1. Immunological profiles affecting stem cell transplant outcome. Recent data suggests that reactivation of cytomegalovirus (CMV) favors relapse free survival after allogeneic stem cell transplantation for acute myelogenous leukemia. We hypothesize that the immune response to CMV through either natural killer cells or T lymphocytes also targets residual leukemia since myeloid-derived cells may be a reservoir for latent CMV. We studied the relationship of CMV reactivation with leukemic relapse in 110 patients with chronic myeloid leukemia (CML) receiving HLA-identical sibling SCT. Seventy nine (72%) were in chronic phase, 5 in second chronic phase, 17 in accelerated phase and 9 in blast phase. CMV reactivation before day 100 was observed in 72 patients (65.5%). At a median follow up of 6 years, CMV reactivation before day 100 was an independent factor associated with decreased relapse. We conclude that CMV reactivation may contribute to a beneficial graft-versus-leukemia effect in CML transplant recipients. 2. Long term follow-up: Our stem cell transplantation program has now accumulated 20 years of follow-up on protocol 05-H-0130. Currently over 140 patients are being followed long term. Our present focus is on outcomes in the second decade after SCT. We found that males have an increased risk of cardiovascular events and have a cardiovascular age 10 years over their actual age. To further investigate this we studied 16 asymptomatic post allo-SCT survivors (11 males;5 females) median age of 45 years at transplant who underwent coronary calcium scoring and contrast enhanced coronary CT angiograms at a median follow up of 5 years post transplant. Framingham cardiovascular risk scores were also calculated at time of screening. Two were high risk, 1 intermediate and 13 low risk. Non-obstructive coronary artery disease was detected in seven (44%) patients. Additionally, four (25%) had aortic root calcification. Coronary artery disease was detected in 4 of 13 (30.8%) low risk patients. We conclude that coronary calcium score with or without CT angiogram is a safe, feasible, highly sensitive study in transplant survivors;and even asymptomatic, low-risk survivors may benefit from screening. 3.Optimizing immune reconstitution after SCT: In 2012 we completed a T cell depletion SCT protocol (protocol 07-H-0248): sixty patients (median age 43 years) with hematologic malignancies underwent SCT from their HLA-identical siblings. G-CSF mobilized peripheral blood stem cells from the donor were CD34+ selected using the Miltenyi CliniMacs system. At a median follow up of 3.6 years, Kaplan-Meier estimates of relapse, non relapse mortality and overall survival were 35%, 33% and 44% respectively. We elected to conduct a further T cell depletion study to address some of the shortcomings of the trial 07-H-0248. In protocol 12-H-0028 the T cell depletion technique was changed so as to negatively select for CD34 cells using a CD3 CD19 antibody magnetic separation approach from Miltenyi Corporation under an investigator IND. The transplant product contains a mixed population of CD34 cells more mature progenitors and NK cells. It was anticipated that hematological reconstitution would be more rapid and robust with this method. Subjects received myeloablative conditioning with cyclophosphamide (60 mg/kg/dose x 2), fludarabine (25 mg/m2/dose x 5) and total body irradiation (12 Gy divided in 8 fractions, with lung shielding to 6 Gy). (ii) No planned donor lymphocyte infusion DLI given;( DLI was reserved only for patients with graft failure or relapsed leukemia) (iii) Older patients receive 600cGy instead of 400cGy to improve engraftment since a number of older patients had engraftment failure in the previous protocol. This protocol accrued 20 patients and is now closed. Hematological recovery was prompt. However we noted increased frequency of grade II-IV acute graft-versus-host disease (GVHD) and graft failure requiring second transplantation in four. Full analysis of outcome awaits longer follow-up. This trial was terminated because of the high frequency of GVHD and graft failure which precluded its use as a platform for T cell therapy. In 2013 a new T cell depletion protocol was initiated Peripheral Blood Stem Cell Allotransplantation for Hematological Malignancies Using Ex Vivo CD34 Selection - A Platform for Adoptive Cellular Therapy. This uses a modified version of protocol 07-H-0248 (incorporating modifications ii and iii as above). Currently two patient are enrolled. 4. Prevention of viral infection after SCT. In collaboration with Dr Catherine Bollard (Baylor College) and Dr David Stroncek (DTM) we have developed a robust system to expand multivirus specific T cells recognizing CMV, EBV, Adenovirus and BK virus which commonly complicate the outcome after SCT. Using a peptide mix of common immunodominant antigens from these viruses we have induced multivirus specific T cells in high frequencies. Currently standard operating procedures have been set up and pre-clinical scale up studies are ongoing in the Cell Processing Section of the Department of Transfusion Medicine. A pre-IND discussion with the FDA has facilitated the completion of a clinical protocol which will be submitted for regulatory approval in 2013. It is anticipated this trial will commence in 2014. 5. Prevention and treatment of GVHD and post transplant complications with mesenchymal stromal cells: In 2013 we completed a phase I trial using third party, early passage, mesenchymal stromal cells (MSC) generated in the NIH Cell Processing Section for patients with steroid-refractory liver or gastrointestinal GVHD, tissue injury or marrow failure following SCT to investigate safety and clinical responses following MSC infusion. Ten subjects were infused 2 x 106 MSC /kg weekly for 3 doses. There was no treatment related toxicity. Eight subjects were evaluable for response assessment at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved complete remission (CR), two of two patients with tissue injury (pneumomediastinum/ pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines occurred after the first MSC infusion. Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD, or CK18 for PM. The GVHD complete responders survived significantly longer (>300 days vs a median of 33 days). Cytokine changes also segregated with survival. These results confirm that MSC are associated with rapid clinical responses and biomarker normalization in steroid-refractory GVHD and PM. Early detection and MSC treatment appear important in patients with refractory GVHD. 6. Prevention of GVHD through induction of regulatory T cells (Tregs) by interleukin-2 (IL-2): We completed a study of low dose IL-2 in healthy volunteers to determine the kinetics of Treg and NK expansion. We found a twofold or greater increase in Tregs and also in natural Tregs by 7 days. IL-2 at the lowest dose of 100,000u/m2 daily for 5 days was optimal. This schedule was without significant side effects. On the basis of these results we now propose to evaluate the protective effect of LD IL2 in haploidentical SCT. Donors will receive LD IL2 together with G-CSF to mobilize stem cells. The transplant will be partially depleted of T lymphocytes to a dose of 1 x 106 CD3 cells/kg . Recipients will receive LD IL-2 from day 2 for 12 weeks. The main study end point will be the development of grade II-IV acute GVHD with stopping rules for unacceptable day 100 mortality.
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