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Interferon gamma receptor-1 (IFNγR1) deficiency is a rare inborn error of immunity. It can be inherited as an autosomal recessive (AR), partial or complete, or autosomal dominant (AD) trait and exhibits high allelic variability. Biallelic deleterious recessive variants of IFNGR1 that result in absent expression or defective downstream signaling of IFNγR1, cause failure to activate macrophages in response to interferon-gamma (IFN-γ), rendering innate immunity ineffective in clearing intracellular pathogens.

The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαβ+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.

Various approaches have been developed for ex vivo T cell depletion in allogeneic stem cell transplantation to prevent graft-versus-host disease (GVHD). Direct comparisons of T cell depletion strategies have not been well studied, however. We evaluated cellular and plasma biomarkers in 2 different graft manipulation strategies, CD3+CD19+ cell depletion (CD3/19D) versus CD34+ selection (CD34S), and their associations with clinical outcomes. Identical conditions, including the myeloablative preparative regimen, HLA-identical sibling donor, GVHD prophylaxis, and graft source, were used in the 2 cohorts. Major clinical outcomes were similar in the 2 groups in terms of overall survival, nonrelapse mortality, and cumulative incidence of relapse; however, the cumulative incidence of acute GVHD trended to be higher in the CD3/19D cohort compared with the CD34S cohort. A distinct biomarker profile was noted in the CD3/19D cohort: higher levels of ST2, impaired Helios− FoxP3+Treg reconstitution, and rapid reconstitution of naive, Th2, and Th17 CD4 cells in the early post-transplantation period. In vitro graft replication studies confirmed that CD3/19D disproportionately depleted Tregs and other CD4 subset repertoires in the graft. This study confirms the utility of biomarker monitoring, which can be directly correlated with biological consequences and possible future therapeutic indications.

Patients with high risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) are commonly older with multiple co-morbidities, rendering them unsuitable for intensive induction chemotherapy or transplantation. We report preliminary cellular immune profiling of four cases receiving sequential clofarabine and lenalidomide for high risk MDS and AML in a phase I study. Our results highlight the potential of immune profiling for monitoring immune-modifying agents in high risk MDS and AML.

Various approaches have been developed for ex vivo T cell depletion in allogeneic stem cell transplantation to prevent graft versus host disease (GVHD). However, direct comparisons between T-cell depletion strategies have not been well studied. We evaluated cellular and plasma biomarkers in two different graft manipulation strategies: CD3(+)CD19(+) cell depletion (CD3/19D) versus CD34(+) selection (CD34S) and their association with clinical outcomes. Identical conditions including the myeloablative preparative regimen, HLA-identical sibling donor, GVHD prophylaxis, and graft source were used for each cohort. Major clinical outcomes were similar between the two groups in terms of overall survival, non-relapse mortality, and cumulative incidence of relapse, however, the cumulative incidence of acute GVHD trended to be higher in CD3/19D compared to CD34S. A distinct biomarker profile was noted in the CD3/19D cohort: higher levels of ST2, impaired Helios(−) FoxP3(+)T(regs) reconstitution, and rapid reconstitution of naïve, Th2, and Th17 CD4 cells in the early post-transplant period. In vitro graft replication studies confirmed that CD3/19D disproportionately depleted T(regs) and other CD4 subset repertoires in the graft. This study confirmed the utility of biomarker monitoring which can be directly correlated to biological consequences and possible future therapeutic indications.

Clinical comorbidity predictive measures in ex vivo T-cell-depleted allogeneic hematopoietic stem cell transplantation

Frequent diagnostic radiology procedures in allogeneic stem cell transplantation (SCT) recipients raise concern about the potential harm from incidental radiation.To determine the cumulative radiation dose from diagnostic studies in allogeneic SCT and its impact on clinical outcome.This retrospective cohort study was conducted to determine the cumulative radiation dose from diagnostic studies following SCT. Sixty-four consecutive patients with hematological malignancies in a single tertiary care institution underwent total body irradiation (TBI)-based myeloablative conditioning followed by six of six human leukocyte antigen (HLA)-identical sibling allogeneic SCT. The median follow-up was 3 years. The cumulative effective dose in mSv from diagnostic radiological studies in the peri-transplant period from day -30 to day +200 was calculated for each patient and its impact on overall survival and non-relapse mortality was determined.The median cumulative radiation exposure from diagnostic radiological procedures was 92 mSv (range 1.2-300), representing about 30× the normal annual background radiation for the population and 10% of the 1200 cGy TBI dose used in conditioning. Sixty-five percent of the cumulative radiation exposure was delivered between day +1 and day 100 and computed tomography scans contributed 88%. In multivariate analysis, diagnostic procedures did not significantly impact clinical outcomes.While radiation exposure from diagnostic procedures did not impact clinical outcomes the risk of secondary cancers in long-term survivors is likely to be increased. Our results indicate that patients who are acutely ill for prolonged periods can receive clinically significant radiation doses during their hospital care. Our findings should prompt attempts to limit radiation exposure from diagnostic procedures in post-SCT recipients.

Introduction: High risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) commonly affect individuals of advanced age with multiple co-morbidities unsuited for curative standard treatments with allogeneic stem cell transplantation (allo-SCT) or intensive chemotherapy (ICT). New reduced intensity therapeutic approaches bringing improved life expectancy are needed for such patients. We conducted a phase I clinical trial to evaluate the safety and activity of sequential therapy with low-dose clofarabine followed by lenalidomide in older high risk MDS or AML. We hypothesized that lympho-depletion by clofarabine could "reboot" lymphocyte reactivity against the malignancy and promote favorable immune modulation by lenalidomide. Here we report preliminary clinical outcomes and associated cellular and molecular immune profiles. Methods: Four subjects (median age 68 years, range-60-79) with relapsed or refractory high-risk MDS (IPSS risk score>intermediate 2) or AML were enrolled to the protocol (12-H-0146, clinicaltrials.gov ID: NCT01629082). All subjects had been previously treated with at least one hypomethylating agent. Subjects received a single course of intravenous low-dose clofarabine 5mg/m2/d for five days, followed by consolidation therapy with oral lenalidomide with dose escalation from 25 mg daily up to 50 mg daily for 2 cycles. In the absence of dose limiting toxicity (DLT) or disease progression, subjects received lenalidomide maintenance 10 mg daily in 28 day cycles, with dose adjustments, for up to 12 cycles. Blood and marrow samples were collected before starting clofarabine, before consolidation with lenalidomide, and during maintenance. Multiparameter flow cytometric analysis was performed to characterize T cell subsets (memory T cells, T regs), natural killer (NK) cells, with functional markers representing T cell exhaustion (PD-1, LAG-3, TIM-3, PDL-1), activating and inhibitory NK cell receptor (KIR, LIR1, NKG2A, CD57). Relative changes in RNA expressions of target genes related to cancer immunology were evaluated by PCR array. Results: Of four subjects enrolled, two subjects showed responses at 28 days post clofarabine and at 28 days post first cycle of lenalidomide. These two responders tolerated subsequent maintenance lenalidomide for 4-6 months. The other two subjects were non-responders. Three subjects were taken off study due to disease progression on day 70, 162, and 206, and 1 subject for DLT on day 69. The clinical trial is now closed without dose escalation beyond the first cohort for reasons of poor accrual and lack of long term responses. In responders immunophenotype analysis showed a decrease in CD4+ %PD-1 expression in blood or bone marrow (0.87±0.29 fold change) after clofarabine and lenalidomide treatment, rising again after lenalidomide discontinuation in one responder (Figure). In contrast, CD4+ %PD-1 expression was increased in non-responders (1.23±0.19 fold change). In pre-treatment samples terminally differentiated CD57+ NK cell with high LIR1 expression (34.6±23.5%) were increased. In responders, both the levels of CD57 and LIR1 expression fell with reciprocal increase in CD56brightNK cells, suggesting restoration of NK cell repertoires after clofarabine. RNA expression profiles in one responder on maintenance lenalidomide showed significant up-regulation of gene expressions in IL-1A, FASLG, ICAM1, IL-27, WT1 (p Conclusion: Sequential treatment of low-dose clofarabine and lenalidomide is feasible for elderly patients with high risk MDS and AML. However individuals vary in the lenalidomide dose tolerated. Immune reconstitution profiles supports a potential immune-rebooting effect of clofarabine leading to restoration of CD4 cell and NK cell repertoires with fewer exhaustion or inhibitory markers. Lenalidomide may further promote a favorable anti-leukemic immune milieu. Immune profiling of T and NK cells can help to guide the application of these immune-modifying agents in high risk MDS and AML. Figure Figure. Disclosures Battiwalla: NIH/NHLBI: Employment.

Background: Allogeneic stem cell transplant (SCT) recipients suffer from a defective T cell mediated immunity causing potentially fatal reactivation of latent viruses. After T cell depleted SCT we have observed over 80% CMV reactivation and significant additional costs ($58-74k/patient). Despite aggressive monitoring and pre-emptive therapy, reactivation and/or positive CMV serology brings a significantly higher risk for non-relapse mortality (NRM). Adoptive transfer of ex vivo generated virus specific donor T cells is effective as a treatment of infection post-SCT but it has not been tested as a prophylaxis of early reactivation. Here in a Phase I study we transferred multi-virus specific T cells (MVSTs) immediately post SCT, targeting CMV, Ebstein-Barr virus (EBV), BK and adenovirus (Ad) as a novel strategy to prevent viral reactivation in the recipients of T cell depleted sibling HLA-matched SCT. Methods: Subjects were eligible if enrolled in HLA-matched T cell depleted transplant protocol (13-H-0144) and deemed at risk for CMV reactivation. MVST cells were manufactured from SCT sibling donors. Elutriated lymphocytes were stimulated with autologous dendritic cells (DCs) pulsed with seven overlapping peptide libraries (pepmixes) spanning the length of immunodominant proteins from CMV (pp65 and IE1), EBV (BZLF1 and EBNA1), BK (LT and VP1) and Ad5. Cultures were maintained in G-Rex flasks for 14 days in presence of IL-7, IL-15 and IL-2 (after 72hrs), tested for sterility, phenotype, potency and cryopreserved. MVST cells were thawed and administered intravenously as early as possible (day 0 to +60) post SCT. A Phase I 3+3 dose escalation design was used at the following dose levels: Cohort 1 - 1x10e5 total nucleated cells (TNC)/kg, Cohort 2 - 5x10e5 TNC/kg, Cohort 3 - 1x10e6 TNC/kg. The primary safety endpoint at day 42 post infusion was the occurrence of dose limiting toxicity (DLT), (Grade IV GVHD or any other severe adverse even (SAE) deemed to be at least "probably" or "definitely" related to the MVST infusion. Patients were followed to day +100 post SCT for secondary outcomes, including efficacy (Figure) and immune reactivity (for donor/recipient pairs). Results: MVST cells recognized the majority of pepmixes, were polyfunctional and robustly proliferated in response the cognate antigens, but minimally against allogeneic targets- suggesting a limited ability to induce GVHD. CDR3 sequencing of T cell repertoire showed a significant reduction in diversity and a striking dominance of a limited number of clonotypes in the final MVSTs. Nine subjects were enrolled and treated with MVST cells. MVSTs were successfully generated for all subjects, meeting the release criteria. Median time from SCT to MVST administration was 16 days (range D +6 to +52 post-SCT). Two subjects received MVST after day +30 due to cardiac instability and scheduling. There were no immediate infusion-related adverse events or DLT by day 42. One subject in cohort II developed a self-limiting grade I cytokine release syndrome in the setting of low-level EBV reactivation. One patient (cohort 1) developed de novo grade III aGVHD post-MVST infusion. CMV reactivation post-MVST occurred in 4 out of 8 evaluable subjects (50%) who completed D+100 post-SCT vs. 45 out of 52 patients (50% vs 87%; p value=0.031) in a historical cohort of recipients of T cell depleted SCT. In all cases CMV reactivation occurred during treatment with high dose steroids. In two cases MVST were generated from CMV seronegative donors and showed minimal activity against pp65 and IE1. In eight evaluable subjects who reached D+100 post-SCT there was no EBV-related disease, but we saw self-limiting low level EBV replication in 6 out of 8 cases. There were no cases of BK or Ad-related disease or viremia. ELISPOT analysis at D+100 revealed robust reconstitution of anti-viral immunity in analyzed recipients (vs. donors, Figure, B) Conclusions: This is the first report demonstrating that it is safe and feasible to use adoptively transferred allo-MVST immediately post-SCT to rapidly reconstitute anti-viral immunity and ameliorate the detrimental impact of the early viral reactivation in SCT recipient. No DLTs were seen. MVSTs had a markedly reduced allo-reactivity and carried a minimal risk of GVHD. Our results also suggest efficacy of this strategy in reducing viral reactivation. A Phase II portion of this study is currently enrolling patients. Figure. Figure. Disclosures Sabatino: Kite: Employment, Equity Ownership.

INTRODUCTION: Ex-vivo T cell depletion strategies have been widely used to reduce the incidence of graft versus host disease (GVHD) in allogeneic stem cell transplantation (allo-SCT). Although several options of ex-vivo graft manipulation strategy are available, direct comparison between strategies along with relevant biomarkers has been lacking. Here we evaluated cellular and plasma biomarkers in two separate graft manipulation strategies, CD3-CD19 depletion versus CD34+ selection using the Miltenyi CliniMACS and their association with clinical outcomes. METHODS: Forty two subjects with hematological malignancies underwent HLA matched sibling allo-SCT at a single center between 2012 and 2015 and received either an ex-vivo CD3-CD19 depleted, CD34+ negatively selected graft (CD3/19D, n=20) or an ex-vivo CD34+ cell positively selected graft (CD34S, n=22). Both cohorts were treated with the same conditioning regimen of cyclophosphamide, fludarabine, and total body irradiation (600-1200 cGy) and GVHD prophylaxis of low dose cyclosporine. Peripheral blood mononuclear cells and plasma samples were collected at days 14 or 30, 60, 100 post-transplant. Post-transplant cellular immune reconstitution was evaluated by multi-color flow cytometry immunophenotyping, characterizing the subsets of memory T cells, regulatory T cells (Tregs), natural killer (NK) cells, and B cells with various functional markers. The plasma levels of ST2, Reg3α, and sTNFR1 were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The median age at transplant was 48 years (range 17-70) in CD3/19D and 45 years (11-73) in CD34S. At a median follow up of 37 months in CD3/19D and 22 months in CD34S, the major clinical outcomes were similar between two groups; the overall survival (70% and 86%), non-relapse mortality (5% and 4.5%), and cumulative incidence of relapse (35% and 39%) at 2 years, respectively. Two subjects in CD3/19D developed late engraftment failure before day 100 but all other subjects achieved primary neutrophil and platelet recovery. Unexpectedly, the cumulative incidence of grade II-IV acute GVHD was higher in CD3/19D (61%) in comparison to the incidence in CD34S (32%, P=0.07, Figure). The cumulative incidence of extensive chronic GVHD was 33% in CD3/19S and 24% in CD34S. The fraction of Helios negative Tregs post-transplant was significantly lower in CD3/19D (median [interquartile range]: 10.4% [7.1-16.4] at day 30; 4.9% [3.0-8.3] at day 60) compared to CD34S (23.8% [10.7-35.8], P=0.03 at day 30; 8.8% [6.8-18.4], P=0.01 at day 60, Figure). Plasma ST2 levels were significantly higher in CD3/19D (45ng/mL [27-67] at day 14; 33ng/mL [27-62] at day 28) in comparison to CD34S (29ng/mL [19-40], P=0.03 at day 14; 25ng/mL [14-33], P=0.03 at day 28, Figure). In addition, significantly higher CD4 naive T cells, lower effector memory and PD-1 bright CD4 T cells were observed in CD3/19D in comparison to CD34S. NK and B cell profiles were not significantly different between the two groups. CONCLUSION: Both methods of ex vivo TCD were associated with extremely low NRM rates (~5%).We observed a higher cumulative incidence of acute GVHD in the recipients of CD3/19 depleted grafts, accompanied with the distinct biomarker profiles of poor Treg reconstitution and high level of ST2. CD3/19 depletion may have disproportionately depleted Tregs in the graft, leading to uncontrolled tissue damage and GVHD evidenced by higher ST2 levels. Further validation is required to confirm the utility of monitoring Treg reconstitution and ST2 level as biomarkers to predict the outcomes of T cell depleted allo-SCT. Figure 1. Figure 1. Disclosures Battiwalla: NIH/NHLBI: Employment.

INTRODUCTION: Haploidentical allogeneic stem cell transplantation (haplo-SCT) incurs a risk of bidirectional immune reactions with either severe acute graft versus host disease (aGVHD) or graft rejection. Induction of immune tolerance with post-graft cyclophosphamide dramatically improves the outcomes of haplo-SCT. However the optimal duration and the combination of systemic immunosuppressive agents in haplo-SCT remain controversial. Ultra-low dose interleukin 2 (ULD IL-2) preferentially expands regulatory T cells (Tregs) and natural killer (NK) cells, promoting both GVHD prevention and graft versus leukemia (GVL) effects. These properties suggest that ULD IL-2 could play a useful role in haplo-SCT. Here we report the outcomes of a pilot study to determine the safety and feasibility of ULD IL-2 as GVHD prophylaxis in haploidentical allo-SCT (14-H-0180, Clinical Trials.gov ID: NCT02226861) METHODS: Ten subjects with high risk hematological malignancies received a myeloablative conditioning regimens of fludarabine 120mg/m2 (day -10 to day -8) and total body irradiation (TBI; 600-1200 cGy, day -10 to day -6). Thereafter the subjects received donor lymphocyte infusion (DLI) products (2x108 CD3+/kg) on day -6, followed by post-DLI cyclophosphamide 120mg/kg on days -3 and -2. CD 34+ selected, peripheral blood stem cell product was infused on day 0. Sirolimus was initiated on day -1 with goal trough level of 5-12ng/mL until day+60. ULD-IL2 (aldesleukin, 100,000 IU/m2) was given subcutaneously daily for 12 weeks starting day +1. Peripheral blood mononuclear cells (PBMC) and plasma samples were collected at days 14, 28, 60, 84, 100 post-transplant. Multi-color flow cytometry immunophenotyping assay were performed to characterize the subsets of memory T cells, Tregs, NK cells, and monocytes with various functional markers. Plasma levels of biomarkers (ST2, Reg3α, sTNFR1, ANG1, IL-6) were measured using a multiplex microfluidic channel assay. RESULTS: The median age at transplant was 35 years (range 20-66). Most subjects had a high risk EBMT score (median 4, range 2-7) and HCT co-morbidity index (median 4, range 2-7). All subjects achieved successful engraftment (neutrophil >500/uL; median 13 days, platelet >20k/uL; median 15 days) and rapid full donor myeloid and lymphoid chimerism by day 21. At median follow up of 6 month, the overall survival was 71%. One subject died of hepatic veno-occulusive disease (VOD) on day 32 and one subject died of relapse on day 178. All evaluable subjects tolerated ULD-IL2 without significant toxicities. Four subjects experienced either de-novo or rapid exacerbation of acute GVHD after discontinuation of ULD IL-2, resulting in the cumulative incidence of grade II-IV aGVHD of 61% and grade III-IV aGVHD of 36% (Figure A). ULD IL-2 expanded and maintained Helios+FoxP3+Tregs population (pre-transplant, 4.7%±3.1%; day 30, 36.2%±23.1%; day 84, 17.4%±10.6%) as well as CD56brightNK cells population (pre-transplant, 10.7%±13.7%; day 30, 49.7%±10.8%; day 84, 26.1%±6.8%). However on discontinuation of ULD IL-2 both populations decreased to low levels within one week. The timing of aGVHD correlated with a fall of %Tregs in PBMC and a sharp increase of ST2 level in plasma (Figure B). CONCLUSION: ULD IL-2 can be safely administered as GVHD prophylaxis after haplo-SCT. Rebound GVHD after discontinuation of ULD IL-2 implies that donor-derived Tregs acquired dependency to exogenous ULD IL-2. Our study is proof of principle that ULD IL-2 induces immune tolerance through Tregs expansion in haplo-SCT, inspiring further clinical and basic researches in human IL-2 biology. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Introduction: Little is known about the lethality of acute GVHD (aGVHD) in T -cell depleted (TCD) allogeneic hematopoietic stem cell transplantation (HSCT). We examined the incidence of aGVHD and its relative contribution to non relapse mortality (NRM) in a cohort of consecutive TCD HSCT at a single institute. Methods: We report 132 consecutive subjects who had undergone TCD HLA-identical sibling HSCT between 2006 and 2016 for hematologic malignancies. All subjects received conditioning with Fludarabine 125mg/m2, Cytoxan 120 mg/kg and 1200 cGy total body irradiation (TBI) ( Thirty-six subjects were over the age of 55 years, 66 were females and 66 were males. Transplant indications were acute leukemia (92), myelodysplastic / myeloproliferative syndromes (26), lymphoid malignancies (7) and chronic myelogenous or myelomonocytic leukemia (7). Fifty-three percent of subjects were at a high risk of relapse. The median follow-up post HSCT was 4.3 years. Results: The incidence of Grade 1-2 and 3-4 aGVHD were 51.6% and 19%, respectively. Of these, 15% of aGVHD was steroid refractory and was treated with infliximab/basiliximab or with mesenchymal stromal cells. Extensive or limited chronic GvHD was observed in 30% and 24% of subjects. 74% of those at risk developed cytomegalovirus (CMV) reactivation. The Kaplan-Meier (KM) estimate for overall survival (OS), NRM and cumulative incidence of relapse (CIR) for the entire cohort was 71%, 14.6% and 24% respectively at one year, and 52%, 32.5% and 39% at 5 years. We considered the impact of CMV reactivation, slow donor lymphoid chimerism and steroid refractory aGVHD on NRM. However there was no significant impact from CMV reactivation or slow (> 31 days) achievement of complete donor lymphoid chimerism. Significantly improved outcomes were noted for those transplanted beyond 2012: OS, NRM and CIR being 82%,6.2% and 20% at one year, and 68%, 6.2% and 41% at 3 years. Cox proportionate hazard modeling identified steroid refractory aGVHD (HR 4.0, P=0.007) and transplant prior to 2012 (HR 6.7, P=0.001) as significant factors impacting NRM. Conclusions: T cell addback after ex vivo TCD HSCT was associated with a significant burden of aGVHD. Steroid refractory aGVHD impacted NRM, but slow lymphoid engraftment, disease risk, CMV reactivation and age did not. Significant improvements in NRM in the current era suggest greatly improved salvage of steroid refractory aGVHD. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Ex vivo T cell depletion (TCD) of the graft by CD34+ selection improves the tolerability of allogeneic hematopoietic stem cell transplantation (HCT). Clinical comorbidity measures enhance the estimates of HCT tolerance and outcomes. However, clinical prediction tools such as the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) have not been validated in TCD transplants. To improve outcome prediction we therefore evaluated published comorbidity measures and other standard clinical biomarkers of outcome in a series of patients with hematological malignancies receiving myeloablative TCD transplants. Pre-transplant factors compared were: HCT-CI, European Group for Blood and Marrow Transplantation (EBMT) risk score, Eastern Cooperative Oncology Group (ECOG) Performance Status, serum C-reactive protein (CRP), albumin (ALB), pre-albumin (PAB), ferritin, absolute lymphocyte counts (ALC), and absolute lymphocyte / monocyte ratio (LMR). All patients received a myeloablative preparative regimen (Flu 125 mg/m2, Cytoxan 120mg/kg, TBI 400 cGy if older than 55 or 1200 cGy if younger than 55) followed by the infusion of a CD34+ selected graft from a HLA-identical sibling containing 1 to 5x10e4 CD3+/kg. Of the 79 recipients, 34 (43%) had standard risk disease, and 45 (57%) recipients had high-risk disease. At a median follow up of ~ 5 years, overall survival (OS) was 42.9% and nonrelapse mortality (NRM) was 32.9%. In the initial analyses we eliminated pre-transplant ECOG, ferritin, ALB, PAB, ALC and EBMT. The independent pre-transplant comorbidity variables identified for further testing were HCT-CI, CRP and LMR. We demonstrated that LMR and HCT-CI score are important independent clinical predictors of OS and NRM in TCD HCT. In univariate analysis of OS, significant factors were HCT-CI scores ≥ 5 (Hazard ratio (HR) 2.125, p= 0.018), CRP (HR=1.016, p=0.058, a trend) and LMR ≤ 1.3 (HR=2.054, p= 0.036). In multivariate models of OS, CRP (HR 1.02, p= 0.029), HCT-CI≥5 (HR 1.966, p= 0.04) and LMR ≤1.3 (HR 2.206, p= 0.029) retained significance. Further refinement of the multivariable model was made by classifying the LMR into a low-risk group (LMR 3-25) versus high-risk groups (LMR< 3 or > 25), then the significant factors were HCT-CI ≥5 (HR 3.008, p= 0.002), LMR < 3 (HR=3.292, p= 0.0014) and LMR > 25 (HR=5.648, p= 0.0008) compared to the LMR 3-25 group. In multivariable analysis of NRM, significant factors were HCT-CI ≥5 (HR 3.283, p= 0.015), LMR < 3 (HR=5.891, p= 0.0008), and LMR > 25 (HR=9.008, p= 0.0011) compared to the LMR 3-25 group. To evaluate the relevance of these factors in predicting the 5-year mortality and NRM beyond traditional risk indicators, we used the continuous net reclassification improvement (NRI) and the C-statistic (area under the receiver operating characteristic (ROC) curve). LMR was found to be the most important risk predictor for both OS and NRM (NRI was 84% and the C-statistic was improved from 0.59 to 0.68 with the addition of LMR to the multivariate model for OS; NRI was 108% and the C-statistic was improved from 0.57 to 0.72 with the addition of LMR to the multivariate model for NRM). Based on our results, we propose a simple novel predictive score for CD34+ selected myeloablative transplantation calculated by summation of scores derived from the HCT-CI [≤4 =Score 0, ≥5=Score 1] and LMR [3-25= Score 0, 25=Score 2]. The scores [range, 0 to 3] differentiate three risk groups for hematopoietic cell transplantation OS (p=0.001) and NRM (p=0.004). In conclusion, this is the first study to explore pre-transplant comorbidity measures after TCD HCT, identifying HCT-CI and LMR as critical predictors of OS and NRM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Clinical comorbidity measures enhance the estimates of HCT tolerance and outcomes, thereby aiding therapeutic decisions. Ex vivo T cell depletion (TCD) of the graft reduces the risk of graft-versus-host disease and improves the tolerability of allogeneic transplantation in patients with impaired pretransplant performance. However, prediction tools such as the hematopoietic cell transplantation-specific comorbidity index (HCT-CI), have only been validated in conventional T-replete HCT. To improve outcome prediction in TCD transplants we therefore evaluated published comorbidity measures and other potential biomarkers of outcome in a series of myeloablative TCD transplant recipients. Pre transplant (week -2) factors measured were: HCT-CI, ECOG performance status, serum C-reactive protein (CRP), albumin (ALB), pre-albumin (PAB), ferritin, absolute lymphocyte counts (ALC), and absolute lymphocyte / monocyte ratio (LMR). CRP was also studied serially post transplant. CRP increased after conditioning, peaked (p =0.0001) in the first week of HCT, with recovery to baseline at 5 weeks post-HCT. We evaluated outcomes in a cohort of 79 patients in our institute with hematological malignancies receiving myeloablative total-body irradiation, and an HLA-identical sibling peripheral blood HCT, T cell depleted using Miltenyi CD34+ selection. The median age of recipients was 43 years (range 13-68 years). Of the 79, 34 (43%) had standard risk disease, and 45 (57%) recipients had high-risk disease. At a median follow up of ∼ 5 years, overall survival (OS) was 42.9% and nonrelapse mortality (NRM) was 33.9%. Comorbidity measures were first screened to eliminate highly-correlated covariates. Univariate Cox regression models were used to identify significant factors (p 0 were found to be significantly associated with the cumulative incidence of relapse (both p=0.01). In conclusion, this is the first study to explore comorbidity scores and biomarkers to predict outcome after ex vivo TCD HCT. Our results suggest that HCT-CI score, preCRP, postCRP and the LMR are important independent clinical predictors of OS and NRM in TCD HCT. Disclosures: No relevant conflicts of interest to declare.