiesis and influencing leukemogenesis. Knockdown of c-Myb causes cell cycle arrest and apoptosis in pre-B-ALL cells. The effects of c-Myb depend on transcriptional regulation of CDK6 and Bcl-2. c-Myb-silenced Ph+ ALL cells exhibit Rb-dependent cell cycle arrest and apoptosis, both of which are rescued by ectopic expression of cyclin D3, CDK6, and Bcl-2 expression. Preclinical studies suggest that the cytotoxic activity of dexamethasone in ALL cells may be due to decreased c-Myb expression and reduced Bcl-2 levels. Thus, the novel combination of palbociclib, a small molecule CDK4/6 inhibitor, and dexamethasone is a logical approach for the treatment of B-cell ALL. Methods: This is a single arm, phase I, dose escalation study with a traditional 3+3 design. Adult patients with relapsed or refractory B-cell ALL are eligible. Patients with Ph+ ALL must be refractory to or intolerant of standard tyrosine kinase inhibitor therapy. Patients receive a 1-week lead-in of palbociclib alone followed by induction with 4 weeks of palbociclib and dexamethasone. If an adequate response is seen, patients move to maintenance therapy, which consists of 1 week of palbociclib plus dexamethasone followed by 3 weeks of palbociclib alone. Treatment continues until disease progression, dose limiting toxicity, or availability of an alternative therapy. The primary endpoints are dose limiting toxicity and maximum tolerated dose of palbociclib and dexamethasone. Correlative studies, which are performed on pretreatment, day +1 and day +8 samples, include RB phosphorylation and FOXM1 expression as measures of palbociclib activity; CD19+ cell gene expression profiling of (1) p21 expression as an indicator of cell cycle activity, (2) S-Phase, Annexin V/Caspase 3 activation as indicators of proliferation and apoptosis and (3) Myb and Bcl-2 expression as indicators of dexamethasone sensitivity. Cohort 1 is currently enrolling. Once a maximum tolerated dose is established, an expansion cohort is planned. Clinical trial information: NCT03472573.

Impact of CAR T-cell product viability on B-cell lymphoid malignancy outcomes

Background & Aim CTL019 (tisagenlecleucel) is an anti-CD19 genetically modified autologous T-cell therapy approved for treatment of relapsed/refractory pediatric and young adult B-cell acute lymphoblastic leukemia (ALL) and adult diffuse large B-cell lymphoma (DLBCL). For CTL019 manufactured at Penn, the product release criteria include a viability assessment. In clinical trials, viability specification for product release was ≥ 70% viable cells; whereas, for commercial tisagenlecleucel, viability specification for product release was ≥ 80%. We examined the relationship between CTL019 viability release testing and clinical outcomes. Methods, Results & Conclusion We analyzed CTL019 viability release testing in patients (pts) treated on prospective single institution clinical trials for non-Hodgkin lymphomas (NHL) (NCT02030834) and pediatric ALL clinical trials (NCT01626495, NCT02906371). The relationship of CTL019 product viability release testing and overall response, progression-free survival, overall survival, sensitivity/specificity of viability testing to predict complete response, and CAR T-cell expansion were examined. For 39 NHL pts, the median product viability was 89.8% (range: 73.7%-97.7%) and 3 pts had products with These results suggest that there is no dose-response relationship between CTL019 product viability (70-98%) and clinical outcomes.

A Step Forward in the Treatment of Adolescent and Young Adult B-cell Acute Lymphoblastic Leukemia

A Step Forward in the Treatment of Adolescent and Young Adult B-cell Acute Lymphoblastic Leukemia

High RASD1 transcript levels at diagnosis predicted poor survival in adult B-cell acute lymphoblastic leukemia patients

B-cell acute lymphoblastic leukemia (B-ALL) in adults remains a highly challenging disease. Identifying new prognostic biomarkers is necessary to help select the best therapeutic schedules and to improve prognosis. We performed bioinformatics analyses of transcriptomic data to identify aberrantly-expressed mRNA transcripts in B-ALL and focused on RASD1 (Ras-related dexamethasone-induced 1). To date, no information is available on the prognostic value of RASD1 in B-ALL. Fifty-three consecutive adults with de novo B-ALL were enrolled in this study. Our data suggested that RASD1 was abnormally overexpressed in B-ALL. High RASD1 transcript levels at diagnosis were associated with lower survival probabilities (44% [20%–61%] vs. 79% [60%–97%]; P = 0.037) and were also an independent prognostic factor in adult B-ALL (HR = 4.9 [1.5–15.9]; P = 0.008). Functional in vitro analyses and bioinformatic analyses indicated that RASD1 promoted cell proliferation, cell cycle progression and chemotherapy resistance and inhibited cell apoptosis. These data demonstrated that RASD1 might serve as a novel prognostic biomarker for adult B-ALL and as a potential therapeutic target in adult B-ALL patients.

A systematic literature review and meta-analysis of minimal residual disease as a prognostic indicator in adult B-cell acute lymphoblastic leukemia.

Minimal (or ‘measurable’) residual disease in acute lymphoblastic leukemia appears to be a prognostic indicator, with potential value in informing individualized treatment decisions. Complete understanding of the strength of the association between minimal residual disease and long-term outcomes is, however, lacking. A systematic literature review and meta-analysis were performed to elucidate the clinical significance of minimal residual disease with respect to relapse-free survival and overall survival in precursor B-cell acute lymphoblastic leukemia. A total of 23 articles and abstracts, most published between 2012 and 2016, were identified for inclusion in the primary meta-analysis. Typically, patients were in their first complete remission at the time of minimal residual disease assessment; in two studies, all patients were in their second, or later, complete remission. The primary analysis revealed improved relapse-free survival across all studies for patients who achieved minimal residual disease negativity (random effects hazard ratio, 2.34; 95% confidence interval, 1.91–2.86). Improved overall survival for patients who achieved minimal residual disease negativity was also observed (hazard ratio, 2.19; 95% confidence interval, 1.63–2.94). There was no observed difference in the impact of minimal residual disease status in subgroups based on disease stage, minimal residual disease sensitivity threshold level, Philadelphia chromosome status, histological phenotype, risk group, minimal residual disease testing location, minimal residual disease timing after induction, or minimal residual disease detection method. Despite heterogeneity in study design and patient populations between the contributing studies, these data provide a compelling argument for minimal residual disease as a clinical tool for assessing prognosis and guiding treatment decisions in precursor B-cell acute lymphoblastic leukemia.

A rare morphological and immunophenotypic presentation of adult B-cell acute lymphoblastic leukemia with blasts in the monocytic zone on CD45 side scatter

<p class="abstract">We present a case of B-acute lymphoblastic leukemia in an elderly patient who presented with severe weakness and pancytopenia. The patient was a 75 year old Female whose blasts had an unusual morphology in form of coarse azurophilic granules and cytoplasmic blebs and on flow cytometry the blasts were present in the bright CD45 zone with a high side scatter. Bone marrow aspirate sample was subjected to multicolour flow cytometry using Beckman Coulter Navios® which is an 8 colour flow cytometer. Flow cytometric analysis of the bone marrow aspirate showed blasts in the monocytic zone with a precursor B cell immunophenotype. Complete blood counts showed pancytopenia with peripheral blood film not showing any blasts. Bone marrow aspirate smears showed 20% blasts with coarse azurophilic granules and cytoplasmic blebs. The position of the blasts in this case which were in monocytic zone giving them a bright expression of CD45 and a high side scatter on the CD45 side scatter. This is not the usual position for blasts in B- acute lymphoblastic leukemia as these blasts are less complex. A bright expression of CD45 by blasts in B- acute lymphoblastic leukemia is known to be associated with a poor prognosis but the clinical significance of blasts being bright CD45 with a high side scatter is a very rare occurrence and more number of cases with a similar presentation are required to determine a prognostic significance.</p>

RAG1 high expression associated with IKZF1 dysfunction in adult B-cell acute lymphoblastic leukemia.

The recombination mediated by recombination activating gene (RAG) is not only the dominant mutational process but also the predominant driver of oncogenic genomic rearrangement in acute lymphoblastic leukemia (ALL). It is further responsible for leukemic clonal evolution. In this study, significant RAG1 increase is observed in the subsets of B-ALL patients, and high expression of RAG1 is observed to be correlated with high proliferation markers. IKZF1-encoded protein, IKAROS, directly binds to the RAG1 promoter and regulates RAG1 expression in leukemic cells. CK2 inhibitor by increasing IKAROS activity significantly suppresses RAG1 expression in ALL in an IKAROS-dependent manner. Patients with IKZF1 deletion have significantly higher expression of RAG1 compared to that without IKZF1 deletion. CK2 inhibitor treatment also results in an increase in IKZF1 binding to the RAG1 promoter and suppression of RAG1 expression in primary ALL cells. Taken together, these results demonstrate that RAG1 high expression is associated with high proliferation markers in B-ALL. Our data for the first time proved that RAG1 expression is directly suppressed by IKAROS. Our results also reveal drive oncogenesis of B-ALL is driven by high expression of RAG1 with IKAROS dysfunction together, which have significance in an integrated prognostic model for adult ALL.

Multivariable Modeling of Disease and Treatment Characteristics of Adults with B-ALL in MRD-Negative CR after CD19 CAR-T Cells Identifies Factors Impacting Disease-Free Survival

Multivariable Modeling of Disease and Treatment Characteristics of Adults with B-ALL in MRD-Negative CR after CD19 CAR-T Cells Identifies Factors Impacting Disease-Free Survival

Molecular Detection of Minimal Residual Disease Precedes Morphological Relapse and Could be Used to Identify Relapse in Pediatric and Young Adult B-Cell Acute Lymphoblastic Leukemia Patients Treated with Tisagenlecleucel

Molecular Detection of Minimal Residual Disease Precedes Morphological Relapse and Could be Used to Identify Relapse in Pediatric and Young Adult B-Cell Acute Lymphoblastic Leukemia Patients Treated with Tisagenlecleucel

CD19-Directed CAR T-Cell (CTL019) Product Viability and Clinical Outcomes in Non-Hodgkin Lymphomas and B-Cell Acute Lymphoblastic Leukemia

Introduction: CTL019 is an anti-CD19 genetically modified autologous T-cell immunotherapy developed at the University of Pennsylvania (Penn) that was recently approved for treatment of relapsed/refractory pediatric and young adult B-cell acute lymphoblastic leukemia (ALL) and adult relapsed/refractory diffuse large B-cell lymphoma (DLBCL) as tisagenlecleucel (Novartis). For ALL, the FDA-approved dose is 0.2 to 5.0 x 106 CAR-positive viable T cells per kg of body weight for patients ≤ 50 kg or 0.1 to 2.5 x 108 CAR-positive viable T cells for pts > 50 kg; for DLBCL, the FDA-approved dose is 0.6 to 6.0 x 108 CAR-positive viable T cells. For CTL019 manufactured at Penn, the dose is determined by flow cytometric staining of CAR-positive T cells, which are cryopreserved in product bags along with replicate aliquots of the final formulation in vials, simultaneously cryopreserved for release testing. The CTL019 product release criteria include a post thaw viability assessment using a vial of replicate aliquot of the final formulation for Trypan blue exclusion or dual fluorescence automated cell counting (Luna-FL, Logos Biosystems). There are no published data examining the relationship between CTL019 viability release testing and clinical outcomes.Methods: We analyzed CTL019 post thaw viability release testing in patients treated on one prospective single institution clinical trial of CD19-expressing non-Hodgkin lymphomas (NHL) (NCT02030834) and two single-institution prospective pediatric ALL clinical trials (NCT01626495 and NCT02906371). Patients were assessed for response to therapy and CAR T-cell expansion. Receiver operating characteristic (ROC) curves were constructed for prediction of complete responses based on sensitivity and specificity of CAR T-cell product post thaw viability release test results.Results: 39 pts with relapsed/refractory NHL (24 diffuse large B-cell lymphoma and 15 follicular lymphoma) were enrolled and received the protocol-specified dose of CTL019. Best response rate was 56% (22/39) complete responses (CR). 123 pts with relapsed/refractory pediatric ALL were enrolled and received the protocol-specified dose of CTL019. Best response rate was 96% (118/123) CR/complete remission with incomplete blood count recovery (CRi). For patients with NHL infused with CTL019, product % viability had a median of 89.8% viability (range: 73.7%-97.7%); product % viability quintiles were as follows: 20%-tile=81.7%, 40%-tile=88.3%, 60%-tile=91.1%, 80%-tile=94.8%). ROC area for NHL patients was 0.47 (95%CI: 0.28-0.65). For patients with ALL infused with CTL019, product % viability had a median of 89.3% viability (range: 56.0%-98.4%); product % viability quintiles were as follows: 20%-tile=82.3%, 40%-tile=87.5%, 60%-tile=90.9%, 80%-tile=94.4%). ROC area for ALL patients was 0.52 (95%CI: 0.32-0.71). For patients with NHL, progression-free survival (PFS) was not significantly influenced by product viability release test results by Cox proportional hazards (HR: 1.0, 95%CI: 0.94-1.09, p=0.7). For patients with NHL, peak CAR T-cell expansion was not significantly correlated with product viability release test results (r2=0.12, p=0.5). Data collection for Cox analysis to investigate the effect of release test viability on PFS and correlation of release test viability with peak CTL019 expansion in ALL is ongoing and will be presented.Conclusions: Our data suggest that, within the ranges obtained in these trials, there is no clear dose-response relationship between CTL019 product viability release test results and clinical response rates in pediatric and young adult ALL or DLBCL. [Display omitted] DisclosuresChong:Novartis: Consultancy. Levine:Cure Genetics: Consultancy; Brammer Bio: Consultancy; CRC Oncology: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Siegel:Novartis: Research Funding. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. June:Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Schuster:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees.

Genomic and Clinical Characterization of Adult Ph-Negative B-Cell Acute Lymphoblastic Leukemia

Although survival rate for children with Acute Lymphoblastic Leukemia (ALL) now exceeds about 90%, the outcome of adult patients with ALL is extremely poor. These differences might be attributed to the lack of insights into pathogenesis and clinical behavior of adult-ALL.Gross chromosomal alterations including chromosome translocations and aneuploidy are considered as early events in ALL and constitute disease subtypes. To identify chromosome translocations underlying adult with Ph-negative B-ALL, we performed RNA-seq analysis on RNA from individuals with B-ALL who had been treated on the Japan Adult Leukemia Study Group (JALSG) ALL202-O protocol (n = 149).We successfully identified chromosome translocations in 100 patients (67.1%). ZNF384 fusions were most frequently detected in 30 patients (20.1%) and they had wide range of fusion partners. DUX4- and MEF2D- fusions were also recurrently found in 7 (4.7%) and 9 (6.0%) patients, respectively. Chromosome translocations activating kinase and cytokine receptor were found in 25 patients (16.8%) with Ph-like gene expression profile. These alterations were almost completely mutually exclusive indicating these are likely to be primary genetic events.For simplicity, here we define (1) fusions involving ZNF384, DUX4, MEF2D, CEBP and PAX5 as well as TCF3-PBX1 and ETV6-RUNX1 as Transcription Factor fusions (TF fusions; 49% of patients), (2) fusions involving CRLF2, JAK2, PDGFRB, EPOR and ABL as Kinase/cytokine-receptor Activating fusions (KA fusions; 15%) and (3) non-recurrent fusions or the absence of fusions/aneuploidy as B-others (30%).First, we analyzed impact of the patient age on types of fusion genes, based of combined data of ALL202-O cohort, childhood B-ALL cohort (Lilljebjörn H, et al. 2016: n = 189) and ALL202-U cohort (Yasuda T, et al. 2016: n = 54). We found that incidence of ZNF384-, CEBP- fusions and B-others increases as patients age, whereas ETV6-RUNX1 and PAX5 fusions were more prevalent in younger patients, exhibiting negative association with age. DUX4 fusions and TF fusions were most prevalent in Adolescent and Young Adult (AYA) generation. JAK2-, PDGFRB-, EPOR- and KA- fusions were positively correlated with age.Next, we analyzed association between patient survival and types of fusions. In Japanese adult B-ALL cohort (ALL202-O and ALL202-U cohort), we observed ZNF384-, DUX4- fusions and TCF3-PBX1 were associated with better disease-free survival than B-others. Furthermore, when combined, MEF2D- (n = 14), CEBP- (n = 4), PAX5- fusions (n = 2) and ETV6-RUNX1 (n = 2) exhibited significantly better disease-free survival than B-others, indicating TF fusions were associated with an improved outcome. In contrast, KA fusions were associated with poorer disease-free survival than B-others. KMT2A fusions were comparable with B-others regarding to patient disease-free survival.These results allowed us to develop a prognostic schema to identify three distinct risk profile groups, based on types of fusion genes and cytogenetics (Table1); favorable-risk (5-year rate of disease-free survival 67.4%), intermediate-risk (5-year rate of disease-free survival 42.5%) and adverse-risk (5-year rate of disease-free survival 9.6%). This prognostic schema predicted the outcome independently of age, sex and methotrexate dose in multivariate analysis (p < 0.001).In conclusion, we promoted a better understanding of the genetic basis of adult B-ALL by focusing on fusion genes. Each chromosome translocations were closely associated with age. ZNF384-, KA fusions and B-others were characteristic for older-adult patients (40-65 years old) with B-ALL. We clearly demonstrated specific primary chromosome abnormalities are strong prognostic marker. Functional properties of primary genetic events (TF fusions vs. KA fusions) might be a key determinant of biological characteristics and clinical outcome. [Display omitted] DisclosuresKiyoi:Novartis Pharma K.K.: Research Funding; Celgene Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Phizer Japan Inc.: Research Funding; Eisai Co., Ltd.: Research Funding. Naoe:Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding.

Comparison of Immunophenotypic Abnormalities in B-Lymphoblastic Leukemia (B-ALL) Minimal Residual Disease (MRD) of Adults and Children Utilizing the Children's Oncology Group (COG)-Validated Multiparameter Flow Cytometry (MFC)- MRD Approach: An Institutional Experience

IntroductionDetection of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) has become an important clinical tool in the treatment of patients with acute leukemia. At our institution, we routinely employ Children's Oncology Group (COG) validated B-lymphoblastic leukemia (B-ALL) MFC-MRD analysis for the detection of MRD in pediatric patients after induction therapy for B-ALL. We have extended this COG-validated assay to the adult B-ALL population. This technique relies on the identification of aberrant immunophenotypes expressed by residual bone marrow B-lymphoblasts compared to the normal pattern of maturing hematogones using a 6-color, 3-tube MFC. This assay was initially designed and validated for the pediatric B-ALL population, but the majority of patients with B-ALL at our institution represent adults. Therefore, we characterized the frequency of abnormal cell surface antigen expression of B-ALL MRD with the COG-validated method in the adult B-ALL population and compared our findings with those of our pediatric population.MethodsThis study was approved by the Institutional Review Board (IRB) of Washington University School of Medicine in St Louis, MO. A total number of 289 bone marrow specimens were submitted for B-ALL MRD analysis over a time period of 14 months. Antibodies employed in the COG-validated method were CD19, CD20, CD10, CD38, CD9, CD45, CD58, CD13/CD33, CD34, CD71 and CD3. At least 1 million cell events per tube (or as many as possible) were acquired using Beckman Coulter Navios flow cytometers, and Kaluza software was used for analysis. Leukemia-associated immunophenotypic aberrancies were identified, and the proportion of aberrant leukemia cells was calculated as percentage of mononuclear cells. We defined any case with an aberrant immunophenotype that encompassed ≤5% of mononuclear cells as an MRD case, and it was included in subsequent analyses. For each case of B-ALL MRD, we examined side scatter along with the above-mentioned COG-validated markers to asses for aberrancies. Aberrant expression was defined as either over- or under-expression for an antigen when compared to the expression pattern of normal hematogones (CD45, CD19, CD10, CD38, CD58, CD9, CD34) or aberrant positivity/negativity (CD20, CD13/33). Side scatter was considered abnormal if higher or lower than normal hematogones. Fisher's Exact test to compare proportions was used for all statistical analyses.ResultsOf the 289 cases, 64 cases (22%) from a total of 46 patients showed positivity for an aberrant B-lymphoblast population, of which 32 cases (50%) represented MRD ≤5%. Of these 32 cases, 22 were from adults and 10 from children. The MRD levels ranged from 0.010% to 3.8%. Next, we assessed the frequency at which each cell surface antigen was aberrantly expressed and compared results between adult and pediatric patients. For the majority of both adult and pediatric patients with B-ALL MRD, we observed concordant abnormal expression between adult and pediatric cases (listed respectively) of CD10 expression (95% vs. 90%, p=0.534), CD38 expression (68% vs. 70%, p=1.0), CD58 expression (64% vs. 60%, p=1.0), CD9 expression (91% vs. 90%, p=1.0), CD34 expression (86% vs. 70%, p= 0.346), and CD45 expression (55% vs. 80%, p= 0.248). Abnormal expression was less frequent for CD19 (27% vs. 40%, p= 0.683) and CD13/CD33 (18% vs. 20%, p=1.0), as well as side scatter changes (45% vs. 10%, p=0.106). Over-expression was the most common immunophenotypic aberrancy for CD10, CD58, and CD34; under-expression was more common for CD38, CD9, and CD45. Adult and pediatric patients had no significant differences in abnormal expression patterns, with the exception of CD20. In adult patients, CD20 expression was abnormal (negative) in 64% of cases, while only 10% of pediatric patients were negative (p=0.0052, Fisher's Exact test).ConclusionThese results indicate that the range of aberrant immunophenotypes seen in adult and pediatric B-cell acute lymphoblastic leukemia minimal residual disease are similar in nature and suggest that the COG-validated assay designed for pediatric B-ALL can be successfully applied to the broader adult population. Because this assay allows for the detection of frank (as well as minimal residual) disease burden, we propose that this flow cytometry approach can be harmonized to accommodate monitoring of B-ALL by flow cytometry regardless of the extent of disease burden. DisclosuresNo relevant conflicts of interest to declare.

Relapse-Initiating Clones Preexisting at Diagnosis in B- Cell Acute Lymphoblastic Leukemia Help Predict Molecular Pathways of Relapse

Disease recurrence remains a significant cause of mortality in B-cell acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples have demonstrated that relapse arises from a minor subclone already present at diagnosis and not the dominant clone in the majority of patients. However, the reasons why only some clones survive therapy and generate relapse are obscure and elucidation of the mechanisms that underlie these differing fates may be revealed by functional analysis of isolated subclones. Previous work has shown that the subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients with varying cytogenetic abnormalities. Diagnosis-specific, relapse-specific, and shared clonal and subclonal variants were identified by whole exome sequencing of the patient samples. Targeted sequencing of these variants in 372 xenografts generated by transplantation of CD19+ cells in a limiting cell dilution assay uncovered clonal variation. This analysis provided for the unequivocal identification of minor subclones ancestral to the relapse, termed diagnosis Relapse-Initiating (dRI) clones, in the diagnostic sample. Our xenografting approach enabled the physical isolation of dRI clones providing a unique opportunity to interrogate their epigenetic and transcriptional landscapes in order to unravel their relapse initiating capacity. To this end, representative diagnosis, dRI and relapse clones from 5 of the 14 patients were subjected to RNAseq and ATACseq (assay for transposase-accessible chromatin using sequencing) analysis. Despite the differences in transcriptional and chromatin openness between patients, principal component analysis of subclones from individual patients positioned the dRI clones as evolutionary intermediates between the diagnosis and relapse clones. Hierarchical clustering of the most significantly differentially expressed genes and open chromatin regions demonstrated that dRI clones shared gene expression and chromatin accessibility signatures with both the dominant diagnosis clone as well as the dominant relapse clone. To gain mechanistic insight into the data we used gene set enrichment analysis (GSEA) and identified common molecular pathways present in all patients that were enriched in dRI clones and persisted at the time of relapse in comparison to the dominant diagnosis clone. dRI and relapse clones converged in the activation of genes involved in cellular functions such as endocytosis, autophagy and innate immune response. In addition, cell surface proteins like ABC transporters and ephrins were also upregulated in dRI and relapse clones. Remarkably, functional interrogation of dRI clones in secondary xenografts, in comparison to more representative diagnosis clones, displayed increased tolerance to standard chemotherapeutic agents (dexamethasone, L-asparaginase and vincristine). Investigation of the molecular pathways and cellular receptors/transporters identified by gene expression analysis are being assessed in vitro and in vivo as potential targets for novel therapeutic approaches and disease monitoring.Overall, we have shown evidence that minor subclones at diagnosis, ancestral to the relapse clone, possess functional advantages and unique properties over other diagnostic subclones prior to treatment exposure. In depth analysis of pathways identified in these dRI subclones will shed light on potential new therapeutic approaches for abrogating and reducing disease recurrence in B-ALL. DisclosuresMullighan:Amgen: Honoraria, Speakers Bureau; Cancer Prevention and Research Institute of Texas: Consultancy; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding.

Genetic Analysis of B-Cell Acute Lymphoblastic Leukemia Dissemination to the Central Nervous System Identifies Clonal Selection and Therapeutic Vulnerability

Without prophylactic therapy, B-cell Acute Lymphoblastic Leukemia (B-ALL) spreads to the leptomeninges of the central nervous system (CNS) in up to 70% of patients. CNS involvement is more common in certain high risk B-ALL subgroups, including patients with KMT2A (MLL)-translocations, and disease relapse in the CNS carries a poor prognosis. The genetic determinants and biology of B-ALL dissemination to the CNS are poorly defined and therefore therapies targeting the drivers of CNS disease are lacking. Whereas B-ALL exhibits significant subclonal diversity that contributes to functional heterogeneity and disease relapse, recent reports suggest similar clonal composition of bone marrow (BM) and CNS disease, with the potential for CNS dissemination being a universal property of B-ALL cells (Williams et al. 2016, Bartram et al. 2018). Furthermore, functional studies of leptomeningeal disease have focused on the invasion of B-ALL cells into the CNS but limited studies have addressed the selection of genetic clones with the ability to grow within the subarachnoid space.To better define the evolutionary history and biology of leptomeningeal B-ALL we performed targeted DNA, SNP copy number, RNA sequencing, and functional analysis on cells isolated from matched BM and CNS tissue of patient derived xenografts (PDX) generated from a cohort of paired diagnosis and relapse samples from 14 pediatric and adult B-ALL patients of varying cytogenetics. The majority of primary patient samples yielded CNS disease 20 weeks after intrafemoral injection into NSG mice. CNS disease burden was higher in PDXs derived from relapsed B-ALL samples. Human B-ALL cells isolated from the CNS of PDXs retained competence to repopulate disease in the BM, spleen, and CNS upon serial transplantation. Targeted DNA sequencing results analyzed using a Bayesian clustering method revealed different genetic clonal composition between matched BM and CNS cells in approximately half of the xenografts. PDXs from relapse samples were more likely to show genetic discordance between the BM and CNS. Copy number analysis also confirmed frequent genetic discordance between cells isolated from the BM and CNS from individual PDXs. Interestingly, in one patient all PDXs generated from the relapse sample displayed chromosome 6p and 17p hemi-deletions that were unique to the CNS. In total, PDXs from four patients showed recurrent enrichment of specific lesions in CNS-engrafting cells, suggesting that transit to and/or survival within the subarachnoid space can be the product of selection for genetic clones with increased CNS tropism.RNA-seq of matched BM and CNS cells derived from 45 of the primary PDXs demonstrated that CNS-isolated cells were transcriptionally distinct from their matched BM. These differences were most pronounced in samples from patients with MLL-AF4 translocations, whose CNS isolated cells grouped together in multi-dimensional scaling. Using GSEA, the most highly CNS-enriched gene sets in MLL samples were related to mRNA translation initiation and polypeptide elongation. Translation-related gene sets are similarly enriched in the blasts of MLL B-ALL patients with CNS disease in the COG 9906 study. CNS-isolated cells from PDXs of MLL patients exhibited altered rates of protein synthesis compared to matched BM-isolated cells. Treatment of PDXs with the clinically-approved translational inhibitor omacetaxine mepesuccinate (OMA) effectively decreased rates of translation in CNS-engrafting cells. Moreover, OMA reduced leukemia burden nearly 4-fold in PDXs bearing established CNS infiltration generated from two MLL patients.Our data represent an advance in the understanding of B-ALL CNS disease. We present a rich resource of genomic and transcriptomic data from xenografts spanning multiple B-ALL subgroups across diagnosis and relapse and have identified selection for genetically and biologically distinct clones in the CNS, contrary to the current model. Furthermore, we demonstrate that in MLL patients, dysregulation of protein synthesis occurs at CNS dissemination and targeting this process is a novel therapeutic paradigm that may benefit patients with CNS disease. DisclosuresMullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding.

CAR-T bridging to allo-HSCT as a treatment strategy for relapsed adult acute B-lymphoblastic leukemia: a case report

BackgroundAdults with relapsed acute lymphoblastic leukemia (ALL) have a poor prognosis, especially in patients who relapsed within 6 months of complete remission 1 (CR1). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the treatment of choice. However, this can only be considered after complete remission 2 (CR2) is achieved. Therefore, bridging treatment is urgently needed.Case presentationIn the present study, we report a relapsed adult B-cell ALL case that achieved CR2 after treatment with CD19-directed chimeric antigen receptor (CAR)-modified T cell (CAR-T) therapy. After subsequent allo-HSCT, the patient acquired 21 months of disease-free survival.ConclusionThe present results confirm that both CAR-T and allo-HSCT are effective for treating refractory or relapsed B-ALL. However, a novel sequential treatment strategy with these two therapeutic methods may achieve longer disease-free survival time.

Impairment of Vα24-Jα18+Vβ11+ natural killer T cells in adult acute lymphoblastic leukemia patients

Type I natural killer T (NKT) cells are attractive candidates for cancer immunotherapy. In this study, we examined the characteristics of type I NKT cells in patients with adult B-cell acute lymphoblastic leukemia (ALL). We first identified type I NKT cells as Vα24-Jα18 and Vβ11 double-positive CD3+ lymphocytes. Using this method, we found that the adult B-cell ALL patients presented significantly lower level of type I NKT cells than the age- and sex-matching control subjects. The expression of IL-21 by type I NKT cells was then examined using intracellular flow cytometry, which showed that with α-GalCer stimulation, the adult B-cell ALL patients presented significantly lower level of IL-21+ type I NKT cells than control subjects. By both flow cytometry and ELISA, we found that the vast majority of IL-21-expressing type I NKT cells expressed IL-21R, which was also reduced in adult B-cell ALL patients. Using an in vitro co-culture system, we demonstrated that IL-21R+, but not IL-21R-, type I NKT cells could promote the IFN-γ, granzyme B, and perforin expression by CD8 T cells in an IL-21-dependent fashion. This type I NKT cell-mediated stimulatory effect was reduced in adult B-cell ALL patients than in control subjects. In addition, we observed a positive correlation between the frequency of IL-21R+ type I NKT cells and the frequencies of IFN-γ-, granzyme B-, and perforin-expressing circulating CD8 T cells in adult B-cell ALL patients directly ex vivo. Overall, this study identified an IL-21-related impairment in type I NKT cells from adult B-cell ALL patients.

Cross-sectional physician survey on the use of minimal residual disease testing in the management of pediatric and adult patients with acute lymphoblastic leukemia

ABSTRACTObjectives: Minimal residual disease (MRD) is a strong prognostic factor in acute lymphoblastic leukemia (ALL), which progresses quickly and is fatal within months if untreated. This study explored use of MRD testing in adult and pediatric B-cell ALL patients, and academic versus community settings.Methods: A survey was administered to US-based hematologists/oncologists currently managing ≥5 B-cell ALL patients and using MRD tests. Descriptive analyses (frequencies and percentages) and Pearson’s chi square testing assessed any differences in various characteristics.Results: 150 adult treaters (treating physicians: 100 community, 50 academic) and 30 pediatric treaters participated. Use of MRD testing was higher among pediatric treaters (93% of patients) than adult treaters (73% of patients) (p < 0.05), and higher among adult treaters at academic centers than in community settings (84% and 67% of patients, respectively; p < 0.01). MRD testing is part of a standard protocol for 93% of pediatric treaters versus 53% of adult treaters. Pediatric treaters most commonly administer an MRD test during/after induction or upon relapse. No consensus on timing among adult treaters was noted.Discussion: MRD testing is an important tool in the prediction of relapse in ALL. Resolving barriers could improve detection of molecular relapse in patients with ALL, particularly among adults and in community settings.Conclusion: MRD testing is fairly common in treatment of ALL, but some barriers still exist in access.

Abstract 5507: RAG1 high expression associated with IKZF1 dysfunction in adult B-cell acute lymphoblastic leukemia

Abstract The recombination activating gene (RAG)-mediated recombination is the dominant mutational process and the predominant driver of oncogenic genomic rearrangement in acute lymphoblastic leukemia (ALL). This then leads to further leukemic clonal evolution. IKZF1 encodes a kruppel-like zinc finger protein, IKAROS that is essential for normal hematopoiesis and acts as a tumor suppressor in ALL. The genetic defects of a single IKZF1 allele are linked to the development of human ALL, characterized by an increased risk of relapses and poorer prognosis. We observed that RAG1 is significantly increased in subsets of B-ALL patients. High RAG1 expression correlates with high proliferation markers. IKAROS directly binds to the RAG1 promoter in B-ALL cells by quantitative chromatin-immunoprecipitation assay. IKAROS suppresses RAG1 promoter activity by luciferase reporter assay. Lentiviral IKAROS expression significantly suppresses RAG1 expression, but IKAROS shRNA promotes RAG1 expression in B-ALL cells. CK2 inhibitor by restoring IKAROS activity, significantly suppresses RAG1 expression in an IKAROS-dependent manner in B-ALL cells. RAG1 expression is significantly higher in patients with IKZF1 deletion, as compared to patients without IKZF1 deletion. Treatment with CK2 inhibitor also results in an increase in IKZF1 binding to the RAG1 promoter and suppression of RAG1 expression in primary B-ALL cells. Taken together, our results demonstrate that high expression of RAG1 correlates with high proliferation markers in B-ALL, and are the first to demonstrate that IKAROS directly suppresses RAG1 expression. Our data suggest RAG1 high expression works together with IKAROS dysfunction to drive oncogenesis of B-ALL, which have significance in an integrated prognostic model for adult ALL. Citation Format: zheng Ge, Qi Han, Jinlong Ma, Yan Gu, Huihui Song, Malika Kapadia, Sinisa Dovat, Chunhua Song. RAG1 high expression associated with IKZF1 dysfunction in adult B-cell acute lymphoblastic leukemia [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 5507.

Ikaros family zinc-finger 1 mutation is an independent factor for the poor prognosis of adult B-cell acute lymphoblastic leukemia, and allogeneic hematopoietic stem cell transplantation canimprove clinical outcomes.

To investigate the prognosis of patients with adult B-cell acute lymphoblastic leukemia (B-ALL) with Ikaros family zinc-finger 1 (IKZF1) mutation and determine the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in improving the clinical outcome, we detected the IKZF1 mutation and BCR-ABL fusion gene at diagnosis in the bone marrow of 164 adult patients with B-ALL, and analyzed the clinical data of these patients retrospectively. Our analysis showed that grade III-IV acute graft-versus-host disease and IKZF1 mutation in the transplantation group and age and IKZF1 mutation in the non-transplantation group were independent factors for poor prognosis by univariate and multivariate analyses.The 3-year overall survival (OS) and leukemia-free survival (LFS) rates were much lower in the IKZF1+/BCR-ABL+ subgroup than in the IKZF1+/BCR-ABL- and IKZF1-/BCR-ABL- subgroups in both the transplantation and non-transplantation groups. The 3-year OS and LFS rates were significantly higher in the transplantation group than in the non-transplantation group with IKZF1 mutation.The study demonstrated that IKZF1 mutation was an independent factor indicating the poor prognosis of adult B-ALL and much worse prognosis in the BCR-ABL+ subgroup in both non-transplantation and transplantation groups. However, allo-HSCT significantly improved the OS and LFS of patients and also their clinical outcomes.

Issue Highlights-May 2018 (94B3).

Issue Highlights-May 2018 (94B3).