ALBERT

Description: Background: Numerous studies from others and our institution have demonstrated that the presence of minimal residual disease (MRD), detected at the time of hematopoietic cell transplantation (HCT), is strongly and independently associated with increased relapse risk and short survival in adults with acute myeloid leukemia (AML) undergoing myeloablative allogeneic HCT in morphologic complete remission (CR). In contrast, very little information is available regarding the prognostic significance of peri-transplant MRD dynamics in these patients. Since bone marrow staging studies with multiparameter flow cytometric (MFC) assessment for MRD are routinely obtained not only before but also at approximately day +28 following transplantation at our institution, we here retrospectively studied the relationship between peri-HCT MRD dynamics and post-transplant outcomes in a large patient cohort. We asked whether persistence or disappearance of MRD might identify cohorts of patients in whom post-transplant therapy was particularly indicated or unnecessary. Patients and Methods: AML patients ³18 years of age were eligible for this retrospective analysis if they were in first or second morphologic CR or CR with incomplete blood count recovery (CRi) irrespective of the presence of MRD, underwent allogeneic HCT with myeloablative conditioning between 2006 and 2014, received peripheral blood or bone marrow as stem cell source, and had pre-HCT bone marrow staging studies available that included 10-color MFC assessments for MRD. MRD was identified as a cell population showing deviation from normal antigen expression patterns compared with normal or regenerating marrow; any level of residual disease was considered MRDpos. We considered post-HCT MRD assessments in patients in whom bone marrow re-staging with MFC MRD analysis were obtained 28±7 days after transplantation. For this analysis, the primary endpoint of interest was overall survival, which was estimated using the Kaplan-Meier method. Results: 311 patients were identified and included in this study. Consistent with our previous analyses, patients with MRD at the time of HCT (MRDpos; n=76) had significantly shorter survival than MRDneg patients (n=234; estimated 3 year post-HCT survival: 26% [95% confidence interval: 17-37%) vs. 73% [66-78%], P

Description: Background. Fanconi anemia (FA) is a rare genetic disorder that often presents with progressive bone marrow failure (BMF) due to an impaired DNA damage response and chronic exposure to elevated levels of proinflammatory cytokines. To date, hematopoietic stem/progenitor cell (HSPC) transplantation remains the only curative treatment for FA-associated BMF. However, donor availability, graft failure, and FA-specific transplant toxicities remain significant hurdles. Androgens have been successfully used but side effects often prevent prolonged therapy. Attempts at genetic correction of FA are underway but clinical efficacy has not yet been demonstrated. In this clinical trial, we investigate whether eltrombopag (EPAG), an FDA-approved mimetic of thrombopoietin that promotes trilineage hematopoiesis in subjects with acquired BMF (Olnes, NEJM 2012; Townsley, NEJM 2017), may offer a novel therapeutic modality for subjects with FA. Our pre-clinical studies indicate that EPAG evades blockade of signal transduction from c-MPL induced by inflammatory cytokines (Alvarado, Blood 2019). Additionally, we found that EPAG enhances DNA repair activity in human HSPCs (Guenther, Exp Hematol 2019). Thus, EPAG may positively influence two of the main known mechanisms leading to BMF in FA. Study Design. This is a non-randomized, phase II study of EPAG given to subjects with FA (NCT03204188). Subjects receive EPAG for 6 months at an oral daily dose adjusted for age and ethnicity. Subjects who cannot tolerate the medication or fail to respond by 6 months are taken off study drug. Subjects who respond at 6 months are invited in the extension phase for an additional 3 years. They continue on the same dose of EPAG until they reach robust count criteria (platelets 〉 50K/μL, hemoglobin (Hgb) 〉 10 g/dL in the absence of transfusions, and absolute neutrophil count (ANC) 〉 1K/uL for 〉 8 weeks) or until they reach steady state response (defined as stable counts for 6 months). Drug dose is tapered slowly to the lowest dose that maintains a stable platelet count and eventually discontinued until they meet off study criteria or the study is closed. Eligibility Assessment. Inclusion criteria: (1) Confirmed diagnosis of FA by a biallelic mutation in a known FANC gene and/or by positive chromosome breakage analysis in lymphocytes and/or skin fibroblasts; (2) One or more of the following cytopenias: platelets ≤ 30K/μL or platelet transfusion dependence in the 8 weeks prior to study entry, ANC ≤ 500/μL, Hgb ≤ 9.0 g/dL or red blood cell (RBC) transfusion dependence in the 8 weeks prior to study entry; (3) Failed or declined treatment with androgens; 4) Age 〉 4 years. Exclusion criteria: (1) Evidence of MDS or AML; (2) Cytogenetic abnormalities associated with poor prognosis in FA; (3) Known biallelic mutations in BRCA2; (4) Active malignancy or likelihood of recurrence of malignancies within 12 months; (5) Treatment with androgens ≤ 4 weeks prior to initiating EPAG. Primary Endpoints. The primary efficacy endpoint is the proportion of drug responders at 6 months. Response to EPAG is defined by one or more of the following criteria: (1) Platelets increase by 20K/μL above baseline, or platelet transfusion independence; (2) Hgb increase by 〉 1.5g/dL or a reduction in the units of RBC transfusions by at least 50%; (3) At least a 100% increase in ANC for subjects with a pretreatment ANC of 〈 0.5 x 109/L, or an ANC increase 〉 0.5 x 109/L. The primary safety endpoint is the toxicity profile assessed at 6 months using the CTCAE criteria. Sample Size and Statistical Methods. Simon's Two-Stage Minimax Design is used, with a response probability of ≤ 20% to terminate the treatment. In the first stage, 12 subjects will be accrued. The study will be stopped if no more than 2 subjects respond to the treatment within 6 months. If 3 or more subjects respond within 6 months in the first stage, then an additional 13 subjects will be accrued, for a total of 25 subjects. Enrollment. Two subjects have been enrolled to date. No drug-related adverse events have been observed. Subject #1 (7YO female) did not respond to 6 months of EPAG, likely due to limited HSPC reserve in the context of profound cytopenias (ANC = 100/µL, Hgb = 6g/dL, Plt = 0K/µL). In contrast, subject #2 (49YO female) showed response to EPAG at 3 months and will continue on the extension phase of the study. Conclusion. This study will provide important clinical information on safety and efficacy of EPAG in subjects with FA. Disclosures Winkler: Agios: Employment.

Description: Purpose. Several studies have shown that allogeneic hematopoietic cell transplant (HCT) in first complete remission (CR1) reduces risk of relapse and improves relapse-free survival in patients with intermediate- and poor-risk AML. Benefits in overall survival (OS) are less obvious. One possible explanation is that once relapse occurs previous receipt of allogeneic HCT in CR1 is associated with shorter survival, with a lower CR rate following salvage therapy for relapse being a possible contributing factor. The aims of our study were to analyze the specific effect of allogeneic HCT in CR1 on (a) second CR rate and (b) OS from first salvage therapy. Patients and Methods. We identified 166 consecutive patients with AML (APL excepted, but including MDS with 10-19% blasts) who after achieving CR1 with initial treatment relapsed and subsequently received first salvage therapy between 2005 and 2013 at our institution. We used chart reviews to collect data on: (1) age, (2) CR1 duration, (3) ECOG performance status at relapse, (4) cytogenetic category (ELN criteria for adverse, intermediate, "favorable", and "missing") at relapse, (5) allogeneic HCT in CR1 (yes vs. no, and then myeloablative vs. reduced intensity vs. no) and (6) intensity of salvage therapy: with high-dose cytarabine containing therapy or myeloablative HCT considered "high intensity", 3+7 +/- other drugs as average intensity, and other salvage treatments as low intensity. CR was defined using conventional criteria. OS was measured from date of first salvage therapy to death from any causes analyzed using the Kaplan-Meier method. Multivariable regression analysis was performed using logistic and Cox models, adjusted for the 6 covariates noted in the preceding paragraph. Results. The median age of the 166 patients was 53.5 years (20 - 80 years). The three groups differed regarding this variable (p 〈 0.001): a median of 47 years (22 - 66) in the 27 patient myeloablative group, 61 years (21 - 75) in the 20 patient reduced intensity group and 54 years (20 - 82) in the 119 patient no-HCT group. The median CR1 duration was 7.9 months, and CR1 duration did not differ statistically (p = 0.46) in the three groups: 11.5 months myeloablative group, 8.8 months reduced intensity group, and 6.4 months no-HCT group. Cytogenetic data were missing in 22 patients; in the remaining 144, 8% had "favorable", 67% intermediate, and 25% adverse risk cytogenetics with no statistically significant differences in distribution between the three groups (p = 0.27 considering all patients). The median PS was 1, with 19% of patients having PS ≥ 2. Patients in the reduced intensity group more often had PS ≥ 2 (40% vs. 19% for myeloablative and 16% for no-HCT) (p = 0.07). 52% received high intensity salvage therapy (including 4 who received myeloablative allogeneic HCT), 18% received average intensity, and 30% received low intensity. The three groups differed regarding intensity of salvage therapy (p = 0.04). 25% of reduced intensity patients received high intensity salvage vs. 44% of myeloablative and 59% of no-HCT. In contrast, 26% of no-HCT patients received low intensity salvage vs. 37% myeloablative group and 40% reduced intensity group. The CR2 rate was 37.9%. The median survival after first salvage was 7.8 months. A multivariable analysis showed that HCT in CR1 had no significant effect on CR2 rate (OR 1.33, p = 0.50) or OS (HR 0.87, p = 0.55). In contrast, shorter CR1 duration and low intensity salvage were independently associated with a lower CR2 rate with favorable cytogenetic at relapse associated with a higher CR2 rate, while PS ≥ 2, low intensity salvage, and, to a lesser extent, shorter CR1 duration were similarly associated with poorer survival with favorable cytogenetic at relapse associated with better survival. An analysis of myeloablative vs. reduced intensity vs. no-HCT led to similar conclusions: myeloablative HCT p = 0.98 and reduced intensity HCT p = 0.29. Together with the Kaplan-Meier plots (Figure 1, log rank p = 0.90) the data suggest that neither myeloablative nor reduced intensity HCT in CR1 has an adverse effect on OS after first salvage. Conclusion.There was no evidence that preceding allogeneic HCT in CR1 was associated with lower probabilities of CR2 or OS after first salvage therapy. We believe our results should encourage use of allogeneic HCT in CR1 with some confidence that this will not unfavorably affect CR2 rate and OS should post HCT relapse occur. Disclosures No relevant conflicts of interest to declare.

Description: Ex vivo culture of human hematopoietic stem/progenitor cells (HSPCs) under either low O2 tension or Notch signal activation driven by Delta1 ligand (Delta1) have been independently shown to primarily expand short-term HSPCs. In contrast, we have previously demonstrated a significant (5-fold) expansion of long-term repopulating HSPCs upon combination of these two strategies, suggesting synergy between Notch and hypoxia signaling pathways. Understanding the molecular mechanisms underlying this synergy in HSPCs could provide new insights to further enhance expansion by combined activation of these pathways. Given the stoichiometric relationship between the number of Notch receptors (Notch 1-4) and Notch signaling intensity, we assessed whether hypoxia regulated Notch receptor expression. G-CSF mobilized human CD34+ cells from 3 healthy subjects were cultured for 0, 4, 24, or 48hrs in the presence of cytokines (SCF, FLT3L, TPO), under hypoxic (1.5-2.0% O2) or normoxic (21% O2) conditions. Using flow cytometry, Notch 3 expression was not detected in CD34+ cells and no differences in the expression level of any of the other Notch receptor isoforms (Notch 1, 2 and 4) were observed regardless of O2 tension. Another unique aspect of Notch signaling is that the receptor must undergo enzymatic cleavage to initiate signal transduction. These cleavage events culminate in the nuclear translocation of the intracellular domain of notch (ICDN) where it binds to Notch promoter regions, leading to the expression of Notch regulated genes. It has been suggested that a shift in energy metabolism from aerobic to glycolytic may lead to an enhancement of these cleavage events. To directly measure the differences in Notch cleavage events, CD34+ cells from 2 healthy donors were cultured for 24 hours under hypoxic or normoxic conditions, followed by a 1-hour exposure to Delta1. Cells were then collected, processed and stained with antibodies specific to the cleaved ICDN of Notch 1, 2, and 4. Using ImageStream analysis, preliminary data suggest that hypoxia accelerates the rate of Notch 2 receptor intracellular signaling by as much as 2-fold at this early time point. Another potential mechanism by which hypoxia may modulate Notch signaling is via protein-protein interaction of the ICDN and HIF1α, a master transcriptional regulator of cellular response to hypoxia. To assess this possible interaction, CD34+ cells were cultured in chamber slides as described above. After exposure to Delta1, cells were fixed, permeabilized, and labeled with antibodies specific for cleaved ICDN and HIF1α. Ten cells per condition (i.e. normoxia or hypoxia, +/- Delta1) were imaged with a Zeiss 780 inverted confocal microscope. Using Imaris and Interactive Data Language (IDL) software, all high resolution images were analyzed to determine the subcellular localization of each protein. As expected, exposure to Delta1 increased the levels of cleaved ICDN, and hypoxia increased the levels of HIF1α detected. Interestingly, the combination of hypoxia and Delta1 exposure resulted in a significant increase in the ICDN-HIF1α Pearson's coefficient of colocalization (PCC = 0.51) compared to control groups without Delta1 (PCC [normoxia] = 0.17, p

Description: Background: Treatment algorithms for allogeneic hematopoietic cell transplantation (HCT) typically consider patients with acute myeloid leukemia (AML) in morphologic complete remission (CR) separately from those with active disease (i.e. ≥5% marrow blasts by morphology), implying distinct outcomes for these two groups. However, it is well recognized that the presence of minimal residual disease (MRD) at the time of transplantation is associated with adverse post-HCT outcomes for patients in morphologic CR. This well established effect of pre-HCT MRD prompted us to compare outcomes in patients in MRDpos CR to those with active AML who underwent myeloablative allogeneic HCT at our institution. Patients and Methods: We retrospectively studied 359 consecutive adults with AML who underwent myeloablative allogeneic HCT from a peripheral blood or bone marrow donor between 2006 and 2014. Pre-HCT disease staging included 10-color multiparametric flow cytometry (MFC) on bone marrow aspirates in all patients. MRD was identified as a cell population showing deviation from normal antigen expression patterns compared with normal or regenerating marrow. Any level of residual disease was considered MRDpos. Results: Three hundred and eleven patients (87%) were in morphologic CR at the time of transplantation, with 76 (21%) in MRDpos CR and 235 (66%) in MRDneg CR. 48 patients (13%) had active disease (7 untreated newly diagnosed AML, 16 untreated relapsed AML, and 25 refractory or relapsed AML who failed salvage therapies). Patients with MRDpos CR or active AML more often had adverse-risk cytogenetics (P=0.001) and secondary leukemias (P

Description: Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

Description: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified 〉20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.