Background Late mortality was investigated in patients with chronic myelogenous leukemia (CML) who underwent blood or bone marrow transplant (BMT) with or without prior tyrosine kinase inhibitor (TKI) therapy. Methods By using data from the Blood or Marrow Transplant Survivor Study, the authors examined late mortality in 447 patients with CML who underwent BMT between 1974 and 2010, conditional on surviving ≥2 years post‐BMT. For vital status information, the medical records, the National Death Index, and the Accurint database were used. Standardized mortality ratios (SMRs) were calculated using general population age‐specific, sex‐specific, and calendar‐specific mortality rates. Kaplan‐Meier techniques and Cox regression were used for all‐cause mortality analyses. Cumulative incidence and proportional subdistribution hazards models for competing risks were used for cause‐specific mortality analyses. Results The 10‐year overall survival rate was 65.7% and 73% for those who underwent transplant with and without pre‐BMT exposure to TKI therapy, respectively. Patients who underwent transplant with and without pre‐BMT TKI experienced SMRs of 6.4 and 6.4, respectively ( P = .8); and the SMRs were 11.6 and 8.1, respectively, for those with high‐risk disease ( P = .2). Independent predictors of non–CML‐related mortality included chronic graft‐versus‐host disease (hazard ratio [HR], 2.8; 95% CI, 1.8‐4.4) and busulfan/cyclophosphamide conditioning (HR, 0.5; 95% CI, 0.3‐0.9; reference, total body irradiation/cyclophosphamide conditioning). The 20‐year cumulative incidence of CML‐related and non–CML‐related mortality was 6% and 36%, respectively, for the entire cohort. Both CML‐related mortality (HR, 1.0; 95% CI, 0.1‐12.6) and non–CML‐related mortality (HR, 1.3; 95% CI, 0.6‐3.1) were comparable for those with and without pre‐BMT TKI therapy. Conclusions The similar late mortality experienced by patients with CML who undergo transplantation with or without pre‐BMT TKIs suggests that allogeneic BMT can be considered in the context of TKI intolerance or nonadherence. The prevention of post‐BMT non–CML‐related mortality could favorably affect long‐term survival.

Dermatologic TherapyVolume 32, Issue 1 e12744 Therapeutic Hotline: Letter Intravenous immunoglobulin for treatment of necrobiotic xanthogranuloma Amrita Goyal, Corresponding Author Amrita Goyal amrita.goyal@gmail.com orcid.org/0000-0003-3790-8242 Department of Dermatology, University of Minnesota, Minneapolis, MN Correspondence Amrita Goyal, Department of Dermatology, University of Minnesota, 516 Delaware St SE Phillips Wangensteen Bldg 14-100 Minneapolis, MN 55401 Email: amrita.goyal@gmail.comSearch for more papers by this authorDaniel O'Leary, Daniel O'Leary Department of Medicine, University of Minnesota, Minneapolis, MNSearch for more papers by this authorGregory Vercellotti, Gregory Vercellotti Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MNSearch for more papers by this authorDaniel Miller, Daniel Miller Department of Dermatology, University of Minnesota, Minneapolis, MNSearch for more papers by this authorPhilip McGlave, Philip McGlave Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MNSearch for more papers by this author Amrita Goyal, Corresponding Author Amrita Goyal amrita.goyal@gmail.com orcid.org/0000-0003-3790-8242 Department of Dermatology, University of Minnesota, Minneapolis, MN Correspondence Amrita Goyal, Department of Dermatology, University of Minnesota, 516 Delaware St SE Phillips Wangensteen Bldg 14-100 Minneapolis, MN 55401 Email: amrita.goyal@gmail.comSearch for more papers by this authorDaniel O'Leary, Daniel O'Leary Department of Medicine, University of Minnesota, Minneapolis, MNSearch for more papers by this authorGregory Vercellotti, Gregory Vercellotti Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MNSearch for more papers by this authorDaniel Miller, Daniel Miller Department of Dermatology, University of Minnesota, Minneapolis, MNSearch for more papers by this authorPhilip McGlave, Philip McGlave Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MNSearch for more papers by this author First published: 17 September 2018 https://doi.org/10.1111/dth.12744Citations: 3Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume32, Issue1January/February 2019e12744 RelatedInformation

Reduced-intensity conditioning (RIC) extends the curative potential of allogeneic hematopoietic cell transplantation (HCT) to patients with hematologic malignancies unable to withstand myeloablative conditioning. We prospectively analyzed the outcomes of 292 consecutive patients, median age 58 years (range, 19 to 75) with hematologic malignancies treated with a uniform RIC regimen of cyclophosphamide, fludarabine, and total body irradiation (200 cGy) with or without antithymocyte globulin and cyclosporine and mycophenolate mofetil graft-versus-host disease (GVHD) prophylaxis followed by allogeneic HCT at the University of Minnesota from 2002 to 6. Probability of 5-year overall survival was 78% for patients with indolent non-Hodgkin lymphoma, 53% for chronic myelogenous leukemia, 55% for Hodgkin lymphoma, 40% for acute myelogenous leukemia, 37% for myelodysplastic syndrome, 29% for myeloma, and 14% for myeloproliferative neoplasms. Corresponding outcomes for relapse were 0%, 13%, 53%, 37%, 39%, 75%, and 29%, respectively. Disease risk index (DRI) predicted both survival and relapse with superior survival (64%) and lowest relapse (16%) in those with low risk score compared with 24% survival and 57% relapse in those with high/very-high risk scores. Recipient cytomegalovirus (CMV)-positive serostatus was protective from relapse with the lowest rates in those also receiving a CMV-positive donor graft (29%). The cumulative incidence of 2-year nonrelapse mortality was 26% and was lowest in those receiving a matched sibling graft at 21%, with low (21%) or intermediate (18%) HCT-specific comorbidity index, and was similar across age groups. The incidence of grades II to IV acute GVHD was 43% and grades III to IV 27%; the highest rates were found in those receiving an unrelated donor (URD) peripheral blood stem cell (PBSC) graft, at 50%. Chronic GVHD at 1 year was 36%. Future approaches incorporating alternative GVHD prophylaxis, particularly for URD PBSC grafts, and targeted post-transplant antineoplastic therapies for those with high DRI are indicated to improve these outcomes.

Abstract Background: Tyrosine kinase inhibitors have become the treatment of choice for CML. However, the high cost and need for life-long treatment contribute to non-adherence and represent a major challenge in their use. Allogeneic HCT is potentially curative, but the very long-term health of the survivors is not known. It is also not clear whether a subgroup of CML patients carries a relatively low risk of long-term morbidity. Methods: We addressed these gaps by studying long-term outcomes in 637 CML patients treated with allogeneic HCT between 1981 and 2010 at City of Hope or Univ MN, and surviving for at least 2y after HCT (median follow-up: 16.7y from HCT); 80% of the cohort was <45y at HCT; 68% received HCT in 1st chronic phase (CP); 63% received matched related [MRD], 34% matched unrelated donor (MUD) and 3% non-myeloablative HCTs; 79% received TBI; 65.8% developed chronic GvHD. Vital status information was collected as of May, 2016, using medical records, National Death Index and Lexis Nexis. US mortality rates were obtained from CDC's National Center for Health Statistics. Thirty percent (n=192) died after having survived at least 2 years after HCT; median time between HCT and death was 8.3y. Of the 445 patients alive at study, 288 (65%) completed the BMTSS health questionnaire used to examine the risk of CTCAE grade 3 (severe) or 4 (life-threatening) chronic health conditions. A sibling comparison group (n=404) also completed the BMTSS questionnaire. Results: Late Mortality: Overall survival was 72.1% at 20y and 69.9% at 30y from HCT. The 20y cumulative incidence of relapse-related mortality was 3.9% (95% CI, 2.6-5.8%) and of non-relapse-related mortality was 18.2% (95% CI, 19.8-28.1%) (Figure 1). 20y cumulative incidence of mortality by cause of death was as follows: infection (7%), chronic GvHD (6%), subsequent malignant neoplasms (SMNs: 3%). HCT recipients were at 4.4-fold increased risk of death (95% CI, 3.8-5.1, p<0.0001) than age-, race-, and sex-adjusted normal populations. For patients transplanted in 1st CP and surviving 15y, mortality rates became comparable with the general population (SMR, 1.5, 95% CI, 0.9-2.3, p=0.1). Among CML patients receiving HCT at <45y with Bu/Cy (n=70), overall survival was 81.5% at 20y from HCT; the 20y cumulative incidence of relapse-related mortality was 2.9% and of non-relapse-related mortality was 14%. This cohort was at 3.3-fold higher risk of death when compared with the general population (95% CI=1.7-5.7, p<0.0001). Late Morbidity: The 20y cumulative incidence of a severe/life-threatening chronic health condition among HCT survivors was 47.2% (95% CI, 39.0-54.9%); the incidence was higher (p=0.0006) for MUD vs. MRD recipients (Figure 2). After adjusting for age, sex, race and SES, HCT survivors were at 2.7-fold higher risk for severe/life-threatening chronic health conditions as compared with siblings (95% CI, 1.8-3.9, p<0.0001). The 20y cumulative incidence of specific conditions experienced by survivors and siblings were: SMNs (10.1% vs. 1.7%, p<0.001); diabetes (11.1% vs. 1.5%, p<0.001) and coronary artery disease (6.9% vs. 3.2%, p<0.001). CML patients receiving MRD HCT at <45y with Bu/Cy were not at increased risk of severe/life-threatening chronic health conditions when compared with the sibling comparison group (HR=0.81, 95% CI, 0.26-2.54, p=0.7). Conclusions: Conditional on surviving the first 2y after HCT, the overall survival exceeds 70% at 20y and remains stable at 70% at 30y after HCT. Non-relapse related mortality (infections, chronic GvHD, SMNs) is by far the major contributor to the late mortality. Conditional on surviving the first 15y, mortality rates are similar to those observed in the general population. HCT survivors are at a 2.7-fold higher risk of severe/life-threatening morbidity when compared with siblings. The more common morbidities include SMNs, diabetes and coronary artery disease. However, CML patients receiving HCT at <45y with Bu/Cy conditioning enjoy survival rates exceeding 81% at 20y from HCT, and their burden of long-term morbidity is comparable to that experienced by siblings. These findings could help inform decisions regarding therapeutic options for management of CML. Disclosures Snyder: BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Forman:Mustang Therpapeutics: Other: Construct licensed by City of Hope.

We studied the safety and clinical outcomes of patients treated with umbilical cord blood (UCB)-derived regulatory T cells (Tregs) that expanded in cultures stimulated with K562 cells modified to express the high-affinity Fc receptor (CD64) and CD86, the natural ligand of CD28 (KT64/86). Eleven patients were treated with Treg doses from 3-100 × 10(6) Treg/kg. The median proportion of CD4(+)FoxP3(+)CD127(-) in the infused product was 87% (range, 78%-95%), and we observed no dose-limiting infusional adverse events. Clinical outcomes were compared with contemporary controls (n = 22) who received the same conditioning regimen with sirolimus and mycophenolate mofetil immune suppression. The incidence of grade II-IV acute graft-versus-host disease (GVHD) at 100 days was 9% (95% confidence interval [CI], 0-25) vs 45% (95% CI, 24-67) in controls (P = .05). Chronic GVHD at 1 year was zero in Tregs and 14% in controls. Hematopoietic recovery and chimerism, cumulative density of infections, nonrelapse mortality, relapse, and disease-free survival were similar in the Treg recipients and controls. KT64/86-expanded UCB Tregs were safe and resulted in low risk of acute GVHD.

The impact of allele-level HLA mismatch is uncertain in recipients of double umbilical cord blood (UCB) transplantation. We report a single-center retrospective study of the clinical effect of using allele-level HLA mismatch HLA-A, -B, -C, -DRB1, and -DQB1 of the 2 UCB units. We studied 342 patients with hematologic malignancy. Donor–recipient pairs were grouped according to the number of matched HLA alleles, with 32 matched at 9-10/10, 202 at 6-8/10, and 108 at 2-5/10 alleles. The incidence of hematopoietic recovery, acute and chronic graft-versus-host disease, and nonrelapse mortality and treatment failure was similar between groups. In an exploratory analysis of 174 patients with acute leukemia, after adjusting for length of first remission and cytogenetic risk group, a 2-5/10 HLA match was associated with lower risk of relapse and treatment failure. These data indicate that a high degree of allele-level HLA mismatch does not adversely affect transplant outcomes and may be associated with reduced relapse risk in patients with acute leukemia.

Haploidentical natural killer (NK) cell infusions can induce remissions in some patients with acute myeloid leukemia (AML) but regulatory T-cell (Treg) suppression may reduce efficacy. We treated 57 refractory AML patients with lymphodepleting cyclophosphamide and fludarabine followed by NK cell infusion and interleukin (IL)-2 administration. In 42 patients, donor NK cell expansion was detected in 10%, whereas in 15 patients receiving host Treg depletion with the IL-2-diphtheria fusion protein (IL2DT), the rate was 27%, with a median absolute count of 1000 NK cells/μL blood. IL2DT was associated with improved complete remission rates at day 28 (53% vs 21%; P = .02) and disease-free survival at 6 months (33% vs 5%; P < .01). In the IL2DT cohort, NK cell expansion correlated with higher postchemotherapy serum IL-15 levels (P = .002), effective peripheral blood Treg depletion (<5%) at day 7 (P < .01), and decreased IL-35 levels at day 14 (P = .02). In vitro assays demonstrated that Tregs cocultured with NK cells inhibit their proliferation by competition for IL-2 but not for IL-15. Together with our clinical observations, this supports the need to optimize the in vivo cytokine milieu where adoptively transferred NK cells compete with other lymphocytes to improve clinical efficacy in patients with refractory AML. This study is registered at clinicaltrials.gov, identifiers: NCT00274846 and NCT01106950.

We previously reported that the infusion of ex vivo expanded umbilical cord blood (UCB)-derived natural regulatory T cells (nTregs) infused immediately after UCB transplantation was associated with a reduced incidence of acute graft-versus-host disease (GVHD) relative to historical control subjects [1Brunstein C.G. Miller J.S. Cao Q. et al.Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.Blood. 2011; 117: 1061-1070Crossref PubMed Scopus (820) Google Scholar]. We did not observe an increased incidence of opportunistic infections or relapse, suggesting the transient nature of nTreg provided sufficient immune suppression to control GVHD without long-term deleterious effects. However, we hypothesized that early side effects might be heightened and could be evaluated at specific time points, such as when adoptively transferred nTregs were detectable in the peripheral blood of patient. All but 1 patient, receiving an nTreg dose of 10 × 106/kg on day +1 and 3 × 106/kg on day 15, have been reported [1Brunstein C.G. Miller J.S. Cao Q. et al.Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.Blood. 2011; 117: 1061-1070Crossref PubMed Scopus (820) Google Scholar]. Patient eligibility, conditioning regimen and immune suppression, and supportive care were reported [1Brunstein C.G. Miller J.S. Cao Q. et al.Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.Blood. 2011; 117: 1061-1070Crossref PubMed Scopus (820) Google Scholar]. In this analysis, the historical control subjects consisted of a 65-patient subset of the 108 reported [1Brunstein C.G. Miller J.S. Cao Q. et al.Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.Blood. 2011; 117: 1061-1070Crossref PubMed Scopus (820) Google Scholar] who had post-UCB transplantation T cell subset phenotype data available using standard procedures. In contrast to prior analyses that compared the cumulative incidence of opportunistic, in this analysis we studied infection density that accounts for multiple infections in an individual patient. We calculated infection density per 1000 patient-days within 180 days by dividing the total number of infections within the time period by total patient-day multiplied by 1000 [2Lin D.Y. Non-parametric inference for cumulative incidence functions in competing risks studies.Stat Med. 1997; 16: 901-910Crossref PubMed Scopus (367) Google Scholar]. In our initial report, we demonstrated that adoptively transferred nTregs were present in the peripheral blood of patients up to 14 days after the infusion of fresh and up to 4 days after the infusion of cryopreserved product [1Brunstein C.G. Miller J.S. Cao Q. et al.Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.Blood. 2011; 117: 1061-1070Crossref PubMed Scopus (820) Google Scholar]. Notably, in the current study we found in this early period (days 0 to +30) that nTregs are present, there was significantly higher cumulative density of OI in Treg (18.06 infections per 1000 patient-days) as compared with historical control subjects (7.71 infections per 1000 patient-days) patients (univariate RR, 5.35, P = .02) (Figure 1A). These were essentially viral reactivations with no effect on the risk of fungal infections. Thus, it is possible that Treg can increase the risk of infection during the period of time they are detectable. This contrasts with our initial report in which we found no difference in the risk OIs as assessed by the cumulative incidence. In contrast to the first 30 days, between days +31 and +180 there was similar OI density in the two groups (7.22/1000 versus 4.22/1000; univariate RR, 0.58, P = .07) (Figure 1A). Notably, the higher risk of early infection did not affect nonrelapse mortality (18% [95% confidence interval [CI], 2% to 34%] versus 10% [95% CI, 2% to 17%], P = .34) or progression-free survival (33% [95% CI, 16% to 52%] versus 35% [95% CI, 24% to 47%], P = .93), as compared with historical control subjects. The viral infections observed in nTreg patients through day +30 were human herpesvirus-6 viremia (HHV6, n = 9), cytomegalovirus (n = 2) viremia, and 1 case of parainfluenza upper respiratory infection. The corresponding viral infections observed in historical control subjects through day +30 were HHV-6 viremia (n = 6), cytomegalovirus reactivation (n = 8), adenovirus gastroenteritis (n = 4), polyoma virus in the urine (n = 4), enterovirus gastroenteritis (n = 1), and upper airway respiratory syncytial virus (n = 1). Three of 9 HHV-6 viremia episodes in the Treg group and 2 of 6 in the historical control group were treated with foscarnet. In our institution, we observed 69% incidence reactivation of HHV-6 by 6 weeks after UCB transplantation, overall [3Betts B.C. Young J.A. Ustun C. et al.Human herpesvirus 6 infection after hematopoietic cell transplantation: is routine surveillance necessary?.Biol Blood Marrow Transplant. 2011; 17: 1562-1568Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. Thus, although a high rate of HHV-6 infections in the first month is not unexpected, the higher density of this infection in the first month as compared with similarly treated patients could be the result of the Treg infusion. However, patients in a phase I clinical trial are monitored more closely than patients receiving the standard of care, and we cannot rule out that our higher infection density is the result of an observation bias and/or the limited number of Treg recipients. A potential benefit of not developing GVHD is the ability to taper immune suppression sooner, leading to potentially faster reconstitution of immune reconstitution. However, we did not observe a significant difference between nTreg patients versus historical control subjects. At day +180, the median absolute number of peripheral blood CD19+ cells was 179/μL (range, 0 to 1160) versus 282/μL (range, 1 to 1632) (P = .54), CD3+/CD8+ was 58/μL (range, 14 to 645) versus 72/μL (range, 8 to 848) (P = .46), and CD3+/CD4+ cells was 255/μL (range, 6 to 527) versus 219/μL (range, 27 to 1179) (P = .48) for nTreg versus historical control subjects, respectively (Figure 1B-D). In contrast, data on the adoptive transfer of nTregs in combination with T effector cells in haploidentical related donor transplantation showed that antigen-specific T cell immunity was observed sooner than in historical control subjects [4Di Ianni M. Falzetti F. Carotti A. et al.Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.Blood. 2011; 117: 3921-3928Crossref PubMed Scopus (816) Google Scholar]. This may be secondary to biological differences between donor types because cord blood recipients may take longer to achieve adequate numbers of virus-specific T cells [5McGoldrick S.M. Bleakley M.E. Guerrero A. et al.Cytomegalovirus-specific T cells are primed early after cord blood transplant but fail to control virus in vivo.Blood. 2013; 121: 2796-2803Crossref PubMed Scopus (31) Google Scholar]. Clearly, longer follow-up and more detailed analysis are required for better understanding of lymphocyte subsets reconstitution. In summary, there is potentially a higher viral infection risk within 30 days of UCB-derived nTregs infusion due to suppression of the immune response; however, there was no adverse effect on the longer term outcomes, including later risk of OIs. Although observation bias is a possibility, our data suggesting higher density of viral infections argues for close observation in studies of the adoptive transfer of nTregs to reduce GVHD in allogeneic hematopoietic cell transplantation and in solid organ transplantation. Financial disclosure: Supported in part by grants from the National Cancer Institute (P01 CA65493 to C.G.B., J.S.M., D.H.M., P.B.M., B.R.B., and J.E.W.), Leukemia and Lymphoma Society Scholar in Clinical Research Award (2417-11 to C.G.B.), the Children's Cancer Research Fund (to J.E.W. and T.E.D. and R01 CA105216 to C.H.J.), National Heart, Lung and Blood Institute (N01 HB037164 to J.S.M., D.H.M., and J.E.W. and HHSN268201000008C to J.S.M., D.H.M., K.L.H., J.C., and J.E.W.), Leukemia and Lymphoma Translational Research (R6029-07 to B.R.B.), and National Marrow Donor Program (13396 AM#2 to B.R.B. and J.E.W.).

Meeting abstracts Adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with AML. Despite lymphodepleting chemotherapy failure may result from suppression by host regulatory T cells (Treg). We report outcomes of 57 refractory AML patients treated with

Double umbilical cord blood transplantation (dUCBT), developed as a strategy to treat large number of patients with hematologic malignancies, frequently leads to the long-term establishment of a new hematopoietic system maintained by cells derived from a single umbilical cord blood unit. However, predicting which unit will predominate has remained elusive. This retrospective study examined the risk factor associated with unit predominance in 262 patients with hematologic malignancies who underwent dUCBT with subsequent hematopoietic recovery and complete chimerism between 2001 and 2009. Dual chimerism was detected at day 21–28, with subsequent single chimerism in 97% of the cases by day +100 and beyond. Risk factors included nucleated cell dose, CD34+ and CD3+ cell dose, colony-forming units-granulocyte macrophage dose, donor–recipient HLA match, sex and ABO match, order of infusion and cell viability. In the myeloablative setting, CD3+ cell dose was the only factor associated with unit predominance (odds ratio (OR) 4.4, 95% confidence interval (CI) 1.8–10.6; P<0.01), but in the non-myeloablative setting, CD3+ cell dose (OR 2.1, 95%CI 1.0–4.2; P=0.05) and HLA match (OR 3.4, 95%CI 1.0–11.4; P=0.05) were independent factors associated with unit predominance. Taken together, these findings suggest that immune reactivity has a role in unit predominance, and should be considered during graft selection and graft manipulation.

Reduced-intensity conditioning (RIC) extends the curative potential of allogeneic hematopoietic cell transplantation (HCT) to patients with hematologic malignancies unable to withstand myeloablative conditioning. We prospectively analyzed the outcomes of 123 patients (median age, 57 years; range, 23-70 years) with hematologic malignancies treated with a uniform RIC regimen of cyclophosphamide, fludarabine, and total-body irradiation (200 cGy) with or without antithymocyte globulin followed by related donor allogeneic HCT at the University of Minnesota between 2002 and 2008. The cohort included 45 patients with acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS), 27 with aggressive non-Hodgkin lymphoma (NHL), 8 with indolent NHL, 10 with Hodgkin lymphoma (HL), 10 with myeloma, and 23 with acute lymphocytic leukemia, chronic myelogenous leukemia, other leukemias, or myeloproliferative disorders. The probability of 4-year overall survival was 73% for patients with indolent NHL, 58% for those with aggressive NHL, 67% for those with HL, 30% for those with AML/MDS, and only 10% for those with myeloma. Corresponding outcomes for relapse in these patients were 0%, 32%, 50%, 33%, and 38%, and those for progression-free survival were 73%, 45%, 27%, 27%, and 10%. The incidence of treatment-related mortality was 14% at day +100 and 22% at 1 year. The incidence of grade II-IV acute graft-versus-host disease was 38% at day +100, and that of chronic graft-versus-host disease was 50% at 2 years. Multivariate analysis revealed superior overall survival and progression-free survival in patients with both indolent and aggressive NHL compared with those with AML/MDS, HL, or myeloma. Worse I-year treatment-related mortality was observed in patients with a Hematopoietic Cell Transplantation Comorbidity Index score >= 3 and in cytomegalovirus-seropositive recipients. These results suggest that (I) RIC conditioning was well tolerated by an older, heavily pretreated population; (2) patients with indolent and aggressive NHL respond well to RIC conditioning, highlighting the importance of the graft-versus-lymphoma effect; and (3) additional peri-transplantation manipulations are needed to improve outcomes for patients with AML/MDS or myeloma receiving RIC conditioning before HCT. Biol Blood Marrow Transplant 17: 1025-1032 (2011) (C) 2011 American Society for Blood and Marrow Transplantation

Abstract Abstract 3611 Adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with refractory myeloid leukemia (AML). However, NK cells do not expand and persist in all patients despite lymphodepleting chemotherapy. In trials of adoptive NK cell therapy in solid tumors or lymphoma, host regulatory T cells (Treg) often expand in response to IL-2 given to stimulate donor NK cell expansion. Although murine studies report that Tregs inhibit NK cells, the influence of human Treg on NK cell proliferation and function is not well characterized. We studied the effect of allogeneic Tregs that were derived from human umbilical cord blood (UCB) as described by our group. Resting CFSE labelled NK cells or Teff were purified from healthy donors, and mixed with UCB Treg at various ratios. Unstimulated NK cells did not proliferate and thus IL-2 or IL-15 were added to the media at concentrations of 0.1, 0.25 and 0.5 ng/ml. In the absence of Treg, both cytokines induced equal NK cell proliferation at 5 days as measured by CFSE dilution in a concentration dependent manner. CFSE dilution was inhibited by Treg at a 1:1 ratio, especially at low cytokine concentrations. There were marked differences between the two cytokine conditions. Following IL-15 induced stimulation, the reduction in NK cell proliferation by Treg ranged from 1–35% (at different concentrations tested), whereas the inhibition of IL-2 stimulated NK cell proliferation ranged from 65–85%. Treg inhibition of NK cell proliferation could be measured at ratios as low as 1:8 in the presence of IL-2, but not IL-15. This inhibitory effect was partially explained by competition from CD25+ Tregs for IL-2. We measured Treg utilization of IL-2 by incubating NK cells with or without Treg in 0.5 ng/ml IL-2 for 4 days. The level of IL-2 with NK cells alone was 40 pg/ml vs. 17 pg/ml with Treg (compared to 330 pg/ml in IL-2-supplemented media without cells). Based on this data, we have incorporated host Treg depletion to enhance NK expansion after adoptive transfer to treat patients with refractory AML. As murine data from Blazar's group shows that CTL therapy is enhanced by Treg depletion, we added one dose of denileukin diftitox (ONTAK®, Eisai Inc) at 12 mg/kg to our lymphodepleting preparative regimen of fludarabine 25 mg/m2 × 5 days, cyclophosphamide 60 mg/kg × 2 days for 12 AML patients. Haploidentical NK cells (CD3- and CD19-depleted PBMCs and overnight activated with IL-2 1000 U/ml) were infused on Day 0, followed by 6 doses subcutaneous IL-2 (9 million units) given every other day to promote in vivo NK cell expansion. Eleven of 12 patients were evaluable, having received at least 4 of 6 planned doses of IL-2. Blood and marrow were collected 7 and 14 days after infusion to assess NK cell and Treg expansion, as well as leukemia clearance. Of the 10 patients with interpretable day 7 chimerism data, 9 had detectable donor DNA (median 68% donor DNA). At day 14, 4 of the 12 patients (33%) had successfully expanded NK cells in vivo, with absolute donor derived NK cell counts of 480, 530, 1470 and 12390 cells/μL blood, improving on our previous 10% rate of in vivo NK cell expansion which was observed with the same regimen, without Treg depletion. In the 4 patients who expanded NK cells in vivo, there were no detectable Treg (defined as a CD25+CD4+FoxP3+ lymphocyte population) at either day 7 or day 14. In contrast, the presence of a bona fide Treg population at either day 7 [range 9.5–53%] or day 14 [27–71%] correlated with a lack of in vivo NK cell expansion at day 14. Clinically, 8 of the 11 evaluable subjects cleared leukemia (72%), 7 of whom recovered neutrophils (63% CRp) and 6 of whom went on to best donor transplant (45%). In summary, we demonstrate in vitro and in vivo suppression of NK cell proliferation by IL-2 stimulated Treg. This effect is not seen in vitro with IL-15. We have shown that the absence of host Treg correlates with in vivo NK cells expansion. Although an increased rate of donor NK expansion was observed with a single dose of denileukin diftitox, it did not completely overcome the IL-2 induced host Treg expansion. Future trials testing additional doses of denileukin difitox or other methods of Treg depletion, as well as the use of IL-15 are planned. Disclosures: No relevant conflicts of interest to declare.

Acute graft-versus-host disease (aGVHD) is associated with high risk of morbidity and mortality and is a common complication after double umbilical cord blood (UCB) transplantation. To reduce these risks, we established a method of CD4(+)CD25(+)FoxP3(+) T regulatory cell (Treg) enrichment from cryopreserved UCB followed by a 18 (+) 1-day expansion culture including anti-CD3/anti-CD28 antibody-coated beads and recombinant human interleukin-2. In a "first-in-human" clinical trial, we evaluated the safety profile of UCB Treg in 23 patients. Patients received a dose of 0.1-30 × 10(5)UCB Treg/kg after double UCB transplantation. The targeted Treg dose was achieved in 74% of cultures, with all products being suppressive in vitro (median 86% suppression at a 1:4 ratio). No infusional toxicities were observed. After infusion, UCB Treg could be detected for 14 days, with the greatest proportion of circulating CD4(+)CD127(-)FoxP3(+) cells observed on day (+)2. Compared with identically treated 108 historical controls without Treg, there was a reduced incidence of grade II-IV aGVHD (43% vs 61%, P = .05) with no deleterious effect on risks of infection, relapse, or early mortality. These results set the stage for a definitive study of UCB Treg to determine its potency in preventing allogeneic aGVHD. This study is registered at http://www.clinicaltrials.gov as NCT00602693.

Although the study of natural killer (NK) cell alloreactivity has been dominated by studies of killer cell immunoglobulin-like receptors (KIRs), we hypothesized that NKG2A and LIR-1, present on 53% +/- 13% and 36% +/- 18% of normal NK cells, respectively, play roles in the NK cell killing of primary leukemia targets. KIR- cells, which compose nearly half of the circulating NK cell population, exhibit tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both acute myelogenous leukemia and acute lymphoblastic leukemia targets were rendered susceptible to lysis by fresh resting KIR- NK cells when inhibitory receptor major histocompatibility class 1 interactions were blocked by pan-HLA antibodies, demonstrating that these cells are functionally competent. Blockade of a single inhibitory receptor resulted in slightly increased killing, whereas combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR- NK cells, demonstrating that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR- NK cells is mediated by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemias.

Despite its common use in nonmyeloablative preparative regimens, the pharmacokinetics of fludarabine are poorly characterized in hematopoietic cell transplantation (HCT) recipients and exposure-response relationships remain undefined. The objective of this study was to evaluate the association between plasma F-ara-A exposure, the systemically circulating moiety of fludarabine, and engraftment, acute GVHD, TRM and OS after HCT. The preparative regimen consisted of CY 50 mg/kg/day i.v. day –6; plus fludarabine 30–40 mg/m2/day i.v. on days –6 to –2 and TBI 200 cGy on day –1. F-ara-A pharmacokinetics were carried out with the first dose of fludarabine in 87 adult patients. Median (range) F-ara-A area-under-the-curve (AUC(0−∞)) was 5.0 μg h/mL (2.0–11.0), clearance 15.3 L/h (6.2–36.6), Cmin 55 ng/mL (17–166) and concentration on dayzero 16.0 ng/mL (0.1–144.1). Despite dose reductions, patients with renal insufficiency had higher F-ara-A exposures. There was strong association between high plasma concentrations of F-ara-A and increased risk of TRM and reduced OS. Patients with an AUC(0−∞) greater than 6.5 μg h/mL had 4.56 greater risk of TRM and significantly lower OS. These data suggest that clinical strategies are needed to optimize dosing of fludarabine to prevent overexposure and toxicity in HCT.

Umbilical cord blood (UCB) transplantation is potentially curative for acute leukemia. This analysis was performed to identify risk factors associated with leukemia relapse following myeloablative UCB transplantation. Acute leukemia patients (n = 177; 88 with acute lymphoblastic leukemia and 89 with acute myeloid leukemia) were treated at a single center. Patients received a UCB graft composed of either 1 (47%) or 2 (53%) partially human leukocyte antigen (HLA)-matched unit(s). Conditioning was with cyclophosphamide and total body irradiation with or without fludarabine. The incidence of relapse was 26% (95% confidence interval [CI], 19%-33%). In multivariate analysis, relapse was higher in advanced disease patients (> or = third complete remission [CR3]; relative risk [RR], 3.6; P < .01), with a trend toward less relapse in recipients of 2 UCB units (RR = 0.6; P = .07). However, relapse was lower for CR1-2 patients who received 2 UCB units (RR 0.5; P < .03). Leukemia-free survival was 40% (95% CI, 30%-51%) and 51% (95% CI, 41%-62%) for single- and double-unit recipients, respectively (P = .35). Although it is known that transplantation in CR1 and CR2 is associated with less relapse risk, this analysis reveals an enhanced graft-versus-leukemia effect in acute leukemia patients after transplantation with 2 partially HLA-matched UCB units. This trial was registered at http://clinicaltrials.gov as NCT00309842.

PurposeTransplantation of hematopoietic stem cells from an unrelated donor (URD) is an option for many patients who do not have an HLA-identical sibling donor (MSD). Current criteria for the selection of URDs include consideration for HLA alleles determined by high resolution typing methods, with preference for allele-matched donors. However, the utility and outcome associated with transplants from URDs compared with those from MSDs remains undefined.Patients and MethodsWe examined clinical outcome after patients received bone marrow transplants (BMTs) from MSDs; HLA-A, -B, -C, and DRB1 allele-matched URDs (8/8); and HLA-mismatched URDs in a homogeneous population of patients with chronic myeloid leukemia (CML) in first chronic phase (CP1) where a strong allogeneic effect and hence a lower risk of relapse is anticipated. Transplantation outcomes were compared between 1,052 URD and 3,514 MSD BMT recipients with CML in CP1.ResultsFive-year overall survival and leukemia-free survival (LFS) after receipt of BMTs from 8/8 matched URDs were worse than those after receipt of BMTs from MSDs (5-year survival, 55% v 63%; RR, 1.35; 95% CI, 1.17 to 1.56; P < .001; LFS, 50% v 55%; RR, 1.21; 95% CI, 1.06 to 1.40; P = .006). Survival was progressively worse with greater degrees of mismatch. Similar and low risk of relapse were observed after receipt of transplant from either MSD or URD.ConclusionIn this homogeneous cohort of good risk patients with CML in CP1, 5-year overall survival and LFS after receipt of transplant from 8/8 allele-matched donors were modestly though significantly worse than those after receipt of transplant from MSDs. Additive adverse effects of multilocus mismatching are not well tolerated and should be avoided if possible.

Nonmyeloablative hematopoietic cell transplantation (HCT) has been used to treat patients with advanced or high-risk lymphoid malignancies. We studied 65 patients (median age 46 years) receiving an umbilical cord blood (UCB) graft after a single conditioning regimen consisting of cyclophosphamide (50 mg/kg) on day −6, fludarabine (40 mg/m2) daily on days −6 to −2, as well as a single fraction of total-body irradiation (TBI) (200 cGy) along with cyclosporine mycophenolate mofetil immunosuppression. Median time to neutrophil and platelet recovery was 7.5 days (range: 0-32) and 46 days (range: 8-111), respectively. Cumulative incidences of grade II-IV, grade III-IV acute, and chronic graft-versus-host disease (aGVHD, cGVHD) were 57% (95% confidence interval [CI]: 43%-70%), 25% (95% CI: 14%-35%), and 19% (95% CI: 9%-29%), respectively. Transplant-related mortality at 3 years was 15% (95% CI: 5%-26%). Median follow-up was 23 months. The progression free-survival (PFS), current PFS and overall survival (OS) were 34% (95% CI: 21%-47%), 49% (95% CI: 36%-62%), and 55% (95% CI: 42%-70%) at 3 years. Based on our data, we conclude that a nonmyeloablative conditioning regimen followed by UCB transplantation is an effective treatment for patients with advanced lymphoid malignancies who lack a suitable sibling donor.