B-lineage Acute Lymphoblastic Leukemia Research Articles

Role of Mitochondrial Apoptotic Pathway in Disulfiram/Copper Mixture-Induced Cell Apoptosis in Human B-Lineage Acute Lymphoblastic Leukemia in Vitro

Backgrounds: The long term prognosis of adult B-lineage acute lymphoblastic leukemia (B-ALL) is poor when compared with pediatric B-ALL. The current therapeutic regimen for adult B-ALL often results in refractory and relapsing diseases. Therefore, it is urgently needed to explore novel approaches to treat adult B-ALL. Disulfiram (DS) has been used clinically as a safe anti-alcoholism drug for over 6 decades. Recent studies demonstrated that disulfiram/cooper mixture (DS/Cu) was cytotoxic to multiple solid cancers, but its effects on B-ALL cells are still unclear. Moreover, the molecular mechanism of the cytotoxicity of DS/Cu to tumor cells was poorly defined. In this study, we investigated the effects of DS/Cu on B-ALL cells in vitro and its related cytotoxic mechanism.Results: Firstly, CCK8 assay indicated that DS/Cu markedly inhibited Nalm6 cell proliferation in a dose-dependent manner. Secondly, colony-forming assay showed that DS/Cu also abolished the clonogenicity of Nalm6 cells (P<0.001). Thirdly, FACS analyses revealed that DS/Cu mixture could induce apoptosis of Nalm6 cells, as well as primary B-ALL cells (n=16) in a dose-dependent manner. We additionally analyzed the relationship between clinical characteristics of B-ALL patients, including age, WBC counts, immunophenotype, cytogenetics, risk stratification and Ph chromosome, with the efficacy of DS/Cu on B-ALL cells. The apoptosis isolated from pro-B and cytogenetic abnormality B-ALL pastients was higher. Therefore, our results demonstrated that DS/Cu mixture could induce significant cytotoxicity against B-ALL cells in vitro.To decipher the cytotoxic mechanism of DS/Cu mixture, JC-1 staining was done and the results showed that DS/Cu mixture could significantly reduce the mitochondrial membrane potential in Nalm6 cells (P<0.01) and 7 cases of primary B-ALL cells (P<0.05). Consistently, Western Blot analysis showed that DS/Cu induced B-ALL cell apoptosis by down-regulating the expression of anti-apoptotic protein Bcl2 and Bcl-XL, as well as activating caspase-3 and its substrate PARP. Hence, our results indicated that DS/Cu induced apoptosis of B-ALL cells at least partly through the intrinsic mitochondrial apoptotic pathway.Conclusion: Our results demonstrated that DS/Cu not only significantly inhibit proliferation and clonogenicity, but also induce apoptosis of B-ALL cells in vitro.The mitochondrial apoptotic pathway might be the molecular mechanism of DS/Cu-induced apoptosis of B-ALL cells. DisclosuresNo relevant conflicts of interest to declare.

Rapid Identification of BCR/ABL1 -like Acute Lymphoblastic Leukemia (ALL) Cases By Quantitative Real Time-PCR (Q-RT-PCR). Generation and Validation of a Predictive Statistical Model

Introduction: Among B-lineage ALL, BCR/ABL1-like cases have clearly emerged as a subgroup with peculiar features. By gene expression profiling (GEP), they have a transcriptional signature similar to that of BCR/ABL1+ cases. Moreover, these cases often have IKZF1 deletions and CRLF2 deregulation, and harbor several lesions involving tyrosine kinases (TKs) that may be patient specific. The incidence varies with age (from 10% of B-lineage ALL in children, to 27% in adolescents/young adults, with a decrease in adults) and they are usually characterized by an inferior outcome, though risk-directed therapy based on minimal residual disease levels has improved prognosis in childhood. Even if GEP promptly recognizes these patients, this technique is expensive, not readily available and not applicable to all centers.Aims: To generate a simple and reliable tool, based on a Q-RT-PCR approach, aimed at the rapid identification of BCR/ABL1-like cases, and to build a statistical predictive model. Finally, to correlate the results obtained with the clinico-biologic features and outcome.Methods and patients: Through a meta-analysis of previously published GEP data focused on BCR/ABL1-like cases, and on our in house results, 9 commonly overexpressed genes were selected and evaluated by Q-RT-PCR; CRLF2 was also included, because of its association with this signature (total=10 genes). Q-RT-PCR was performed using the ABI PRISM 7300 Sequence Detection System and FastStart Universal SYBR Green Master (Rox). Overall, 149 B-lineage ALL cases, negative for known major rearrangements, i.e. BCR/ABL1, ETV6/RUNX1, E2A/PBX1 and ALL1 (B-NEG ALL), were tested. Median age was 28.8 years (range 1-78), 59 were females and 90 males. To generate a predictive model for BCR/ABL1-like identification, one round of cross-validation was performed; the full dataset was divided into two panels (discovery and screening). The discovery panel included 52 cases previously evaluated for GEP analysis and comprised 26 BCR/ABL1-like and 26 B-NEG ALL samples, while the screening panel (n=97) was based on 81 cases including 16 B-NEG ALL samples harboring JAK2, IL7R or CRLF2 mutations, tested as internal control, given their association with a BCR/ABL1-like signature.Results: The statistical model was built on the discovery panel and was validated, in terms of sensitivity and specificity, on the screening panel. Q-RT-PCR values of the 10 genes were grouped in the discovery panel according to principal component analysis to reduce multicollinearity and a logistic regression model was used to examine the association among the first three principal components (accounting for more than 80% of the total variance) and BCR/ABL1-like cases. Finally, a score was computed and validated in the screening panel. The model had a sensitivity and specificity of 93.8% and 93.8%, respectively, with a positive predictive value of 75%. Within the screening panel, 10/81 (12.3%) and 6/16 JAK2, IL7R or CRLF2 mutated cases (37.5%) were predicted as BCR/ABL1-like (total=16.5%). GEP was performed on 14/16 BCR/ABL1-like samples and confirmed the similarity with BCR/ABL1+ ALL cases by unsupervised and supervised analysis. Molecular screening, comprising the screening of JAK1/2, CRLF2, IL7R (JAK/STAT pathway) mutations and IKZF1 deletions, confirmed a significant enrichment of the JAK/STAT pathway (48% vs 15%, p=0.001) and of IKZF1 deletions (82 vs 46%, p=0.006) in BCR-ABL1-like cases as opposed to the other B-NEG ALL cases. Finally, BCR-ABL1 -like patients showed a significantly higher white blood cells (WBC) count (46x109/l vs 12.8x109/l, p=0.003) and poorer prognosis compared to the other B-NEG ALL patients, in terms of complete remission achievement (70.3 vs 87%, p=0.07), overall survival (41.1% vs 59.1%, p=0.06) and disease-free survival (DFS) (31.1 % vs 46%, p=0.008).Conclusions: We describe a Q-RT-PCR approach and a statistical model capable of rapidly identifying BCR-ABL1-like cases. We confirm a significant association with JAK/STAT mutations, IKZF1 deletions and poorer outcome. The prompt recognition of BCR-ABL1-like ALL at presentation can: i) identify cases in which additional lesion/s involving a TK - that might sustain the BCR/ABL1-like signature - should be further investigated; ii) drive therapeutic decisions, since these patients might benefit by the addition of targeted therapies, particularly TK inhibitors. DisclosuresFoà:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

An Integrated Genomic Classification That Includes Copy Number Alterations Is Highly Predictive of Post Induction Minimal Residual Disease (MRD) Status in Childhood Precursor B Lineage Acute Lymphoblastic Leukemia

IntroductionChildhood precursor B lineage ALL (B-ALL) is a genetically heterogeneous disease where the underlying genetics is an important determinant of outcome. Copy number alterations (CNA) have been described in B-ALL, which in conjunction with chromosomal abnormalities drive leukemogenesis. Some, especially IKZF1 deletions are prognostically relevant and influence disease outcome. However, there is no consensus on how these CNAs can be incorporated in a clinical setting. Recently, an integrated genomic classification has been proposed for ALL which includes CNA as well as cytogenetics based risk prediction. However, there have not been many studies which validated these suggestions or correlated them with immunophenotyping based MRD. Using end induction MRD as a surrogate marker of outcome we demonstrate that the integrated genomic profile is highly predictive of MRD clearance.Patients and Methods91 cases of childhood B-ALL (WHO 2008 criteria) were prospectively accrued over a 4 months. NCI risk was calculated as per standard recommendations. FISH detected recurrent cytogenetic abnormalities as well as iAMP(21); conventional karyotyping and flow cytometry determined the ploidy status. SALSA MLPA P335 was used to detect CNA in BALL following the manufacturers recommendations. Data was analyzed on the Coffalyzer software. Patients were divided into good and poor risk genetic abnormalities to stratify them according to the integrated genetic profile. The former included good risk cytogenetic (ETV6-RUNX1 or high hyperdiploidy) as well as good risk CNA profiles (no deletion of IKZF1, CDKN2A/B, PAR1, EBF1, ETV6 or RB1; isolated deletion of ETV6, PAX5 or BTG1 or ETV6 deletion with single deletion of BTG1, PAX5, or CDKN2A/B). Poor risk genetic abnormalities included high risk cytogenetic groups (BCR-ABL1, MLL rearranged, near haploidy, low hypodiploidy or iAMP21) as well as intermediate and poor risk CNA profiles (IKZF1/ PAR1/EBF1/RB1 deletion or any other CNA profiles) Cytogenetic abnormalities took precedence over CNA abnormalities as has been described (Moreman AV et al Blood 2014). MRD was detected using 9 colour flow cytometry (CD19, CD20, CD10, CD45, CD38, CD66c, CD123, CD34, CD58) on an end of induction bone marrow sample. In every case attempt was made to acquire 10,00,000 events. Syto 16 dye was used to correct the MRD value. Flow cytometry data was analyzed with Kaluza (v1.3). Two-tailed fishers exact test and chi squared test were applied for statistical analysis.ResultsMedian age was 5 years (range: 1-14), male predominant (58 males). Majority patients had good risk (50.5%) followed by intermediate (40.7%) and poor risk cytogenetics (8.8%). The frequencies of CNA were as follows; CDKN2A/B (23.1%), ETV6 (19.8%), IKZF (18.7%), PAX5 (14.3%), EBF1 (4.4%), BTG1 (4.4%), RB1 (3.3%). Using these data patients were classified into good risk (47.3%), intermediate (30.8%) and poor risk CNA profiles (22%). The cytogenetic and CNA risk profiles were compiled together into good risk genetic (74.7%) and poor risk (25.3%) profiles. MRD positivity (28.6%) ranged from 0.01% to 48.4% where as the rest were negative (71.4%). The CNA risk profiles showed a tendency for correlation with MRD status (p=0.08) whereas the integrated genetic profile showed a very high correlation with the MRD status (in which good risk patients were associated with MRD negative status) and NCI risk. In addition, the integrated genomic profile also predicted the MRD status in the intermediate cytogenetic group. (Table 1)ConclusionThis data seems to indicate that in addition to cytogenetics, CNA should be incorporated into routine clinical testing and risk algorithms for B-ALL. The integrated genomic classification is of prognostic relevance and offers an additional avenue for prognostication and risk adapted therapy. Table 1Correlation of immunophenotypic MRD, NCI Risk, Prednisolone Response and intermediate cytogenetics with integrated genetic profile.Variable TestedGood Genomic ProfilePoor Genomic ProfileStatistical SignificanceMRD StatusEnd Induction MRD Positive1214Significant (p=0.0003)End Induction MRD Negative569NCI RiskHigh NCI Risk1812Significant (p=0.03)Standard NCI Risk5011D+8 Prednisolone Response (n=83)Good Response5818Not Significant (p=1)Poor Response61Intermediate Cytogenetic RiskEnd Induction MRD Positive36Significant (p=0.05)End Induction MRD Negative197 DisclosuresNo relevant conflicts of interest to declare.

Minimal Residual Disease Evaluation By Real-Time Quantitative Polymerase Chain Reaction for Wilms Tumor 1 Gene Expression and Multicolour Flow Cytometry, in Pediatric B Lineage Acute Lymphoblastic Leukemia

Introduction: Minimal residual disease (MRD) determination involves the measurement of very low levels of leukemia using sensitive techniques which at present are complex, time consuming and require expertise for performance and interpretation. A ''panleukemic''marker such as Wilm's tumor 1 gene (WT1) which is frequently over expressed in acute leukemia could simplify MRD detection and serve as a useful prognostic marker.Aim of study: To evaluate the usefulness of WT1 gene expression, as a marker for MRD in B-lineage acute lymphoblastic leukemia (B-ALL).Method and Material: Flow cytometric immunophenotyping (FCMI) and real time-polymerase chain reaction (RQ-PCR) for WT1 gene expression were performed usingbone marrow at diagnosis and at day 15 (mid-induction). Of the 23 patients recruited, day 15 MRD analyses by both these methods was performed on 11 bone marrow samples of patients who showed WT1 over expression at day 0.Results:WT1 over expression at diagnosis was found in 69.5% cases (16/23). MRD was detectable in 54.5% cases by WT1 RQ-PCR and by FCM in 72% cases. A statistically significant correlation was seen between WT1 normalized copy number (NCN) at diagnosis with MRD levels detected by FCM.Conclusion:WT1 represents a candidate MRD and prognostic marker. The significant correlation between WT1 over expression at diagnosis and MRD positivity by flow cytometry at day 15 (mid induction) of chemotherapy suggests that high WT1 expression could correlate with unfavourable outcome in childhood ALL.However, as not all patients of B-ALL over-express WT1 at diagnosis, quantitative assessment of WT1 transcripts can be used as useful molecular marker for MRD detection, but only in a subset of patients. DisclosuresNo relevant conflicts of interest to declare.

In Philadelphia-Chromosome-Negative Acute Lymphoblastic Leukemia, Late Relapses Are Not Uncommon, Occur Mostly in Patients at Standard Risk and Have a Relatively Favorable Outcome. Results of the International ALL Trial: MRC Ukallxii/ECOG E2993

This study was coordinated by the ECOG-ACRIN Cancer Research Group (Robert L. Comis, MD and Mitchell D. Schnall, MD, PhD, Group Co-Chairs) and the Medical Research Counsel, United Kingdom, and supported in part by Public Health Service Grants CA180820, CA180794, CA180790, CA189859, CA180853, CA180791, and from the National Cancer Institute, National Institutes of Health and the Department of Health and Human Services. Its content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute.Background: Late relapse in acute leukemia is considered a relatively rare event. Patients with acute myeloid leukemia (AML) are often considered cured of the disease at 3 years, but information regarding adult acute lymphoblastic leukemia (ALL) patients is scarce. Data are presented from one of the largest prospective adult ALL studies, the MRC UKALLXII/ECOG E2993, to evaluate the rate and characteristics of late relapse in ALL. For this purpose, late relapse was defined, arbitrarily, as relapse 3 years post achievement of complete remission (CR) and very late relapse was defined as relapse > 5 years from CR.Methods: The UKALLXII/ECOG E2993 was an international ALL trial conducted jointly by the MRC in the United Kingdom and ECOG in the United States. All patients received identical induction therapy, followed by central nervous system prophylaxis. Patients with a sibling donor (or a matched unrelated donor in Philadelophia-chromosome-positive ALL) were assigned to receive an allogeneic hematopoietic stem cell transplant (HSCT); all others were randomized to undergo an autologous transplant or protracted standard consolidation/ maintenance therapy. The study accrued 2109 patients from 1993 to 2008. Following relapse, patients were followed for survival. For this report only patients registered before the tyrosine kinase inhibitors era are included in the analysis.Results: 1518 study patients were eligible for this analysis, 1208 (79.6%) Philadelphia-chromosome negative (Ph-neg) and 267 (17.5%) Philadelphia-chromosome positive (Ph-pos). 1381 (91%) of the patients achieved CR; 93% of the Ph-neg and 82% of the Ph-pos. 572 patients (37.7%) underwent allogeneic HSCT. The median duration of follow-up of patients who achieved CR was 10 years. Among the 1381 patients who achieved CR, 626 (45.3%) had a documented relapse; 566 (90.4%) relapsed within 3 years of CR and 60 (9.6%) relapsed beyond 3 years ('late relapse') (Figure 1). Among these 60 patients, 18(2.9%) relapsed after 5 years ('very late relapse').TablePatientsnCRAll relapsesRelapses< 3 yearsRelapses≥ 3 yearsRelapses≥ 5 yearsAll patients15181381 (91%)626 (45.3%)566 (90.4%)60 (9.6%)18 (2.9%)Ph-neg1208 (79.6%)1123 (93%)485 (40.1%)429 (88.5%)56 (11.5%)17 (3.5%)Ph-pos268 (17.5%)219 (82%)124 (56.6%)122 (98.4%)2 (1.6%)1 (0.8%)Relapse beyond 3 years occurred in 4.3% of all who achieved CR, in 5% of Ph-neg and 0.01% of Ph-pos patients.Among the 60 late relapses, the median time to relapse was 46 months. 61.7% of the late-relapse patients were males, median age was 32 years, 88.3% were B-lineage ALL and the median white cell count at diagnosis was 6000/ul. 56.7% were in cytogenetic standard risk, 8.3% at high risk and the data of 35% are unknown.The median survival for the late relapse patients was longer than for those who relapsed within 3 years. The overall survival (OS) of the 56 Ph-neg patients who relapsed beyond 3 years is shown in Fig 2. TableRelapse > 3 yearsRelapse > 3 yearsMedian survival from relapse (months)5.411.23-year OS from relapse6.5%29%5-year OS from relapse5.6%19%Conclusions: Late relapses in adults with Ph-neg ALL are not uncommon. About 10% of relapses occur beyond 3 years and 4.3% of all ALL patients who achieved a CR can expect to have a late relapse. These data are in contrast to AML where only 1% of patients relapse beyond 3 years (Watts JM et al, 2014). Most of the late relapse patients were at standard risk and appeared to have a relatively favorable outcome post relapse. Patients with ALL, particularly those who are Ph-neg, cannot be considered as cured at 3 years and need to be closely followed. [Display omitted] [Display omitted] DisclosuresRowe:Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy. Douer:Gilead: Consultancy.

CDKN2 Gene Deletion As Poor Prognosis Predictor Involving in the Progression of Adult B-Lineage Acute Lymphoblastic Leukemia Patients

Background: Genes CDKN2A (MTS1) and CDKN2B (MTS2), which encode for p16INK4a/p14ARF and p15INK4b respectively from the 9p21.3 locus, are thought to be an important growth suppressor gene. INK4a/ARF regulates p53 tumor suppressor function by interacting with MDM2. P15/INK4b works as a CDK inhibitor, which inhibits phosphorylation of Rb by inactivating cyclin/ CDK-4/6 complexes and thereby controls cell-cycle G1 progression. Deletion of CDKN2A/B was well known in many hematologic malignancies, but only few report investigated this deletion effect on clinical prognosis.Methods: This study performed analysis of the CDKN2 deletion in 215 adult B-ALL patients, and relation with cytogenetic prognostic factors (BCR/ABL; E2A/PBXl; TEL/AML1; MLL rearrangement; MYC, IGH translocation).Results: The prevalence of CDKN2 deletions in all study population was 28.4%. There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells(WBC) count, BM blast percentage,extra infiltration and induction complete remission(CR) rate. Analysis at relapse patients revealed increased rate of CDKN2 loss (44.6%) compared with the rate in all patients (28.4%) (p=0.006). Deletions of CDKN2 were significantly associated with poor outcomes including decreased overall survival (OS) (p<0.001), lower disease free-survival (DFS) (p<0.001), and increased cumulative incidence of relapse (p=0.002); Also, CDKN2 deletions were strongly associated with IGH translocation (p=0.021); and had an adverse effect on patients who with BCR-ABL fusion gene or MLL rearranged. Among of patients with the CDKN2 deletions , the subgroup of received Allo-HSCT treatment had a better OS and DFS compared with the subgroup received chemotherapy only (OS: p<0.000; DFS: p<0.000).Conclusion: Patients with CDKN2 gene deletion were benefited from allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Deletion of CDKN2 gene was commonly observed through leukemia progression and was a possible poor prognostic marker in long-term outcomes. DisclosuresNo relevant conflicts of interest to declare.

PP2A Is Required for B Cell Survival and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

Background: The Ser/Thr Protein phosphatase 2A (PP2A) attenuates activity of RAS-ERK and PI3K-AKT signaling pathways and functions as important tumor suppressor in chronic myeloid leukemia (CML) by downregulating output of oncogenic BCR-ABL1 signaling. Restoration of PP2A activity has been proposed for the treatment of CML and eradication of TKI-resistant leukemia CML-stem cells (Neviani et al., 2013). While the tumor suppressor function of PP2A was independently confirmed by multiple groups, our analyses of PP2A function during early B cell development suggested a role for PP2A to contribute to B cell survival. Therefore, we studied whether PP2A has different roles in B-lineage ALL and CML and whether these mechanistic differences are relevant for patients with B-lineage and B-lymphoid blast crisis CML.Results: Consistent with a divergent role of PP2A in B-lineage ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A but also the catalytic subunit C and was paralleled by near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of p70S6K and S6 ribosomal protein, which indicated elevated mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. In agreement with previous studies, however, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. To verify that the observed difference between B cell and myeloid disease was indeed lineage-dependent, we reprogrammed B cell lineage ALL cells into myeloid CML using inducible overexpression of CEBPα. As expected, CEBPα-driven myeloid lineage conversion prevented cell death upon deletion of PP2A, demonstrating that PP2A-function represents a B cell-specific vulnerability.We next transplanted luciferase-labeled Ppp2r1afl/fl ALL cells into recipient mice and monitored cell growth and leukemia progression. Cre-mediated deletion significantly prolonged overall survival of recipient mice that were transplanted with Ppp2r1afl/fl ALL cells. Although mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles in all cases studied demonstrating that fatal leukemia arose from few subclones that had escaped Cre-mediated deletion of PP2A.Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in B cell lineage Ph+ ALL but not CML-like cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted ALL cells, together with decreased anti-oxidant gene expression, increased H2AX phosphorylation and p53 expression indicated impaired balance of glycolytic flux may account for increased death of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells.The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation.Conclusion: Cre-mediated ablation of PP2A in mouse Ph+ ALL cells induced rapid cell death through excessively oxidative stress but not in myeloid CML cells. We confirmed this pro-survival role of PP2A in human Ph+ ALL-patients derived leukemia cells through both CRISPR-Cas9 mediated genetic study and PP2A inhibition by a novel small molecule LB-100 (Jie Lu et al., 2009). These findings highlight PP2A as a therapeutic target with potential relevance in Ph+ ALL and B-lymphoid blast crisis progression of CML. DisclosuresNo relevant conflicts of interest to declare.

CD4 CAR T Cells Mediate CD8-like Cytotoxic Anti-Leukemic Effects Resulting in Leukemic Clearance and Are Less Susceptible to Attenuation By Endogenous TCR Activation Than CD8 CAR T Cells

Adoptive immunotherapy using T cells armed with chimeric antigen receptors (CAR) has proven extremely effective against CD19+ B-lineage acute lymphoblastic leukemia (ALL) with remission rates as high as 70-90% in recent clinical trials in relapsed/refractory patients. CD8 T cells are typically thought of as the primary antitumor effector cells in an adoptively transferred product due to their potent cytolytic capabilities, whereas CD4 T cells are thought to primarily provide "help" to enhance CD8 T cell activity via cytokine production. However, CARs are synthetic constructs that likely alter the functionality of T cells. Further, CAR T cells are endowed with 2 specificities, one through the CAR and one through the endogenous T cell receptor (TCR). To evaluate the biology of CAR T cells, we sought to evaluate the impact of CAR expression on the functionality of T cells and to study the impact of TCR on CAR T cell activity using both human CAR T cells in a xenograft model and murine CAR T cells in a syngeneic murine model. A human or murine second-generation anti-CD19 scfv/CD28/CD3ζ CAR was transduced into human or mouse CD8 (CAR8) and CD4 (CAR4) T cells, respectively, and tested against pre-B ALL cell lines in human murine xenografts or syngeneic models. Surprisingly, human CAR4 cells alone had equivalent ability to eradicate the Nalm6 ALL in vivo as CAR8 cells. Although CAR8 cells more rapidly cleared leukemia (2 vs 4 days), relapse eventually occurred. In contrast, CAR4 cells eradicated leukemia more slowly, but persisted longer and prevented relapse. In mice receiving CAR4+CAR8 cell products, only CAR4 cells were detectable at day 55. Next we utilized our syngeneic murine system to study CAR4 and CAR8 activity in an immunocompetent system using TCR transgenic T cells with known TCR specificity against the male histocompatibility antigen, HY. As expected, when stimulated through the TCR, CAR8 cells produced cytokines and degranulated, as manifested by CD107a expression, whereas CAR4 cells only demonstrated a cytokine production response. However, murine CAR4 cells activated through the CAR receptor develop "CD8-like" cytolytic features characterized by degranulation and were able to completely eradicate leukemia in vivo when administered without CAR8 cells, similar to the human CAR4 cells. We then evaluated the effect of TCR antigen in vivo on CAR T cell function by comparing activity in male (HY+) vs female (HY-) recipients. CAR4 cells were curative in both male and female recipients. However, CAR8 treatment has no anti-tumor activity in male recipients indicating that CAR8 cells are very susceptible to the negative effects of TCR signaling. TCR antigen availability in vivo greatly increased the number of infused CAR4 cells (P=0.003, male vs female recipients), while they significantly decreased the number of CAR8 cells (P=0.0023, male vs female recipients) after 1 week. Interestingly, we found that there was significant down-regulation of CAR expression on both CAR4 and CAR8 cells in male recipients (HY+). PD-1 and Tim3 were expressed at low levels on CAR4 cells and CAR8 cells in female recipients. However, there was significantly higher PD-1 (P=0.0079) and TIM3 (p=0.014) expression on CAR8 cells in male vs female recipients, whereas there was no difference in PD-1 or Tim3 on CAR4 cells recovered from male vs female recipients. These data indicate that CAR4 cells are more functional and become less exhausted than CAR8 cells in the presence of TCR antigen. Finally, we evaluated the long-term persistence of CAR4 and CAR8 cells in our syngeneic model in which there is no xenogeneic reactivity allowing for long-term monitoring. At day 60 and day 80, CAR4 cells were detectable in bone marrow and spleen, whereas CAR8 cells had completely disappeared. Despite the long-term persistence of CAR4 cells in recipients with or without TCR antigen, CAR4 cells are not completely immune to effects of TCR activation. In male recipients after 60 days, CAR4 cells also start to express high PD-1 levels and have decreased CAR T cell counts. These findings have important implications for CAR therapy, particularly in the allogeneic setting where the presence of TCR antigen may be more likely to be present in the recipient. In addition, these results suggest that CAR4 cells alone are cytolytic, equally potent to CAR8 cells at eradicating leukemia in vivo, and may be superior due to better persistence and reduced susceptibility to exhaustion. DisclosuresNo relevant conflicts of interest to declare.

Targeting Heme Oxygenase-1 Improves Poor Therapeutical Outcomes of BCR-ABL-Positive Adult B-Allwithikaros Isoform 6

Genomic profiling studies have demonstrated that the dominant-negative Ikaros isoform 6 (IK6) is overexpressed in BCR-ABL1-positive B-lineage acute lymphoblastic leukemia (BCR-ABL1-positive B-ALL), and it is strongly associated with a poor outcome. In addition, IK6 expression leads to BCR-ABL-positive B-ALL patients insensitive to tyrosine kinase inhibitors (TKIs). Moreover, a study has revealed that Heme oxygenase-1 (HO-1), an essential survival factor, could be a potential target in treating ALL. Therefore, we were wondering whether targeting HO-1 could improve poor therapeutical outcomes of BCR-ABL-positive adult B-ALL with IK6. Firstly, we detected that IK6 existed in 20 of 42 (47.6%) adult BCR-ABL1-positive B-ALL by using reverse transcribed polymerase chain reaction (PCR) and nucleotide sequencing. We also found that early clinical response was poor in BCR-ABL1-positive B-ALL patients with IK6, and they were usually high WBC counts at diagnosis and a high relapse rate. Compared to healthy donors' CD34+ cells, the levels of HO-1 expression were higher in primary CD34+ cells derived from adult BCR-ABL1-positive B-ALL patients with IK6 by qRT-PCR and western blot studies. And there was a strong correlation between the expression of IK6 and HO-1. In primary CD34+ leukemic cells derived from our IK6-positive patients' pool, silencing HO-1 resulted in the reduce of proliferation and a substantial proapoptotic effect, including a significantly enriched subG1 population, an increase of Annexin V-positive cells and of caspase3 cleavage. Furthermore, silencing HO-1 also increased their sensitivity to TKIs. This phenomenon was also supported by the observation that ZnPP IX substantially augmented the growth-inhibitory effects of imatinib on primary leukemic cells. Finally, we found that IK6 led to STAT5 activiation, and HO-1 was one of the downstream target genes of STAT5. In conclusion, the existence of extra IK6 seems to exacerbate poor prognosis and risks of relapse in adult BCR-ABL1-positive B-ALL. HO-1 plays an important role as a survival factor in BCR-ABL1-positive B-ALL with IK6, and targeting HO-1 can probably improve the clinical outcomes. DisclosuresNo relevant conflicts of interest to declare.

Impact of GvHD and Other Patient-, Disease-, Donor and Transplantation-Related Factors on 5 Year Relapse after Unrelated Cord Blood Transplantation for Children with Acute Lymphoblastic Leukemia in Remission

Relapse remains a major barrier to the long-term overall survival (OS) of pediatric patients with acute lymphoblastic leukemia (ALL). Allogeneic HSCT is a potentially curative option for these patients and its mechanism relies on graft-versus-leukemia (GvL) and delivery of high-dose chemotherapy. However, the mechanism and degree of GvL in curing ALL is not fully understood. Unrelated cord blood (CB) is an attractive donor option for these patients due to rapid procurement and lenient HLA matching without increased graft-versus-host disease (GvHD). Despite less GvHD, relapse is not increased when compared to other donor sources. Therefore, through collaboration between Eurocord, PDWP-EBMT and Duke University, we performed a retrospective analysis to identify risk factors associated with relapse, and the role of GvHD (as time dependent co-variate) in preventing relapse after unrelated CB transplantation (CBT) for children with ALL.We analyzed 640 children (<18 years (y)) with ALL in complete remission (CR) who received a single-unit CBT in either first (n=257, 40%) or second (n=383, 60%) CR from 2000-2012. Most patients were diagnosed with B-cell (79%) or T-cell (18%) lineage ALL and had no CNS involvement (92%). Cytogenetic data was available for 411 patients (CR1=171, CR2=240), and 37% were considered intermediate risk and 36% poor risk. Pre-transplant, 49% of patients were CMV seropositive. All patients received myeloablative regimens and most received anti-thymocyte globulin (88%). Regimens included total body irradiation (TBI; 69%) with either cyclophosphamide (Cy)+Fludarabine (Flu; 10%) or other agents (59%), Busulfan (Bu)+ Cy (16%), or Thiotepa+Bu+Flu (8%). Most patients received HLA mismatched CBs at 1 (50%) or 2+ (34%) loci. GvHD prophylaxis included cyclosporine with either steroids (72%) or mycophenolate mofetil (21%), or other agents (6%).Considering CR1 patients only, the median age at CBT was 5.3 y. Most were diagnosed with B-cell ALL (75%) and approximately half of those with known cytogenetics were considered poor risk. The median duration from diagnosis to CBT was 6.7 months and median follow up was 47 months. Estimated OS and LFS at 5 y for CR1 patients was 59% and 52%, respectively. Improved OS and LFS were associated with TBI-containing regimens and younger age at time of CBT (Table 1). The CI of relapse at 5 y was 23% for CR1 patients. In a multivariate analysis (MVA) the presence of acute GvHD (grade II-IV) and TBI-containing regimens were both protective of relapse (Table 1). No impact of GvHD was observed on OS, LFS or NRM.In CR2 patients, the median age at CBT was 6.9 y. Disease was primarily B-lineage ALL (82%) with intermediate or poor risk cytogenetics in 40% and 28% of patients, respectively. Median time from diagnosis to CBT was 36 months and median follow up was 54 months. Estimated OS and LFS in CR2 patients were 46% and 44%, respectively. In MVA, younger age and longer duration from diagnosis to CBT (>30 months) was associated with improved OS and LFS (Table 2). Conversely, the use of ATG was associated with lower OS.In CR2 patients, the CI of relapse was 28%. In MVA, longer duration from diagnosis to CBT (>30 months) and receiving TBI were both associated with less relapse. Importantly, receiving a fully matched HLA CB graft was a strong risk factor for increased relapse compared to mismatched CB recipients. Acute GvHD (II-IV) in this cohort was associated with higher mortality but not with relapse.In this large retrospective collaborative study, we investigated the impact of GvHD as a marker of GvL in pediatric ALL patients after CBT. In CR1 patients, acute GvHD and use of TBI were protective of relapse. However in CR2 patients, use of TBI, longer duration from diagnosis to CBT and HLA mismatched grafts were associated with decreased incidence of relapse but not with the presence of GvHD. These results showed that the impact of GvHD as a GvL marker is more evident in CR1 patients than CR2. Whereas, the impact of HLA mismatch CB on relapse is more important in CR2 patients. Decreasing immunosuppression more rapidly for CR1 patients or avoiding HLA identical CB for CR2 patients should be further investigated. In both cohorts, TBI has shown to be an important protective factor for relapse. [Display omitted] [Display omitted] DisclosuresBader:Riemser: Other: Institutional grants; Neovii: Other: Institutional grants; Medac: Other: Institutional grants; Amgen: Consultancy; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy.

The PI3Kdelta Inhibitor, CAL-101, Inhibits Migration of Primary Pre-B ALL to SDF1alpha: Treatment Implications for Overcoming Cell-Adhesion-Mediated Drug Resistance

BACKGROUND: ALL cells are physically anchored in the bone marrow (BM) microenvironment through a network of adhesion molecules. Adhesion also creates intracellular signals that regulate proliferation and cell death. We have previously identified integrin α4 (α4) as a critical molecule for such cell-adhesion-mediated drug resistance in pre-B ALL. In chronic lymphocytic leukemia (CLL), α4-mediated activation of the PI3K/AKT pathway was reported. In ALL, stromal cell protection of B-lineage ALL cells has also been shown to require active Akt. In addition, we have shown that treatment with CAL-101, a commercially available PI3Kδ inhibitor, de-adheres pre-B ALL cells from the α4-counter-receptor VCAM1, downregulates p-Akt and induces apoptosis in primary pre-B ALL cells. However, the microenvironmental stimuli that activate PI3K/AKT in ALL remain unknown. PI3Kδ can be activated by extracellular signals upon engagement with extracellular matrix, however specific ligands remain elusive, the identification of which would be critical to understand the mechanism of PI3Kδ inhibition. These studies were designed to determine which extracellular ligands activate PI3K/AKT in patient-derived (primary) pre-B ALL cells and whether CAL-101 affects adhesion and migration of ALL cells.METHODS: Four samples ofpatient-derived (primary) pre-B ALL cells, co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells were treated with a commercially available PI3Kδ inhibitor, CAL-101 (Selleckchem), or its vehicle DMSO control. Culture plates with and without transwells were used for in vitro assays. Annexin V/7-AAD staining was used for viability determination by flow cytometry and Western Blot was used for determination of total Akt and p-Akt expression.RESULTS: To determine which ligands activate p-Akt, ALL cells were adhered to fibronectin (FN), VCAM-1 (both counter-receptors of integrin α4), laminin-1 (hLam-1; counter-receptor for integrin α6) and albumin (BSA). OP9 cells were used in direct contact with ALL cells or separated through a transwell insert. α-MEM or RPMI were used as media controls. hVCAM-1 and hLam-1 activated p-Akt in one out of three samples. In all 3 samples, OP9 cells activated p-Akt compared to media control, but only in direct contact, not when separated through a transwell. This suggests that direct adhesion of ALL cells to stromal cells rather than diffusible factors are associated with p-Akt activation. Next, we determined if CAL-101 de-adheres ALL cells independently from the ligands ALL cells are bound to. Primary pre-B ALL cells were plated in triplicates onto FN, VCAM-1, hLam1-coated 96-well plates and incubated with CAL-101 (10µM) ± HUTS-21 antibody (20µg/mL). HUTS-21 is an antibody that binds only to the active form of integrin β1, CD29, and can activate outside-in signaling via integrin β1 stimulation. CAL-101 inhibited cell-adhesion from FN (15±4.2% vs. 51±9.9%, p=0.04) and VCAM-1 (54.6 x±8.6%vs. 95.8±3.8%, p=0.03), but not from hLam1 indicating that it acts specifically on the VCAM-1/FN-mediated adhesion of ALL cells. HUTS-21 restored cell-adhesion of ALL cells to FN (67.0±1.4% vs. 81±7.1%, p=0.19) and VCAM-1 (42.5±16.2% vs. 50.6±8.6%, p=0.59) indicating adhesion recovering upon HUTS-21 addition to CAL-101-treated cells. Next, we determined if CAL-101 has an impact on migration of ALL cells. ALL cells were treated with CAL-101 (10μM) or DMSO control for 24 hours and added to a migration assay. We observed that migration of ALL cells to stromal cell-derived factor 1alpha (SDF-1alpha; CXCL12) was significantly decreased (1.7 x103 ±0.3x103 vs. 3.1x103 ±2.4x103 p=0.004), while migration to OP9 cells was partially inhibited compared to DMSO control (2.1 x103 ±0.5x103 vs. 4.2 x103 ±0.5x103 p=0.005).CONCLUSION: Taken together, our data demonstrate that the PI3Kδ/AKT pathway is stimulated by extracellular matrices and that PI3Kδ inhibition with CAL-101 inhibits adhesion of ALL cells to FN/VCAM-1 but not Lam1, which can be restored using HUTS-21. Data derived from further studies will determine the potential of the FDA-approved PI3Kδinhibitor Idelalisib as a novel therapy for pre-B ALL. DisclosuresWayne:NIH: Patents & Royalties; Medimmune: Honoraria, Other: travel support, Research Funding; Kite Pharma: Honoraria, Other: travel support; Pfizer: Honoraria; Spectrum Pharmaceuticals: Honoraria, Other: travel support, Research Funding.

CD25 (IL2RA) Orchestrates Negative Feedback Control and Stabilizes Oncogenic Signaling Strength in Acute Lymphoblastic Leukemia

Background and hypothesis: CD25 (IL2RA) represents the α chain of the interleukin 2 receptor on T cells and plays an important role in the maintenance of regulatory T (Treg) cells, hence preventing T cell autoimmunity. In a comprehensive gene expression analysis, we found that CD25 is specifically upregulated by pre-B cell receptor (pre-BCR) signaling during early B cell development and oncogenic tyrosine kinase that mimic pre-BCR signaling (e.g. in Ph+ ALL and Ph-like ALL). In adults with Ph+ ALL (ECOG; MDACC) and children with Ph-like ALL (P9906) patients with CD25 expression at the time of diagnosis have a particularly poor outcome (n=416; P =0.005). For these reasons, we studied the function of CD25 in B cell development and leukemia in a series of genetic experiments.Results: Unlike T cells, CD25 (IL2RA) does not function as IL2 receptor chain in B cells and B-lineage ALL: CD25 expressed on B-lineage cells did not pair with IL2Rb and g-chains and was not responsive to IL2. Il2ra-/- B cells were arrested at the pre-B cell stage with hyperactive pre-BCR downstream signaling including SRC, BTK and ERK. In the presence of CD25, Il2ra+/+ B cells responded to engagement of the pre-BCR with phosphorylation of pre-BCR downstream tyrosine kinases and coordinated release of Ca2+ from cytoplasmic stores. In the absence of CD25 (Il2ra-/-), the pre-BCR signals autonomously, resulting in uncoordinated Ca2+ oscillations of variable duration. While CD25 does not function as IL2 receptor chain in B cells, it coordinates pre-BCR-dependent signal transduction and regulates its intensity.The pre-BCR related tyrosine kinase BTK is phosphorylated by BCR-ABL1 in Ph+ ALL and other tyrosine kinase oncogenes in Ph-like ALL (Chen et al., 2015). Interestingly, overexpression of a constitutively active form of BTK resulted in strong upregulation of CD25 surface expression. Conversely, the BTK-inhibitor ibrutinib abolished CD25 expression suggesting that feedback control between pre-BCR signaling and CD25 requires BTK.The ability of CD25 to stabilize oncogenic signaling strength in Ph+ ALL and Ph-like ALL was important for leukemia-initiation and development of fatal disease. In the absence of CD25, Il2ra-/- ALL cells showed impaired proliferation and colony formation. Serial transplantation experiments revealed a profound defect of Il2ra-/- ALL cells to initiate leukemia. 100-times more cells were required to cause fatal disease. In addition, CD25 expression mediated drug-resistance in ALL cells: In patient-derived pre-B ALL cells with heterogeneous CD25 expression, vincristine selectively induced apoptosis in CD25Low cells but spared CD25High ALL cells. Combination with an anti-CD25 immunotoxin efficiently eradiated CD25High leukemia cells and sensitized the ALL cell population to treatment with vincristine.To elucidate the mechanism of how CD25 coordinates negative feedback control of pre-BCR signaling or its oncogenic mimics, we focused on its short (13aa) cytoplasmic tail, which includes two phosphorylation sites (S268 and T271) that are known substrates for serine/threonine protein kinase, PKCα. To identify cytoplasmic interaction partners of CD25, we overexpressed a Flag-tagged truncated form of CD25 including a myristoylation signal for constitutive membrane localization, transmembrane domain and cytoplasmic tail. Immunoprecipitation (IP; Flag) followed by 2D mass spectrometry revealed strong interactions of PP2A with cytoplasmic tail of CD25. Western blots showed additional strong interactions of the cytoplasmic tail of CD25 with inhibitory phosphatases PTEN, SHP1 and SHIP1. Importantly, reconstitution of myristoylated CD25 tail but not a mutant construct lacking the serine/threonine motif (S268A/T271A) rescued proliferation and survival defects of Il2ra-/- ALL cells.Conclusion: We identified CD25 as a surface receptor that mediates membrane recruitment of PP2A, PTEN, SHP1 and SHIP1, which balances fluctuations in signaling output from a pre-B cell receptor or its oncogenic mimic in ALL cells (e.g. BCR-ABL1 in Ph+ ALL). We propose that CD25-mediated negative feedback control stabilizes oncogenic tyrosine kinase signaling and mediates drug-resistance in Ph+ ALL and Ph-like ALL cells. Targeted inhibition using CD25-directed immunotoxins may be useful in new approaches to overcome drug-resistance in Ph+ ALL and Ph-like ALL. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetic Profile of De Novo B lineage Acute Lymphoblastic Leukemia: Determination of Frequency, Distribution Pattern and Identification of Rare and Novel Chromosomal Aberrations in Indian Patients.

Chromosomal aberrations identified in acute lymphoblastic leukemia (ALL) have an important role in disease diagnosis, prognosis and management. Information on karyotype and associated clinical parameters are essential to physicians for planning cancer control interventions in different geographical regions. In this study, we present the overall frequency and distribution patterns of chromosomal aberrations in both children and adult de novo B lineage ALL Indian patients using conventional cytogenetics, interphase FISH and multiplex RT-PCR. Among the 215 subjects, cytogenetic results were achieved in 172 (80%) patients; normal karyotype represented 37.2% and abnormal 62.8% with a distribution as follows: 15.3% hypodiploidy; 10.3% hyperdiploidy; 15.8% t(9;22); 9.8% t(1;19); 3.7% t(12;21); 2.8% t(4;11); 2.8% complex karyotypes. Apart from these, we observed several novel, rare and common chromosomal rearrangements. Also, FISH studies using LSI extra-signal dual-color probes revealed additional structural or numerical changes. These results demonstrate cytogenetic heterogeneity of ALL and confirm that the incidence of chromosomal abnormalities varies considerably. To the best of our knowledge, this is one of the largest reported series of cytogenetic investigations in Indian B-lineage ALL cases. In addition, ongoing cytogenetic studies are warranted in larger groups of B-lineage ALL cases to identify newly acquired chromosomal abnormalities that may contribute to disease diagnosis and management.

Meta-analysis and meta-regression analysis to compare the outcomes of chemotherapy for T- and B-lineage acute lymphoblastic leukemia (ALL): the use of dexamethasone, l-asparaginase, and/or methotrexate may improve the outcome of T-lineage ALL

The effects of intensive regimens and the roles of drugs used might differ between T- and B-lineage acute lymphoblastic leukemia (ALL). We performed a literature search for clinical studies published from January 1998 to March 2013. Studies were eligible for inclusion in the analyses if they included more than 80 patients with adult ALL who were treated with a uniform regimen and compared T- and B-lineage ALL. Studies that included only adolescent or elderly patients were excluded. We identified 11 clinical studies, which included a total of 381 and 1366 patients with T- and B-lineage ALL, respectively, and performed meta-analyses using the selected studies. Nine studies included patients with Philadelphia chromosome-positive (Ph+) ALL. A meta-analysis using the random-effect model demonstrated superior survival in patients with T-lineage ALL compared to those with B-lineage ALL (hazard ratio 1.78, 95% confidence interval 1.50-2.11), though the inclusion of patients with Ph+ ALL in B-lineage ALL must have influenced this result strongly. We performed meta-regression analyses, adjusted according to whether or not patients with Ph+ ALL were included in each study. Use of dexamethasone (Dex), higher dose of methotrexate (MTX), and higher dose of L-asparaginase (L-asp) were associated with a significant trend toward a better outcome in T-lineage ALL. A meta-regression analysis including Dex and the dose of L-asp or MTX together as covariates showed that these factors were independently significant. In conclusion, the use of Dex and high-dose L-asp or MTX may improve the outcome of T-lineage ALL. This hypothesis should be tested in a prospective study including only patients with Ph-negative ALL.

Abstract 1124: Transcriptional control of B cell identity restricts metabolic fitness in human leukemia

Abstract Oncogenic lesions in multi-potent progenitor cells often give rise to either B-cell or myeloid lineage leukemia. While transformed by the same oncogenes (e.g. BCR-ABL1, RAS), B-lineage and myeloid leukemias are distinct diseases. Given that oncogenic tyrosine kinase signaling (e.g. BCR-ABL1) imposes significant metabolic requirements on energy supply, biogenesis and metabolic fitness, we studied whether the divergent characteristics of myeloid and B-lineage leukemias have a metabolic basis. Metabolic analyses revealed that B-lineage acute lymphoblastic leukemia (Ph+ ALL) cells proliferate at maximum capacity of their glycolytic machinery. In contrast to myeloid leukemia (CML), B-lineage ALL cells lack metabolic adaptive fitness in response to metabolic fluctuations. C/EBPα-mediated reprogramming of B-lineage cells into the myeloid lineage induced glycolytic gene expression (Insr, Slc2a1, G6pdx, G6pd2, and Hk3). Frequent genetic lesions of transcription factors that determine B cell identity (IKZF1, PAX5, EBF1) partially mitigate B cell-instrinsic metabolic liability. Reconstitution of PAX5 expression in patient-derived B-lineage ALL cells reduced metabolic fitness by impacting glucose metabolism. Using genetic and metabolic experiments, we identified the metabolic liability observed in B-lineage ALL is in part dependent on the serine/threonine kinase LKB1. In agreement with previous studies, Cre-mediated deletion of Lkb1 induced proliferation in myeloid leukemia. Surprisingly, Lkb1 deletion led to apoptosis and decreased leukemogenic capacity in B-lineage leukemia. Consistent with the above observations, Arf, p53 and p27 levels were reduced in Lkb1-deficient myeloid leukemia cells, while Lkb1 deletion in B-lineage ALL cells up-regulated Arf, p53 and p27 levels. Enhanced glucose consumption and lactate production were observed in Lkb1-deficient myeloid leukemia cells. In contrast, loss of Lkb1 led to defective glycolytic and mitochondrial activity in B-lineage ALL. Lkb1 deletion in B-lineage ALL caused global accumulation of metabolites, suggesting that LKB1 is required for maintaining metabolic homeostasis. Moreover, loss of Lkb1 decreased protein levels of mitochondrial, anti-apoptotic BCL-2 family proteins, BCL-xL and MCL1, in B-lineage ALL. Reverse Phase Protein Array analyses revealed that LKB1 levels positively correlated with BCL-xL and MCL1 in patient-derived Ph+ ALL samples (n = 51) as well as other subtypes of B-lineage ALL (n = 183; MDACC, 1983-2007). Importantly, C/EBPα-mediated reprogramming of B-lineage ALL cells to the myeloid linage relieved dependency on LKB1. Taken together, we showed that transcriptional control of B cell identity causes unique metabolic liability. B-lineage ALL cells exhibit unique reliance on LKB1 for metabolic homeostasis and survival. Our findings revealed LKB1 as a potential therapeutic target in B-lineage ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Daniel Braas, Christian Hurtz, Seyedmehdi Shojaee, Huimin Geng, Valeria Cazzaniga, Carina Ng, Behzad Kharabi Masouleh, Yi Hua Qiu, Nianxiang Zhang, Kevin R. Coombes, Thomas Ernst, Giovanni Cazzaniga, Andreas Hochhaus, Steven Kornblau, Thomas Graeber. Transcriptional control of B cell identity restricts metabolic fitness in human leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1124. doi:10.1158/1538-7445.AM2015-1124

Abstract 1339: Preclinical combination activity of SGN-CD19A and CVAD in patient-derived B-lineage acute lymphoblastic leukemia models

Abstract Combining targeted therapies with standard of care chemotherapy is a proven method for improving outcomes for cancer patients. SGN-CD19A is an antibody-drug conjugate (ADC) targeting the B-lineage specific marker CD19, and it is currently in phase 1 clinical trials for treatment of B-lineage acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). HyperCVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone) is a common frontline chemotherapy regimen used to treat adult ALL. To explore the potential for combining SGN-CD19A with these agents (CVAD) ALL patient-derived xenografts (PDX) were developed, as these models are known to more closely reflect clinical disease. PDX were established by intravenous implant of 1-5×106 unsorted ALL patient bone marrow isolate cells into irradiated NOD scid IL2Rgammanull (NSG) mice. Initially, patient isolates were comprised of a wide-ranging (20-77%) population of CD19+/CD10+/CD45low blast cells and a varied population (5-70%) of CD19+/CD10+/CD45high cells. After implant, cells homed to and expanded in the bone marrow, thereafter progressing into the spleen and peripheral blood. After passage in mice, phenotyping revealed an enrichment of the CD19+/CD10+/CD45low blast cells and a loss of the CD19+/CD10+/CD45high population. These passaged cells were utilized for subsequent activity experiments. To evaluate PDX sensitivity to SGN-CD19A, a single dose at 1, 3 or 10 mg/kg was administered when bone marrow in sentinel animals contained between 5-24% blast cells. Activity was measured by the extent of ALL blast cell reduction in the bone marrow compared to untreated mice. SGN-CD19A elicited a dose-dependent depletion (71-99%) of blast cells in bone marrow by day 14 post dose. Like SGN-CD19A, CVAD also produced a dose-dependent depletion of the ALL blasts. Next, PDX models were used to examine SGN-CD19A in combination with CVAD. SGN-CD19A + CVAD produced a statistically superior depletion of blast cells when compared to SGN-CD19A or CVAD alone. To better understand these combined therapies, NALM-6 and RS4;11 cell line disseminated xenografts were also evaluated. SGN-CD19A + CVAD significantly prolonged survival compared to either single treatment alone in these cell line derived xenografts as well. Remarkably, SGN-CD19A in combination with vincristine (NALM-6, RS4;11) or dexamethasone (RS4;11) resulted in a survival advantage that was similar to that of SGN-CD19A + CVAD. When SGN-CD19A was tested in combination with CVAD and CVAD components in the PDX models, it was observed that SGN-CD19A potentiated the single-agent activity of vincristine, cyclophosphamide and doxorubicin. Together, these data suggest that SGN-CD19A can combine with a frontline ALL chemotherapy regimen and potentially improve outcomes for patients with ALL. Citation Format: Ivan J. Stone, Tina Albertson, Che-Leung Law. Preclinical combination activity of SGN-CD19A and CVAD in patient-derived B-lineage acute lymphoblastic leukemia models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1339. doi:10.1158/1538-7445.AM2015-1339

Eradication of B-ALL using chimeric antigen receptor–expressing T cells targeting the TSLPR oncoprotein

AbstractAdoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell–associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.

Can we be SMaRT-er in our approach to cancer therapy?

Can we be SMaRT-er in our approach to cancer therapy?

Prognostic Significance of Copy Number Alterations in B-lineage Adult Acute Lymphoblastic Leukemia Patients Enrolled in Risk-adapted Protocols from the PETHEMA Group

Abstract Introduction In the last years genome wide profilings have identified recurrent Copy Number Alterations (CNA) in genes potentially involved in the pathogenesis of Acute Lymphoblastic Leukemia (ALL). These studies have identified deletions in B-cell development genes (IKZF1, EBF1, PAX5, TCF3, etc.), cell cycle regulation genes (CDKN2A/B, RB1, TP53, etc.), glucocorticoid resistance genes (BTG1, CREBBP) and growth factor receptors genes (CRLF2, CSF2RA, IL3RA) among others. Some of these CNA (i.e. IKZF1, CDKN2A, CRLF2) have been reported to have prognostic significance in several pediatric series but there are very few data regarding their impact in B-lineage adult ALL. Our aim was to analyze the frequency and prognostic significance of CNA in a series of 125 B-lineage adult ALL patients treated according to risk-adapted protocols from the Spanish PETHEMA Group. Methods Bone marrow or peripheral blood (with significant blast burden) samples from 125 B-lineage adult ALL patients enrolled in risk-adapted protocols from the PETHEMA Group were analyzed at diagnosis. MLPA assays (MRC-Holland) were performed for the following genes: IKZF1, IKZF2, IKZF3, EBF1, CDKN2A/B, PAX5, ETV6, BTG1, RB1, hsa-miR-31, X/Y PAR1 region genes (CRLF2, CSF2RA, IL3RA) and 14q32.33 region genes (IGH D, MTA1, KIAA0284). Fragment analysis was made by Genescan in an ABI-3130 sequencer (Applied Biosystems). Data normalization provided a value indicative of the presence or absence of CNA: 0-0.20 homozygous deletion, 0.21-0.70 heterozygous deletion, 0.71-1.30 normal, 1.31-1.70 heterozygous duplication and 1.71-2.20 homozygous duplication. Results The median age [range] was 40 [15-74] years, 71 (57%) males, median WBC count 12.11 x109/L [0.4-388]. Immunophenotype: pro-B 14 (11%), common 71 (58%), pre-B 26 (21%), mature-B 10 (8%), unavailable 2 (2%). Cytogenetics: normal 16 (13%), hyperdiploid 6 (5%), hypodiploid 2 (2%), t(9:22) 20 (16%), t(1;19) 8 (6%), 11q23/MLL 11 (9%), 8q24/C-MYC 7 (5%), complex 1 (1%), iAMP21 2 (2%), other translocations or deletions 31 (25%), no growth 20 (16%). CNA frequencies of the 125 patients are shown in the table. Table . Deletion Duplication Normal IKZF1 38 (30%) 1 (1%) 86 (69%) EBF1 11 (9%) 7 (5%) 107 (86%) CDKN2A/B 44 (35%) - 81 (65%) PAX5 33 (26%) 4 (4%) 88 (70%) hsa-miR-31 23 (18%) - 102 (82%) ETV6 7 (6%) 1 (1%) 117 (93%) BTG1 11 (9%) 3 (2%) 111 (89%) RB1 20 (16%) - 105 (84%) 14q32.33 region 20 (16%) 11 (9%) 94 (75%) X/Y PAR1 6 (5%) 16 (13%) 103 (82%) IKZF2 - 4 (3%) 121(97%) IKZF3 2 (2%) 8 (6%) 115 (92%) IKZF1 deletions were significantly associated with EBF1 deletions, high WBC count and Philadelphia (Ph) chromosome. In the IKZF1 deleted cohort whole gene deletions were as frequent as Ik6 isoforms (28% each). A high codeletion rate was detected in genes located in 9p (CDKN2A/B with PAX5, CDKN2A/B with hsa-miR-31 and PAX5 with hsa-miR-31). CDKN2A/B also showed concomitant deletions with ETV6 while PAX5 showed codeletions with BTG1. CDKN2A/B and PAX5 deleted patients had higher WBC counts than non-deleted individuals. Clinical follow-up data was available for 123 patients of the whole series and for the 105 patients of the Ph-negative cohort. Multivariate analysis showed that advanced age, BTG1 deletions and EBF1 deletions were negative prognostic factors for achieving Complete Remission (CR) and WBC count and IKZF1 deletions significantly reduced CR duration in both cohorts. Interestingly, there were significant differences in relapse rates between whole and partial gene IKZF1 deletions. IKZF1 haploinsufficient patients had a probability of CR duration at 3 years of 83% ± 30% vs. 6% ± 12% of partial gene deletion carriers. Advanced age and IKZF1 deletions were predictors for overall survival in the Ph-negative cohort and age>30 years, IKZF1 deletions and hsa-miR-31 deletions were associated with poor prognosis in the whole series. Conclusions In B-lineage adult ALL, deletions of IKZF1, EBF1, BTG1 or hsa-miR-31 are markers with prognostic significance in addition to age and WBC count. Patients with partial IKZF1 gene deletions have a significantly higher probability of relapse than those with whole gene loss. These genetic abnormalities could help to better define prognostic subgroups in adult patients with B-lineage ALL. Supported by the grants PI10/01417 and RD12-0036-0029 from Instituto Carlos III and a grant from the Spanish Society of Hematology and Hemotherapy (2012). Disclosures: No relevant conflicts of interest to declare.

CD20 Expression in Adult Patients with B-lineage Acute Lymphoblastic Leukemia and Its Prognostic Significance

To explore the relationship between the expression of CD20 antigen and clinical characteristics in adult patients with B-acute lymphoblastic leukemia(B-ALL). The CD20 expression of 126 acute lympho-blastic leukemia patients in our hospital from July 2009 to July 2012 were determined by flow cytometry. The characteristics, examination results and outcome were analyzed retrospectively. The complete remission rate (CR rate), relape rate, 2-year survival rate and 2-year event-free survival (EFS) of patients with CD20 positive and negative after the first cycle of chemstherapy were compared. Positive rate of CD20 antigen expression in 126 patients was 24.4% (31 cases), negative rate of CD20 antigen expression in 126 patients was 75.6% (95 cases). No significant relationship was found between CD20 antigen expression and sex, age, peripheral blood leucocytes count and chromosomal changes. The relapse rate, 2-year survival rate (OS) and 2-year event-free survival (EFS) of adult patients with B-ALL in CD20 positive and negative groups were 53.3% and 38.0%, 52.1% and 92.3%, 33.7% and 70.8% respectively. Expression of CD20 in adult patients with B-ALL did not related with clinical features, but related with poor prognosis.