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Purpose: To evaluate survival and neurocognitive outcomes in pediatric acute lymphoblastic leukemia (ALL) patients with central nervous system (CNS) involvement treated according to an institutional protocol with stem cell transplantation (SCT) and a component of craniospinal irradiation (CSI) in addition to total-body irradiation (TBI) as preparative regimen. Methods and Materials: Forty-one pediatric ALL patients underwent SCT with TBI and received additional cranial irradiation or CSI because of CNS leukemic involvement. Prospective neurocognitive testing was performed before and after SCT in a subset of patients. Cox regression models were used to determine associations of patient and disease characteristics and treatment methods with outcomes. Results: All patients received a cranial radiation boost; median total cranial dose was 24 Gy. Eighteen patients (44%) received a spinal boost; median total spinal dose for these patients was 18 Gy. Five-year disease-free survival (DFS) for all patients was 67%. Those receiving CSI had a trend toward superior DFS compared with those receiving a cranial boost alone (hazard ratio 3.23, P=.14). Patients with isolated CNS disease before SCT had a trend toward superior DFS (hazard ratio 3.64, P=.11, 5-year DFS 74%) compared with those with combined CNS and bone marrow disease (5-year DFS 59%). Neurocognitive testing revealed a mean post-SCT overall intelligence quotient of 103.7 at 4.4 years. Relative deficiencies in processing speed and/or working memory were noted in 6 of 16 tested patients (38%). Pre- and post-SCT neurocognitive testing revealed no significant change in intelligence quotient (mean increase +4.7 points). At a mean of 12.5 years after transplant, 11 of 13 long-term survivors (85%) had completed at least some coursework at a 2- or 4-year college. Conclusion: The addition of CSI to TBI before SCT in pediatric ALL with CNS involvement is effective and well-tolerated. Craniospinal irradiation plus TBI is worthy of further protocol investigation in children with CNS leukemia. [ABSTRACT FROM AUTHOR]

A major complication of mismatched unmanipulated hematopoietic stem cell transplant (HSCT) is Graft-versus-Host Disease (GvHD), which results in significant morbidity and increased non-relapse mortality. Novel strategies to reduce GvHD and improve long-term tolerance between mismatched donor-host pairs include regulatory T cell therapy. We are investigating a new cell product named T-allo10 which contains suppressive anergic cells and is enriched in IL-10 producing regulatory type 1 T cells (Tr1). The main advantage of adoptive immunotherapy with Tr1 compared to other regulatory T cells is the host alloantigen specificity that is established in the donor Tr1 during in vitro culture in the presence of IL-10 and host tolerogenic dendritic cells. Here we report the preliminary results of our phase I trial (IND 17292) on the use of escalating doses of T-allo10 cells for patients aged 3-45 years affected by hematological malignancies. At present, we completed the first cohort of the phase I portion of the study (Table 1). The donors were class I mismatched for patient 1 and class II HLA-mismatched for patients 2 and 3, respectively. The GvHD prophylaxis consisted of Sirolimus and Mycophenolate and was serotherapy and Calcineurin inhibitory-free. Patients received 1 × 106 T-allo10 cells/Kg on Day-1. No adverse events were observed after the T-allo10 cell infusion. All 3 patients met the safety criteria and are alive and disease-free at 2 years, 15 months and 2 months post-HSCT, respectively. Tr1, phenotypically defined as CD4+CD45RA−CD49b+LAG3+ were detectable in the peripheral blood shortly after the infusion at 21% and 26% in patient 2 and 3, respectively. At day 28 post-HSCT, Tr1 represented 12% of circulating memory CD4+ T cells in patient 1, 3% in patient 2 and 2% in patient 3. TCR sequencing of T-allo10 cells prior and after infusion showed a restricted clonotype diversity with persistence of certain clonotypes in vivo, demonstrating Tr1 cell recirculation and survival. These data show that the T-allo10 cell infusion is safe and well tolerated and demonstrate that T-allo10 cells are detectable in the recipients after the infusion and traceable by TCR clonotype analysis. [ABSTRACT FROM AUTHOR]

Adoptive immunotherapy using banked virus-specific T-lymphocytes (VSTs) derived from partially HLA-matched donors has been successful in restoring antiviral immunity after hematopoietic stem cell transplantation (HSCT), although broad availability of this therapy is currently limited, and best methods for donor-recipient matching remain unclear. To determine the feasibility and efficacy of third-party VSTs for the treatment of immunocompromised pediatric patients with refractory viral infections in a multicenter phase I/II consortium study. There are two arms to this study. Arm A are pediatric subjects who had infections with CMV, EBV, and/or adenovirus after hematopoietic stem cell transplant (HSCT) and had failed antiviral therapy, or who had toxicities related to anti-viral drug-related. Arm B enrolls patients with primary immunodeficiency disorders with refractory viral infection prior to HSCT. A VST bank containing products generated from 40 donors at Children's National was utilized, and antiviral HLA mapping was applied to identify the most suitable product for each patient referred. Patients received a dose of 2 × 10^7 VSTs/m2, with optional additional doses given 28 days after the initial VST infusion. Viral PCRs were monitored for efficacy, and VST persistence and expansion in vivo was measured via IFN-g ELISpot and flow cytometry. Twenty-three patients were enrolled from 11 study sites to date. Twenty-two were treated in Arm A (after HSCT), at a median time of 95 days post-HSCT (range 16-476). Fourteen patients were treated for adenovirus and 9 for CMV. Patients received a median of 1 infusion (range 1-3). Infused VSTs were matched at a median of 3/10 HLA alleles (range 2-5), and 22 had confirmation of antiviral activity through at least one shared HLA allele. Of 18 evaluable patients, 14 had antiviral responses at 1-month post-infusion (4/5 with CMV, and 10/13 with adenovirus, Figure 1), including 7 who had complete resolution of viremia. No GVHD was observed after infusion, and 17 of 23 (74%) are virus free up to 15 months post-infusion. Virus-specific T-cell activity was higher by day 45 (mean 177 IFN-g SPW) post-infusion than at baseline (mean 13 IFN-g SPW, p=0.045). VSTs remained detectable for up to 3 months via flow cytometry staining of discordant HLA alleles (Figure 2). Third-party VSTs utilized from a single bank can be distributed and administered in a multicenter consortium setting, and are safe and feasible for the treatment of refractory viral infections in pediatric patients. Preliminary efficacy data shows high rates of response and long-term survival. Studies of targeting strategies and characterization of T-cell epitopes are ongoing. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-matched donor, either related or unrelated, has been extensively used to treat patients with genetic disorders. However, only 25% of patients eligible to receive allogeneic HSCT have an HLA-identical sibling, and an HLA-matched unrelated donor can be identified for less than 60% of other patients. Mismatched HSCT has been associated with a high risk of graft loss, graft-versus-host disease (GvHD), and transplant-related mortality. Graft manipulation strategies have been used over the last decade to reduce these risks. HLA-haploidentical HSCT after ab T-cell/CD19 B-cell depletion (abhaplo-HSCT) has been shown to be effective in curing up to 90% of children with a variety of non-malignant disorders [Bertaina A, Blood 2014]. However, a scarcity of data are available for patients with rare and complex genetic diseases, including Tregopathies, an emerging new class of primary immunodeficiencies. Here we report preliminary data on the single center experience developed at Lucile Packard Children's Hospital, Stanford, CA, USA on the use of abhaplo-HSCT for rare and complex monogenic diseases (Table 1). All the cell products were manufactured at the Laboratory for Cell and Gene Medicine (LCGM) at Stanford (Table 2). Between 05/2018 and 08/2019, 5 patients with complex monogenic disorders were referred to our Center for HSCT including: a 23 year-old with Fanconi Anemia, two with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) and two with Schimke Immuno-Osseous Dysplasia (SIOD). Remarkably, all these patients were previously considered not eligible for a HSCT, and one of the IPEX patients was in palliative care. At a median follow-up of 240 days (range 39-520), all patients are alive and disease-free. One patient (SIOD) developed Grade I/stage II skin only graft-versus-host disease (GvHD), while none of the evaluable patients had developed chronic GvHD. Five months after abhaplo-HSCT and with 100% of the circulating T cells being donor derived, the first SIOD patient received a living kidney transplant from the stem cell donor (mother) using minimal immunosuppression with tacrolimus and steroid. Four weeks after the kidney transplant all immunosuppression was stopped, and the patient remains off immunosuppression 12 weeks post-kidney transplantation. Our preliminary data on the use of abhaplo-HSCT in patients with rare and complex monogenic diseases provide a strong rationale for expanding this therapeutic approach to diseases not previously considered as an indication for allogeneic HSCT, to patients in very poor clinical condition and to patients requiring both HSCT and solid organ transplant. [ABSTRACT FROM AUTHOR]

Patients who fail to adequately reconstitute the donor-derived immune system after allogeneic hematopoietic stem cell transplantation (HSCT) are at increased risk for infections and leukemia relapse. In the past, pan T-cell depleted haploidentical grafts were associated with delayed immune reconstitution (IR). Recently, the majority of patients receiving αβ T-cell/B-cell depleted haploidentical HSCT (αβhaplo-HSCT) reach a threshold of 200 CD3+ T cells/mcl by 100 days after HSCT (Bertaina A et al. Blood 2014 Jul 31;124(5):822-6). However, a proportion of patients experience a slower IR with consequent higher morbidity and mortality. Early prediction of delayed IR may permit prompt clinical intervention such as infusion of donor lymphocytes or of virus-specific cytotoxic T cells. Flow cytometry, the most widely applied approach for IR analysis, suffers from intrinsic limitations, such as high lymphocyte number requirement, degradation of samples, and insufficient standardization due to technical and operator variability. To overcome these limitations, we used a DNA methylation-based quantitative PCR that detects the epigenetic signature of different peripheral blood immune cell subsets (epigenetic quantification). This technique provides relative and absolute immune cell counts applicable to fresh, frozen, or paper-spotted dried blood. Epigenetic measurements are based on a per cell DNA copy number and provide a clear positive or negative signal rather than arbitrarily defined thresholds for "positivity" as in flow cytometry. We hypothesize that epigenetic quantification at day 15 after αβhaplo-HSCT could predict flow-based IR at day 100. Patients were consented at Lucile Packard Children's Hospital (Stanford, CA). Blood was collected between days 10-17 for epigenetic quantification and days 82-124 for flow cytometry. Bisulfite treated DNA underwent qPCR quantification of cell type-specific DNA regions of de-methylation (Baron U et al. Sci Transl Med 2018 Aug 1;10(452):eaan3508). Flow cytometry was performed using directly conjugated antibodies. Absolute cell counts were determined, plotted, and then analyzed using a linear regression model. In the first 5 αβhaplo-HSCT patients evaluated, we found a direct correlation between the epigenetic quantification at day 15 and flow cytometry at day 100 for CD4+ T cells (P=0.01), while the early epigenetic quantification of CD3+ and CD8+ T cells was not informative (Fig. 1). Preliminary data suggest that the use of epigenetic quantification early after αβhaplo-HSCT can predict the IR of CD4+ T cells at day 100 in αβhaplo-HSCT recipients. Ongoing analysis on a larger cohort of both αβhaplo-HSCT and unmanipulated HSCT recipients, will confirm if epigenetic quantification results obtained early post-HSCT can be used as a clinical biomarker of delayed IR and guide physicians in the use of post-HSCT adoptive immunotherapy. [ABSTRACT FROM AUTHOR]