CD34+ Cell Dose Influences Survival after Allogeneic Peripheral Blood Stem Cell Transplantation with Post-Transplant Cyclophosphamide

Introduction: Higher infused total nucleated cell dose (TNC) in allogeneic bone marrow transplant (BMT) with post-transplant cyclophosphamide (PTCy) resulted in improved overall survival (OS) in a recent report (McCurdy, et al. BBMT. 2018). As many centers prefer peripheral blood stem cell grafts (PBSCT) with PTCy, the optimal cell dose with this platform requires elucidation. Methods: We retrospectively evaluated 144 consecutive adult patients with hematologic malignancies treated at the Moffitt Cancer Center between 2012-2018 with allogeneic T-cell replete PBSCT followed by PTCy-based graft-versus-host disease (GVHD) prophylaxis. Data were extracted from the Moffitt BMT Research & Analysis Information Network (BRAIN) database. Both myeloablative (n=87) and reduced intensity (n=57) conditioning regimens were included. For analyses, CD34+ cell dose was stratified into low (<5 x106/kg), intermediate (5-10 x 106/kg) and high (>10x106/kg). TNC was stratified as higher or lower than the median. CD3+ T cell dose was evaluated as a continuous, linear variable. Associations with transplant related survival outcomes were assessed using univariate and multivariate Cox proportional hazard survival models. Fine and Gray regression models were used to assess associations of transplant related endpoints with competing risks. Kaplan Meier curves and cumulative incidence function curves were also plotted. RESULTS: The median follow-up for the entire cohort was 24 months. The median age at PBSCT was 58 (range: 21-76) years. Donors were haploidentical (n=72, 50%), matched unrelated (n=39, 27%), matched related (n=18, 13%), and mismatched unrelated (n=15, 10%). Median CD34+ cell dose was 8.12 x 106/kg (range: 2.27 - 22.4 x 106/kg). Stratified CD34+ dose was low for 16 patients (11.1%), intermediate for 93 patients (64.6%), and high for 35 patients (24.3%). Median CD3+ dose was 2.89 x 108 (range: 2.33 x 105 - 6.54 x 108/kg). Median TNC was 9.66 x 108/kg (range: 3.82 x 108 - 1.04 x 109/kg). Overall, successful neutrophil engraftment by day 30 was achieved in 94% (95% CI 91-98%) and platelet engraftment (>20,000/mL) by day 100 in 87% (95% CI 81-93%) of all patients. The likelihood of neutrophil recovery was similar in the high and low CD34+ cell groups, while it was higher for the intermediate CD34+ cell group (HR=1.52, 95% CI 1.0-2.2; p=0.03). Platelet recovery was similar in the intermediate and high CD34+ cell groups, but significantly lower in low CD34+ cell group (HR=0.38, 95% CI 0.2-0.8; p=0.01). CuI of grade II-IV acute GVHD at day 100 was 39% (95% CI 32-48%) and of grade III-IV acute GVHD was 10% (95% CI 6-17%). CuI of chronic GVHD at 2 years was 50% (95% CI 41-59%). The CD34+ cell dose had no significant impact on either acute or chronic GVHD. Nonrelapse mortality (NRM) at 1 year was 19% (95% CI 13-27%). By CD34+ cell dose, the rates of NRM for low, intermediate, and high dose were 46% (95% CI 25-85%), 15% (95% CI 10-25%), and 15% (95% CI 7-34%), respectively (p=0.002). Relapse rate was 30% at 2 years with no differences across dose levels. Progression free survival (PFS) probability at 2 years was 47% (95% CI 39-57%) for the entire group, 0% (95% CI 0%) for the CD34+ low group, 53% (95% CI 42-65%) for the intermediate group and 51% (95% CI 35-73%) for the high dose group (p=0.0003). Two-year OS for the entire group was 57% (95% CI 49-67%), and was inferior in the low group (0%, 95% CI 0%) with no difference between the intermediate (61%, 95% CI 50-73%) and high (67%, 95% CI 52-87%) CD34+ cell dose groups (p=0.0002). In multivariable modeling, the low CD34+ cell dose group was associated with worse NRM (HR=4.1, 95% CI 1.2-14.5, p=0.03), PFS (HR=2.9, 95% CI 1.3-6.5, p=0.01), and OS (HR=3.2, 95% CI 1.4-7.6, p=0.01) compared with the high group, while there were no differences between the high and intermediate groups. Increasing CD3+ dose was also associated with improved NRM (HR=0.8, 95% CI 0.69-0.97, p=0.02), PFS (HR=0.7, 95% CI 0.5-0.9, p=0.02), and OS (HR=0.7, 95% CI 0.5-0.9, p=0.004). TNC had no impact on survival outcomes. CONCLUSION: In patients receiving allogeneic PBSCT with PTCy, CD34+ cell doses below 5 x 106 cells/kg yielded inferior OS and PFS, attributable to higher NRM. Doses of 5-10 x 106 cells/kg and >10 x 106 cells/kg resulted in comparable outcomes. Higher CD3+ dose/kg was also associated with improved survival outcomes. Overall, this study supports targeting a CD34+ dose of >5 x 106 cells/kg for allogeneic PBSCT with PTCy. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Bejanyan:Kiadis Pharma: Other: advisory board.

Cost effectiveness of G-CSF after allogeneic peripheral blood stem cell transplant.

e18540 Background: In spite of the fact G-CSF has been used post stem cell transplant (SCT) to accelerate neutrophil engraftment its use post allogeneic SCT remains controversial. ASCO does not recommend its use after allogeneic SCT. To further evaluate the effectiveness of its use, we compared outcomes in pts who underwent related and unrelated peripheral blood SCT(PBSCT) either with or without the use of G-CSF post SCT. Methods: This is a retrospective study comparing early outcomes in pts who received G-CSF starting on day + 6 post SCT until engraftment with pts who did not receive a planned course of G-CSF. Pts who underwent Allogeneic PBSCT between 2012-2014 at our institution were included. Pts who received marrow, haploidentical or cord blood transplants were excluded. Associations with survival outcomes were assessed by univariable and multivariable Cox proportional regression models. Results: A total of 162 patients were evaluated. Sixty-five pts received G-CSF post SCT and 97 did not. The only difference between the two groups was that more pts in the G-CSF group received myeloablative-conditioning (MAC) regimen (78% vs. 55%, p = 0.008). Other pt characteristics were not significantly different. Length of hospital stay was significantly lower in the G-CSF group (24 vs. 27 days P = 0.002). Pts who received G-CSF had earlier neutrophil engraftment (median, days 11 vs. 14 p = < 0.001). The median day of platelet engraftment was 15 days in both groups. There was no significant difference between the 2 groups in re-admissions in the first 100 days, and the incidence of acute or chronic GVHD. In multivariate analysis use of G-CSF did not significantly impact non- relapse mortality, relapse free survival and overall survival. However, relapse rate was significantly lower in G-CSF group in multivariable analysis (hazards ratio = 0.44, p = 0.03). Conclusions: Use of G-CSF post allogeneic PBSCT is associated with earlier neutrophil engraftment, shorter hospital stay and a suggestion of a reduced relapse rate after PBSCT. Our experience suggests that use of G-CSF (on average for approximately 5 days) in this setting is cost effective as it reduces hospitalization duration without adversely impacting post-transplant outcomes.

Calcineurin Inhibitor-Free GVHD Prophylaxis with Post-Transplant Cyclophosphamide and Brief-Course Sirolimus Results in Low Rates of Non-Relapse Mortality and Chronic GVHD Following Matched Related and Unrelated Donor Peripheral Blood Stem Cell Transplantation (PBSCT)

Calcineurin inhibitors (CNIs) form the foundation of current GVHD prophylaxis regimens. Although advances in immunosuppressive regimens have had a significant impact on the incidence and severity of acute GVHD, it is noteworthy that CNIs have had little impact on chronic GVHD. We hypothesized that a CNI-free regimen consisting of post-transplant cyclophosphamide (Cy) and brief-course sirolimus would decrease the risk of chronic GVHD and non-relapse mortality and improve outcomes following reduced intensity allogeneic PBSCT. Twenty-seven patients with high risk hematologic malignancies were enrolled in the study: median age 61 years (25-73). All patients had a 10/10 locus matched donor; MRD=18, MUD=9. Conditioning consisted of fludarabine 30mg/m2 on days -9 to -6, IV busulfan 130 mg/m2 on days -5 to -4, and Cy 14.5 mg/kg on days -3 and -2 followed by unmanipulated PBSCT. Post-grafting immunosuppression consisted of Cy 50 mg/kg/day on days 3 and 4 and sirolimus starting day +5 and completing day+90 in the absence of GVHD. Donor engraftment occurred in all patients with a median time to neutrophil and platelet recovery of 15 and 30 days, respectively. The median day +90 donor T cell and myeloid chimerism was 94% (40-100%) and 100% (11-100%) respectively. Three patients received donor lymphocyte infusions for incomplete donor T cell chimerism. The cumulative incidence of grade II-IV acute GVHD, grade III-IV acute GVHD, all chronic GVHD, and severe chronic GVHD was 41%, 15%, and 32%, and 12% respectively. Non-relapse mortality (NRM) and relapse incidence at 2 years was 4% and 17% respectively. With a median follow-up of 18 months, the estimated 2 year overall and disease-free survival was 71% and 80% respectively for the whole cohort, while it was 87% and 89%, respectively in the subgroup of 18 patients receiving MRD transplants. Good immune reconstitution was evidenced by low cytomegalovirus reactivation rates, occurring in only 4 of 19 at-risk patients (21%). Transplant-related toxicity included BK virus-associated cystitis in 33% of patients and a non-fatal hepatotoxicity syndrome in three patients consisting of transaminase elevation and ascites, with resolution following discontinuation of sirolimus. CNI-free GVHD prophylaxis with post-transplant Cy and brief-course sirolimus achieves consistent donor engraftment, low rates of GVHD and NRM, and excellent outcomes in recipients of HLA-identical donor allogeneic PBSCT. Disclosures: Off Label Use:fludarabine, cyclophosphamide, and sirolimus are not FDA-labeled for stem cell transplantation.

Early Clinical Experience of aAPC Activated NK-DLI Following Allogeneic PBSCT in Pediatric Solid Tumor Patients.

Abstract 3013 Background: Allogeneic Hematopoietic Stem Cell Transplant (alloHSCT) is effective in some hematologic malignancies and studies of alloHSCT for ultra high-risk pediatric solid tumors have shown some promise. Preclinical data demonstrates that activated NK cells readily kill pediatric solid tumors and leukemias, large numbers of activated NK cells can be generated ex vivo using artificial APCs (aAPCs) and the post-transplant period may be favorable for expansion and survival of adoptively transferred NK cells. Methods: We initiated a Phase I trial to assess feasibility and toxicity of escalating doses of donor-derived activated NK cell donor lymphocyte infusions (NK-DLI) on days 7 and 35 days following HLA-matched T cell depleted PBSCT in children and young adults with ultra-high risk solid tumors and leukemias. Donors underwent a single apheresis for filgrastim mobilized PBSC. The product was T cell depleted and CD34 and CD56 selected prior to cryopreservation, and the CD56+ fraction was cultured for 9–11 days with a K562 based aAPC expressing 4-1BBL and IL-15Ra plus rhIL-15 to generate the NK-DLI. NK-DLI consistently expressed high levels of natural cytotoxicity receptors NKp44 and NKp46 and mediated potent cytotoxicity ex vivo. T cell addback to the CD34 selected graft was performed to administer a T cell dose of 0.8–1.4 × 10e4 T cells/kg. Except for two pilot patients where NK-DLI was infused following engraftment, NK-DLI were administered ∼Day 7 and 35 post-transplant. Patients received a reduced intensity preparative regimen (fludarabine 30 mg/m2 and cyclophosphamide, 1200 mg/m2 on days –6 to -3; and melphalan 100 mg/m2 on day -2), and no prophylactic immunosuppression. Results: Seven patients with sarcomas were transplanted with NK-DLI infusion (1×10e5 CD56+ cells/kg/dose). Engraftment was brisk (median platelet recovery at 8d and neutrophil recovery at 9d). Patients had full donor myeloid chimerism by day 14 and all but one had >80% lymphoid chimerism by day 28. Patient #1 received NK-DLI on day +24 following alloHSCT and experienced aGVHD within 24 hours of NK-DLI with bullous skin rash and voluminous diarrhea. Prior to infusion the patient had what appeared to be a viral rash and fevers (skin biopsy negative for GVHD), but in retrospect likely GVHD, which was exacerbated by NK-DLI. Patient #2 received NK-DLI on Day +15 with no adverse effects. Subsequent patients enrolled onto cohort 1 with planned NK-DLI at days 7 and 35 (+7). Two patients developed aGVHD (rash and diarrhea), one with a cytokine-type reaction (fevers, hypotension) within 48 hours of NK-DLI infusion and one with aGVHD starting 21 days post NK-DLI. All 3 patients with matched unrelated donors experienced aGVHD after NK-DLI, whereas the 5 patients with matched sibling donors had no apparent adverse reactions nor GVHD. Persistence/engraftment of infused NK-DLI cannot be definitively determined, however, mean NK cell counts measured pre-alloHSCT and day +28 are 165/mm3 and 668/mm3 in the present cohort (n=6) compared to 149/mm3 and 395/mm3 in a historical alloHSCT population that did not receive NK-DLI (n=24). Antitumor effects via PET imaging were observed in patients with measurable disease and those without disease at the time of transplant have remained NED. Conclusions: Ex-vivo, aAPC expanded NK-DLIs do not interfere with stem cell engraftment but may contribute to or induce aGVHD, particularly in patients with matched, unrelated donors. Preliminary results suggest that aAPC NK-DLI expand in vivo and mediate antitumor effects following allogeneic PBSCT. Disclosures: No relevant conflicts of interest to declare.

5–azacitidine Treatment of Imminent Relapse Defined by Decreasing Donor CD34+ Progenitor Subset Chimerism in Patients with CD34+ High-Risk Myelodysplastic Syndromes (MDS) or Acute Myeloid Leukemia (AML) after Allogeneic Stem Cell Transplantation.

Besides graft versus host disease (GVHD), disease relapse is one of the major challenges in the care of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) undergoing allogeneic peripheral blood stem cell transplantation (PBSCT). However, we and others have shown that relapse can be predicted in case of CD34-expression on the malignant clone by a sensitive chimerism analysis in sorted CD34+ peripheral blood cells. If the percentage of donor cells in this compartment drops below 80%, leukemia relapse is inevitable within 4–8 weeks in the absence of clinical interventions like immediate cessation of immunosuppressive drugs or the administration of donor lymphocyte infusions. However, both approaches often result in clinically significant GVHD. Therefore, new strategies are warranted in order to treat imminent relapse in MDS or AML patients. We report results of an ongoing phase II clinical trial evaluating the efficacy of 5-azacitidine (5-aza) to prevent hematological relapse in patients with CD34+ AML or MDS and a decreasing CD34-donor chimerism after allogeneic PBSCT. Therefore, a total of 23 patients with CD34+ MDS (n=3) or AML (n=20) were prospectively screened on day 56, 84, 112, 140, 184, 365 and at later time points after PBSCT for a decreasing chimerism of donor CD34+ cells in the peripheral blood. In case of imminent relapse, defined as a drop of donor CD34+ cells below 80%, 5-aza was given at a dose of 75mg/m2/d s.c. day 1–7. A total of 4 cycles every 28 days were allowed in the absence of hematological relapse or toxicity. A median of 226 days after PBSCT, 9 out of 23 patients screened entered the treatment phase of the study with a median of 31% (range 0–53%) CD34+ donors cells in the peripheral blood (PB). However, a complete overall PB donor chimerism and less than 5% marrow blasts were documented in all patients before 5-aza treatment. Reversible neutropenia and thrombocytopenia grade 3/4 occurred in 50% of the patients. Only three patients still had immunosuppression prior 5-aza, which in two of them was slowly tapered during the period of 5-aza administration. With a median follow-up of 186 days after starting 5-aza all nine patients are eligible for response evaluation. Of these, CD34-chimerism was reverted to complete donor type (&gt;90%) in 6 (66%) patients. Two patients showed a further decrease of donor CD34+ cells and relapsed shortly after having completed the 1st or the 4th cycle of 5-aza, respectively. One patient showed an increase of CD34-chimerism after two cycles, however, died from non-relapse mortality. No hematological relapse occurred in the responders and in the screening cohort without decreasing CD34+ chimerism. Two patients (one without immunosuppression) developed limited cGVHD during 5-aza treatment. Preemptive treatment of minimal residual disease defined by decreasing donor CD34+ subset chimerism with 5-aza seems to be a potent strategy to prevent hematological relapse of CD34+ myeloid malignancies after allogeneic PBSCT.

Late Onset Eosinophilia (beyond Day 100), Rather Than Early Onset Eosinophilia, Has a Significant Prognostic Impact on Transplant Outcomes after Allogeneic Peripheral Blood Stem Cell Transplantation (PBSCT).

Background: The significance of eosinophilia after allogeneic stem cell transplantation (SCT) is presently unknown. The eosinophil activation pathway involves Th2 cytokines and thus eosinophilia after PBSCT may reflect Th2 pathway activation. Previous data suggest that blood eosinophilia precedes or coincides with the development of acute or chronic graft-verus-host disease (aGVHD or cGVHD). In a recent report, Aisa et al reported that patients with peripheral eosinophilia within 100 days post-SCT had a superior overall survival (OS) and transplant-related mortality (TRM) along with a lower incidence of cGVHD compared to those who did not (Transplant Int 2007). Methods: A retrospective analysis of 237 recipients who received allogeneic PBSC from related donors at PMH was performed (myeloablative:reduced intensity conditioning 162:75). The present study evaluated the impact of early (before day 100: EEo) or late onset (beyond day 100: LEo) eosinophilia (≥0.5x109/L in peripheral blood) on transplant outcomes after allogeneic PBSCT. The patient population included 237 recipients of allogeneic PBSC from related donors (male:female 137:100, median age 51 years-old). Results: Biphasic pattern of eosinophilia with 2 peaks before day 100 and beyond day 100 was observed after PBSCT. The cumulative incidence of early onset eosinophilia was 43% at day 100. EEo was not associated with the development of grade 3–4 aGVHD (p=0.1), but there was a trend in the development of grade 2–4 aGVHD (p=0.053). In univariate analyses, the group with EEo showed favorable OS (p=0.002), TRM (p=0.05) and a lower relapse incidence (p=0.05) compared to those without EEo but did not have improved GVHD-specific survival (GSS). EEo did not predict the grade of aGVHD or response to steroid therapy for aGVHD. EEo was significantly associated with the development of late onset eosinophilia (p=0.000004 by chi-square test, p=0.00007 by McNemar test). The cumulative incidence of LEo was 62% at 2 years with median onset of 1 year after SCT. There was an association with the onset of cutaneous (p=0.001), oral (p=0.04), ocular (p=0.02) or pulmonary cGVHD (p=0.03). In univariate analyses, with median follow-up durations of 773 days (patients with LEo) and 679 days (patients without LEo), significant differences were observed in favor of those with LEo in terms of 3 year OS (86% vs 41%, p=0.00000000005), TRM at 3 years (10% vs 37%, p=0.000003), 3 year relapse rate (11% vs 31%, p=0.00003) and 3-year GSS (90% vs 64%, p=0.00001). In multivariate analyses, the LEo, rather than EEo, was identified as a significant favorable prognostic factor for OS (p=0.000006, hazard ratio [HR] 0.21), TRM (p=0.0006, HR 0.24), relapse incidence (p=0.003, HR 0.28) and GSS (p=0.001, HR 0.26). Conclusion: The late onset eosinophilia (beyond day 100), not EEo, seemed to have an important prognostic implication for outcome after allogeneic PBSCT. Further studies are warranted to validate its prognostic impact on SCT outcomes. Figure 1. Comparisonof overall survival (A) and GVHD-specific survival (B) between patients with and without late onset eosinophilia after allogeneic PBSCT Figure 1. Comparisonof overall survival (A) and GVHD-specific survival (B) between patients with and without late onset eosinophilia after allogeneic PBSCT