ALBERT

Description: Key Points Increased immune suppressors and PD-1 abrogates effector responses in CML patients at diagnosis. Enhanced net effector immune responses and decreased PD-1 and immune suppressors may promote sustained deep molecular response in CML.

Description: We hypothesized that immune responses contribute to deep BCR-ABL molecular responses in chronic phase chronic myeloid leukaemia (CML) patients on tyrosine kinase inhibitors (TKI). We studied 32 CML patients; 16 at diagnosis, patients treated with imatinib (n=20), nilotinib (n=9) or dasatinib (n=3). Methodology: The effector immune responses of Natural Killer (NK) cells were characterized by flowcytometry and functional analysis by CD107a degranulation assay. Cytotoxic T lymphocyte (CTL) responses to leukaemia-associated antigens (LAAs) WT1, BMI-1, PR3 and PRAME were quantified by interferon-gamma ELISPOT using peptide libraries of 15-mer peptides overlapping by 11 amino acids spanning the entire protein, or HLA-A0201 specific peptides in HLA-A0201+ patients. Immune suppressor regulatory T cells (Treg), Myeloid Derived Suppressor Cells (MDSC), Programmed cell death-1 (PD-1) expression on T cells, NK cells, B cells and monocytes, and major B cell subsets were extensively characterized by flowcytometry. Results: Patients in deep molecular response (MR4.5; BCR-ABL

Description: Introduction:An attempt at tyrosine kinase inhibitor (TKI) withdrawal in deep molecular remission leads either to treatment free remission (TFR) or early molecular relapse (MolR) in chronic myeloid leukaemia (CML) patients. We hypothesise that immune responses promote sustained TFR and immunological markers may predict response following TFR attempt. Methodology: We studied 54 CML patients (from ALLG trials CML 8, median follow-up 66 mo, and CML 10, median follow-up 24 mo) at baseline on TKI (minimum 24 mo MR4.5) and 3 mo and 6 mo following TKI discontinuation. MolR was defined as any single sample on follow-up with BCR-ABL1 〉0.1% or two consecutive BCR-ABL1 positive samples at any value. Effector immune responses of CD56dim natural killer (NK) cells and NK cell receptor repertoire were characterised by flow cytometry and cytotoxic T lymphocyte (CTL) responses to leukaemia-associated-antigens (LAAs) WT1, BMI-1, PR3 and PRAME by interferon-gamma ELISPOT. Immune suppressor regulatory T cells (Treg; CD4+CD25brightCD127-FoxP3+), Granulocytic and Monocytic Myeloid-Derived Suppressor Cells (MDSCs; HLA-DR-Lin-CD11b+CD33+CD66b+CD15+ and HLA-DR-Lin-CD11b+CD33+CD66b-CD14+, respectively), Programmed cell death-1 (PD-1) expression on T cells, NK cells, B cells and Monocytes, and major B cell subsets were characterized by flow cytometry. Results: TFR patients displayed increased CD3-CD56dimCD16bright cytolytic NK cells as a proportion of total lymphocytes at baseline (n=23, 27.1% ± 2.9) vs MolR (n=23, 19.1% ± 2.0, p=0.02). TFR patients displayed a more mature CD56dim CD57+ NK cell phenotype at baseline (74.5% ± 2.2 of total NK cells) vs MolR (66.3% ± 2.7, p=0.04). Extensive characterisation of NK cell receptor repertoire revealed NKG2D activating receptor expression was increased in TFR patients (baseline= 56.8% ± 3.8, 3 mo= 61.4% ± 5.0, 6 mo= 49.9% ± 5.8) vs MolR (baseline= 44.2% ± 3.7, 3 mo= 42.2% ± 5.5, 6 mo= 22.0% ± 8.3, all p=0.02). KIR2DL2/DL3/DS2-positive NK cells were increased in MolR patients at 3 and 6 mo vs TFR. (MolR; 3 mo= 44.8% ± 4.6, 6 mo= 48.8% ± 4.9. TFR; 3 mo= 31.5% ± 4.0 p=0.05, 6 mo= 31.1% ± 2.1, p=0.001). No significant differences were observed in CD56brightCD16-/dim immunoregulatory NK cells, C-type lectin receptor expression (CD94/NKG2A/NKG2C, CD161, CD69), Natural cytotoxicity receptors (NKp30, NKp44, NKp46), CD62L (on T cells and NK cells) and KIR2DL5 expression. No difference in NK Cell-mediated K562 degranulation as a surrogate marker of NK cell function was observed between TFR and MolR patients. Functional CTL immune responses were observed in TFR and MolR patients. BMI-1 CTL responses were increased at baseline in TFR (23%) vs MolR (9%). PR3 CTL responses were not detected in TFR at baseline, 3 mo or 6 mo (0%) vs MolR (baseline= 18%, 3 mo= 50%, 6 mo= 50%). No difference was observed in WT1 or PRAME CTLs. Quantification of immune suppressor cell types revealed decreased Monocytic MDSCs in TFR patients at baseline (10.0% ± 2.3) vs MolR (17.7% ± 3.1, p=0.02). There was no difference in granulocytic MDSCs or Treg between TFR and MolR. No difference in PD-1 expression was observed on NK cells, T cells, B cells and Monocytes. Extensive characterisation of B cell subsets revealed no difference in TFR vs MolR (Table 1). Conclusion: In keeping with STIM and EURO-SKI trials, a threshold level of particular NK cell subsets may be important in maintaining TFR. We found additionally that enhanced NK and CTL effector responses and decreased inhibitory NK KIR2DL2/DL3/DS2 expression, in combination with reduced monocytic MDSC may promote sustained TFR. Methods to enhance nett immune effector responses, such as mature CD56dimCD57+ NK cells and BMI-1 CTL responses or targeting inhibitory KIR may increase TFR success rates. Disclosures White: Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria. Yong:Celgene: Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Description: Chimeric antigen receptor T-cells (CAR Tc) have yielded impressive remission rates in treatment-refractory B-cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in the first FDA approved CAR Tc therapies, Kymriah and Yescarta. However, the translation of these results for other cancer entities remains a challenge. Pre-clinical studies using second-generation CAR Tc against the interleukin-3 receptor alpha chain (CD123) engendered strong anti-leukemic activity. CD123 CAR Tc clinical studies resulted in transient responses, or complete remission but at the expense of on-target off-tumor toxicities. Our studies employing third-generation anti-CD123 CAR Tc demonstrate strong anti-leukemic activity with no adverse effects in vivo. However, the leukemia was not completely eradicated. Combining anti-CD123 CAR Tc with DNA hypomethylating (HMA) agents may enhance the anti-leukemic effect and survival. HMAs such as azacytidine (Aza) activate key epigenetically silenced pathways in AML cells, inhibiting cell proliferation while enhancing cell immunogenicity. We hypothesized that Aza will increase the expression of CD123 on AML cells resulting in long-term disease eradication by anti-CD123 CAR Tc. The anti-leukemic efficacy, survival advantage, safety and feasibility of the combination treatment with Aza and anti-CD123 CAR Tc were evaluated in vivo. HL-60 (CD123med), MLL-2 (CD123lo), MOLM-13 (CD123hi), primary de novo and relapsed/refractory (r/r) AML cells were cultured for 0-8 days in the presence of Aza (0µM-5µM) and analysed for their CD123 expression by flow cytometry, quantitative western blot and RNAseq. The anti-CD123 CAR was constructed with the humanized CSL362-based ScFv and the CD28-OX40-CD3ζ signaling domain, encoded in a third-generation lentiviral vector and expressed in CD3+ Tc from healthy donors. Rag2γc-/- mice (n=12-16/ group) were engrafted with 1x105 MOLM13/ffLuc AML cells and treated with PBS, 5x106 Non-transduced (NTD) Tc orCAR Tc, 4x 2.5mg/kg Aza, or 5x106 CAR Tc following 4x Aza (2.5mg/kg). Leukemic burden was assessed weekly by bioluminescence imaging. Tc activity and immunophenotyping was performed using flow cytometry at day 35 post engraftment, and survival was monitored. HL-60, MLL-2 and MOLM-13 cells showed significant increases in HLA-DR, PD-L1, STAT1 and IRF7 expression, as well as CD123 when exposed to Aza (Fig 1A,B). Interestingly, the increased effect was seen from day one regardless of concentration. This was similarly reflected in AML patient cells. Aza treatment also arrested cell proliferation and decreased viability in both cell lines and patient cells suggesting Aza can aid in the anti-leukemic effect. Rag2γc-/- mice engrafted with MOLM-13 and treated with Aza and CD123 CAR Tc demonstrated suppressed growth, and eradication of MOLM-13 cells compared to mice treated with CD123 CAR Tc or Aza alone. Additionally, a significant decrease in residual CD123+ cells in the bone marrow (BM) of dual treated mice was seen (Fig 1C). A higher frequency of residual CD8+ T-cells in the BM, and CD4+ Tc in the peripheral blood (PB) and BM of dual treated mice was observed compared to CAR Tc only treated mice. Most prominently, we found a significantly higher mean number of stem cell-like and central memory CD8+ Tc in the BM of dual treated mice (232 cells/µl and 208cells/µl, respectively) compared to the CAR Tc only group (55 cells/µl and 23 cells/µl, respectively). Assessment of immune checkpoint markers on residual CAR Tc of dual treated mice revealed significantly decreased levels of CTLA-4, PD-1 and TIM-3 in the BM, and CTLA-4 in the PB compared to the CAR Tc only group. While CAR Tc treatment alone demonstrated a survival advantage compared to PBS, NTD or Aza treated mice, Aza and CAR Tc treatment had a significantly higher survival rate compared to the CAR Tc only group (92% vs. 46% at day 50, p

Description: Introduction Pegaspargase (PEG) is a critical component of therapy for pediatric acute lymphoblastic leukemia (ALL); however, complete asparaginase treatment may be hampered by the development of hypersensitivity reactions. Inadequate exposure to asparaginase has been shown to result in inferior outcomes (Gupta 2020). Although Erwinia asparaginase can be utilized as a replacement in patients with PEG allergy, recent shortages of Erwinia leave some patients who develop allergy to PEG without an alternative. Premedication with antihistamines and corticosteroids, as well as decreasing the pegaspargase infusion rate have been proposed as approaches to reduce hypersensitivity reactions (Cooper 2019, Bade 2019, Stock 2019). We evaluated the episodes of hypersensitivity reactions to PEG during three time periods with differing premedication and infusion practices at a single institution. Methods We utilized pharmacy records to conduct a retrospective chart review on PEG administration at Children's Healthcare of Atlanta from June 2017 to May 2020. Abstraction captured data on clinical hypersensitivity reactions to PEG over 3 time periods. PEG 2500 units/m2 was delivered as an intravenous infusion according to the schedules used in Children's Oncology Group ALL protocols. In the first time period (P1, June 22, 2017 to June 21, 2018) PEG was infused over 1 hour without premedication. In the second period (P2, June 22, 2018 to May 19, 2019) PEG was infused over 1 hour following premedication with ranitidine and diphenhydramine. In the final period (P3, May 20, 2019 to May 19, 2020) PEG was infused over 2 hours with normal saline piggyback intravenous fluids following premedication with ranitidine (or famotidine), diphenhydramine and hydrocortisone. Asparaginase activity was measured 7-10 days after PEG doses in consolidation and subsequent phases in P1 and P2, and also after induction in P3. Silent inactivation was defined as asparaginase activity

Description: Key Points Immune responses to FVIII sequence variants encoded by ns-SNPs do not contribute appreciably to inhibitor development in African Americans. African American HA subjects with an intron-22 inversion had a 2- to 3-times-higher inhibitor incidence than whites with the same mutation.

Description: Key Points A novel clinical syndrome of CSA, B-cell immunodeficiency, periodic fevers, and developmental delay is described. Bone marrow transplant resulted in complete and durable resolution of the hematologic and immunologic manifestations.

Description: We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL–independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P 〈 .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Description: Background: Allogeneic SCT is considered standard treatment for patients with advanced phase CML (accelerated phase, blast crisis), de novo Ph+ ALL, or patients in chronic phase (CP) resistant or intolerant to at least 2 tyrosine kinase inhibitors (TKI). Ponatinib is FDA and EMA approved for the treatment of CML or Ph+ ALL in patients with the BCR-ABL1 T315I mutation or for whom no other TKI therapy is indicated. In patients harboring the T315I mutation, ponatinib currently represents a suitable alternative treatment option to allogeneic SCT. However, differences in outcomes between patients treated with ponatinib and allogeneic SCT have not been analyzed. Objective: To compare overall survival (OS) among CML and Ph+ ALL patients with the BCR-ABL1 T315I mutation treated with ponatinib (in PACE) versus allogeneic SCT (in the EBMT database). Methods: Data from a Phase II trial of ponatinib (PACE trial; Cortes et al., New Engl J Med 2013; NCT01207440) and European Bone Marrow Transplant (EBMT) registry were pooled to conduct an indirect comparison of ponatinib with allogeneic SCT. Both ponatinib and allogeneic SCT cohorts comprised patients with the T315I mutation age 18 years or older in any phase of CML or with Ph+ ALL. All patients harbored the T315I mutation detected by Sanger sequencing, DHPLC, PCR-RFLP, or other equivalent tests. Allogeneic SCT patients in their second CP phase were excluded, and no patients in the EBMT database were treated with ponatinib prior to receiving allogeneic SCT. The date of intervention (ponatinib or SCT) served as the index date. Baseline demographic and clinical characteristics were compared between the two intervention groups. OS was compared between the two groups using adjusted Kaplan-Meier (KM) survival curves and multivariate Cox proportional hazards models; all comparisons were adjusted for age (as a continuous variable), gender, geographic region (Europe, Asia, and Australia vs. North America), time from CML diagnosis to intervention, and CML phase or Ph+ ALL at intervention to control confounding by these variables. Results were presented overall and stratified by phase of CML or Ph+ ALL. Results: A total of 184 (128 ponatinib, 56 allogeneic SCT) patients were included in the analysis: 90 were in CP-CML, 26 were in accelerated phase (AP-CML), 41 were in blast phase (BP-CML), and 27 had Ph+ ALL. On average, ponatinib patients were older than allogeneic SCT patients on the date of intervention (median age 53 vs. 45 years, p=0.006). In addition, a larger proportion of patients in the ponatinib group were from North America than in the allogeneic SCT group (43.8% vs. 26.8%, p=0.030). Median time from diagnosis to intervention was longer for patients treated with ponatinib compared with those treated with allogeneic SCT in CP-CML (58 vs. 32 months, p=0.029), but not significantly different in AP-CML (80 vs. 49 months, p=0.075) nor Ph+ ALL (17 vs. 10 months, p=0.212). This period was nominally shorter for the ponatinib cohort in BP-CML (26 vs. 43 months, p=0.340). Over 93% of patients in both treatment cohorts in all disease phases reported previous use of imatinib. Adjusted median OS was significantly longer in CP-CML patients treated with ponatinib as opposed to allogeneic SCT patients (KM median: not reached [NR] vs. 103.3 months, p=0.013), with a hazard ratio (HR) of 0.37 (95% CI: 0.16, 0.84, p=0.017). Median OS was not significantly different between the two treatment groups in patients with AP-CML (NR vs. 55.6 months, p=0.889; HR=0.90 [95% CI: 0.20, 4.10, p=0.889]). However, among patients with BP-CML, ponatinib was associated with significantly shorter OS compared with allogeneic SCT: median 7.0 vs. 10.5 months (p=0.026), HR=2.29 (95% CI: 1.08, 4.82, p=0.030). Ph+ ALL patients treated with ponatinib had nominally shorter median OS than allogeneic SCT (6.7 vs. 32.4 months, p=0.119; HR=2.77 [95% CI: 0.73, 10.56, p=0.136]). See Figures 1a-1d for adjusted KM survival curves. Conclusion: AllogeneicSCT remains a potential curative therapy for patients with BP-CML. However, ponatinib was associated with significantly longer OS than allogeneic SCT in patients with CP-CML that harbor the T315I mutation and could represent a promising therapeutic alternative in this setting, although follow-up remains short to date. OS was similar between intervention groups in AP-CML and longer for allogeneic SCT patients in BP-CML and Ph+ ALL. Disclosures Nicolini: Ariad Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Basak:MSD: Consultancy, Honoraria; Astellas: Honoraria; Sanofi: Honoraria; Pierre-Fabre: Honoraria. Kim:Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Il-Yang: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Pinilla-Ibarz:BMS: Consultancy, Speakers Bureau; Novartis: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy, Speakers Bureau. Apperley:ARIAD: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hughes:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Mauro:Ariad: Consultancy; Pfizer: Consultancy; Novartis Pharmaceutical Corporation: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy. Chuah:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Chiltern International: Honoraria. Hochhaus:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Martinelli:Novartis: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; AMGEN: Consultancy. DerSarkissian:ARIAD: Research Funding. Kageleiry:ARIAD: Research Funding. Yang:ARIAD: Employment. Huang:ARIAD: Employment, Equity Ownership. McGarry:ARIAD: Employment, Equity Ownership. Cortes:Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; BerGenBio AS: Research Funding; Teva: Research Funding; Novartis: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.