ALBERT

Description: Backgroud: Despite advances in therapy and improved survival, relapsed and refractory B-cell precursor acute lymphoblastic leukemia (r/r BCP-ALL) in pediatric and adult patients still remains a problem. Chimeric antigen receptor T cells against CD19 (CD19 CAR T) show promising results in patients with r/r BCP-ALL. However, relapse of the disease still occurs with appreciable frequency even with this novel therapy. As a significant number of relapses post-CAR T lack surface CD19 expression, further CD19-directed therapy is not an option for these cases. Hypothesis: Sometimes despite CAR T engraftment and establishment of B-cell aplasia, relapse still occurs. We hypothesized that, similarly to cell adhesion mediated chemotherapeutic drug resistance (CAM-DR), cell adhesion mediated CAR T-cell resistance (CAM-CART-R) can contribute to relapse of ALL. Results: To test our hypothesis, primary ALL cells were treated with CD19 CAR T cells either with murine calvaria-derived bone marrow stromal cells, OP9, or cultured only with media in short term cultures. We observed B-ALL cells treated with CD19 CAR T on OP9 has 10-20% higher viability compared to B-ALL and CD19 CAR T co-culture in medium alone, supporting the notion of CAM-CART-R. We also determined that soluble factors in OP9 primed medium may contribute to CAM-CART-R. However, the direct stromal contact mediated significant protection again CAR T induced apoptosis of B-ALL cells. To determine the molecular mechanisms underlying the survival promoting effects of stromal cells on CD19-, these cells were starved in serum-free media for 4hours and then treated with PI3Kδ inhibitor CAL-101 or DMSO and co-cultured with OP9 cells for 1 hour. We found that p-Akt is upregulated by stromal contact in CD19-negative B-ALL cells post-CAR T therapy and that PI3Kδ inhibition using can downregulate p-Akt in CD19-negative B-ALL patients. Critically, we investigated whether CD19 CAR T cells were functional under these conditions. For this purpose, we determined if stromal contact of ALL cells or stromal contact of CAR T cells changes the intracellular cytokine milieu of CD19 CAR T cells and found that intracellular IL-6, TNF- α and IFN-γ were reduced upon stromal contact supporting our hypothesis of a role of stromal cells in CAM-CART-R. We also determined that immune checkpoints molecules on T cells are unaffected by OP9 cells. Despite the reduction of cytokine level in T cells upon co-culture with B-ALL cells on OP9, PD-1, TIM-3 and LAG3 expression on CD19 CAR T cells after 2 days of co-culture was not altered as determined by flow cytometry. Resistance of ALL cells to CD19 CART cells was not mediated through checkpoint inhibition, since the PD-1/PD-L1 inhibitor Nivolumab failed to enhance ALL killing. Phenotypic profiling of thirteen cases of primary ALL relapse post-CD19 CAR T cell therapy showed high expression of adhesion molecules including integrin α4. Phenotypic analysis also revealed high expression of integrins is retained in primary ALL cells after CD19 knockout in one case. To explore possible solutions to overcome CAM-CART-R, we examined a strategy of blocking specifically integrin α4. We have previously shown that blocking integrin α4 can de-adhere CD19-negative B-ALL relapse post-CAR T cell therapy from their respective counter-ligands in vitro and that these cells can benefit from integrin blocking therapy in vivo. We have now confirmed this in NSG mice injected with CD19-negative B-ALL cells from a patient with post-CAR T cell relapse. Mice were treated intraperitoneally (n=6/group) with total immunoglobulin (Ig) control or humanized anti-human integrin α4 antibody Natalizumab (NZM). As a result, Natalizumab monotherapy significantly prolonged survival of leukemic mice compared to control Ig group (66 days (Ig) vs 85 days (NZM) p

Description: MRD detected by flow cytometry (FC) or PCR has been associated with key outcomes after HCT for ALL. In a prospective multicenter trial (NCT02646839; Pediatric Blood and Marrow Transplant Consortium [PBMTC] ONC1401), we performed a planned sub-study of NGS-MRD to predict outcomes pre- and post-HCT for ALL patients (n=57, median follow-up 523 [range 58-1198] days post-HCT). In addition, we performed analyses of relevant clinical factors to assess their relationship to EFS. The larger trial studying the role of KIR favorable haploidentical vs. other transplantation approaches for children diagnosed with ALL, acute myeloid leukemia and myelodysplastic syndrome will be reported separately. We evaluated baseline blast samples from 74 patients for dominant BCR/TCR rearrangements and to follow MRD by NGS. Dominant rearrangements were identified in 100% of B-ALL patients, 96.8% (61/63) in BCR and 3.2% in TCR gamma. For T-ALL patients, rearrangements were identified in 62.7% (7/11), with the remaining 37.3% being polyclonal. Patients proceeded to HCT only if they were in morphological remission. Pre-HCT NGS-MRD from bone marrow (BM) was highly predictive of EFS (n=29 P=0.027, Figure 1) and although NGS-MRD from peripheral blood (PB) did not reach statistical significance due to decreased sample size (n=27, P=0.17, Figure 2), it trended similarly. In BM NGS-MRD negative patients, relapse was exceptionally low with all events due to transplant related mortality (TRM). There did not appear to be a benefit of acute graft-vs-host-disease (aGVHD) in NGS-MRD- patients (Figure 3) or chronic (c)GVHD, but sample size was a limitation. Pre-HCT, 10% of the BM samples were MRD+ by FC, but 35% were MRD+ by NGS. Direct comparison of NGS-MRD in BM and PB with FC MRD pre- and post-HCT showed improvements positive and negative predictive power (data not shown). Alpha/beta depleted haploidentical grafts had similar outcomes to other stem cell sources (Figure 4) with decreased incidence of GVHD [aGVHD 〉 grade 2: n=1 (3.3%) and extensive cGVHD: n=1(3.3%)]. TBI (total body irradiation) based myeloablative conditioning (TBI/TT [Thiotepa]/CY [Cyclophosphamide], TBI/CY, or TBI/VP16; ± anti-thymocyte globulin [ATG]) and non-TBI reduced toxicity (Flu [Fludarabine]/Mel [Melphalan]/TT; ± ATG) had identical EFS (P= 0.31). TRM was very low 8.7%(n=5) in this population (n=57)) and rescue of relapse was high for the duration of follow up to date, resulting in similar OS for MRD- vs. MRD+ patients (P= 0.15), likely due to rescue with cell/immunotherapy. We next examined the interaction of obesity, using body mass index (BMI) based on height/weight pre-HCT, in the context of NGS-MRD on EFS. The BMI score was converted to a percentile through population norms for age and gender and defined thresholds published by the Centers for Disease Control and prevention (CDC). Lean patients (〈 85th percentile [%]) overall had better survival than the overweight (OW)/obese (85-94%/≥95%) (Figure 5: P=0.016). But among the lean patients, NGS-MRD still had an effect, with NGS-MRD+ patients had poorer EFS. Same was observed with the OW/obese group, where being NGS MRD+ adds further reduction in survival. Overweight/Obese patients who were pre-HCT MRD- had survival closer to the lean/MRD+ patients. We observed decreased EFS by weight category and NGS-MRD (Figure 6: P= 0.02). In conclusion, NGS-MRD was highly predictive for EFS regardless of HCT preparative approach or graft Alpha/Beta Depletion. NGS improved predictive power for MRD positivity compared to FC. NGS was able identify rearrangements suitable for MRD tracking in all B-ALL and in most of T-ALL samples. In BM NGS-MRD negative patients, relapse was exceptionally low. There did not appear to be a benefit of aGVHD or cGVHD in NGS-MRD- patients. Alpha/beta depleted haploidentical grafts had similar outcomes to other stem cell sources with decreased incidence of severe GVHD. We observed decreased EFS by weight category and NGS-MRD. Lean patients had better survival, after HCT, than OW/Obese patients. But among the lean patients, NGS-MRD+ patients had poorer EFS. Disclosures Abdel-Azim: Adaptive: Research Funding. Dvorak:Jazz Pharmaceuticals: Consultancy; Alexion Inc: Consultancy. Bunin:PRA Health Sciences: Other: Immediate family member employed. Walters:Editas Medicine: Consultancy; TruCode: Consultancy; AllCells, Inc: Consultancy. Cairo:Jazz Pharmaceuticals: Other: Advisory Board, Research Funding, Speakers Bureau; Miltenyi: Other: MTA; Osuka: Research Funding. Kitko:Novartis: Consultancy, Honoraria; Mallinckrodt: Honoraria. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Wing:Miltenyi Biotec: Employment. Pulsipher:Medac: Honoraria; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Other: Education for employees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Key Points The genetic cause of SCID impacts on survival and immune reconstitution and should be considered in tailoring HCT for individual patients. Total and naive CD4+ cell counts in SCID patients 6 and 12 months post-HCT predict long-term survival and sustained immune reconstitution.

Description: Background. Even though remarkable progress has been made in the treatment of childhood acute lymphoblastic leukemia (ALL), salvage of relapse patients remains a challenge. The role of the bone marrow (BM) microenvironment is critical to protect leukemia cells from chemotherapy. The BM microenvironment promotes cell adhesion-mediated drug resistance (CAM-DR) in ALL.We and others have shown that the adhesion molecule integrin α4, referred to hereafter as α4, mediates drug resistance of B-ALL. In our previous studies, we showed that both α4 blockade by natalizumab and inhibition by the small molecule α4 antagonist TBC3486 can sensitize relapsed ALL cells to chemotherapy. However, no α4 targeting therapy is currently clinically available to treat leukemia. Here, we preclinically evaluate a novel non-peptidic small molecule antagonist, AVA4746, which has been safely used in clinical studies, as a potential new approach to combat drug resistant ALL. Method. Six refractory or relapsed primary pre-B ALL cases were used for in vitro studies. Viability was assessed by trypan blue counts or annexin V/7AAD flow cytometric analysis and metabolic activity was evaluated by Cytoscan WST-1 assay. For in vivo evaluation a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL (LAX7R) was used.AVA4746 (15mg/kg) was administered by oral gavage twice a day continuously for 14 days, and vincristine, dexamethasone, L-asparaginase (VDL) was given intraperitoneally (weekly) for 4 weeks. Overall survival was determined by Kaplan-Meier Survival analysis. Results. AVA4746 caused a significant decrease in mean fluorescence intensity (MFI) of α4 expression in six out of six ALL cases at doses of both 5μM and 25μM after 24 hours and 96 hours compared to DMSO control. Interestingly, decreased protein expression of α4 was also observed by Western Blot analysis all six ALL cases. We tested next in two cases (LAX53, ICN13), if AVA4746 de-adheres ALL cells from its counter receptor VCAM-1. The percentages of adherence after treatment with AVA4746 (25μM) were significantly lower than after DMSO treatment (10.3%±4.9% vs. 99.9%±7.6%, p= 0.00007 for LAX7R; 8.1%±1.0% vs. 100.1%±13.6%, p= 0.0003 for LAX53; 9.0%±1.6% vs. 100.0%±14.0%, p=0.0004 for ICN13). AVA4746 was not associated with apoptosis in vitro alone or in combination with chemotherapy (VDL). Metabolic activity as assessed by WST-1 assay was markedly decreased by AVA4746 in two of two ALL cases. AVA4746 also decreased ALL proliferation in two out of two ALL samples tested. In vivo, AVA4746 in combination with VDL chemotherapy treatment led to significant prolongation of overall survival (n=6) compared with the VDL only treated group (n=6) (MST= 78.5 days vs MST= 68 days; P

Description: 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. Disclosures Wayne: 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.

Description: Abstract 3052 The initial analysis of the impact of cGVHD on thymic function used TREC analysis and demonstrated a sustained decrease in thympoiesis in hematopoietic stem cell transplant (HSCT) recipients with a history of cGVHD (Blood 97:1458, 2011). We have reported that HSCT recipients, who had the clinical resolution of their cGVHD, had increases in their resting regulatory T lymphocytes (rTreg) as compared to HSCT recipients with active cGVHD [ASH 2011]. The increase in rTreg lymphocytes suggested that clinical resolution of cGVHD might be due to improved thymic function, which resulted in the increased production of rTreg lymphocytes. To measure the thymic function of pediatric HSCT recipients with cGVHD, we determined the frequency of recent thymic emigrants (RTE; CD4+, CD45RA+, CD31+) in the whole CD4 T lymphocyte population and the CD4 regulatory T (Treg; CD127-, CD25+) and CD4 conventional T lymphocytes (Tcon; CD127+, CD25+) subpopulations of normal individuals, HSCT recipients without a history of cGVHD (n =16) and HSCT recipients with a history of cGVHD (n=20). The frequency of RTE in total, Treg, and Tcon lymphocytes of the HSCT recipients with cGVHD was decreased (P=0.001) compared to HSCT recipients without a history of cGVHD but was the same as normal individuals. Thus, pediatric HSCT recipients with cGVHD have thymic function that is similar to normal individuals, but have decreased thymic reserves compared to HSCT recipients without cGVHD. However, since rTreg lymphocytes (CD4+, CD127-, CD25+, CD45RA+, FoxP3+) represent only 20% of total Treg lymphocytes, we specifically determined the thymic contribution to rTreg lymphocytes. The RTE content of rTreg lymphocytes was determined in HSCT recipients with active or resolved cGVHD. The RTE content of the rTreg lymphocytes (as a percentage of total CD4 T lymphocytes) was similar in HSCT patients with both active and resolved cGVHD. Thus, the increase in rTreg lymphocytes present in HSCT recipients with the clinical resolution of their cGVHD is not due to an increase in the frequency of RTE in their rTreg lymphocyte population. Therefore, the increase in rTreg lymphocytes, that is associated with the clinical resolution of cGVHD, may be due to either their decreased conversion to Th17 lymphocytes or their decreased apoptosis. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Obesity is associated with increased risk for persistent minimal residual disease after induction therapy for pediatric BP-ALL.

Description: Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Description: We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (〈 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

Description: Key Points Reduced intensity and myeloablative regimen results in comparable survival after allogeneic transplantation.