Abstract: Vacuolar protein sorting 45 homolog (VPS45), a member of the Sec1/Munc18 (SM) family, has been implicated in the regulation of endosomal trafficking. VPS45 deficiency in human patients results in congenital neutropenia, bone marrow fibrosis, and extramedullary renal hematopoiesis. Detailed mechanisms of the VPS45 function are unknown. Here, we show an essential role of mammalian VPS45 in maintaining the intracellular organization of endolysosomal vesicles and promoting recycling of cell-surface receptors. Loss of VPS45 causes defective Rab5-to-Rab7 conversion resulting in trapping of cargos in early endosomes and impaired delivery to lysosomes. In this context, we demonstrate aberrant trafficking of the granulocyte colony-stimulating factor receptor in the absence of VPS45. Furthermore, we find that lack of VPS45 in mice is not compatible with embryonic development. Thus, we identify mammalian VPS45 as a critical regulator of trafficking through the endosomal system and early embryogenesis of mice.

Abstract: Clonal hematopoiesis (CH) is associated with age and an increased risk of myeloid malignancies, cardiovascular risk, and all-cause mortality. We tested for CH in a setting where hematopoietic stem cells (HSCs) of the same individual are exposed to different degrees of proliferative stress and environments, ie, in long-term survivors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their respective related donors (n = 42 donor-recipient pairs). With a median follow-up time since allo-HSCT of 16 years (range, 10-32 years), we found a total of 35 mutations in 23 out of 84 (27.4%) study participants. Ten out of 42 donors (23.8%) and 13 out of 42 recipients (31%) had CH. CH was associated with older donor and recipient age. We identified 5 cases of donor-engrafted CH, with 1 case progressing into myelodysplastic syndrome in both donor and recipient. Four out of 5 cases showed increased clone size in recipients compared with donors. We further characterized the hematopoietic system in individuals with CH as follows: (1) CH was consistently present in myeloid cells but varied in penetrance in B and T cells; (2) colony-forming units (CFUs) revealed clonal evolution or multiple independent clones in individuals with multiple CH mutations; and (3) telomere shortening determined in granulocytes suggested ∼20 years of added proliferative history of HSCs in recipients compared with their donors, with telomere length in CH vs non-CH CFUs showing varying patterns. This study provides insight into the long-term behavior of the same human HSCs and respective CH development under different proliferative conditions.

Abstract: Background After induction therapy for acute myelogenous leukemia (AML), the presence of minimal residual disease (MRD) by targeted next-generation sequencing (NGS) during complete remission (CR) predicts relapse and survival, particularly after exclusion of pre-leukemic mutations. MRD assessment is not routinely performed for AML prior to transplant, partly because consensus regarding assay methodology, appropriate timing, interpretation of results, and therapeutic value prior to SCT is lacking. We therefore sought to describe the rates of mutational clearance and correlate these with relapse rates post-transplant. Methods We conducted a retrospective review of sequential AML or myelodysplastic syndrome (MDS) patients undergoing allogeneic hematopoietic cell transplant (alloHCT) at our institution between 2014 and 2017. There were 119 patients with AML/MDS who were treated with either myeloablative or reduced intensity conditioning regimens. Of the 119 patients transplanted, 60 had both pre- and post-treatment NGS results and were included in the analysis. 56 patients had somatic mutations on initial NGS and were therefore eligible for mutational clearance analysis. Twelve patients were in active disease and excluded from further analyses. The remaining patients (n=44) represent the core dataset. Blood and/or marrow specimens were analyzed via a clinical NGS panel targeting 68 leukemia-associated genes. Median coverage (across 88 samples) was 2817 reads. Mutations were considered persistent if present at variant allele frequencies (VAF) ≥ 1% for single nucleotide variants (SNV) or ≥ 2 copies for insertions and deletions (indels). Validated laboratory reporting practice at our institution reports VAF 〉 4% for SNVs and ≥ 1% for indels with a minimum of 250 total reads. We therefore defined three levels of mutational clearance on the basis of the VAF of residual mutations: VAF for SNV 〈 1% (and/or indels ≤1 copy), between 1-4% (and/or indels 〈 1% and ≥ 2 copies), and 〉 4% (and/or indels 〉 1%). Patients with ≥ 1 mutation meeting these thresholds were designated NGS(-), NGS-low and NGS(+), respectively. The median follow-up was 332 days. Results On review of NGS data, 120 mutations were present in initial sequencing, with 64 mutations persistent in pre-transplant samples from 26 patients. The most commonly mutated genes from initial samples were FLT3 (18), ASXL1 (11), TET2 (10), NPM1 (9), RUNX1 (8), SRSF2 (8), and DNMT3A (7) (Figure 1A). Mutational clearance varied widely, with the putative pre-leukemic genes DNMT3A, TET2, and ASXL1 (DTA) demonstrating low rates of mutational clearance (Figure 1A). Mutations persisting below the validated reporting threshold were present in 20 patients, including 10 patients otherwise negative by NGS. There were 16 patients categorized as NGS(+), 10 NGS-low, and 18 NGS(-), with relapse rates of 31%, 22%, and 30%, respectively. No difference in relapse risk was observed between NGS(-) and NGS-low subgroups (p = 0.72), and no RFS benefit was observed for patients without persistent mutations 〉 4% relative to the NGS(+) subgroup (p = 0.56, Figure 1B). Recent work has shown a survival benefit in AML patients in CR without persistent mutations that is enhanced when DTA genes were excluded from the analysis (Jongen-Lavrencic, NEJM 2018). In our cohort, after exclusion of DTA mutations, 6 patients were reclassified by mutational clearance status, and 2 were excluded from the analysis as they had only DTA mutations in pre-treatment samples. Similar to the more comprehensive cohort, no RFS benefit based on NGS status was observed in the post-transplant period (p = 0.42, Figure 1C). Conclusions There were similar outcomes regardless of molecular MRD findings by NGS for patients with advanced myeloid malignancies who were in morphologic CR prior to alloHCT. These results contrast with those in the published literature that address a more uniform patient population of clinical trial participants, not all of whom went on to transplant. Further detailed analyses from larger more homogeneous populations will be useful to determine the prognostic significance of MRD by NGS prior to allogeneic HCT. Figure 1 Figure 1. Disclosures Frey: Servier Consultancy: Consultancy; Novartis: Consultancy. Perl:Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Daiichi Sankyo: Consultancy. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding; Amgen: Consultancy; Janssen: Consultancy. Porter:Genentech: Other: Spouse employment; Kite Pharma: Other: Advisory board; Novartis: Other: Advisory board, Patents & Royalties, Research Funding. Gill:Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Equity Ownership; Novartis: Research Funding.

Abstract: Background UCART19 is an allogeneic, genetically modified CAR T-cell product (anti-CD19 scFv- 41BB-CD3ζ) manufactured from healthy donor T cells, in which TRAC and CD52 genes have been knocked out to allow its administration in non-HLA matched patients (pts). Aims Preliminary safety/anti leukemic and cellular kinetics data of UCART19 administered to pediatric and adult patients with R/R B-ALL are reported. Methods Data of patients included in the ongoing CALM study (adult) and PALL study (pediatric) have been pooled. CALM is a dose-escalation study (approximate dose level [DL] 1: 1x105 cells/kg,DL2: 1x106 cells/kg, DL3: 3x106 cells/kg) and PALL is testing a unique dose of 1.1 to 2.3x106 cells/kg. Eligible patients presented with a morphological disease ( 〉 5%) or MRD load ≥1x10-3. A lymphodepleting regimen (LD) combining cyclophosphamide (1500 mg/m² in CALM, 120 mg/kg in PALL) and fludarabine (90 mg/m2 in CALM, 150 mg/m2 in PALL) without (FC) or with alemtuzumab (FCA) (1 mg/kg) was administered one week before UCART19 infusion on Day 0 (D0). Cellular kinetics of UCART19 was assessed by flow cytometry (flow) in CALM and by vector copy number in PALL. Results As of 15 July 2018, 20 pts had received at least one UCART19 infusion, including 13 pts in CALM (6 at DL1; 6 at DL2 and 1 at DL3) and 7 pts in PALL. Seventeen pts had completed D28 evaluation (1 pt died at D15, 2 pts have not reached D28 yet). Prior to LD, blasts % in the bone marrow (median [range]) was 6% [0-68%] in PALL and 25% [0-96%] in CALM. Seventeen pts received LD with FCA and 3 pts received LD with FC. Safety was evaluable in 18/20 pts. Cytokine release syndrome (CRS) was reported in 17/18 pts and was mild and reversible in the majority of cases (2 G1, 12 G2, 2 G3, 1 G4). One pt died in context of CRS G4 and neutropenic sepsis. According to data available to date, the severity of CRS does not seem to follow the levels of serum cytokines (IL-6, IL-10 and IFNγ). Mild self-limited neurotoxicity events were reported in 6 pts (5 G1 and 1 G2). Two pts (1 infant and 1 adult) developed G1 acute skin GvHD (non-biopsy proven for 1 pt) that was reversible with steroids. Grade 1-4 viral infections were reported in 8/18 pts. The majority of events recovered, except in 2 pts who died after allo-SCT in context of prolonged cytopenia (defined as persistent G4 beyond D42 post UCART19 infusion): 1 child with BK virus infection and 1 adult with adenovirus infection. A further 4 pts developed prolonged cytopenia and all recovered after allo-SCT. Cellular kinetic data was available for 18/20 pts. UCART19 was detectable in blood from D7. Peak expansion was observed in 72% (13/18) pts between D10 and D17, mostly at D14, with a median persistence duration of 28 days. Expansion occurred at each dose tested; without consistent relationship between administered dose and magnitude of expansion. One patient treated at DL2 presented a high expansion and a long persistence (very low detection by flow at D120). UCART19 persistence could not be assessed beyond D42-D56 in 3 pts because the remaining CARs were ablated by the transplant conditioning regimen. No expansion was observed in 5/18 pts in whom early lymphocyte recovery was detected from D14. Of these 5 pts, 3 did not receive alemtuzumab. The role of clinical status, tumor burden and lymphodepleting regimen on UCART19 expansion is under investigation. Anti-leukemic activity was evaluable in 16/20 pts (1 pt died at D15, 1 pt was not assessed at D28, 2 pts have not yet reached D28). After UCART19 infusion, 88% of evaluable pts (14/16) achieved CR or CRi by D28 or D42 and 86% (12/14) of these pts were MRD negative (MRD- stands for 〈 1x10-4 copies) by flow or qPCR. Two out of 16 pts had no expansion and showed refractory disease. Among 12 pts achieving MRD-, 5 pts remain in molecular remission 4.5 to 16.4 months post UCART19. In total, 11 pts underwent allo-SCT (5 in PALL and 6 in CALM). Preliminary data suggests that in the majority of pts, anti-leukemic activity is linked with CAR expansion. Conclusion Pooled data of 20 pts show an acceptable and manageable safety profile of UCART19. Severe CRS was reported in 15%. Only 2 G1 cutaneous acute GvHD were observed and no severe neurotoxicity was reported. In this heavily pre-treated population, 88% pts of evaluable pts (14/16) achieved CR or CRi of which 86% (12/14) achieved MRD-. All pts who achieved MRD- had evidence of UCART19 expansion. Updated data, including data for the highest dose level in CALM study, will be presented (NCT 02746952, NCT02808442). Disclosures Benjamin: Servier: Research Funding; Amgen: Honoraria; Takeda: Honoraria; Novartis: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Pfizer: Research Funding. Graham:Servier: Research Funding. Yallop:Pfizer: Consultancy; Servier: Other: Travel funding. Jozwik:Servier: Honoraria, Research Funding. Ciocarlie:Servier: Research Funding. Jain:Cellectis: Research Funding; Seattle Genetics: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; ADC Therapeutics: Research Funding; Astra Zeneca: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Research Funding; Servier: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologioes: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; Adaptive Biotechnologioes: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Pharmacyclics: Research Funding; Cellectis: Research Funding; Pfizer: Research Funding; Verastem: Research Funding; BMS: Research Funding; Servier: Research Funding; Infinity: Research Funding; Astra Zeneca: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Abbvie: Research Funding; Pfizer: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maus:adaptimmune: Consultancy; novartis: Consultancy; windmil therapeutics: Consultancy; agentus: Consultancy, Research Funding; crispr therapeutics: Consultancy, Research Funding; kite therapeutics: Consultancy, Research Funding. Boissel:Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees. Larghero:Gilead: Consultancy. Baruchel:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Amgen: Consultancy; Servier: Consultancy; Celgene: Consultancy. Mohty:Takeda: Honoraria, Speakers Bureau; Molmed: Consultancy; Servier: Consultancy; Jazz Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Janssen: Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol Myers: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Bloor:Janssen: Research Funding; AbbVie: Research Funding. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Konto:Pfizer: Equity Ownership. Veys:Pfizer: Honoraria; Servier: Research Funding; Novartis: Honoraria. Qasim:Bellicum: Research Funding; Autolus: Equity Ownership; Orchard: Equity Ownership; Servier: Research Funding.