Rate Of CMV Reactivation Research Articles

Very Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells Is a Safe Strategy for Prevention of Viral Infection after Allogeneic T Cell Depleted Stem Cell Transplantation

▪Background: Reactivation of latent viruses post-allogeneic stem cell transplant (SCT) negatively affects outcomes and increases non-relapse mortality. We have reported over 80% early CMV reactivation rate and significant additional costs in recipients of T cell depleted (TCD) SCT. Ex vivo generated multi-virus specific T cells (MVSTs) are effective as a therapy of active infection, but have not been evaluated as prophylaxis early after transplant. In a Phase I study (NIH 14-H-0182) we transferred MVSTs targeting immunodominant viral proteins of CMV, Epstein-Barr virus (EBV), BK and adenovirus (Ad) immediately post SCT as a novel approach to prevent viral reactivation post-SCT.Methods: All subjects were enrolled in HLA-matched T cell depleted (TCD) transplant protocol (NIH 13-H-0144). MVSTs cells were manufactured from SCT sibling donors by stimulation of elutriated lymphocytes for 14 days with seven overlapping peptide libraries (pepmixes) pulsed onto autologous dendritic cells (DCs) in presence of IL-7, IL-15 and IL-2. MVSTs were infused as early as possible (day 0 to +60) post SCT. A Phase I 3+3 dose escalation design was used as follows: Cohort 1 - 1x10e5 total nucleated cells (TNC)/kg, Cohort 2 - 5x10e5 TNC/kg, Cohort 3 - 1x10e6 TNC/kg. Three additional subjects received MVST cells manufactured using pepmix-pulsed mononuclear cells as stimulators (Cohort 3A; 1x10e6TNC/kg) under an amended protocol. The primary safety endpoint at day 42 post infusion was the occurrence of dose limiting toxicity (DLT) defined as 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 of efficacy, immune reconstitution and GVHD biomarkers (ST2, REG3). CDR3 sequencing (ImmunoSEQ) was performed on selected MVST products and peripheral blood samples post MVST and compared to control SCT recipients. GVHD biomarkers were analyzed pre- and post-treatment.Results: Twelve subjects were treated: nine received MVSTs generated using DCs and three subjects using mononuclear cells (cohort 3A). Median time from SCT to MVST administration was 13 days (range D +2 to +52 post-SCT). Median time to MVST for subjects in Cohort 3A was 3 days (range 2-7). There were no immediate infusion-related adverse events or DLT at the highest dose level. De novo grade II-III aGVHD post-MVST infusion was seen in three subjects (one in Cohort 1 and two in Cohort 3A), but GVHD biomarker elevation predated MVST infusion. CMV reactivation post-MVST occurred in 6 out of 12 subjects (50%) vs. 45 out of 52 patients (50% vs 87%) in a historical group of recipients of T cell depleted SCT. In all cases CMV reactivation occurred in the context of high dose steroids and in two subjects MVSTs were derived from CMV seronegative donors with minimal anti-CMV activity. One subject experienced rapidly rising AdV viremia (asymptomatic) and received an additional infusion of MVSTs. We saw self-limiting low level EBV replication in 8 cases and one BK viremia, but no disease. CDR3 sequencing of MVST products and serial peripheral blood from subjects revealed a robust contribution of ex vivo expanded cells to the overall repertoire, in contrast to untreated controls where the repertoire of (sham) MVST cell products generated from the transplant donors did not significantly overlap with the immune repertoire in peripheral blood of TCD-SCT recipients in the early post SCT period. Only at day +180 some convergence of repertoires became visible indicating spontaneous immune reconstitution (Figure). Detailed CDR3 analysis of cytokine-captured CMV pp65 and IE-1 specific CD4+ and CD8+ T cells was performed in a representative subject clearly demonstrating the detrimental effect of high-dose steroids on frequency of anti-viral T cells, precipitating CMV reactivation.Conclusions: This is the first report demonstrating safety and feasibility of using 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 and a minimal risk of aGVHD was observed, as there was no correlation with GVHD biomarkers. As revealed by serial CDR3 sequencing, MVSTs robustly contributed to the T cell repertoire. Our results suggest efficacy of this strategy in reducing viral reactivation. A Phase II study is warranted. DisclosuresNo relevant conflicts of interest to declare.

96 - A Modified Intensive Strategy to Prevent CMV Disease in Seropositive Umbilical Cord Blood Transplant Recipients

Abstract We previously demonstrated a lower rate of CMV reactivation and disease among seropositive umbilical cord blood transplant (CBT) recipients receiving an intensive prophylaxis strategy consisting of ganciclovir on days -8 to -2 pre-transplant, high-dose valacyclovir post-transplant, and twice-weekly serum CMV PCR testing. We hypothesized that a modified intensive strategy excluding pre-transplant ganciclovir would be similarly effective. We compared the risk of CMV reactivation, occurrence of CMV disease, and duration of anti-CMV therapy by day 100 post-CBT in patients receiving the modified intensive and intensive strategies. Forty patients received the modified intensive strategy, and 43 received the intensive strategy. There was no difference in the hazard for CMV reactivation (HR, 1.1; p = 0.77). No patients developed CMV disease in the modified intensive cohort compared to 2 patients in the intensive cohort ( p = .53). There was no difference in the hazard for early (≤30 days post-CBT; p = .76) or high-level (>1000 IU/ml; p = .37) CMV reactivation. Patients in the modified intensive cohort had marginally higher CMV viral loads and percentages of days of CMV detection and treatment, although the contribution of pre-transplant ganciclovir to these changes is unclear. The overall percentage of treatment days was 32% in both cohorts after accounting for pre-transplant ganciclovir. In conclusion, exclusion of prophylactic ganciclovir before CBT did not impact the risk for CMV reactivation or disease, although CMV kinetics appeared to differ by prevention strategy. Best practices for CMV prevention will need further study as new prophylactic strategies become available.

Addition of ATG to Myeloablative Haplo Conditioning with Post-Transplantation Cyclophosphamide Might Decrease the Risk of Gvhd and TRM without Increasing the Risk of Relapse

Introduction: The use of a Myeloablative (MA) regimen followed bypost-transplantation high dose Cyclophosphamide (PT-CY) has been adopted to overcome the increased relapse risk following nonmyeloablative conditioning regimen and unmanipulated Haploidentical Bone Marrow Transplantation, in patients with high-risk hematological malignancies, with acceptable TRM and risk of GVHD. We added ATG to our Myeloablative regimen with PT-CY after noticing significant incidence of grade II-IV aGVHD with the first few cases. Here we compare the outcomes of the 12 patients who received ATG to the outcomes of the first 11 patients who were transplanted without ATG.Patients and Methods: our haploidentical program was started in 2013 as a phase I/II prospective clinical trial. After reviewing the first 11 cases enrolled on the trial (MA regimen with PT-CY, without ATG), we noticed significant incidence of high grade aGVHD (54.55%). We amended our protocol in early 2015. Rabbit ATG was added at a dose of 3 mg/kg (1.5 mg/kg day -3 and day -2) to our conditioning regimen [thiotepa (5mg/kg/day on day -8 and -7), busulfan (3.2mg/kg/day IV day-6, -5 and -4), fludarabine (50mg/m2/day on day -6, -5 and -4 )] or [TBI 1000 cGy (200cGy twice a day on days -10, -9 and one dose on day -8), fludarabine (30mg/m2/day on Days -7, -6, -5 and -4)]. Graft-versus-host disease (GVHD) prophylaxis consisted of PT-CY (50mg/kg/day) on days +3 and +5, cyclosporine (starting day +4), and mycophenolate mofetil (starting day +1). Table 1 summarizes the disease-type and disease-status at transplant. Patient characteristics were comparable at baseline between the 2 groups.Results (Table 2): We enrolled patients with high risk hematologic malignancies in need for SCT but have no matched donor (MSD, MUD). Despite the small sample size, our results showed a statistically significant difference in the rate of acute GVHD between the 2 groups (p= 0.0161) in favor of the ATG group, chronic GVHD was more frequent in the non-ATG group however the difference did not reach statistical significance probably due to the small sample size. There was no statistically-significant difference in the risk of relapse, CMV reactivation or Hemorrhagic cystitis between the 2 groups. However, there was a trend of higher relapse rate (33.3% vs 18.18%), a higher rate of Hemorrhagic cystitis (50% vs 18.18%) and a higher rate of CMV reactivation (100% vs 81.82%) in the ATG group. The cumulative incidence of TRM at day 100 was in favor of the ATG group (figure 1), with a trend toward a better DFS in these patients 6 months post-transplant (figure 2). To be noted the follow up period was shorter for the ATG group because ATG was added later on. Figure 3 shows the survival for ATG vs non ATG group.Conclusion: The use of ATG with myeloablative Haplo conditioning can significantly reduce the risk of acute GVHD and early TRM. We have seen more relapses, higher rate of CMV reactivation, and hemorrhagic cystitis with the addition of ATG but these did not reach statistical significance probably due to the small sample size. A lower dose of ATG might be the way to go to strike a careful balance and improve the outcomes of myeloablative haploidentical transplant. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Survival Benefit of Intravenous Busulfan (120 mg/M2) and Fludarabine Myeloablative Regimen for Treatment of Myeloid Malignancies

Background: Intravenous fludarabine and busulfan 130 mg/m2 (Flu/Bu) conditioning regimen has induced the long term survival with a low treatment related mortality rate. However, there have been few reports on long term survival of patients undergoing allo-HSCT with intravenous Flu/Bu regimen. Therefore, we conducted a retrospective study of 42 patients diagnosed with myeloid malignancies received allo-HSCT with intravenous fludarabine and busulfan (120 mg/m2) regimen between 2006 and 2015 at Ramathibodi hospital. The aim of our study was to observe the long term survival and the complication after transplantation. Findings: Thirty- four, three and five patients were AML, MDS and CML-CP respectively. With a median followup of 95 months, 1- year EFS and 8- year EFS were 82 and 70% respectively. Overall survival (OS) rate at 1 and 8 years were equally observed in 88%. Patient younger than age 45 years had significantly longer OS than patients aged 45 years and older (96 vs 70%, p=0.019). Eight- year OS in AML, MDS and CML were 88, 67 and 100% respectively. Acute and chronic graft versus host disease were found in 29 and 46.3% of 41 evaluable patients respectively. Whereas the rates of sinusoidal obstruction syndrome, sepsis, CMV reactivation, cyclosporine and tacrolimus induced thrombotic microangiopathy were 2, 10, 12, 5 and 2% respectively. Non- relapse mortality rate at day +100, 1 and 8 year were only 9.5, 13.8 and 13.8% respectively. There was no neurological toxicity, severe mucositis, secondary malignancy or therapy related MDS syndrome in this study. Conclusion: Allogeneic hematopoietic stem cell transplantation with intravenous fludarabine and busulfan at the dose of 120 mg/m2 was well tolerated and demonstrated impressive treatment outcomes in young adult patients diagnosed with myeloid malignancies.

Pre-Emptive Donor Lymphocyte Infusion with CD19-Directed, CAR-Modified T Cells Infused after Allogeneic Hematopoietic Cell Transplantation for Patients with Advanced CD19+ Malignancies

Background: Allogeneic hematopoietic cell transplantation (HCT) can be curative in a subset of patients with advanced lymphoid malignancies but relapse remains a major reason for treatment failure. Donor-derived, non-specific lymphocyte infusions (DLI) can confer an immune anti-malignancy effect but can be complicated by graft-versus-host-disease (GVHD). Chimeric antigen receptor (CAR)-modified T cells directed toward CD19 have demonstrated dramatic efficacy in patients with refractory ALL and NHL. However, responses are often associated with life-threatening cytokine release syndrome. Aim: We hypothesized that infusing CAR-modified, CD19-specific T-cells after HCT as a directed DLI would be associated with a low rate of GVHD, better disease control, and a less severe cytokine release syndrome since administered in a minimal disease state. Methods: We employed a non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a CD19-specific CAR (designated CD19RCD28 that activates via CD3z & CD28) in donor-derived T cells for patients with advanced CD19+ lymphoid malignancies. T-cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T-cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The AaPC were derived from K562 cells and genetically modified to co-express CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound version of IL-15. Results: To date, we have successfully treated 21 patients with median age 36 years (range 21-62) with advanced CD19+ ALL (n=18) or NHL (n=3); 10 patients had active disease at time of HCT. Donor-derived CAR+ T cells (HLA-matched sibling n=10; 1 Ag mismatched sibling n=1; haplo family n=8; cord blood n=2) were infused at a median 64 days (range 42-91 days) following HCT to prevent disease progression. Transplant preparative regimens were myeloablative, busulfan-based (n=10) or reduced intensity, fludarabine-based (n=11). All patients were maintained on GVHD prophylaxis at time of CAR T-cell infusion with tacrolimus, plus mycophenolate mofeteil for cord, plus post-HCT cyclophosphamide for haplo donors. The starting CAR+ T-cell dose was 106 (n=7), escalated to 107 (n=6), 5x107 (n=5), and currently at 108 (n=3) modified T cells/m2 (based on recipient body surface area). Patients have not demonstrated any acute or late toxicity to CAR+ T cell infusions. Three patients developed acute grades 2-4 GVHD (liver n=1, upper GI n=1, skin=1) which was within the expected range after allogeneic HCT alone. Of note, the rate of CMV reactivation after CAR T cell infusion was 24% vs. 41 % previously reported for our patients without CAR T cell infusion (Wilhelm et al. J Oncol Parm Practice, 2014, 20:257). Nineteen patients have had at least 30 days follow-up post CAR T-cell infusion and are evaluable for disease progression. Forty-eight percent of patients (n=10) remain alive and in complete remission (CR) at median 5.2 months (range 0-21.3 months) following CAR T cell infusion. Importantly, among 8 patients who received haplo-HCT and CAR, 7 remain in remission at median 4.2 months. Conclusion: We demonstrate that infusing donor-derived CD19-specific CAR+ T cells, using the SB and AaPC platform, in the adjuvant HCT setting as pre-emptive DLI may provide an effective and safe approach for maintaining remission in patients at high risk for relapse. Graft-vs-host disease did not appear increased by administration of the donor derived CAR-T cells. Furthermore, the add-back of allogeneic T cells appears to have contributed to immune reconstitution and control of opportunistic viral infection. Disclosures Huls: Intrexon and Ziopharm: Employment, Equity Ownership. Singh:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Olivares:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Su:Ziopharm and Intrexon: Employment. Figliola:Intrexon and Ziopharm: Equity Ownership, Patents & Royalties. Kumar:Ziopharm and Intrexon: Equity Ownership. Jena:Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential roylaties (Patent submitted); Intrexon: Equity Ownership, Patents & Royalties: Potential royalties (Patent submitted). Ang:Intrexon and Ziopharm: Equity Ownership. Lee:Intrexon: Equity Ownership; Cyto-Sen: Equity Ownership; Ziopharm: Equity Ownership.

Comparable Immune Reconstitution Between Ex Vivo Amplification and Un-Manipulated Umbilical Cord Blood Transplantation

Umbilical cord blood transplantation (UCBT) is an alternative in the absence of related or unrelated HLA-matched donor. Defects in the mechanisms of immuno-surveillance, due to the absence of antigen-experienced lymphoid subsets transferred in the cord blood unit (CBU), render recipients more prone to viral infections. Our center conducted a clinical trial testing the benefit of ex vivo amplification of the CD34+ fraction (using SCF, FLT3L, TPO and GCSF) enriched from one CBU on hematopoietic reconstitution, while the CD34- fraction was infused at the same time as the expanded fraction (NCT01034449). We intended to simultaneously evaluate the potential impact of such a procedure on the immune reconstitution following freeze-thaw cycle for the CD34- fraction and on the expansion potential of lymphoid progenitors from CD34+ fraction.We prospectively analyzed fresh patient samples from the clinical trial (called the EVEX), at different time points (Day 42, D100, D180, D360), and, in parallel, from patients receiving one or two un-manipulated CBU during the same period (Control group - CTRL). Immune monitoring included flow cytometry analysis of T-cells (CD3+, CD4+, CD8+) and sub-compartments, B-cells (CD19+), NK cells (CD3-CD16+CD56+) and Dendritic cells (DC).Sixteen patients were included in EVEX, of which 12 were analyzed (4 excluded: 1 not grafted, 1 graft infection, 2 primary rejections before Day 42) and 12 patients in CTRL. In the EVEX group, all patients received reduced intensity conditioning, compared to 6 out of 12 in the CTRL group. GVHD prophylaxis was similar. EVEX showed a shorter duration of neutropenia compared to the CTRL (8.5 versus 17 days, p=0.02). NK and B-cell subsets recovered earlier than T-cells, reaching median normal values at 3 and 6 months, respectively. NK cell count tended to be lower in EVEX, starting at 3 months until 1 year. B-cell counts were lower for EVEX at D360. Both groups recovered T-cells within 12 months, but, interestingly, EVEX recovered earlier, reaching Healthy Donor (HD) median values as soon as D180. The CD4+ T-cells (CD4+T) recovered within 12 months for both groups, with EVEX values tending to plateau at low normal values (Fig 1). CD8+ T-cells (CD8+T) reached HD values within 12 months with an earlier recovery for EVEX (Fig 1). We characterized CD4+T and CD8+T compartments with CD45RA and CD27 delineating: naïve (TN: CD45RA+CD27+), central memory (TCM: CD45RA-CD27+), effector memory (TEM: CD45RA-CD27-) and late effector (TEMRA: CD45RA+CD27-) T-cells. Among the CD4+T, TN and TCM gradually increased until D360 for CTRL, while EVEX reached a plateau at D180. EVEX showed a higher TEMRA CD4+T count at D360. Thus, the plateau observed in the whole CD4+T at D180 in EVEX suggested an altered capacity to induce new CD4+T (thymic output or peripheral expansion), or an exhaustion phenomenon. To this end, we evaluated thymic function via CD31 expression among TN CD4+T and TREC analysis by qPCR. Both methods showed a decreased thymic function in EVEX after D180, probably related to the age difference observed (median: 52 vs. 26 years old for EVEX and CTRL, respectively). TEM and TEMRA CD8+T subsets were higher starting at D180 for the EVEX group. There was no difference between groups for myeloid or plasmacytoid DC.Group outcomes differed in terms of chronic GVHD (cGVHD) (1 vs. 6 patients for EVEX and CTRL respectively, p=0.024). Among the subsets playing a role in cGVHD pathogeny, no difference was found in Treg/Tcon ratio (Treg = CD25hiCD127lo CD4+T, Tcon = non-Treg CD4+T), but memory B-cell subset tended to be greater in CTRL from D42 to D180 (median time of cGVHD onset = 186.5 days).CMV reactivation rate was similar in both groups, occurring around 42 days post transplant. Vdelta-2 negative γd T-cells were greater in EVEX early after infection (D42), suggesting a comparable anti-CMV innate function. However, when looking at adaptive anti-CMV immunity with ELISpot, we found reduced IFNg-secreting cells in EVEX, although group size was small.In conclusion, patients receiving ex vivo expanded CBU showed faster hematopoietic reconstitution than un-manipulated CBU and comparable recovery of the main immune subsets. Baseline clinical differences observed between groups may have impacted on some of the immune findings. A prospective and randomized trial would help to better analyze if the expansion procedure has an impact on immune reconstitution. [Display omitted] DisclosuresMilpied:Celgene: Honoraria, Research Funding.

Maturation and Mip-1β Production of Cytomegalovirus-Specific T Cell Responses in Tanzanian Children, Adolescents and Adults: Impact by HIV and Mycobacterium tuberculosis Co-Infections.

It is well accepted that aging and HIV infection are associated with quantitative and functional changes of CMV-specific T cell responses. We studied here the expression of Mip-1β and the T cell maturation marker CD27 within CMVpp65-specific CD4+ and CD8+ T cells in relation to age, HIV and active Tuberculosis (TB) co-infection in a cohort of Tanzanian volunteers (≤16 years of age, n = 108 and ≥18 years, n = 79). Independent of HIV co-infection, IFNγ+ CMVpp65-specific CD4+ T cell frequencies increased with age. In adults, HIV co-infection further increased the frequencies of these cells. A high capacity for Mip-1β production together with a CD27low phenotype was characteristic for these cells in children and adults. Interestingly, in addition to HIV co-infection active TB disease was linked to further down regulation of CD27 and increased capacity of Mip-1β production in CMVpp65-specific CD4+ T cells. These phenotypic and functional changes of CMVpp65-specific CD4 T cells observed during HIV infection and active TB could be associated with increased CMV reactivation rates.

Viral Infections in Renal Transplant Recipients in Belarus

The presented study had its aim in establishing the prevalence and etiological structure of post-transplant viral complications and the kinetics of infection development. Two groups of recipients were examined: Group 1 comprised patients without any clinical signs of infection (n=84) monitored for a year, Group 2 included recipients with a clinical presentation of infection (n=92). 591 blood samples and 446 urine samples from 196 patients (20 donors and 176 renal transplant recipients) were analyzed. All donor-recipient pairs were tested for CMV, EBV, and HSV-1, 2. Latent viral infection reactivation was detected by the presence of viraemia (DNAemia) induced by CMV, EBV, HSV-1, 2, HHV-6, VZV, PVB19, or viruria (DNAuria) induced by BKV. The percentage of Group 1 patients with viral DNA detected amounted to 41.9% for CMV, 30.4% for EBV, 17.4% for BKV, 5.6% for HHV-6, 1.9% for AdV, and 1.7% for PVB19. HSV-1, 2 and VZV infections in this group were not observed. The rate of CMV, EBV, and BKV infection reactivation, determined by the viraemic patients percentage, increased with time and peaked 1, 1.5 and 2 months after transplantation for EBV, BKV, and CMV, respectively. Three to six months after transplantation, the percentage of patients with active CMV, EBV, or BKV infection decreased significantly; and later on, viraemia was only occasional finding in Group 1 patients. Examination of Group 2 patients revealed that the predominant etiological agents of post-transplant viral complications were CMV (24.4%), EBV (18.8%), and BKV (17.6%). The current study is the first complex investigation into the etiological structure and activation kinetics of viral infections in renal transplant recipients in Belarus. The results have major implications for early prevention of potential viral complications and adjustment of the therapy regimens for kidney transplant recipients currently in use.

Intravenous Immunoglobulin Post Allogeneic Stem Cell Transplantation Is Associated with Lower Levels of CMV Reactivation

▪IntroductionCytomegalovirus (CMV) reactivation is a common complication of allogeneic stem cell transplantation (ASCT), particularly within the first 100 days after stem cell infusion (D+100). It results in significant cost, morbidity and potentially mortality in the case of severe CMV disease.Intravenous polyvalent immunoglobulin (IVIg) has been used to support immunoglobulin levels post-ASCT in an attempt to reduce opportunistic infection and graft versus host disease (GVHD). Historically we employed routine IVIg therapy (0.5g/kg weekly for the first 12 weeks) as standard post-ASCT care. In July 2012 funding was withdrawn for IVIg for this indication.Since 2001 we have been using weekly quantitative polymerase chain reaction (PCR) analysis to monitor for CMV reactivation during the first 100 days after ASCT.We investigated whether the omission of post-ASCT IVIg altered the rate or consequences of CMV reactivation in the immediate post-ASCT setting.MethodsWe analysed sequential ASCT recipients from 2010 to 2014 and identified 100 patients who met the predetermined eligibility criteria. These subjects were then divided into two uniformly treated populations: ASCT recipients who either received (Group A, n=50) or did not receive (Group B, n=50) IVIg as part of their post-ASCT care.Inclusion criteria: matched adult unrelated or sibling donor source, age ≥18 years and CMV positive recipient by serology within 3 months prior to ASCT or CMV positive donor by serology.Exclusion criteria: CMV negative donor and recipient (D-/R-), umbilical cord donor source, death before day 100 or incomplete data available for analysis.CMV reactivation was defined as any detectable level by quantitative CMV PCR in the peripheral blood. The lower limit of detection for the assay was 20 copies/mL.A plasma CMV viral load of ≥400 copies/mL resulted in treatment with intravenous ganciclovir (5mg/kg twice daily) which was continued for a minimum of 2 weeks and was thereafter guided by weekly CMV viral load response. Patients with a CMV viral load of <400 copies/mL were observed with no specific treatment.CMV reactivation was recorded as minor (not requiring ganciclovir treatment) if CMV viral load remained <400 copies/mL and as major (requiring treatment) if CMV viral load reached ≥400 copies/mL within a single episode of infection. Time to first CMV reactivation and cumulative days of major CMV reactivation were also recorded.ResultsA total of 169 consecutive subjects were screened for study entry. Abstract 2482. Table 1:Cohort compositionsTotal screenedD-/R-Death <D+100Umbilical cord donorIncomplete dataTotal includedGroup A (IVIg)84206*8050Group B (non-IVIg)85215#8150*: 1 subject had active CMV infection at time of death#: 2 subjects had active CMV infection at time of deathDemographics were similar between Group A and Group B in terms of age (median 51.0 vs 48.5 years), sex (male: 33 vs 29) and transplant indication (acute leukaemia/MDS 28 vs 28; lymphoma 11 vs 11) and D+/R- transplants (9 vs 11).Comparing Group A and Group B, the number of subjects who had a CMV reactivation was 37 vs 39 and for minor reactivations the number was 28 vs 25. The median time to CMV infection was likewise similar: D+38 vs D+35.The number of patients with a major reactivation, however, was significantly different between Group A and Group B (13 vs 25 – RR 0.52, 95% CI [0.30-0.90], p 0.018), as was the cumulative number of days of major reactivation (181 vs 604 – RR 0.30, 95% CI [0.25-0.35], p <0.0001). The median number of days of major reactivation per patient was 13 vs 21 respectively.ConclusionRoutine IVIg use in the immediate post-ASCT period did not reduce the rate or latency of CMV reactivation but was associated with a lower magnitude of CMV reactivation and a reduced requirement for anti-CMV therapy. These findings suggest that the use of IVIg post-ASCT may assist in reducing CMV viral burden. DisclosuresNo relevant conflicts of interest to declare.

High Proportions of Regulatory T Cells in Peripheral Blood Stem Cell Grafts Are Associated with Improved Total Lymphocyte, CD8+ T Cell and CD19+ Cell Recovery Following Allogeneic Haematopoietic Stem Cell Transplantation

Co-infusion of donor regulatory T-cells (Tregs) with conventional T-cells (Tcons) in allogeneic haematopoietic stem cell transplantation (HSCT) may improve immune reconstitution and suppress graft-versus-host disease (GvHD) without inhibiting the graft-versus-tumour (GvT) response. While phase I/II studies have focused on co-infusion of ex-vivo isolated Tregs, few have examined the impact of Tregs within peripheral blood stem cell (PBSC) grafts. In previous work, we observed that high proportions of Tregs (CD4+CD25+FOXP3+CD127-/dim T-cells; expressed as percentage of CD4+ T-cells) in PBSC grafts were associated with improved myeloid and platelet recovery and decreased non-relapse mortality (NRM). In this current study, we therefore hypothesised that patients transplanted with high proportions of Tregs within allogeneic PBSC grafts will also have improved lymphocyte recovery.Ninety-four allogeneic HSCT transplants were recruited from Oxford University Hospitals NHS Trust. The median age of the recipients was 48 years (range, 18-74). Forty-seven (50%) of the transplants were performed for acute leukaemia and 80 (85%) were performed using a reduced intensity conditioning regimen. Thirty-eight (40%) PBSC grafts were obtained from sibling donors (37 HLA-matched; 1 single HLA-mismatch (9/10)) and 56 (60%) from unrelated donors (39 HLA-matched (10/10); 17 single HLA-mismatch (9/10)). The cellular contents of the grafts were analysed using multi-colour flow cytometry. The median total nucleated cell (TNC), CD34+, CD3+, CD19+ and CD3-CD56+ cell dose per kg recipient body weight was 10.1 x 108/kg (range, 2.7-37.6), 6.3 x 106/kg (range, 1.1-19.8), 280 x106/kg (range, 87-801), 62 x 106/kg (range, 13-245) and 35 x106/kg (range, 8-101) respectively. Tregs accounted for a median of 2.96% of CD4+ T-cells (range, 0.81-8.56%) within the grafts with a median Treg dose of 4.7 x 106/kg (range, 0.8-20.6). There were no significant correlations between the proportion of Tregs in the grafts and the TNC, CD34+, CD3+, CD19+ or CD3-CD56+ cell dose.The cumulative incidence of total lymphocyte (>1.0 x 109/l), CD3+ (>0.7 x 109/l), CD19+ (>0.1 x 109/l) and CD3-CD56+ (>0.1 x 109/l) cell recovery by 6 months was 71.3% (95% CI, 60.8-79.4%), 51.2% (95% CI, 40.6-62.3%), 62.4% (95% CI, 50.8-72.0%) and 91.0% (95% CI, 82.0-95.7%) respectively. The median time to total lymphocyte, CD3+, CD19+ and CD3-CD56+ recovery was 47 days (range, 11-455), 40 days (range, 33-265), 62 days (range, 27-262) and 34 days (range, 27-173) respectively. Patients transplanted with PBSC grafts containing higher proportions of Tregs (>2.96% CD4+ T-cells (median)) had improved lymphocyte recovery (P<0.001), with a cumulative incidence at 6 months of 83.0% (95% CI, 68.1-91.3%) compared to 59.6% (95% CI, 43.9-72.2%) for those receiving less. In multivariate analysis, high proportions of Tregs in the graft (%CD4+ T-cells) (HR 1.95 (95% CI, 1.24-3.06), P=0.004), use of Alemtuzumab during conditioning (HR 0.31 (95% CI, 0.17-0.56), P<0.001) and use of female donors (HR 1.65 (95% CI, 1.01-2.56), P=0.03) were independent predictors of total lymphocyte recovery. When analysing the major lymphocyte populations, high proportions of Tregs in the grafts were significantly associated with improved recovery of CD8+ T-cells (HR 1.98 (95% CI, 1.12-3.50), P=0.02) and CD19+ cells (HR 2.02 (95% CI, 1.23-3.06), P=0.004). In relation to clinical markers of immune recovery, there was no significant difference in the rates of CMV reactivation (P=0.89) between the two groups. However, less acute GvHD (grade II-IV) was observed in those patients receiving higher proportions of Tregs in the graft (23.4% (95% CI, 12.5-36.3%) vs 38.3% (95% CI, 24.5-51.9%) at 100 days), although this did not reach statistical significance (P=0.11). NRM at 3-years was lower (P=0.02) in patients receiving high proportions of Tregs in the grafts (12.8% (95% CI, 5.1-24.0%) [GvHD (n=1), infection (n=2), other (n=3)] vs 34.7% (95% CI, 21.2-48.5%) [GvHD (n=9), infection (n=4), other (n=3)]). The proportion of Tregs in the graft (HR 0.30 (0.11-0.85), P=0.02) was an independent predictor of NRM.These data support the hypothesis that higher proportions of donor Tregs in PBSC grafts may also enhance lymphoid reconstitution following allogeneic HSCT. However, larger studies will be required to understand the potential mechanisms involved and to confirm our findings. DisclosuresNo relevant conflicts of interest to declare.

Long-term outcomes of cytomegalovirus reactivation in patients with moderate to severe ulcerative colitis: a multicenter study.

Background/AimsCytomegalovirus (CMV) reactivations are frequently observed in patients with active ulcerative colitis (UC), and ganciclovir therapy is effective in patients with steroid-refractory UC. This study aimed to determine the long-term outcomes of CMV reactivation and the long-term therapeutic efficacy of ganciclovir treatment.MethodsThis retrospective multicenter study included a cohort of 72 patients with moderate-to-severe UC who were evaluated for CMV reactivation at the time of their initial UC flare. Colectomy, disease relapse, and the recurrence rate of CMV reactivation were investigated.ResultsThe mean duration of follow-up for the 72 patients was 43.16±19.78 months (range, 1 to 67 months). The cumulative colectomy (log-rank, p=0.025) and disease flare-up rates (log-rank, p=0.048) were significantly higher in the CMV-positive group. Of the 11 patients who were successfully treated with ganciclovir in the initial treatment, three patients (27.3%) experienced CMV reactivation, and six patients (54.5%) experienced poor outcomes, such as the need for colectomy or a steroid-dependent state.ConclusionsThe patients who had CMV-reactivated UC showed poor outcomes at the long-term follow-up, and the long-term efficacy of ganciclovir therapy was marginal. Careful assessment is necessary for patients who exhibit evidence of CMV reactivation.

The Addition Of Interleukin-6 Inhibition To Standard Gvhd Prophylaxis Prevents Acute Gvhd: Interim Results Of a Phase I/II Clinical Study

We and others have demonstrated the dysregulation of interleukin-6 (IL-6) early after experimental bone marrow transplantation (BMT) and protection from acute GVHD following the administration of an anti-IL-6 receptor (IL-6R) antibody. In these models, where GVHD prophylaxis is not administered, systemic IL-6, IFNγ and TNF levels peak 7 days after BMT before returning to baseline by the third week. We have determined cytokine dysregulation in a large clinical cohort of allogeneic stem cell transplant (SCT) recipients conditioned with myeloablative Cy/TBI (12 Gy, n = 25) or reduced intensity Flu/Mel (120mg/m2, n = 25) receiving standard GVHD prophylaxis with cyclosporine and MTX (d 1 at 15mg/m2, d 3, 6, 11 at 10mg/m2). IL-6 levels rose from pre-transplant levels of 6.4 ± 0.7 pg/ml to a peak of 58.8 ± 8.8 pg/ml at day 7 (P < 0.0001) with a fall at day 14 to 39.0 ± 12.5 pg/ml (P < 0.0001) and return to baseline by day 30 (6.2 ± 0.9 pg/ml), consistent with the preclinical data. IL-6 dysregulation was not different in recipients of matched sibling or unrelated donor grafts but was proportional to the intensity of conditioning (day 7 levels after Cy/TBI vs. Flu/Mel: 83.3 ± 12.2 pg/ml vs. 31.0 ± 10.1 pg/ml, P< 0.0001). In contrast to preclinical mouse data, no systemic increases were seen in any other cytokine including IFNγ, TNF, IL-17, IL-4, IL-13 and IL-10.We thus initiated a phase I/II study whereby a human neutralizing monoclonal antibody (mAb) against the IL-6R was administered on day -1 to patients receiving Cy/TBI or Flu/Mel conditioned allogeneic SCT from HLA (10/10)–matched sibling or unrelated donors with standard cyclosporine/MTX GVHD prophylaxis. There was no T cell depletion. The primary endpoint was the incidence of grade II-IV acute GVHD and the study has achieved its planned enrollment (n = 48). There was no toxicity attributable to IL-6R antibody administration. Pharmacokinetic analysis confirmed high levels of IL-6R Ab at day 3 (mean 64.7 ug/ml) which persisted in all patients 3 weeks after BMT (mean = 9.8 ug/ml) and remained above the level of detection (0.1ug/ml) in 75% of patients at day 30 (mean = 1.9 ug/ml). IL-6 levels were dramatically increased (relative to baseline) in patients receiving antibody due to the inability to excrete the inactive IL-6 – soluble IL-6R antibody complex (peak IL-6 levels at day 7 = 773.6 ± 207.9 pg/ml; P < 0.0001) and remained increased at day 30 (60.9 ± 24.4 pg/ml; P < 0.0001), returning to baseline by day 60 (9.5 ± 1.7 pg/ml), consistent with antibody clearance. Soluble IL-6R levels also rose over the first month of SCT and levels at day 30 correlated with residual antibody levels (r2 = 0.72, P = 0.02). Neutrophil (> 0.5x109/L) and platelet (> 20x109/L) recovery was normal relative to a matched untreated control cohort at a median of 16 and 18 days respectively. Donor chimerism and immune reconstitution (conventional T, regulatory T and B cells) was equivalent at day 30 in recipients of IL-6R inhibition versus the control cohort. In contrast, changes in innate immunity were seen in patients receiving IL-6R inhibition with increases in plasmacytoid DC (P = 0.002), CD1c+ conventional DC (P = 0.04) NKT cells (P =0.03) and marked reductions in inflammatory (CD14+CD16+) monocytes (P < 0.0001). Transcriptional profiling of T cell subsets is underway. With 36 patients evaluable (beyond day 100, median follow up of 297 days), the incidence of grade II-IV GVHD is 11.1% in recipients of IL-6R inhibition versus 39.6% in the matched (n = 53) control cohort (P = 0.004). The incidence of grade III/IV acute GVHD is 5.6% in recipients of IL-6R inhibition versus 20.8% in the control cohort (P = 0.045). Protection from grade II-IV acute GVHD was noted in patients receiving both Cy/TBI (7.7% vs. 40.7%, P=0.045) and Flu/Mel conditioning (13.0% vs. 38.5%, P = 0.044). Rates of CMV reactivation were very low in the IL-6R neutralized patients (16.7% vs. 35.8% in controls, P = 0.04), likely due to the prevention of acute GVHD and its' consequent therapy. At one year, the relapse incidence and disease free survival in patients receiving IL-6R inhibition versus the control cohort is 21.2% vs. 30.0% (P = 0.28) and 73.1% vs. 62.4% (P = 0.14) respectively. IL-6 is thus the principal inflammatory cytokine dysregulated after clinical allogeneic SCT and its inhibition appears to offer profound protection from acute GVHD despite robust immune reconstitution, without compromise of the GVL effect. Disclosures:Off Label Use: The use of Tocilizumab to prevent GVHD is experimental and an off label use.

Effect of intensive granulocyte and monocyte adsorptive apheresis in patients with ulcerative colitis positive for cytomegalovirus

Background and aimCytomegalovirus (CMV) exacerbates ulcerative colitis (UC) refractory to immunosuppressive therapies. The conditions under which CMV reactivation occurs in patients with UC, however, is unclear. In addition, the diagnostic and treatment strategies for UC positive for CMV have not been established. Granulocyte and monocyte adsorptive apheresis (GMAA) is natural biological therapy for UC in which the granulocytes/macrophages producing inflammatory cytokines are removed. We investigated the rate of colonic CMV reactivation and the efficacy of GMAA in active UC patients positive for CMV without concomitant corticosteroid (CS) therapy. MethodsFifty-one active UC patients without concomitant CS therapy were enrolled. Colonic CMV reactivation was examined by real-time polymerase chain reaction (PCR) using biopsy specimen and/or histological examination. All patients were treated with intensive GMAA (twice per week). Rates of clinical remission and mucosal healing were compared between UC patients positive and negative for CMV. ResultsOf 51 patients, 15 (29.4%) were diagnosed as CMV positive. The clinical remission rates following intensive GMAA did not differ between UC patients positive and negative for CMV (73.3% vs 69.4%, p=0.781). Proportion of patients achieving mucosal healing was also similar between these two groups. CMV-DNA became negative in all UC patients positive for CMV who achieved clinical remission 1week after completion of intensive GMAA. ConclusionsIntestinal inflammation might trigger CMV reactivation in a subpopulation of active UC patients without CS treatment. GMAA could be a promising option for active UC positive for CMV.

CMV Serostatus of Donor-Recipient Pairs Influences the Risk of CMV Infection/Reactivation in HSCT Patients

CMV donor/recipient serostatus was analyzed in 200 patients allografted in our institution from unrelated (122 patients) donors and 78 sibling donors in the years 2002–2011 in relation to posttransplant complications. On a group basis independently of the CMV serostatus of donor-recipient pairs sibling transplantations and those from unrelated donors that matched 10/10 at allele level had a similar rate of CMV reactivation (17/78 versus 19/71, P = ns). The rate of CMV reactivation/infection was higher in patients grafted from donors accepted at the lower level of matching than 10/10 (18/38 versus 36/149, P = 0.008). The incidence of aGvHD followed frequencies of CMV reactivation in the tested groups, being 40/156 and 25/44 in patients grafted from sibling or unrelated donors that 10/10 matched and in those grafted from donors taht HLA mismatched, respectively (P = 0.001). Regarding the rate of reactivation in both groups seropositive patients receiving a transplant from seronegative donors had more frequently CMV reactivation as compared to those with another donor-recipient matching CMV serostatus constellation (22/43 versus 32/143, P = 0 < 0.001). Multivariate analysis revealed that seropositivity of recipients with concomitant seronegativity of donors plays an independent role in the CMV reactivation/infection (OR = 2.669, P = 0.037; OR = 5.322, P = 0.078; OR = 23.034, P = 0.023 for optimally matched and mismatched patients and the whole group of patients, resp.).

A Phase I Study of Alemtuzumab Dosing for Steroid-Refractory Chronic Graft-Versus-Host Disease

AbstractAbstract 744 Introduction:There are no definitive therapies for chronic graft-versus-host disease refractory to steroid therapy. Alemtuzumab transiently depletes B and T cells and can decrease the incidence of acute and chronic GvHD when part of pre-transplant conditioning. In steroid-refractory acute GvHD, alemtuzumab has induced complete response rates of 20–35%, but optimal dosing in acute or chronic GvHD treatment has not been established. We report a phase 1, dose-escalation study assessing alemtuzumab as therapy for steroid-refractory chronic GvHD (cGvHD). Methods:Patients with cGvHD refractory to prednisone at > 0.5mg/kg/day were eligible. The primary objective was to determine maximum tolerated dose (MTD) and toxicity. The secondary objective was to determine efficacy. Subjects received alemtuzumab on Days 1, 3, 5, 8, 15 and 22. Three regimens were investigated in a 3+3 dose escalation design: 3 mg × 6 (Dose level 1); 3 mg × 1, then 10 mg × 5 (Dose level 2); and 3 mg × 1, 10 mg × 1, then 30mg × 4 (Dose level 3). Dose-limiting toxicities were Grade 4 or 5 toxicities occurring within 12 weeks of initiating therapy. During the study period, doses of other immunosuppressants were held constant; antiHSV, antiPJP and antifungal prophylaxis was required. CMV reactivation was treated pre-emptively. GvHD severity was assessed by NIH consensus criteria at initiation and 12 weeks.Results: Safety:13 subjects were enrolled: 3 at Dose level 1, 6 at Dose level 2 and 4 at Dose level 3. All were evaluable for toxicity. 6 subjects had moderate and 7 subjects had severe cGvHD at initiation. The median number of organs involved was 4 (range 1–7). A median of 5 systemic immunosuppressants other than steroids (range 2–7) had been previously used. Dose level 2 was the MTD. Subjects experiencing > Grade 3 infections in the first 12 weeks were 0 at Dose level 1, 3 at Dose level 2 (1 death), and 3 at Dose level 3 (2 deaths). (Table) Bacterial pathogens included E.coli, pseudomonas and corynebacterium. Viral pathogens included parainfluenza, influenza A, BK virus and adenovirus. The CMV reactivation rate was 30.8%; there were no parenchymal CMV infections. Between 12 and 24 weeks, significant infections were seen in 1 of 10 subjects. Subjects experiencing > Grade 3 neutropenia +/− thrombocytopenia, were 0 in Dose level 1, 1 in Dose level 2 and 2 in Dose level 3 in the first 12 weeks. Monocyte and lymphocyte numbers fell immediately upon treatment at all dose levels. Monocyte numbers returned to starting levels in 9 of 13 subjects at time of last censoring; lymphocytes numbers returned to starting levels in 5 of 13 subjects. Time to restoration of lymphocyte numbers ranged from 6 to >27 months. Efficacy:Ten of 13 subjects were evaluable for response at Week 12. The overall response rate was 70%. The complete response rate was 30%, all observed at the MTD. (Table) Responses were seen predominantly in skin and joint/fascia/muscle. 4 of 10 subjects reduced steroid dose or discontinued an immunosuppressant at 12 weeks. At 24 weeks, only 2 of 10 subjects had increased steroids or initiated new GvHD therapies. At 1 year, 4 of 8 subjects evaluable were on < 7.5mg prednisone daily with no new GvHD therapies introduced since study initiation. There were no relapses of malignancy over a median of 32.2 months follow up (range 7.4–44.8). Median overall survival was 23.9 months from study initiation. Conclusions:Our dose-escalation study establishes an MTD for alemtuzumab of 3 mg × 1, then 10 mg × 5 doses over a 4 week period. We observed significant rates of infectious complications, but they occurred early in the course of therapy, despite prolonged lymphopenia, and in a dose-dependent manner. Our response rate of 70% suggests efficacy for alemtuzumab in the treatment of steroid-refractory chronic GvHD in patients who have failed multiple therapies. The use of alemtuzumab in this context deserves study in larger phase II trials.TableToxicities and responses in first 12 weeks of therapyDose LevelcGvHD responseViral ReactivationViral InfectionBacterial InfectionCytopenias + Other1Partial----1Partial----1PartialCMV---2CompleteOral HSV---2Complete----2Stable-Grade 3Grade 3-2Progression----2CompleteOral HSV-Grade 3-2DeceasedCMVGrade 3 Grade 5Grade 3Grade 4 N+T3PartialOral HSV CMV---3Deceased--Grade 5-3StableCMVGrade 3-Grade 3 N Grade 3 TMA3Deceased--Grade 3 Grade 5Grade 4 N+TN neutropenia; T thrombocytopenia; TMA thrombotic microangiopathy Disclosures:Off Label Use: Alemtuzumab used in off-label indication for treatment of chronic graft-versus-host disease in the setting of a Phase 1 clinical trial.

38-OR

Aim Reactivation of Cytomegalovirus is one most frequent viral complication that affects renal transplant recipients. In this regard, role of NK cells becomes important by virtue of their anti-viral activities as well as the fact that currently used immunosuppressive drugs significantly impair T cells. Functioning of these NK cells are regulated by series of receptors known as Killer Immunoglobulin like receptors (KIR) that exist in activating and inhibitory forms. Here, we assessed whether KIR gene profile of kidney transplant recipients influence the rate of CMV reactivation after transplantation. Methods A total of 192 renal transplant recipients were genotyped for a set of 16 KIR genes by a Luminex based multiplex KIR genotyping assay. Humans have various combinations of these 16 genes that normally segregate in two haplotypic forms – ‘A’ (containing 1 activating receptor gene) and ‘B” (containing 2-6 activating receptor genes). PCR based method was used for CMV reactivation assessment. Logistic regression analysis was performed to assess the association of recipient KIR gene profile with CMV reactivation. Results An activating KIR gene profile of the recipient was found significantly associated with protection from CMV infection. Renal tranaplant recipients carrying Haplotype B (p = 0.02), ⩾5 activating genes (0.01) or presence of 2DS2 activating KIR (0.01) were significantly associated with low rate of CMV reactivation. Conclusions Our findings strongly support the role of NK cells in general and that of activating KIR profile in particular in the immune surveillance against cytomegalovirus after renal transplantation.

Cytomegalovirus-Specific T Cells Are Elicited Early After Umbilical Cord Blood Transplant but Fail to Expand In Vivo and Control Virus Replication

Abstract 1974Cytomegalovirus (CMV) is a major infectious complication in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) and has been linked to deficiencies of virus-specific T cell immunity. Compared to bone marrow or peripheral blood stem cell transplants, recipients of single or double umbilical cord blood transplants (UCBT) receive lower numbers of donor T cells that have not previously been primed to CMV and are at increased risk for early and recurrent CMV infections. At our institution, the rate of CMV reactivation in CMV seropositive patients undergoing CBT is close to 100% with standard dose Acyclovir as prophylaxis [Delaney unpublished data]. Here, we systematically analyzed the kinetics of recovery, durability, and specificity of CMV-specific CD8+ and CD4+ T cell responses in UCBT recipients. CD8 T cell responses to CMV were analyzed by interferon γ (IFN-γ) intracellular cytokine staining after stimulating recipient peripheral blood mononuclear cells (PBMC) obtained at various time points after CBT with autologous patient fibroblasts infected with the RV798 virus, which is a mutant CMV strain that lacks the viral US genes that downregulate class I MHC and can present all potentially immunogenic epitopes of the virus. The mean absolute CD8 T cell counts were 59, 93 and 213 cells/μl and the mean CD4 T cell counts were 154, 223 and 397 cells/μl in PBMC at day 56, 180 and 365 respectively. Direct assays of PBMC after a 4–6 hour stimulation with RV798-infected fibroblasts did not detect a significant frequency of IFN-γ+ CD8+ T cells in CBT recipients, in contrast to normal CMV+ donors that exhibited frequencies of CD8+ T cells of 2–10%. However, IFN-γ+ CMV specific CD8 T cells were readily detectable in PBMC obtained as early as day 42 after UCBT from 8 out of 8 CMV positive CBT recipients after a 10 day stimulation with RV798 infected fibroblasts. These responses were sustained at multiple time points through day 365 post transplant. This result was not a consequence of in vitro priming of CD8 T cells by prolonged stimulation with RV798 since we did not detect a CMV-specific T cell response in 3 out of 3 CMV seronegative recipients at any time through day 365 with the same assay. To assess CD4+ T cell responses, we performed lymphoproliferative assays (LPA) by stimulating patient PBMC obtained at the same time points with whole CMV antigen. The proportion of patients with a positive response at day 56, 80, 180 and 365 was 0.38, 0.50, 0.88, and 1.0 respectively. All of the CMV positive CBT recipients in our study had multiple occurrences of CMV reactivation throughout the first year post CBT requiring antiviral drug therapy.The CMV-specific CD8 T cell response in normal CMV+ individuals recognizes a large number of distinct dominant and subdominant antigens and a potential explanation for the failure to control CMV after CBT is that the T cell response may not be sufficiently diverse. We analyzed the specificity of CMV specific CD8+ T cells that developed after CBT in 4 recipients by assessing recognition of COS cells transfected with the class I HLA restricting alleles and with a CMV plasmid library consisting of 142 ORFs, subdivided into pools. A response was seen in 3 out of 4 patients to at least 3 different CMV antigens by day 80 post CBT, including previously defined dominant epitopes in pp65 and this diversity was maintained through 6–12 months post transplant. One patient had a less diverse response early post CBT and the response changed over time to include recognition of new epitopes.Collectively, our results demonstrate that CD8+ and CD4+ T cells are primed to CMV antigens very early after CBT despite the infusion of limited numbers of naïve T cells and the administration of post transplant immunosuppression. The inability to control CMV infection may be due to a quantitative deficiency of CMV-specific T cells resulting from the inability of CMV-specific T cells to expand in vivo to numbers sufficient to eliminate virus replication. Disclosures:No relevant conflicts of interest to declare.

A Randomized Trial of Sirolimus-Based Graft Versus Host Disease (GVHD) Prophylaxis After Hematopoietic Stem Cell Transplantation (HSCT) in Selected Patients with CR1 and CR2 ALL: Results From Children's Oncology Group Study ASCT0431

Abstract 837Based upon demonstrated activity of mTOR inhibitors in preclinical models of ALL and a promising pilot study, we hypothesized that the use of sirolimus added to a standard GVHD prophylaxis regimen would decrease relapse and improve survival after HSCT for children with high-risk CR1 and CR2 ALL. The COG performed a randomized phase III trial comparing tacrolimus/methotrexate (tac/mtx) with tac/mtx plus sirolimus. Sirolimus was initiated on day 0 of transplant and continued at therapeutic levels for 6 months. All patients received a preparative regimen consisting of fractionated TBI (1200–1320 cGy), and cyclophosphamide (60mg/kg × 2) +/− thiotepa (5mg/kg × 2) or VP-16 (1500mg/m2). Patients with primary induction failure who attained CR, hypodipoid ALL (<44 chromosomes), and Ph+ ALL were eligible for HCT in CR1 (intermediate risk); eligible CR2 patients included B-lineage with early bone marrow (BM) relapse (<36m from diagnosis) or T-lineage with BM relapse at any time (high risk, HR) and B-lineage with late BM relapse or isolated extramedullary relapse occurring <18m from diagnosis (intermediate risk, IR). HLA-matched sibling donors, 7–8/8 HLA matched related or unrelated donors, and 4–6/6 HLA matched cord blood stem cell sources were allowed. Randomization was stratified by donor type and relapse risk group. Events included relapse and treatment related mortality (TRM); the study was designed to enroll 259 patients to detect a two-year event free survival difference of 16% with 80% power. Results: A total of 146 patients enrolled, with 142 providing data to the analysis. As of July 1, 2011, there were 27 events in the 69 control arm patients and 31 events in the 73 experimental arm patients. The study was closed on May 10, 2011 when a stopping rule for futility of the primary endpoint (improved EFS) was met. The rates of grade III–IV acute GVHD, relapse, and TRM were 22%, 28% and 12% in control and 15%, 29%, and 14% in experimental arm patients (p=NS). Rates of CMV reactivation, venoocclusive Disease (VOD), and transplant associated microangiopathy (TAM) were 19%, 9%, and 1% in control arm patients and 12%, 17%, and 8% in experimental arm patients (p=0.26, 0.13, 0.10, respectively). Multistate modeling results indicate that the treatment group experienced less grade 2 to 4 aGVHD, but relapse rates in aGVHD patients were a quarter of those in non-aGVHD patients (p=<.001) while TRM rates were 1.5 times that of patients with no or grade I aGVHD (p=NS, see table). Survival after sibling donor transplantation was identical to URD transplantation. There was a trend toward increased risk of relapse in recipients of cord blood (p=0.14); further analysis of HLA type, pre-HSCT minimal residual disease (MRD), and patient risk is underway to clarify this finding.NaGVHD (III–IV)RelapseTRM1 year EFSaGVHD (II–IV) not present9333 (31%)10 (11%).45a (.32–.61)aGVHD (II–IV) present497 (15%)8 (16%).79a (.60–.89)P value<0.0010.38Matched Sib7513 (17%)20 (27%)5 (7%).58 (.42–.71)URD/Other RD387 (18%)7 (18%)7 (18%).54 (.32–.71)Cord296 (21%)13 (45%)6 (21%).33 (.16–.51)P value0.720.140.30 Conclusions:The addition of sirolimus to tac/mtx resulted in a trend toward higher rates of non-fatal TAM and VOD, but identical rates of severe VOD and TRM. There was a lower rate of grade II–IV aGVHD in the experimental arm, but no decrease in relapse. For the combined cohort, grade II–IV acute GVHD was associated with significantly less relapse and improved survival, suggesting that GVL is important in decreasing relapse after HSCT for pediatric ALL. The cytotoxic effect of sirolimus against ALL was insufficient to decrease relapse when given to very high risk ALL patients after HSCT as part of GVHD prophylaxis, possibly because of the offsetting effect of decreasing GVL. Disclosures:Off Label Use: The trial used sirolimus for graft vs. host disease prophylaxis in children. Sirolimus is approved for kidney transplantation in children. Borowitz:BD Biosciences: Research Funding. Grupp:Children’s Hospital of Philadelphia: Patents & Royalties.

Anti-CD25 Monoclonal Antibody for Graft-Versus-Host Disease Prophylaxis Following Unrelated Donor Peripheral Blood Stem Cell Transplantation,

Abstract 4070 Introduction:Enhanced risk of graft-versus-host disease (GVHD) makes one of major critical barriers to successful unrelated donor hematopoietic stem cell transplantation (URD-HSCT). Antithymocyte globulin (ATG) has been combined to standard GVHD prophylaxis regimens and significantly reduced GVHD in URD-HSCT. But ATG is associated with increases of infections and post-transplant lympholiferative disease (PTLD). Thus it is valuable to find alternative immunosuppressive agent for GVHD prophylaxis in URD-HSCT. Basiliximab and daclizumab, two anti-CD25 monoclonal antibodies, prevent graft failure in renal transplantation, which also effectively treat steroid-refractory graft-versus-host disease (GVHD). However, only few researches report that anti-CD25 monoclonal antibodies prevent GVHD. Here we firstly retrospectively explored the prophylactic effects of basiliximab or daclizumab against GVHD in 93 patients with hematological malignancies following unrelated donor peripheral blood stem cell transplantation (URD-PBSCT). Patients/methods:Between April 2004 and May 2011, 93 patients with hematological malignancies received basiliximab or daclizumab additional to standard GVHD prophylaxis regimens and underwent URD-PBSCT in our department. Their clinical data were retrospectively collected and analyzed. All patients received GVHD prophylaxis regimen consisting of cyclosporine A, short-course methotrexate, mycophenolate mofetil and an anti-CD25 monoclonal antibody. Basiliximab was administrated to 71 patients at a dose of 20 mg, while daclizumab was given to 22 patients at a dose of 1 mg/kg on day 0 (2 hours before transplantation) and day +4. Eighteen patients were 8/8 identical, 41 were 7/8 identical and 34 were 6/8 identical at HLA-A,-B,-Cw and -DRB1. The median number of infused nucleated cells and CD34+ cells were 7.5×108/kg and 5.8×106/kg, respectively. Results:i) All the recipients achieved engraftment. The median time to neutrophil recovery and platelet recovery were 12 days and 15 days, respectively. The rates of grade II-IV and III-IV acute GVHD (aGVHD) were 35.5% and 18.3%, respectively. Chronic GVHD (cGVHD) developed in 43.4% of evaluable patients. The limited cGVHD rate was 27.6% and the extensive cGVHD rate was 15.8%. The transplantation-related mortality (TRM) was 19.4% while relapse rate (RR) was 10.8%. The 2-year overall survival (2-yr OS) reached 74.2% and disease free survival (2-yr DFS) accumulated to 69.6% during a median follow-up of 24 months.ii)The side effects of basiliximab and daclizumab were moderate and tolerable. The infectious rate was 66.7% including 44.1% bacterial infection, 10.8% probable or proven invasive fungal infection, and 11.8% mixed infection. The infection-related mortality was 7.5%. The CMV reactivation rate was 46.2% and only one patient suffered CMV pneumonia. Moreover there was no clinical evidence of PTLD.iii) There were no significant differences in aGVHD onset and survival between daclizumab and basiliximab group. However, basiliximab presented superior prophylactic effects on cGVHD than daclizumab (cGVHD rate, 31.1% versus 66.7 %, P=0.007).iiii) The aGVHD rate, cGVHD rate, RR, TRM, 2-yr OS and 2-yr DFS were compared among different HLA matching groups. There were significant differences in the occurrence of grade II-IV aGVHD (11.1% versus 36.6%, P=0.047) and cGVHD(18.8% versus 51.6%, P=0.03) between HLA 8/8 identical group and HLA 7/8 identical group. Comparing HLA 8/8 identical group versus HLA 6/8 identical group, there were not only significant decreases in the rate of grade II-IV aGVHD (11.1% versus 47.1%, P=0.01) and grade III-IV aGVHD (5.6% versus 32.4%, P=0.039), but also a significant increase in the RR (22.2% versus 2.9%, P=0.043). There were no significant differences between HLA 7/8 identical group and HLA 6/8 identical group. Interestingly, the survival was not significantly different among three HLA matching groups. Conclusion:Basiliximab or daclizumab prevents GVHD efficiently and feasibly following URD-PBSCT, especially in HLA identical or only one allele mismatched recipients. Furthermore, anti-CD25 monoclonal antibodies benefit the outcome, even for those recipients with two or more HLA disparities. Basiliximab has similar effects on aGVHD prophylaxis but superior effects on cGVHD prophylaxis than daclizumab. Further prospective and randomized control studies are needed. Disclosures:Off Label Use: Drug: basiliximab (Simulect, Novartis Pharmaceuticals); daclizumab (Zenapax, Roche Pharmaceuticals). Purpose: for graft-versus-host disease prophylaxis following unrelated donor peripheral blood stem cell transplantation.

Selective Depletion of T Cell Alloresponses Using a Photodepletion Strategy Preferentially Targets CD4+ T Cells.

AbstractAbstract 1467P-glycoprotein (Pgp) is the product of the multidrug-resistance-1 (MDR1) gene and actively transports various molecules across the extracellular membrane. Previous studies demonstrated that activated lymphocytes decrease Pgp activity and preferentially retain the photosensitizing agent 4, 5-dibromorhodamine 123 (TH9402, Kiadis Pharma, NL). We evaluated a photodepletion technique to achieve selective depletion (SD) of graft-versus-host disease (GVHD) alloreacting T cells in 24 HLA-identical sibling stem cell transplants. Donor lymphocytes were activated by 72 hr exposure with irradiated in-vitro expanded recipient T lymphocytes and pulsed with TH9402. Alloactivated T cells preferentially retaining the photosensitizer were eliminated by light exposure. The transfused cell products were then analyzed for T cell subset frequency. After SD preferential CD4+ T cell depletion occurred with an inversion of the CD4+/CD8+ ratio. Within the CD4+ compartment a significant depletion of naïve (CD27+ CD45RO-), central memory (CD27+ CD45RO+), and effector (CD27-CD45RO+) populations was noted. Conversely, CD8+ naïve and effector subsets was relatively conserved with depletion occurring predominantly in the central memory population. Additionally, an 80% reduction in B cells occurred, with a 60% reduction of cytotoxic NK cells (CD56+CD16+) and a relative expansion of regulatory NK cells (CD56+CD16-). We compared outcomes of 24 SD transplants with 34 patients receiving a T-cell depleted transplant. All patients with hematological malignancies were conditioned with fludarabine, cyclophosphamide, and total body irradiation and received a CD34-selected stem cell allograft from an HLA identical sibling. SD recipients also received 5×106/kg SD donor T cells. Low-dose cyclosporine was used as the only post-transplant immunosuppression. Median follow up was 14.5 months. The probability of severe acute GvHD (grade III-IV, 13%) and relapse (25%) was low for all patients. On day 100 post-transplant SD transplant recipients had lower absolute CD4+ counts, and increased rates of CMV reactivation requiring treatment (median 2 vs 1 reactivations within 100 days, p<0.01), and more chronic GvHD (70% vs 31%, p=0.04). Previous studies have shown that Pgp efflux of rhodamine-123 in quiescent T cells varied between subsets, with a preferential retention occurring in the CD4+ and central memory compartments (Br J Haematol. 1998 Jun;101(4):722-7). Our data is in accordance with these observations. In conclusion, while SD appeared to eliminate alloactivated T cells resulting in low frequencies of severe acute GVHD, differences in Pgp activity and dye retention led to the preferential non-specific elimination of CD4+ and central memory T cells which may have contributed to greater viral reactivation and chronic GVHD. Disclosures:No relevant conflicts of interest to declare.