Bone Marrow Group Research Articles

Superiority of BM over PBSC for recipients with pre-transplant lung dysfunction in HLA-matched allogeneic HCT

Background aimsPre-transplant lung dysfunction is known to be a risk factor for non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (allo-HCT). It is unclear which cell source gives better outcomes for patients with pulmonary dysfunction. MethodsWe analyzed 3289 adult patients with standard-risk disease who had received HLA-matched allo-HCT, and compared outcomes between those who received peripheral blood stem cell (PBSC) vs. bone marrow (BM) in two cohorts based on the presence of a lung score by the Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI): the Lung-scored (LS) and non-LS cohorts. ResultsIn the LS cohort, the 2-year overall survival (OS) in the BM group tended to be higher than that in the PBSC group (72.4% vs. 61.4%; P = 0.044). In the non-LS cohort, there was no significant difference between the two groups (71.7% vs. 73.2%; P = 0.13). Multivariate analyses confirmed that PBSC was significantly associated with inferior OS in the LS cohort (hazard ratio [HR], 1.66; 95% CI, 1.09-2.54; P = 0.019). On the other hand, the cell source did not affect OS in the non-LS cohort (HR, 0.92; 95% CI, 0.76-1.12; P = 0.41). We found that PBSC was associated with an increased risk of NRM in the LS cohort (HR, 2.17; 95% CI, 1.16-4.05; P = 0.016), while the cell source did not significantly affect NRM in the non-LS cohort. PBSC was not identified as a risk factor for relapse in either cohort. ConclusionsOur results suggest that BM might be beneficial for recipients with lung dysfunction in HLA-matched allo-HCT.

Alemtuzumab, Dual Graft-versus-Host Disease Prophylaxis, and Lower CD3+ T Cell Doses Equalize Rates of Acute and Chronic Graft-versus-Host Disease in Pediatric Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation with Matched Unrelated Donor Peripheral Blood Stem Cells or Bone Marrow Grafts

Data comparing hematopoietic stem cell transplantation (HSCT) using bone marrow (BM) or peripheral blood stem cell (PBSC) grafts in children after alemtuzumab-based conditioning are lacking. We investigated whether in vivo T cell depletion using alemtuzumab could reduce the risk of severe acute graft-versus-host disease (aGVHD) and chronic GVHD (cGVHD) after HSCT with matched unrelated donor (MUD) BM or PBSCs. This retrospective multicenter study included 397 children (BM group, n = 202; PBSC group, n = 195) who underwent first MUD HSCT at 9 pediatric centers in the United Kingdom between 2015 and 2019. The median age at transplantation was 7.0 years (range, .1 to 19.3 years), and the median duration of follow-up was 3.1 years (range, .3 to 7.5 years). The 3-year overall survival was 81% for the entire cohort (BM group, 80%; PBSC group, 81%). The incidence of grade II-IV aGVHD was significantly higher in the PBSC group (31%) compared to the BM group (31% versus 19%; P = .003), with no difference in the incidence of grade III-IV aGVHD (BM, 7%; PBSC, 12%; P = .17). CD3+ T cell dose >5 × 108/kg and the use of PBSCs were independent predictors of grade II-IV aGVHD. When considering CD3+ T cell dose and GVHD prophylaxis, PBSC transplantation with a calcineurin inhibitor (CNI) and mycophenolate mofetil (MMF) and a CD3+ T cell dose ≤5 × 108/kg had a comparable grade II-IV aGVHD to BM transplantation plus a CNI (20% versus 18%; P = .52). PBSC transplantation was associated with a lower incidence of cGVHD compared to BM transplantation (6% versus 11%; P = .03). Within the limits of this study, we identified a potential strategy to reduce the risk of severe GVHD in pediatric PBSC recipients that includes a combination of in vivo T cell depletion using alemtuzumab and dual GVHD prophylaxis (with a CNI and MMF) and limiting the CD3+ T cell dose to ≤5 × 108/kg.

Bone Marrow Versus Peripheral Blood in HLA-Matched Sibling Transplantation: Does the Stem Cell Source Matter in Severe Aplastic Anemia?

Background Selecting a graft source is critical in HLA-matched sibling donor hematopoietic stem cell transplantation (MSD-HCT) for severe aplastic anemia (SAA). Since graft-versus-host-disease (GVHD) is the major cause of mortality after MSD-HCT, and SAA patients do not need the graft-versus-leukemia effect, the selection of a suitable source of stem cells becomes critical. Although bone marrow (BM) is the preferable stem cell source based on the registry-based studies, mobilized peripheral blood stem cells (PBSC) may offer faster engraftment with rapid immune reconstitution. Moreover, the recent advances in conditioning regimens have significantly reduced transplantation-related complications. Here, we compared the long-term outcomes of BM vs PBSC as a graft source in adult patients with SAA who received MSD-HCT with the reduced intensity conditioning (RIC) regimen. Method We conducted a retrospective analysis of 171 consecutive SAA adult patients who received MSD-HCT with Flu6/Cy2/ATG4 RIC regimen (Flu, 30 mg/m 2 IV for 6 days: Cy, 50 mg/kg IV for 2 days; rabbit ATG, 2.5 mg/kg IV for 4 days) or total nodal irradiation (TNI) of 750 cGy combined with ATG as GVHD prophylaxis (TNI-750/ATG) from March 2002 to March 2021 at Seoul St. Mary's Hospital, Seoul, Korea. BM was the primary graft source, but PBSC was used in donors who refused BM harvest, were unsuitable for general anesthesia, or received TNI-750/ATG conditioning. The primary endpoint of this study was to compare the overall survival (OS), and the secondary endpoints included speed of hematologic recovery, primary or secondary graft failure (GF), and failure-free survival (FFS) between the BM and PBSC group. Results The median patient age in the BM group was 38 years (range, 15 - 59) and 44 years (range, 19 - 64) in the PBSC group (p=0.005). There were no differences of baseline characteristics between the two groups except for a donor-to-recipient sex discrepancy (female to male, BM vs. PBSC; 16.4% vs. 30.9%, p=0.029). The majority of BM group (114/116, 98.2%) received the Flu6/Cy2/ATG4 RIC regimen, while 38.2% (21/55) of PBSC group received TNI-750/ATG for the conditioning (p<0.001). The PBSC contained a median 4.71 (range, 1.22 - 17.03) x 10 6/kg CD34 + cells and 368.82 (range, 25.12 - 1234.70) x 10 6/kg CD3 + cells which were about 1.7-fold (BM 2.80 x 10 6/kg CD34 + cells; range, 0.25 - 10.05) and 10-fold (BM 39.61 x 10 6/kg CD3 + cells; range, 1.34 - 463.68) higher than the BM graft (p<0.001). Consequently, the PBSC group exhibited faster engraftment of neutrophils (median 13 vs. 11 days, p<0.001) and platelets (median 18 vs. 12 days, p=0.012). However, there were no differences in the incidence of secondary GF (19.0% vs. 12.7%, p=0.386) and donor-type aplasia (4.3% vs. 10.9%, p=0.178) between two groups. Furthermore, the incidence of acute grade I-II (8.6% vs. 10.9%, p=0.619) and moderate to severe chronic graft-versus-host disease (GVHD) (3.5% vs. 5.5%, p=0.542) were also not significantly different between two groups. However, the BM group has shown more CMV reactivation (44.0% vs. 24.9%, p=0.005), and higher incidence of respiratory infection (14.7% vs. 3.6%, p=0.037) than those of the PBSC group. The incidences of transplantation-related complications between the BM and PBSC group are summarized in Table. The 10-year OS (93.8% vs. 98.1%, p=0.246) and FFS (65.7% vs. 69.0%, p=0.455) were comparable (Figure). Moreover, there was no significant difference in FFS between BM and PBSC group by potential risk factors of patient and donor age at MSD-HCT, sex incompatibility, ABC mismatch, PNH clone presence, HCT-CI ≥3, infused CD34 + cell count, interval from diagnosis to transplant, prior IST history, heavy transfusion before allo-HCT, or severity of AA. Conclusions Our results suggest that survival outcomes of MSD-HCT showed no difference between BM and PBSC as graft sources, and using PBSC may be an efficient alternative offering faster engraftment without significant difference in graft failure or transplantation-related adverse events such as GVHD, and survival. Current analysis and results were drawn from the Asian population with a fludarabine-based conditioning regimen and therefore, need to validate in the Western population. In addition, well-designed prospective studies in a larger cohort are required to confirm our findings.

Outcomes after Bone Marrow Versus Peripheral Blood Haploidentical Hematopoietic Stem Cell Transplantation Using Post-Transplant Cyclophosphamide-Based Gvhd Prophylaxis

Background: Patients who do not have a human leukocyte antigen (HLA)-matched donor can undergo hematopoietic stem cell transplantation (HSCT) by utilizing haploidentical (haplo) family donors and post-transplant cyclophosphamide (PTCy) for Graft-versus-host disease (GVHD) prophylaxis. Originally, Haplo transplants utilized a bone marrow (BM) source because of concern of higher incidence of GVHD with peripheral blood stem cells (PBSC) graft. Recent years have seen an increase in PBSC graft utilization. Some published studies have reported comparable survival between the two graft sources. These studies also reported lower rate of chronic GVHD and higher quality of life with BM and a lower relapse rate and faster engraftment with PBSCs. We aimed to investigate the outcomes following BM versus PBSC haplo HSCT using (PT-Cy)-based GVHD prophylaxis. Methods: In this single-center retrospective study, we included all (n=176) adult patients who underwent haplo HSCT from August 2016 to July 2021 and had at least one year of follow-up. Bivariate analyses, using the chi-square and t-test, were performed. Kaplan-Meier and regression analyses were conducted. Data were analyzed using SPSS version 28. Statistical significance was considered at p<0.05. Results: The study included 176 haplo transplants including 65% peripheral blood and 35% bone marrow grafts. The median age was 54 (18-74) years and 68% of recipients were males. Ethnic minorities comprised 30% of recipients. Hematologic diagnoses included myeloid disease (65%), lymphoid disease (29%), and others (6%). 31 % of the patients underwent a Myeloablative transplant. Hematopoietic cell transplantation comorbidity index of 3 or higher was noted in 53% of patients. With a Median follow-up 21 (0-73) months, Overall survival (OS) and disease-free survival (DFS) was not reached in either BM or PBSC haplo HSCT group. 1-year OS of 75% and 74% (p-0.898) and 1-year DFS of 63% and 63% (p-0.994) were noted in BM and PBSC group respectively. In PBSC versus BM haplo recipients, an earlier neutrophil engraftment (17 days vs 18 days, p=0.022) was seen. Similar incidences of grade II-IV acute GVHD (50% vs 51%, p=0.875), relapse (22% vs 26%, p=0.579), non-relapse mortality (NRM, 17% vs 20%, p=0.682), platelet engraftment (28 vs 31 days, p<0.092) and 1- year chronic GVHD (35% vs 38%, p=0.410) were noted. Conclusions: In this study, Haploidentical allogeneic hematopoietic stem cell transplant recipients demonstrated similar rates of overall and disease-free survival, relapse, non-relapse mortality, acute and chronic GVHD, and platelet engraftment regardless of graft source. Neutrophil engraftment was observed one day earlier in peripheral stem cell recipients. This supports the use of either PBSC or Bone marrow as the preferred source of Haploidentical transplants.

The Comparison between BM and PBSC for the Patients with Pre-Transplant Pulmonary Dysfunction in HLA-Matched Allo-HCT: Subgroup Analysis Exploring the Impact Based on HCT Factors

Introduction Pre-transplant pulmonary dysfunction is known as an important risk factor for lung complications and subsequent non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (allo-HCT). However, it remains unknown which cell source would be optimal for the patients with pre-transplant pulmonary dysfunction and whether the impact of cell source would differ according to recipient age, donor relation, and conditioning regimens. Therefore, we evaluated the impact of stem cell sources in patients with pre-transplant pulmonary dysfunction and in the subgroups. Patients and methods We performed a retrospective analysis of clinical data on 3374 allo-HCT in Japan between 2016 and 2020 using Japanese registry database. All patients had standard-risk disease and underwent their first allo-HCT from an HLA-matched donor. Their clinical outcomes were compared between peripheral blood stem cells (PBSCs) and bone marrow (BM) grafts individually in two distinct cohorts, namely the Lung-scored (LS) cohort and the normal cohort, based on the presence of lung scores determined by the Hematopoietic Cell Transplantation-specific Comorbidity Index. In addition, we conducted subgroup analyses to explore potential differences in the impact of cell sources on these outcomes according to the recipient age (≥50 or < 50), donor relation (related or unrelated), and conditioning regimens (cyclophosphamide [CY] / total body irradiation [TBI]-based myeloablative conditioning [MAC], busulfan [BU]-based MAC, or reduced-intensity conditioning [RIC]). Results In the LS cohort, the 2-year overall survival (OS) tended to be higher in the BM group than that in the PBSCs group (71.9% vs 61.4%; P = 0.052). However, in the normal cohort, there was no significant difference between both the cell source types (71.6% vs 73.1%; P = 0.13). Multivariate analyses showed that PBSCs were significantly associated with inferior OS in the LS cohort (hazard ratio [HR], 1.60 [95% CI, 1.06-2.42], P = 0.026). On the other hand, cell source type did not significantly affect OS in the normal cohort (HR, 0.94 [95% CI, 0.78-1.14], P = 0.55). We also found that PBSCs were significantly associated with an increased risk of NRM in the LS cohort (HR, 1.90 [95% CI, 1.03-3.52], P = 0.041), while cell source types did not affect NRM in the normal cohort (HR, 0.85 [95% CI, 0.64-1.13], P = 0.26). PBSCs were not identified as a risk factor for relapse in both cohorts. When we focused on the LS cohort alone, the Figure 1 summarized the impact of the use of PBSCs in the subgroups according to age, donor relation, and conditioning regimens. Briefly, the use of PBSCs exhibited a significant adverse impact on OS (HR, 3.07 [95% CI, 1.16-8.11], P = 0.024) in patients younger than 50 years old. For recipients aged 50 years or older, PBSCs were not associated with inferior OS (HR, 1.49 [95% CI, 0.91-2.42], P=0.11), although it tended to be associated with an increased risk of NRM with a borderline significance (HR, 1.94 [95% CI, 0.99-3.78], P = 0.053, Figure 1). Focusing on the subgroup stratified according to their donor relation, the use of PBSCs were not associated with inferior OS or an increased risk of NRM in both allo-HCT groups from a related donor or unrelated donor. Furthermore, focusing on the subgroups stratified according to conditioning regimens, the use of PBSCs had a significantly negative impact on OS in the subgroup with CY/TBI-based MAC (HR, 7.48 [95% CI, 2.87-19.51], P < 0.001). On the other hand, the use of PBSCs had no significant impact on OS or NRM in the subgroups of BU-based MAC or RIC. Regarding relapse incidences, the impacts of the use of PBSCs were not different in any of the individual subgroups. Conclusion The use of PBSCs were associated with inferior survival in the LS cohort, but not in the normal cohort. When we focused on the subgroups of the LS cohort alone, the use of PBSCs were associated with inferior survival in the subgroup of younger recipients or those with CY/TBI-based MAC. These patients in the LS cohort might benefit from the use of BM. These results should be validated in another registry study, and further research would be warranted for the purpose of preventing adverse impact of PBSCs or the optimal cell source selection.

Outcomes after Bone Marrow Versus Peripheral Blood Stem Cell Matched Unrelated Donor Hematopoietic Stem Cell Transplantation

Background: Matched unrelated donors (MUD) have become an acceptable alternative donor for patients requiring potentially curative hematopoietic stem cell transplantation (HSCT) and lacking a matched sibling donor. Patients may undergo a bone marrow (BM) or peripheral blood stem cell (PBSC) graft, with previous studies showing similar survival outcomes. However, BM grafts have shown a lower incidence of chronic graft-versus-host disease (GVHD) but a higher relapse rate and slower engraftment. We aimed to compare the outcomes of BM versus PBSC in patients undergoing MUD HSCT. Methods: For this retrospective single-center study, we included all (n=276) adult MUD HSCT recipients at the University of Kansas Medical Center who had at least one year of follow-up (August 2016 to July 2021). We conducted a bivariate analysis using chi-squared and ANOVA tests. The Kaplan-Meier and regression analyses were also performed. Data analyses were conducted using SPSS version 28. Statistical significance was considered at p<0.05. Results: Our study included 276 MUD patients including 110 PBSC (40%) and 166 BM (60%) grafts. The median age was 55 (range 21-77) years, and 158 patients (57%) were males. Eighteen patients (6%) were ethnic minorities, and 89 (32%) patients had a Karnofsky performance status of ≥90%. 118 (43%) patients had a hematopoietic cell transplantation-specific comorbidity index of 3 or higher. Hematologic diseases included acute myeloid leukemia (n=119, 43%), myeloid disorders (n=77, 28%), acute lymphoblastic leukemia (ALL) (n=35, 13%), Lymphoma (n=15, 5%), and others (n=30, 11%). Sixty-two percent of patients were in complete remission at the time of transplantation, and patients not in remission included myelodysplastic syndrome, myelofibrosis, and lymphoma patients. Reduced-intensity conditioning (RIC, n=162, 58%) and myeloablative conditioning (MAC) (n=114, 41%) were used. The median follow-up was 55 (21-73) months. Overall survival (OS) was not reached in either BM or PBSC MUD HSCT group, while a median disease-free survival of 45 and 49 months was noted in PBSC and BM groups respectively (p=0.915). We observed similar rates of relapse (25% vs 27%, p=0.413), one-year overall survival (75% vs 72%, p=0.679), one-year disease-free survival (63% vs 62%, p=1.00), and one-year non-relapsed morality (NRM, 23% vs 21%, p=0.779) after PBSC or BM graft. Bone marrow graft recipients had delayed engraftment of neutrophils (20 days vs 16 days, p<0.001) and platelets (>20 K/uL: 24 days vs 16 days, p<0.001; >50 K/uL: 28 days vs 18 days, p=0.006) compared to PBSC recipients. Bone marrow graft recipients had similar rates of grade II-IV acute GVHD (50% vs 52%, p=0.714) and grade III-IV GVHD (12% vs 11%, p=0.847), but lower rates of 1-year chronic GVHD (39 % vs 55%, p=0.014) compared to PBSC recipients. In the subgroup analysis stratified by conditioning intensity, faster engraftment of neutrophils (median 16 vs 21 days, p<0.001) and platelets (>20 K/uL: 18 days vs 27 days, p<0.001; >50 K/uL: 18 days vs 33 days, p<0.001) was observed with PBSC as compared to BM graft in myeloablative transplant recipients, while there was no statistically significant association with the rates of acute or chronic GVHD, NRM, Relapse, DFS, and OS after a PBSC or BM graft. Among the RIC transplant recipients, faster neutrophil engraftment (mean 17 vs 20 days, p<0.001) and higher 1-year extensive chronic GVHD (52% vs 32%, p=0.015) were observed with PBSC compared to BM graft whereas no statistically significant difference was noted in platelet engraftment, acute GVHD, NRM, Relapse, DFS, and OS after a BM or PBSC graft. Conclusions: Allogeneic matched unrelated donor hematopoietic stem cell transplant recipients had similar rates of overall and disease-free survival, relapse, non-relapse mortality, and acute GVHD after a bone marrow or peripheral blood stem cell graft; however, delayed neutrophil and platelet engraftment with a lower incidence of chronic GVHD was noted after a bone marrow graft compared to peripheral blood stem cell graft.

Pre-Leukemic Transformation of the Bone Marrow Microenvironment Induced By AML1-ETO Fusion Protein

Objective: The bone marrow (BM) microenvironment, especially stromal cells (SCs) and cytokines, plays a critical role in supporting normal hematopoiesis. However, in cases of hematological malignancies such as acute myeloid leukemia (AML), leukemia cells can reshape the BM microenvironment to promote their survival. Despite this, there is still limited understanding of the changes in AML1-ETO fusion protein induced pre-leukemia microenvironment niche. In this study, the alterations of the BM microenvironment in the Aml1 Eto/+; Mx1-Cre (AE KI) mouse model are thoroughly explored and analyzed. This investigation will encompass the study of SCs, cytokines, their sources, and their potential impacts on the occurrence and progression of AML induced by AML1-ETO. Methods: After 4 weeks of PIPC induction, proportion changes of endothelial cells (ECs), osteoblasts, mesenchymal stem cells (MSCs) in the BM of Aml1 Eto/+; Mx1-Cre mice (experimental group) and Aml1 Eto/+; w/o Mx1-Cre mice (control group) were analyzed by flow cytometry. Bulk RNA sequencing (RNAseq) and single cell RNA sequencing (scRNAseq) techniques were applied for analysis of abnormal hematopoietic Lin -Sca-1 +c-Kit + (LSK) stem cells. Cytokine levels were detected using ELISA and Luminex bead assay. Additionally, CRISPR-Cas9 technology was used to knock out the target genes and their functions were validated. Results: Following 4 weeks of PIPC induction, the experimental group exhibited a considerable reduction in the proportion and absolute number of ECs, osteoblasts and MSCs, with ECs being the most significantly reduced. The Luminex assay revealed a substantial increase in cytokine release in the BM of the experimental group, including IL4, IL10, TNFa, Cxcl10, Cxcl11, Ccl1, Ccl2, Ccl4, Ccl7, etc. Bulk RNAseq studies of LSK cells showed that the inflammatory pathway in the experimental group was significantly upregulated. To further explore the release sources of cytokines in the BM and heterogeneity in LSK cell cytokine secretion, scRNAseq analysis of LSK cells was conducted in the AE KI group, which were classified into 9 subgroups. Subgroups 1 and 5 exhibited a significant upregulation of Ccl2, Ccl4, Cxcl2, and IL15. Subgroups 4 and 8 showed upregulation of Ccl2 and IL4. Subgroups 0 and 2 exhibited upregulation of Ccl4, while subgroups 6, 7, and 9 showed upregulation of Cxcl2. Notably, subgroups 1 and 5 also displayed increased expression of Ccr5, which is the receptor for Ccl2. To further validate the origins of cytokines, LSK cells were sorted from experimental group and the control group for in vitro culture, respectively. Subsequently, the cell culture supernatants were collected for Luminex assay, revealing a notable upregulation of Ccl2 and Ccl7 in the experimental group. Furthermore, when comparing m-cherry + LSK cells (AML1-ETO positive) with m-cherry - LSK cells (AML1-ETO negative) from the same mouse, a significant increase in the Ccl2 levels in the m-cherry + LSK cell culture supernatant was observed. Flow cytometry analysis of LSK cells further revealed high expression of Ccl2 and its receptor Ccr5 in Ccl2 + LSK cells. In order to gain a deeper understanding of the functions of Ccl2 and Ccl7, CRISPR-Cas9 technology was used to knockout Ccl2 and Ccl7 in c-kit + cells, which were then transplanted into mice for further investigation, respectively. After 3 months, partial restoration of ECs was observed compared to that of the scramble control group. Conclusion: Our findings suggested that significant changes occurred in the BM microenvironment in AML1-ETO-induced pre-leukemia stage. These changes were characterized by a decrease in SCs and an increase in pro-inflammatory cytokine levels. Moreover, our study identified the heterogeneity in cytokine transcriptional profiles among AE KI LSK cells. The secretion of Ccl2 and Ccl7 by AE KI LSK cells with a blockage in hematopoietic differentiation seemed to be involved in the reduction of stromal cell population. These results provided valuable insights for further exploration of the intricate interactions between abnormal hematopoietic stem cells and the BM microenvironment in AML1-ETO-induced pre-leukemic and leukemic states. Understanding these interactions is crucial for advancing our knowledge of the underlying mechanisms involved in the development and progression of AML.

Impact of the source of hematopoietic stem cells on immune reconstitution after transplantation: A systematic review.

Hematopoietic stem cell (HSC) transplantation's success lies in its ability to induce immune reconstitution. To date, there is no review published to compare the immune reconstitution among the three sources of HSC: umbilical cord blood (UCB), bone marrow (BM), and peripheral blood (PB). The review aims to analyze the kinetic of immune reconstitution among UCB, PB, and BM in HSC transplant patients by focusing on natural killer (NK) cells, B and T lymphocytes, and neutrophils. A systematic review was conducted through five databases, searching for clinical trials and randomized control trials (RCTs) which analyze the kinetics of immune reconstitution in at least two sources. Selected studies were assessed with Cochrane RoB 2.0. This review included 14 studies, with a total of 2539 subjects. The PB group achieved the fastest time to neutrophil recovery, while the B-cell count was the highest in the UCB group. The T-cell count is the lowest in the BM group, and the NK-cell count does not differ significantly among the three HSC sources. Among the three sources of HSC, there is no superior HSC source for any immune reconstitution parameter. More studies must be conducted to compare the immune reconstitution and clinical outcomes of all HSC sources in specific diseases.

CD84 Is a Therapeutically Targetable Driver of Leukemogenesis Via Disruption of Energy Supply in Acute Myeloid Leukemia

CD84 is a member of the signaling lymphocyte activation molecule (SLAM) family; it forms homophilic dimers by self-association. CD84 is reported as an important survival receptor in chronic lymphocytic leukemia and its activation upregulates PD-L1 expression on leukemia cells. We and others recently reported that CD84 is highly expressed on myeloid derived suppressor cells, but its function in myeloid malignancies has yet to be investigated. Using publicly available microarray data (GSE13509), we found that CD84 is overexpressed on acute myeloid leukemia (AML) cells compared to normal hematopoietic counterparts (AML, n=542 vs. normal, n=73, p<0.0001). and that its upregulation is associated with poor prognosis in AML patients (GSE10358; p=0.0105). Knocking down CD84 expression in AML cell lines (THP1 and HEL) by shRNAs caused a substantial inhibition of cell viability and extended THP1-luciferase xenografted mice survival in vivo (median survival: shCtrl, 38 days vs shCD84, 53 days, p=0.0015, n=5). Moreover, we observed that CD84 depletion in AML primary patient cells dramatically induced cell apoptosis (shCtrl: 13.0±1.84% vs shCD84: 29.33±1.3%; n=3; p=0.02) and inhibited colony formation (shCtrl: 68.44±11.65 vs shCD84: 15.56±6.32; n=3; p=0.01). In light of highly expressed CD84 in MLL-r AML and inv(16) AML, we employed MLL-AF9 and CBFB-MYH11(CM) AML mouse models, and found that CD84 knockdown robustly dampened cell growth and decreased colony-forming units in vitro. In both models, bone marrow (BM) transplantation shown that CD84 deficiency significantly decreased leukemic engraftment in BM (MLL-AF9: shCtrl: 95.92±0.61% vs shCD84: 76.42±4.11%, p=0.0016, n=5/group; inv(16): shCtrl: 39.02±2.98% vs shCD84: 13.66±1.24%, p<0.0001, n=5/group), spleen, and peripheral blood (PB). In AML patient derived xenografts (PDX), we observed AML burden was markedly reduced in BM (shCtrl:51.22±10.29% vs shCD84: 2.304±0.16%; n=5/group, p=0.0014) and spleen (shCtrl: 8.790±2.989% vs shCD84: 0.8420±0.13%; n=5/group, p=0.0255) in the CD84 knockdown group. Mechanistically, CD84 deletion depressed mTOR signaling, induced mitochondrial stress, and disrupted energy supply chains based on RNA-seq analysis. We demonstrated CD84 deletion caused mitochondrial dysfunction as indicated by decreasing oxidation phosphorylation (shCtrl vs shCD84, p<0.0001) and fatty acid oxidation (shCtrl vs shCD84, p=0.0004). Next, we determined that CD84 deletion caused disruption of mitochondrial matrix morphology and loss of mitochondrial cristae in AML. We also observed a dramatic loss of mitochondrial membrane potential upon CD84 deletion. We generated a novel murine-human chimeric anti-human CD84 monoclonal antibody (mAb) with high affinity and specificity. In the presence of NK cells, anti-CD84 mAb induced strong antibody-dependent cell-mediated cytotoxicity in CD84-expressing AML lines (n=2) and primary samples (n=2), but not in healthy donor CD34+ cells (n=2). Next, the luciferase-expressing THP1 was transplanted into NSG mice. Mice with comparable tumor burden were randomized into 2 groups: control human IgG or anti-CD84 mAb (5mg/kg, i.v, bid, n=6/group), every week together with 3x106 human PB mononuclear cells (PBMCs) as effector cells (for 4 weeks). Anti-CD84 mAb significantly suppressed leukemia development and extended median survival (IgG: 30 days vs anti-CD84 mAb: undefined, p=0.0018). However, without PBMCs, human IgG or anti-CD84 mAb did not induce significant differences in tumor burden or median survival (n=5/group; p=0.5082). Finally, we developed two AML PDXs in NSG mice. Following engraftment >1% in PB, mice were divided into 2 groups (n=6/group). We maintained the same treatment strategy as applied above. In both AML PDXs, the BM engraftment of CD45+CD33+ cells markedly decreased in anti-CD84 mAb treated mice (AML #1: IgG: 94.25± 0.41% vs anti-CD84 mAb: 71.52±3.96%, p= 0.0002; AML #2: IgG: 88.74±0.7% vs anti-CD84 mAb: 53.98±5.61%, p=0.0003). Anti-CD84mAb treatments in secondary transplantation of BM harvested from the first treatment groups resulted in further significant reduction in BM engraftment (IgG: 86.48± 0.67% vs anti-CD84 mAb: 29.74±6.02%, n=5, p<0.0001), and extended survival (IgG, 28 days vs anti-CD84 mAbs, 36 days, p=0.0026, n=5) following third BM transplantation. The results indicated our pharmacological targeting of CD84 inhibits leukemogenesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Haplo-Peripheral Blood Stem Cell Plus Cord Blood Grafts for Hematologic Malignancies Might Lead to Lower Relapse Compared with Haplo-Peripheral Blood Stem Cell Plus Bone Marrow Grafts

To compare the outcomes between peripheral blood stem cell (PBSC)+cord blood and PBSC+bone marrow (BM) grafts in the setting of haploidentical donor (HID) transplantation, 110 patients were enrolled in this retrospective study, including 54 recipients of haplo-PBSC+cord transplants and 56 recipients of haplo-PBSC+BM transplants. Chimerism analyses revealed that by day 30 post-transplantation, 94.3% of surviving patients in the haplo-PBSC+cord group had achieved full haploidentical chimerism and 5.7% had <10% cord chimerism, whereas 100% of surviving patients in the haplo-PBSC+BM group had achieved full donor chimerism. The cumulative incidence of platelet engraftment at 30 days was 92.6% in the haplo-PBSC+cord group versus 89.3% in the haplo-PBSC+BM group (P =.024), that of grade II-IV acute graft-versus-host disease (GVHD) at 100 days was 31.5% versus 48.2% (P =.060), and 1-year relapse was 13.0% versus 25.0% (P =.027), nonrelapse mortality was 9.3% versus 12.5% (P =.76), disease-free survival (DFS) was 77.7% versus 62.5% (P =.028), and overall survival (OS) was 81.4% versus 69.6% (P =.046). Multivariate analysis identified haplo-PBSC+cord transplantation as a protective factor for relapse (hazard ratio [HR], .31; P =.007), DFS (HR, .40; P =.007), and OS (HR, .44; P =.016). Overall, haplo-PBSC+cord transplantation led to faster platelet engraftment, lower relapse, and superior DFS and OS compared with haplo-PBSC+BM transplantation and thus might be a better transplant mode in the setting of HID transplantation.

Impact of bone marrow involvement in patients with peripheral T-cell lymphoma undergoing autologous stem cell transplant.

7550 Background: Nodal-based T-cell lymphomas (PTCL) are treated with induction therapy and consideration for autotransplant (autoSCT) in first remission. The impact of bone marrow (BM) involvement at diagnosis and at autoSCT is unclear. Methods: We performed a retrospective review of consecutive patients with PTCL-NOS, AITL, and ALCL who received autoSCT in first remission at Memorial Sloan Kettering Cancer Center from 1997-2021. For BM involvement at baseline, patients were classified as positive (≥5% by histology), any involvement (by histology and/or at least 1 high-sensitivity [HS] assay, referring to flow or clonal TCR gene rearrangement), or negative (no involvement by histology and at least 1 negative HS assay). Patients with no baseline BM biopsy were excluded. At end of therapy (EOT, after induction but prior to autoSCT), BM involvement was considered positive (≥5% by histology), minimal residual disease positive (MRD+, no histological involvement but at least 1 positive HS assay), and MRD- (no histological involvement and at least 1 negative HS assay). Patients with no EOT BM but negative baseline BM were included as a separate cohort. Patients with no baseline BM and no EOT BM were excluded, as were those with baseline BM involvement and no EOT BM. The Kaplan-Meier method was used to estimate survival and compared using log-rank tests. Results: A total of 135 patients were included. Median follow-up after autoSCT for survivors was 56 months. The 5-year PFS/OS for the entire cohort was 45% and 65%, respectively. By histology, 5-year PFS/OS for PTCL-NOS (N = 27), AITL (N = 78), ALK+ (N = 8) and ALK- (N = 21) ALCL was 36%, 37%, 75%, 74% (p = 0.005), and 51%, 59%, 100%, 85% (p = 0.01). Among those with adequate baseline BM evaluation (N = 98), 31 were positive, 52 had any involvement, and 44 were negative. Five-year PFS and OS in the positive, any involvement, and negative groups was 46%, 42%, 44% (p = 0.82), and 63%, 61%, 74% (p = 0.27). Of those with adequate EOT BM evaluation (N = 72), 1 was positive, 17 were MRD+, and 43 were MRD-; 32 patients had no EOT BM but negative baseline BM. Five-year PFS and OS by the EOT positive, MRD+, MRD-, and no EOT BM but negative baseline groups was 0%, 37%, 45%, 45% (p = 0.86), and 0%, 92%, 59%, 71% (p = 0.39). Of the 17 patients with MRD+ EOT BM, 14 (82%) had AITL. Two-year PFS/OS in the AITL only cohort by EOT BM was 0%, 54%, 48%, 33%, and 0%, 90%, 75%, 72%, for the positive, MRD+, MRD-, and no EOT BM but negative baseline groups (PFS: p = 0.4; OS: p = 0.3). Conclusions: In patients treated with upfront induction and autoSCT in first remission, neither BM involvement at diagnosis nor the presence of MRD at the time of autoSCT resulted in worse survival outcomes compared to those with no BM involvement. For patients with PTCL-NOS, AITL, or ALCL in first remission, baseline BM involvement or pre-autoSCT BM MRD+ should not dissuade use of autoSCT consolidation.

Co-Targeting E-Selectin/CXCR4 with GMI-1359 Facilitates AML Stem Cell Mobilization and Protects BM Niches from Anti-Leukemia Therapy

Acute myeloid leukemia (AML) is characterized by the heterogeneous clonal expansion of undifferentiated myeloid cells in the bone marrow (BM). AML cells compete with normal hematopoietic cells and rewire the BM microenvironment into niches that selectively support leukemia stem cells (LSC). The leukemic niche produces soluble factors that facilitate the retention of LSC and provide protection from cytotoxic and targeted agents. The vascular adhesion molecule, E-selectin is expressed on endothelial cells (EC) in the perivascular niche where therapy-resistant AML cells have an increased affinity to E-selectin compared to normal hematopoietic stem cells (HSC) (Winkler et al., 2020). We previously demonstrated (Chang et al., ASH 2020) that E-selectin blockade by the pharmacological antagonist, GMI-1271 (uproleselan; GlycoMimetics, Inc) sensitized therapy-resistant LSC to Bcl-2 targeted therapy. Efficacious eradication of LSC in the BM however requires blocking multiple receptors and/or associated signaling pathways. A more optimal dislodgement of LSC from the BM could be attained by combining an E-selectin antagonism with blockade of the CXCR4/SDF-1α axis. The dual antagonist of E-selectin and CXCR4, GMI-1359 (GlycoMimetics, Inc.), has been tested in a phase 1 clinical trial (NCT02931214). Previously, we showed that GMI-1359 in combination with a FLT3-ITD inhibitor, improved survival in a xenograft model of FLT3-ITD + AML (Zhang et al., 2016). Hence, we hypothesized that co-targeting E-selectin/CXCR4 more efficiently mobilizes AML cells from BM niches and synergizes with the anti-leukemia activity of venetoclax/hypomethylating agent (Ven/HMA).Intra-vital 2-photon imaging and tracking of individual leukemia cells in triple reporter mice (Blood: dextran-TRITC; Host T-cells: DsRed; Host myeloid CD11 cells: EYFP) injected with AML cells carrying a turquoise fluorescent protein reporter gene suggested that dual inhibition of E-selectin/CXCR4 with GMI-1359 significantly enhanced AML cell motility (Fig 1. from 2.2 um/min to 5.4 um/min, p<0.001). Individual cells were dislodged from the niche and traveled long-distance. The combined inhibition of E-selectin and CXCR4 depleted BM leukemia cells in vascular niche areas. In a patient-derived primary AML xenograft (PDX) model (harboring mutations in JAK2 and c-Kit), combinatorial treatment of GMI-1359 with Ven/HMA significantly reduced BM retention of LSC compared to control cohorts or to Ven/HMA alone (p = 0.02 and p=0.003, respectively).In order to better understand how the augmented AML mobilization improves the efficacy of AML therapy, BM cells from PDX mice treated for 2 weeks with GMI-1271, GMI-1359, Ven/HMA, and their combinations were analyzed by single-cell proteomics (CyTOF). Blockade of E-selectin alone or dual E-selectin/CXCR4 inhibition in combination with Ven/HMA diminished levels of E-selectin ligand, mTOR, pFAK, pRb, cMyc, while increasing p21 and cleaved caspase3, which was associated with significant reduction of BM-resident LSC compared to Ven/HMA alone (CD45+34+CD38-CD123+, p= 0.03). AML blasts from the BM of the combinatorial treatment groups showed altered signaling including decreased Ki67, pRb, pNFkB, pPI3K, and E-selectin ligand, and increased levels of cleaved caspase 3. We further found that Ven/HMA significantly diminished CD31+ EC in the BM compared to control cohorts (p= 0.009). However, pharmacological antagonists of E-selectin or E-selectin/CXCR4 protected EC from Ven/HMA-induced detrimental insults through upregulation of survival signaling cascades including pAKT, pERK, pMAPK and decreased eNOS expression in EC compared to Ven/HMA treatment alone. Both EC and MSC were protected by dual inhibition of E-selectin/CXCR4 with GMI-1359. We also observed upregulated pro-survival signaling pathways such as phosphorylation of AKT-MAPK-ERK along with increased Bcl-xL, Bcl-2, and Idu expression in MSC from the GMI-1359 + Ven/HMA treated PDX mice compared to Ven/HMA single treatment cohorts.Collectively, our results provide strong evidence that co-targeting E-selectin/CXCR4 with GMI-1359 profoundly reduces BM retention of LSC as well as protects BM niche component cells from apoptosis induced by targeted therapy, resulting in improving the anti-leukemia activity of Ven/HMA therapy in AML. [Display omitted] DisclosuresFogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Carter: Ascentage: Research Funding; Syndax: Research Funding. Andreeff: Oxford Biomedica UK: Research Funding; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Aptose: Consultancy; Glycomimetics: Consultancy; Medicxi: Consultancy; Senti-Bio: Consultancy.

COMPARABLE OUTCOMES OF ALLOGENEIC PERIPHERAL BLOOD VERSUS BONE MARROW HEMATOPOIETIC STEM CELL TRANSPLANTATION IN CHILDREN

Hematopoietic stem cell transplantation (HSCT) is used in many malignant and non-malignant diseases in pediatric patients. Peripheral blood (PB), bone marrow (BM) or cord blood can be used as a graft source. In this study, it was aimed to compare the transplantation results of patients who used bone marrow as a graft source and those who used peripheral blood in pediatric patients who underwent allogeneic HSCT. We retrospectively analyzed the transplant results of 349 pediatric patients who received a transplant between April 2010 and August 2021 considering their stem cell source as a comparative variable. Engraftment days, development of acute graft versus host disease(aGVHD) or chronic graft versus host disease (cGVHD), development of relapse and overall survival of patients were evaluated. The source of stem cells was BM in 240 and PB in 109 patients. The mean age of patients was 96.8±60 and 94.5±63 months in BM and PB group, respectively. The mean myeloid and platelet engraftment time was statistically significantly earlier in PB group (p<0.001). Acute GVHD was statistically significantly higher in PB group (p<0.001). The relapse rate was statistically significantly higher in the PB group (p:0.02). The mean follow-up period was 49.2±41.6 months. The 5-year overall survival rate was 83.4% in the BM group and 68.5% in the PB group (p:0.003). In our study, in accordance with the literature, it was observed that myeloid and platelet engraftment was earlier if the source is PB in HSCT in pediatric patients, but acute GVHD was more frequent. In the survival analysis, the 5-year survival of the bone marrow transplant group was found to be higher. Peripheral blood could be an alternative stem cell source in patients but it would be more appropriate to decide the stem cell source according to the primary diagnosis of the patients.

Bone Marrow versus Peripheral Blood Grafts for Haploidentical Hematopoietic Cell Transplantation with Post-Transplantation Cyclophosphamide

In the coronavirus disease 19 (COVID-19) pandemic era, the number of haploidentical hematopoietic cell transplantations (HCTs) with peripheral blood (PB) grafts increased significantly compared with HCTs with bone marrow (BM) grafts, which may be associated with adverse outcomes. We compared outcomes of HCT in BM graft and PB graft recipients age ≥18 years with hematologic malignancies who underwent T cell- replete haploidentical HCT and received graft-versus-host disease (GVHD) prophylaxis with post-transplantation cyclophosphamide, tacrolimus, and mycophenolate mofetil. Among the 264 patients, 180 (68%) received a BM graft and 84 (32%) received a PB graft. The median patient age was 50 years in both groups. The majority (n = 199; 75%) received reduced-intensity conditioning. The rate of acute leukemia or myelodysplastic syndrome was higher in the BM graft recipients compared with the PB graft recipients (85% [n = 152] versus 55% [n = 46]; P < .01). The median times to neutrophil and platelet engraftment and the incidence of grade II-IV and grade III-IV acute GVHD (aGVHD) were comparable in the 2 groups. Among the patients with grade II-IV aGVHD, the rate of steroid-refractory aGVHD was 9% (95% confidence interval [CI], 5% to 18%) in the BM group versus 32% (95% CI, 19% to 54%) in the PB group (hazard ratio [HR], 3.7, 95% CI, 1.5 to 9.3; P = .006). At 1 year post-HCT, the rate of chronic GVHD (cGVHD) was 8% (95% CI, 4% to 13%) in the BM group versus 22% (95% CI, 14% to 36%) in the PB group (HR, 3.0; 95% CI, 1.4-6.6; P = .005), and the rate of systemic therapy-requiring cGVHD was 2.5% (95% CI, 1% to 7%) versus 14% (95% CI, 7% to 27%), respectively (HR, 5.6; 95% CI, 1.7 to 18; P = .004). The PB group had a significantly higher risk of bacterial and viral infections, with no appreciable advantage in the duration of hospitalization, immune reconstitution, relapse, nonrelapse mortality, or survival. Our data suggest a benefit of the use of BM grafts over PB grafts for haplo-HCT.

Graft-Versus-Host Disease (GVHD)-Free Relapse-Free Survival (GRFS) and Chronic Gvhd (CRFS) in Alternative Donor Hematopoietic Cell Transplantation (HCT) in Adults

BACKGROUNDGRFS is a composite endpoint developed by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) to reflect the major outcomes post HCT. Events in GRFS include grade 3-4 acute GVHD (aGVHD 3-4), systemic therapy-requiring chronic GVHD (cGVHD), relapse, or death. We reported that bone marrow (BM) grafts from matched sibling donor (MSD) led to best GRFS at 1-and 2-years compared with peripheral blood (PB) grafts from any donor or umbilical cord blood (UCB). (Holtan et. al. Blood 2015 & Mehta et.al. Haematologica. 2016) These studies excluded haploidentical (haplo) and mismatched (MM)-HCT. The outcomes of this composite endpoint in alternative donor HCT is unknown. Here, we analyzed GRFS and chronic GVHD-free relapse-free survival (CRFS) among alternative donor HCT (UCBT, haplo, one-antigen MM (7/8)-BM or 7/8-PB HCT) for patients with no MSD or matched unrelated donor. METHODSPatients with acute myeloid leukemia (AML) or acute lymphoblastic leukemia in remission, chronic myeloid leukemia or myelodysplastic syndrome who received alternative donor HCT from 2000-2014 were included. Only those haplo patients who received post-HCT cyclophosphamide and only those UCBT done using fludarabine, cyclophosphamide and total body irradiation myeloablative (MA) or reduced intensity (RIC) with or without antithymocyte globulin (ATG) were analyzed. Exclusion criteria were prior autologous or allogeneic HCT, ex-vivo T-cell depletion or CD34 selection and those with UCB unit <4/6 HLA match. Multivariable analysis was done using Cox's proportional hazards (PH) modeling with time-dependent adjustments as needed for stepwise modeling, selecting factors using a threshold of 0.05 for both entry and retention in the model. CRFS was defined survival without cGVHD or relapse. RESULTS2198 patients were analyzed, including UCBT (n=838), haplo (n=159), 7/8-BM (n=241) and 7/8-PB (n=960). Groups (except haplo) were further divided by conditioning and ATG use [Table 1]. AML was most common diagnosis (52%). As expected, patients who received MA conditioning were younger (median 35-43 years) than those who got RIC (median 58-61 years). 81% of UCB group received double cord. Most patients in haplo group received BM graft (71%), and a majority (82%) were from 2012-2014; consequently, follow-up of haplo group (median 25 months) was shorter than others (49-96 months).In multivariate analysis of early post-HCT outcomes (within 4.5 months) adjusted for year of HCT and other covariates, as compared to haplo, UCB, 7/8-BM (MA, no ATG) and 7/8-PB had inferior GRFS. However, beyond 4.5 months, haplo, UCB and 7/8-BM (MA +/- ATG) had similar GRFS while 7/8-PB had inferior GRFS. [Fig 1] Likewise, beyond 4.5 months, as compared to haplo, CRFS was similar in UCB and 7/8-BM (MA +/-ATG), while 7/8-PB had inferior CRFS.As compared to Haplo, risk of aGVHD was significantly higher in all groups, except UCB (RIC + ATG) and the risk of cGVHD (beyond 4.5 months) was higher in 7/8-BM (MA, no ATG) and 7/8-PB but similar in UCB and 7/8-BM (MA + ATG). As compared to haplo, risk of relapse was significantly lower after UCB (MA), 7/8-BM (MA +ATG) and 7/8-PB (MA, no ATG), similar in UCB (RIC + ATG), 7/8-BM (MA, no ATG), 7/8-PB (MA + ATG) and 7/8-PB (RIC +/-ATG) and higher in UCB (RIC, no ATG). Haplo had superior disease free survival compared to UCB, 7/8-PB (MA, no ATG) and 7/8-PB (RIC + ATG), but it was similar in 7/8-PB (MA + ATG) and 7/8-BM (MA +/-ATG). Similarly, haplo had superior overall survival compared to any other group. CONCLUSIONLong-term morbidity and mortality as measured by GRFS and CRFS are similar in UCBT, haplo and 7/8 BM groups. The use of 7/8-PB should be avoided given substantially poor GRFS and CRFS. As 7/8-BM group included only younger patients who received MA conditioning, no conclusion can be drawn about its outcome in older patients, for whom either haplo or UCB remain as the preferred donor source. These outcomes may also be a reflection of different GVHD prophylaxis regimens rather than donor type. Ongoing randomized comparison of UCBT versus haplo in the BMT CTN 1101 trial will prospectively evaluate these two groups. [Display omitted] DisclosuresHoltan:Incyte: Other: One-time advisory board member. Arora:Takeda Oncology: Consultancy.

Wave-Complex-Dependent Actin Dynamics Controls Hematopoietic Stem Cell Bone Marrow Retention and Orderly Differentiation into Mature Hematopoietic Lineages

BackgroundSpatiotemporally controlled actin dynamics are crucial for a vast number of cellular functions including migration, proliferation, attachment or immunological synapse formation. Environmental signals activate cognate surface receptors which in turn engage specific small GTPases. Signal integrators (e.g. WASP, WAVE complex or WIP) propagate the signal to Arp2/3 complex and lead to immediate actin polymerization. Here we show that disruption of one of the actin polymerization pathways through specific targeting of WAVE complex disturbs hematopoietic stem and progenitor cells (HSPC) bone marrow retention and their sequential differentiation towards mature blood cells.MethodsWe made use of the recently described mouse model of Hem1 ablation. Hem1 is a strictly hematopoietic specific component of WAVE complex; its loss disrupts the entire WAVE complex exclusively in cells of hematopoietic origin. An array of in vitro assays was employed to test cell intrinsic effects of Hem1 ablation on actin polymerization dependent cellular functions. In vivo transplantation experiments were performed to study the impact of Hem1 ablation on immature and in consequence on mature hematopoiesis.ResultsHem1-/- HSPC display a bone marrow (BM) retention defect evidenced by strongly elevated numbers of circulating HSPC and 50% reduced BM cellularity. In line with that, migration towards CXCL12, a key BM anchor for HSPC, was completely abrogated in Hem1-/- HSPC. Attachment to feeder cells was also decreased by 35% compared to WT HSPC, although stress fiber formation upon CXCL12 stimulation or general ability to polarize was not affected. Next we tested whether this cellular phenotype had any impact on HSPC performance in vivo. Homing, the initial interaction of transplanted HSPC with BM environment, was not affected by Hem1 ablation, while engraftment, the ability to repopulate all mature hematopoietic lineages, was strongly diminished. Platelet and RBC engraftment was delayed, whereas Hem1-/- neutrophils did not engraft at all (Figure 1). Hem1-/- mice presented with chronic inflammation, hyperleukocytosis and elevated pro-inflammatory cytokine plasma levels. To rule out inflammation as causal for numeric and functional Hem1-/- HSPC depletion we performed co-transplantation experiments. Hem1-/- or WT BM was transplanted in a four-fold excess over GFP+ competitor BM. In Hem1-/- transplanted recipients, already 4 weeks after transplantation 60 to 90% of both myeloid and lymphoid compartments were GFP+, whereas, as expected, in the control experiment WT BM out-competed GFP+ BM over time. A similar picture emerged in secondary recipients. Again Hem1-/- BM completely lost against, whereas WT BM prevailed over GFP+ competitor. We furthermore confirmed that the Hem1-/- HSPC cell intrinsic defect is already apparent during embryonic development: As in BM of adult Hem1-/- mice, HSPC content in E14.5 old fetal livers (FL) was reduced by half compared to WT littermates. Also the competitiveness of FL HSPC was strongly impaired, similar to Hem1-/- adult BM HSPC. Whereas short term engraftment was provided by more mature competitor GFP+ BM in both recipients groups, already 4 weeks after transplantation, as expected, WT FL derived HSPC took over blood cell production and completely outcompeted GFP+ BM. In Hem1-/- FL recipients, by contrast, the contribution of competitor GFP+ BM to hematopoiesis remained high for the whole period tested without significant contribution of Hem1-/- FL hematopoiesis (Figure 2). Taken together, Hem1 (and, by extension, WAVE complex) governs HSPC BM retention and proper engraftment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Acute Lymphoblastic Leukemia Cells Bypass the Blood Brain Barrier to Enter the CNS Via a Neuronal Developmental Pathway

Background: In the absence of prophylactic treatment, CNS disease involvement will occur in 30-70% of acute lymphoblastic leukemia (ALL) patients. CNS metastasis is observed in all ALL subtypes, suggesting that lymphoblasts utilize a conserved mechanism to invade the CNS. CNS ALL relapse predicts a poor outcome, but treatment options remain limited due to a minimal understanding of the biologic processes involved.PI3K regulates in many cell activities, including proliferation, migration, and apoptosis. The δ isoform of PI3K is preferentially expressed in immune cells. The PI3Kδ inhibitor, idelalisib, is approved for use in combination with rituximab for treatment of chronic lymphocytic leukemia. Previously, the PI3Kd tool compound, GS-649443 (443), which has more favorable murine pharmacokinetic properties, was evaluated in in vivo models of acute lymphoblastic leukemia (Yao H, et al. ASH meeting, 2016). Here we report on the mechanisms of ALL CNS invasion and the potential for PI3Kd inhibition to block CNS disease.Results: Our prior study of 443 in Nalm-6 (N6) B-ALL mice demonstrated that PI3Kd inhibition prevents ALL CNS disease progression. All control mice showed symptoms of CNS involvement including hind limb paralysis, but only 17% of 443-treated mice developed paralysis at their clinical endpoint. There was no difference in ALL burden or apoptotic rate in bone marrow (BM) or spleen of treated vs. control mice at matched time points. In contrast, cerebrospinal fluid (CSF) blast counts were significantly decreased in 443-treated mice.To confirm our results were not isolated to the N6 model, we tested the efficacy of PI3Kδ inhibition in mice engrafted with primary human ALL (hALL). Treated mice demonstrated a modest decrease in peripheral disease burden. Similar to the results in N6 mice, 443 markedly decreased CNS disease burden in the hALL model (Fig. 1).Examination of 443 pharmacokinetics as well as mitotic and apoptotic indices of CSF blasts isolated from 443 vs. vehicle-treated mice indicated that PI3Kd inhibition did not directly impact growth of ALL in the CNS. We therefore investigated whether 443 prevented migration of blasts from the periphery into the CNS. We found that 443 decreased Transwell® (TW) migration of N6 and hALL cells toward SDF-1 or human CSF. We next interrogated the signaling pathways downstream of PI3K that are implicated in cell motility and found that 443 suppressed the phosphorylation of myosin light chain 2 in ALL cells. To identify candidate regulatory molecules upstream of this pathway, we performed a microarray analysis of N6 cells isolated from GS-443 vs. vehicle-treated mice. This showed that multiple genes within focal adhesion and contractility pathways were downregulated in vivo in 443 treated blasts, including integrin α6.α6, a subunit of cell surface laminin receptors, is expressed in the majority of ALL cases. Laminin deposition is found in the extracellular matrix (ECM) of CNS microvessels and meninges, where it coordinates α6-dependent neuronal progenitor cell pathfinding. Using confocal microscopy, we determined that N6 and hALL cells in circulation are unable to breach the blood brain barrier. Rather, we identified that ALL cells migrate into the CNS along vessels that pass directly between vertebral or calvarial BM and the subarachnoid space, and that the ECM of these bridging vessels is enriched in laminin (Fig. 2). In in vitro invasion assays, we confirmed that ALL migration to CSF is enhanced by laminin in an α6-dependent manner. Lastly, the in vivo effects of α6 were investigated in the N6 model. Mice were treated with a6 neutralizing antibodies beginning on post-engraftment day 1 through the development of clinical symptoms requiring sacrifice. Anti-α6-treated mice showed cachexia at their clinical endpoint but none had severe CNS symptoms, whereas 100% of isotype control-treated mice showed hind limb paralysis. No difference in ALL burden or apoptotic rate was observed in the BM or spleen of anti-α6 vs. isotype control groups. In contrast, there was a significant decrease in the number of blasts harvested from the CSF of anti-α6-treated mice (Fig. 3).Conclusion: Our results identify a novel ALL metastasis mechanism. Expression of integrin α6, common in ALL, allows cells to co-opt neural migratory pathways to invade the CNS. The potential for PI3Kδ inhibition to block development of CNS ALL warrants further investigation in a clinical study. [Display omitted] DisclosuresYao:Gilead Sciences, Inc.: Research Funding. Warner:Gilead Sciences, Inc.: Research Funding. Price:Gilead Sciences, Inc.: Research Funding. Olivere:Gilead Sciences, Inc.: Research Funding. Cantelli:Gilead Sciences, Inc.: Research Funding. Ridge:Gilead Sciences, Inc.: Research Funding. Ngo:Gilead Sciences, Inc.: Research Funding. Ravichandran:Gilead Sciences, Inc.: Research Funding. Tannheimer:Gilead Sciences, Inc.: Employment. Sipkins:Gilead Sciences, Inc.: Research Funding.

Graft-Versus-Host Disease (GVHD)-Free Relapse-Free Survival (GRFS) and Chronic Gvhd (CRFS) in Alternative Donor Hematopoietic Cell Transplantation (HCT) in Pediatric Patients

BACKGROUNDGRFS is a composite endpoint developed by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) to reflect the major outcomes post HCT. Events in GRFS include grade 3-4 acute GVHD (aGVHD 3-4), systemic therapy-requiring chronic GVHD (cGVHD), relapse, or death. We reported that bone marrow (BM) grafts from matched sibling donor (MSD) led to best GRFS at 1-and 2-years compared with peripheral blood (PB) grafts from any donor or umbilical cord blood (UCB). (Holtan et. al. Blood 2015 & Mehta et.al. Haematologica. 2016) These studies excluded haploidentical (haplo) and mismatched (MM)-HCT.Here, we analyzed GRFS and chronic GVHD-free relapse-free survival (CRFS) among alternative donor HCT (UCBT and one-antigen MM (7/8)-BM HCT) for pediatric patients who did not have a MSD or matched unrelated donor. However, because too few patients received 7/8-PB or haplo with post HCT cyclophosphamide, these were excluded. METHODSData were obtained from the CIBMTR. Patients <18 years of age with acute myeloid leukemia or acute lymphoblastic leukemia (ALL) in remission who received UCBT or 7/8-BM HCT between 2000 and 2014 after myeloablative conditioning were included. Exclusion criteria were prior autologous or allogeneic HCT, ex-vivo T-cell depletion or CD34 selection and those with UCB unit <4/6 HLA. Multivariable analysis was done using Cox's proportional hazards (PH) modeling with time-dependent adjustments as needed for stepwise modeling, selecting factors using a threshold of 0.05 for both entry and retention in the model. CRFS was defined as absence of cGVHD, relapse and death. RESULTS1613 patients were analyzed, including UCBT (n=1441) and 7/8-BM (n=172). Overall, most patients had ALL (63%), 64% received total body irradiation based conditioning, about 60% received ATG or alemtuzumab and 86% UCBT used a single unit. Methotrexate based GVHD prophylaxis was more commonly in 7/8-BM (79%) than in UCBT (15%). Other characteristics were similar in two groups. Median follow-up was 71 months in UCBT and 96 months in 7/8-BM groups.In multivariate analysis, 7/8-BM had similar GRFS (HR 1.19, p=0.1) and CRFS (HR 1.11, p=0.36) as UCBT [Fig 1a & 1b]. Risks of aGVHD 3-4 (HR 1.68, p=0.007) at any time and cGVHD (HR 7.11, p<0.0001) beyond 14 months, but not before, were significantly higher in 7/8-BM than UCBT. 7/8-BM group had similar risk of relapse (HR 1.26, p=0.11) as UCBT. Disease free survival (DFS; HR 0.87, p=0.4) was similar in both groups up to 14 months beyond which 7/8-BM had inferior DFS (HR 2.33, p=0.0008). Overall survival (OS; HR 1.08, p=0.6) was similar in both groups. CONCLUSIONUCBT and 7/8 BM groups yield similar long term morbidity and mortality as measured by GRFS and CRFS, but 7/8-BM had higher risk of aGVHD 3-4, late cGVHD, and inferior late DFS. No preference of alternative donor source can be made; based upon this analysis, both are acceptable options. Improvements in GVHD control could broaden the applicability of partially matched BM grafts. [Display omitted] DisclosuresHoltan:Incyte: Other: One-time advisory board member. Arora:Takeda Oncology: Consultancy.

The Application of and Factors Influencing, the NB5 Assay in Neuroblastomas.

PurposeThe NB5 assay was performed in bone marrow (BM) and peripheral blood (PB) to detect neuroblastomas (NBs) with micrometastases. The sensitivity and factors influencing the NB5 assay were preliminarily evaluated.MethodsThe NB5 assay uses RT-PCR to detect the co-expression of five mRNAs from the neuroblastoma-associated genes, CHGA, DCX, DDC, PHOX2B, and TH. We enrolled 180 cases of neuroblastoma and 65 cases of non-neuroblastoma. Bone marrow and peripheral blood were collected from every patient. The gold standard for the diagnosis of NB was pathological evaluation of solid tumor specimens or bone marrow biopsies (BMBs) from hematological tumors. STATA version 15 and SPSS version 17 software were used for analysis.ResultsWe found that 17 patients were BMB (+), and they were diagnosed as the International Neuroblastoma Staging System (INSS) stage IV and the high-risk group. All 17 patients were BM (+), while 15 patients were PB (+) (15/17, 88.2%). Among the 163 children who were BMB (−), 56 were BM (+), 40 were PB (+), and 36 were BM (+) and PB (+). The sensitivity of the NB5 assay in BM (40.5%) and PB (30.5%) was significantly higher than the sensitivity of BMB (9.4%, P = 0.000). In the non-NB group, four cases were BM (+) and one case was PB (+). The specificity of the NB5 assay in BM and PB was 93.8% and 98.5%, respectively. The sensitivity of the NB5 assay in both BM and PB in INSS stage IV patients was significantly higher than that in INSS stage I–II patients (P <0.05). The sensitivity of the NB5 assay in both BM and PB in the high-risk group was significantly higher than that in the middle-low-risk groups (P = 0.0001). Logistic regression analyses indicated that liver metastases and bone metastases were the primary factors influencing the sensitivity of the NB5 assay in BM and PB (P <0.05).ConclusionsThe NB5 assay had significantly higher sensitivity than the pathological analysis of BMB in detecting NB with micrometastases. The NB5 assay had higher sensitivity in INSS stage IV or the high-risk group. Liver metastases and bone metastases were the primary factors that affected the sensitivity of the NB5 assay.

CD34+ Stem Cell Selection and CD3+ T Cell Add-Back from Matched Unrelated Adult Donors in Children with Primary Immunodeficiencies and Hematological Diseases

Less than 25% of children who require hematopoietic stem cell transplantation (HSCT) for primary immunodeficiencies (PIDs) or genetic hematological diseases have an HLA-identical sibling. For them, a matched unrelated donor (MUD), although baring a greater risk of graft failure, delayed engraftment and immune reconstitution, and severe graft-versus-host disease (GvHD), represents a valid alternative. The stem cell source is also important, as unprocessed peripheral blood stem cells (PBSCs) contain 5 to 10 times more T cells than bone marrow (BM)-derived grafts, a major risk especially for small children with PID. A CD34+ positive selection can mitigate HLA compatibility issues, but the resulting CD3+ T cell depletion hampers engraftment and facilitates infections. To mitigate those problems, we decided to add back a certain number of T cells (30×106 cells/kg body weight [BW]) to the positive CD34+ selection derived from MUD BM or PBSCs and report the results in terms of time to engraftment and immune reconstitution, GvHD incidence, infections, and survival. Our aim was to show not only the feasibility and clinical efficacy of this addback but also that PBSC-derived CD34+ selected grafts with calibrated T cell addback would be equivalent to BM-derived grafts. We analyzed retrospectively our single-center cohort of 76 children (median age, 1.9 years) affected by PID (61) and hematological diseases (15) who received a total of 79 MUD HSCTs with CD34+ selection and addback of 30×106 CD3+ cells/kg BW between 2001 and 2019. We used descriptive and analytic statistics (chi-square, Student's t-test, Mann-Whitney U test, as appropriate) and constructed Kaplan-Meier curves using the log-rank test to compare patients grafted with BM or PBSC-derived inocula. The two groups showed no statistically significant differences in terms of age, sex, HLA-mismatch, or amount of CD3+ cells/kg BW added back to the CD34+ selection. However, the latter being higher in the PBSC group (P=.0001). Overall engraftment rate was 96% (73/76) and occurred faster in the PBSC group than in BM recipients: polymorphonuclear cells, 16 versus 21 days (P=.006); platelets, 15 versus 22 days (P=.001). GvHD incidence was low. No acute GvHD was diagnosed in 24 children, whereas grades I, II, III, and IV occurred in 19, 28, five, and three children, respectively (P not significant). Chronic GvHD was seen in only two children. The CD4+ count at six months after HSCT was higher in PBSC recipients as compared to those receiving BM (184 versus 88 CD4+ cells; P=.003). Overall survival for the whole cohort was 80% at 10 years, with no significant difference between the two stem cell sources (P not significant). Viral infections occurred among five of the PBSC grafted children and 14 in the BM group (P not significant), and no patient suffered from post-transplant lymphoproliferative disorder (PTLD). The results we present show that an addback of 30×106 donor CD3+ cells/kg recipient BW to a MUD BM or PBSC-derived CD34+ selection gives promising results in infants and young children undergoing HSCT for PID or hematological diseases. Furthermore, with this manipulation the inherent limits of PBSC-derived grafts can be overcome, allowing both swift engraftment and immune reconstitution without an increase in GvHD, infections, or PTLD.