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The current approach for preventing hemolysis of red blood cells (RBCs) in major ABO-incompatible bone marrow (BM) grafts after infusion is to deplete RBCs from BM products before transplantation. Traditionally, manual density separation (MDS) using Ficoll-Hypaque (Cytiva Sweden AB, Uppsala, Sweden has been used to accomplish RBC depletion. This process yields good CD34+ cell recovery, but it requires open manipulation and is labor-intensive and time-consuming. We hypothesized that an alternative automated method using Haemonetics Cell Saver 5+ (Haemonetics Corporation, Boston, MA, USA) would offer equivalent RBC depletion and CD34+ cell recovery. Small marrow volumes from pediatric donors can be processed using Cell Saver (CS) without adding the third-party RBCs necessary for other automated methods. This retrospective analysis comprised data from 58 allogeneic BM grafts. RBC depletion and CD34+ cell recovery from BM using MDS (35 grafts) were compared with CS (14 grafts). Nine products underwent RBC depletion using CS with Ficoll (CS-F) when RBC volume was less than 125 mL. Linear regression analysis of log transformation of CD34+ cell recovery adjusted for log transformation of both baseline CD34+ cell content and baseline total volume showed no significant difference between MDS and CS (estimated coefficient, –0.121, P = 0.096). All products contained an RBC volume of less than 0.25 mL/kg post-processing. CD34+ cell recovery with CS-F was comparable to MDS and CS and suitable for pediatric recipients of allogeneic hematopoietic cell transplantation. We provide evidence that an automated method using Haemonetics Cell Saver 5+ achieves RBC depletion and CD34+ cell recovery comparable to MDS when adjusting for baseline factors. [ABSTRACT FROM AUTHOR]

Background: Relapse remains a challenge after transplantation in pediatric patients with hematological malignancies. Myeloablative regimens used for disease control are associated with acute and long-term adverse effects. We used a CD45RA-depleted haploidentical graft for adoptive transfer of memory T cells combined with NK-cell addback and hypothesized that maximizing the graft-versus-leukemia (GVL) effect might allow for reduction in intensity of conditioning regimen. Methods: In this phase II clinical trial (NCT01807611), 72 patients with hematological malignancies (complete remission (CR)1: 25, ≥ CR2: 28, refractory disease: 19) received haploidentical CD34 + enriched and CD45RA-depleted hematopoietic progenitor cell grafts followed by NK-cell infusion. Conditioning included fludarabine, thiotepa, melphalan, cyclophosphamide, total lymphoid irradiation, and graft-versus-host disease (GVHD) prophylaxis consisted of a short-course sirolimus or mycophenolate mofetil without serotherapy. Results: The 3-year overall survival (OS) and event-free-survival (EFS) for patients in CR1 were 92% (95% CI:72–98) and 88% (95% CI: 67–96); ≥ CR2 were 81% (95% CI: 61–92) and 68% (95% CI: 47–82) and refractory disease were 32% (95% CI: 11–54) and 20% (95% CI: 6–40). The 3-year EFS for all patients in morphological CR was 77% (95% CI: 64–87) with no difference amongst recipients with or without minimal residual disease (P = 0.2992). Immune reconstitution was rapid, with mean CD3 and CD4 T-cell counts of 410/μL and 140/μL at day + 30. Cumulative incidence of acute GVHD and chronic GVHD was 36% and 26% but most patients with acute GVHD recovered rapidly with therapy. Lower rates of grade III-IV acute GVHD were observed with NK-cell alloreactive donors (P = 0.004), and higher rates of moderate/severe chronic GVHD occurred with maternal donors (P = 0.035). Conclusion: The combination of a CD45RA-depleted graft and NK-cell addback led to robust immune reconstitution maximizing the GVL effect and allowed for use of a submyeloablative, TBI-free conditioning regimen that was associated with excellent EFS resulting in promising long-term outcomes in this high-risk population. The trial is registered at ClinicalTrials.gov (NCT01807611). [ABSTRACT FROM AUTHOR]

Simple Summary: This study addresses the challenge of respiratory motion in pediatric patients undergoing proton therapy for total lymphoid irradiation (TLI), an essential component of reduced-intensity conditioning regimens for hematopoietic cell transplant (HCT) patients. The primary concern is the interplay effect, which can cause deviations from the planned dose during respiratory motion. The research utilized static and 4D CT images from ten patients to simulate the interplay effect and assess its impact on treatment planning. The study concludes that 4D plan evaluation and robust optimization techniques can help address respiratory motion challenges in proton TLI treatments, especially in cases where other motion management strategies like breath-holding are not feasible due to the patient's young age, the need for anesthesia, or medical conditions. Patient-specific respiratory motion evaluations are crucial to ensure adequate dosimetric coverage in proton therapy for pediatric patients. (1) Background: The most significant cause of an unacceptable deviation from the planned dose during respiratory motion is the interplay effect. We examined the correlation between the magnitude of splenic motion and its impact on plan quality for total lymphoid irradiation (TLI); (2) Methods: Static and 4D CT images from ten patients were used for interplay effect simulations. Patients' original plans were optimized based on the average CT extracted from the 4D CT and planned with two posterior beams using scenario-based optimization (±3 mm of setup and ±3% of range uncertainty) and gradient matching at the level of mid-spleen. Dynamically accumulated 4D doses (interplay effect dose) were calculated based on the time-dependent delivery sequence of radiation fluence across all phases of the 4D CT. Dose volume parameters for each simulated treatment delivery were evaluated for plan quality; (3) Results: Peak-to-peak splenic motion (≤12 mm) was measured from the 4D CT of ten patients. Interplay effect simulations revealed that the ITV coverage of the spleen remained within the protocol tolerance for splenic motion, ≤8 mm. The D100% coverage for ITV spleen decreased from 95.0% (nominal plan) to 89.3% with 10 mm and 87.2% with 12 mm of splenic motion; (4) Conclusions: 4D plan evaluation and robust optimization may overcome problems associated with respiratory motion in proton TLI treatments. Patient-specific respiratory motion evaluations are essential to confirming adequate dosimetric coverage when proton therapy is utilized. [ABSTRACT FROM AUTHOR]

Disease relapse remains a major cause of treatment failure in pediatric patients receiving allogeneic hematopoietic cell transplantation (HCT) for high-risk acute leukemias (AL) or myelodysplastic syndrome (MDS). Comprehensive data on prognostic factors and outcomes after post-transplant relapse in pediatric patients is lacking. The goal of this study was to identify factors associated with survival following post-transplant relapse. We performed a retrospective study of pediatric patients who experienced disease relapse following their 1st HCT for AL or MDS between 1990 – 2018 at our institution. Patient, disease and transplant-related characteristics were extracted from the medical record. Overall survival (OS) was estimated by the Kaplan-Meier (KM) method and compared using the log-rank test. Cox and logistic regression were performed to identify factors statistically associated with outcomes after post-transplant relapse. During the study period, 703 patients received 1st HCT, of which 221 (31%) relapsed at a median of 3.9 months after HCT. Of these relapsed patients, 60% (n = 132) received some chemotherapy or supportive care after post-transplant relapse, 13% (n = 28) received donor lymphocyte infusion only and 28% (n = 61) were able to proceed to a 2nd HCT. Patients who received a 2nd HCT were more likely to have been in first remission at the time of 1st HCT (P = 0.02), experienced relapse more than 6 months after 1st HCT (P < 0.001), experienced acute or chronic GVHD (P = 0.05 and 0.01, respectively) and relapsed in the current decade (2011 – 2018) (P = 0.001). Of the 61 patients who received a 2nd HCT, 18 (30%) were alive at last follow-up, compared to 7 (4%) of 160 patients who had received other therapies. In a multivariable model, longer interval from HCT to relapse (P < 0.001), being able to receive a 2nd HCT (P < 0.001) and receiving a HCT in the current decade (P < 0.001) were found to be significantly associated with OS (Fig 1 & 2). Factors that are typically associated with poor outcomes after HCT, such as disease status or type of HCT, donor and graft source were not predictive of survival following post-transplant relapse. Duration of remission before post-transplant relapse was identified as the strongest independent predictor of a patient's ability to receive a 2nd HCT as well as survival after relapse. The 3-year OS for patients who received a 2nd HCT was 6 times higher than that for patients who did not receive a 2nd HCT. Although the outcomes after 2nd HCT are not commendable, they are improving with time. Our data supports the argument that, when feasible, pediatric patients experiencing post-transplant relapse should be considered for 2nd HCT. [ABSTRACT FROM AUTHOR]

Donor T-cell reconstitution following hematopoietic cell transplantation (HCT) is critical for infection and leukemic control. Donor lymphocyte infusions (DLI) provide additional donor T cells, but may cause graft-versus-host-disease (GVHD) from alloreactive T-cells. As GVHD risk increases with increasing HLA-mismatch, conventional haploidentical DLI doses are often limited to 1 × 105 T cells/kg. Since the majority of alloreactive T cells reside in the naïve (CD45RA-positive) subset, CD45RA-depleted DLI may provide a diverse lymphocyte repertoire to aid in infection and leukemic disease control, with decreased GVHD risk. Herein, we report on feasibility, safety, and early efficacy of escalating doses of haploidentical memory DLI. Donors underwent leukapheresis without mobilization; 2 total blood volumes were collected. Depletion of CD45RA-positive cells was performed using the CliniMACS device (Miltenyi Biotec). DLI dosing typically started at 1 × 105 T cells/kg, with dose escalation every 4 weeks based on clinical need. All donors collected successfully in one attempt. Following CD45RA-depletion, median residual T cell dose was 45 × 106/kg and CD45RA-positive T cell dose was <0.001 × 106/kg (>3.9 log depletion), allowing multiple doses to be stored for future use (Figure 1). Twelve patients received a total of 29 DLIs (median infusions=2, range 1–5; Figure 2). Indications for DLI included detectible leukemia (6), mixed chimerism (6), and lymphopenia/viral infection (3); 3 recipients had multiple indications. First DLI was infused a median of 101.5 days post-HCT (range 41-356). Twenty-three doses had no detectible CD45RA-positive T cells (<0.001 × 106/kg) and 6 contained 0.001-0.002 × 106 CD45RA-positive T cells/kg. No infusion related acute adverse events occurred. One patient developed acute GVHD (clinical grade IV, stage 3 skin/stage 4 gut), 21 days post-DLI (dose=1 × 105 T cells/kg). After DLI, the majority of patients showed some clinical response (chimerism improvement/stability, decreased viral load, and/or rise in peripheral CD3 count). Eradication of detectable leukemia however was not successful, with all 6 patients eventually having disease progression (1 rapid progression; 5 frank relapse 2–6 months after first DLI). With median length of follow-up after last DLI of 243 days (range 20-924), no patients have developed chronic GVHD. CD45RA-depletion is feasible and effective in removing naïve donor T-cells. Haploidentical CD45RA-depleted DLIs were well tolerated and associated with low incidence of acute GVHD, even with T-cell doses as high as 10 × 106/kg. The majority of patients showed clinical benefit from DLI, with the exception of leukemia disease control. We are currently further investigating the efficacy and safety of CD45RA-depleted DLI as part of a prospective clinical haploidentical HCT trial. [ABSTRACT FROM AUTHOR]

Background Genes conferring carbapenem resistance have disseminated worldwide among Gram-negative bacteria. Here we present longitudinal changes in clinically obtained Escherichia coli isolates from 1 immunocompromised pediatric patient. This report demonstrates potential for antibiotic resistance genes and plasmids to emerge over time in clinical isolates from patients receiving intensive anticancer chemotherapy and broad-spectrum antibiotics. Methods Thirty-three isolates obtained over 7 months from 1 patient were included. Clinical data were abstracted from the medical record. For each isolate, studies included phenotypic antibacterial resistance patterns, sequence typing, bacterial isolate sequencing, plasmid identification, and antibiotic resistance gene identification. Results Sites of isolation included blood, wound culture, and culture for surveillance purposes from the perianal area. Isolates were of 5 sequence types (STs). All were resistant to multiple classes of antibiotics; 23 (69.6%) were phenotypically resistant to all carbapenems. The blaNDM-5 gene was identified in 22 (67%) isolates, all of ST-167 and ST-940, and appeared to coincide with the presence of the IncFII and IncX3 plasmid. Conclusions We present unique microbiologic data from 33 multidrug-resistant E. coli isolates obtained over the course of 7 months from an individual patient in the United States. Two E. coli sequence types causing invasive infection in the same patient and harboring the blaNDM-5 gene, encoded on the IncX3 plasmid and the IncFII plasmid, were identified. This study highlights the emergence of multidrug-resistant bacteria on antibiotic therapy and the necessity of adequate neutrophil number and function in the clearance of bacteremia. [ABSTRACT FROM AUTHOR]