Donor and recipient plasma follistatin levels are associated with acute GvHD in Blood and Marrow Transplant Clinical Trials Network 0402.

Follistatin is an angiogenic factor elevated in the circulation after allogeneic hematopoietic cell transplantation (HCT). Elevations in follistatin plasma concentrations are associated with the onset of and poor survival after acute GvHD (aGvHD). Using data from the Blood and Marrow Transplant Clinical Trials Network 0402 study (n=247), we sought to further quantify the longitudinal associations between plasma follistatin levels in transplant recipients, as well as baseline HCT donor follistatin levels, and allogeneic HCT outcomes. Higher recipient baseline follistatin levels were predictive of development of aGvHD (P=0.04). High donor follistatin levels were also associated with the incidence of aGvHD (P<0.01). Elevated follistatin levels on day 28 were associated with the onset of grade II-IV aGvHD before day 28, higher 1-year non-relapse mortality (NRM) and lower overall survival. In multivariate analyses, individuals with follistatin levels >1088 pg/mL at day 28 had a 4-fold increased risk for NRM (relative risk (RR)=4.3, 95% confidence interval (CI) 1.9-9.9, P<0.01) and a nearly three-fold increased overall risk for mortality (RR=2.8, 95% CI 1.5-5.2, P<0.01). Given the multiple roles of follistatin in tissue inflammation and repair, and the confirmation that this biomarker is predictive of important HCT outcomes, the pathobiology of these relationships need further study.

Low EGF in myeloablative allotransplantation: association with severe acute GvHD in BMT CTN 0402.

Epidermal growth factor (EGF) is a recently described biomarker of acute GvHD (aGvHD). Whether low plasma EGF prior to hematopoietic cell transplantation (HCT) predisposes to the development of aGvHD, or whether EGF levels fall because of severe aGvHD, is unknown. To evaluate this, we tested plasma samples collected at pre-HCT baseline, day +28 and day +100 during the course of the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) 0402. We found that baseline EGF plasma concentrations were three-fold lower in HCT recipients compared to donors (24.3 vs 76.0 pg/mL, P<0.01). Ninety-one patients (43%) had a markedly low plasma EGF at pre-HCT baseline, defined as <2.7 pg/mL-an optimal cutpoint associated with development of grade III-IV aGvHD. Patients with these low EGF levels at pre-HCT baseline had a 2.9-fold increased risk of grade III-IV aGvHD by day +100. Patients with low EGF at day +28 after HCT had an increased risk of death (relative risk 2.3, P=0.02) by 1 year due to transplant-related toxicities, especially aGvHD. Our results suggest that very low plasma EGF early in the HCT process may predispose patients to an increased risk of death, potentially due to epithelial damage and limited repair capacity.

In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines.

Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)-2 in vitro. Although in vivo IL-2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2-supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.

Upper GI GVHD: similar outcomes to other grade II graft-versus-host disease.

The significance of upper gastrointestinal tract (UGI) acute GVHD (aGVHD) compared with other grade II aGVHD is not clearly defined. We compared the outcomes of patients with grade II aGVHD with or without biopsy-proven UGI involvement in three groups: grade II aGVHD without UGI (n=178), grade II aGVHD with UGI and other sites (n=102) and isolated UGI aGVHD (n=32). The overall response (ORR) to steroids at day 28 differed among the three groups (76, 67 and 91%, respectively, P=0.01), but was only marginally different in direct comparison with those without or with UGI aGVHD (P=0.07) or with isolated UGI aGVHD (P=0.06). In multivariate analysis, as compared with grade II aGVHD patients without UGI involvement, those with UGI involvement and those with isolated UGI aGVHD had similar risks of chronic GVHD, relapse and non-relapse mortality and similar disease-free survival and overall survival. Our data suggest that patients with UGI aGVHD have similar outcomes as those without UGI involvement, supporting the view that UGI aGVHD should still be included as a grade II-defining event.

GvHD after umbilical cord blood transplantation for acute leukemia: an analysis of risk factors and effect on outcomes.

Using the Center for International Blood and Marrow Transplant Research (CIBMTR) registry, we analyzed 1404 umbilical cord blood transplantation (UCBT) patients (single (<18 years)=810, double (⩾18 years)=594) with acute leukemia to define the incidence of acute GvHD (aGvHD) and chronic GvHD (cGvHD), analyze clinical risk factors and investigate outcomes. After single UCBT, 100-day incidence of grade II-IV aGvHD was 39% (95% confidence interval (CI), 36-43%), grade III-IV aGvHD was 18% (95% CI, 15-20%) and 1-year cGvHD was 27% (95% CI, 24-30%). After double UCBT, 100-day incidence of grade II-IV aGvHD was 45% (95% CI, 41-49%), grade III-IV aGvHD was 22% (95% CI, 19-26%) and 1-year cGvHD was 26% (95% CI, 22-29%). For single UCBT, multivariate analysis showed that absence of antithymocyte globulin (ATG) was associated with aGvHD, whereas prior aGvHD was associated with cGvHD. For double UCBT, absence of ATG and myeloablative conditioning were associated with aGvHD, whereas prior aGvHD predicted for cGvHD. Grade III-IV aGvHD led to worse survival, whereas cGvHD had no significant effect on disease-free or overall survival. GvHD is prevalent after UCBT with severe aGvHD leading to higher mortality. Future research in UCBT should prioritize prevention of GvHD.

A risk-adapted approach to acute GVHD treatment: are we there yet?

Acute GVHD (aGVHD) is an immunologic complication of allogeneic hematopoietic cell transplantation (HCT) that can range from mild to life-threatening. Models to predict patients at risk of poor outcomes have been developed using both clinical and laboratory data, and the time to test these models in clinical trials has arrived. However, each modeling method has its potential advantages and limitations. In this mini-review, we summarize recent refinements to these models. We also suggest avenues for improving risk stratification through further studies of a patient's healing capacity and predisposition to endothelial damage, two factors that impact aGVHD outcomes but are absent from the current risk stratification models.

Similar outcomes between adolescent/young adults and children with AML following allogeneic hematopoietic cell transplantation.

We recently reported that adolescents and young adults (AYAs) with B-cell ALL receiving allogeneic hematopoietic cell transplantation (allo-HCT) have inferior survival compared with children, primarily because of greater TRM. We therefore hypothesized that in the setting of allo-HCT for AML, similar inferior outcomes would be observed in AYA patients as compared with children. We reviewed outcomes of 168 consecutive patients (ages 0-30 years) with AML undergoing allo-HCT at our institution. Of these, 60% (n=101) were <15 years of age and 40% (n=67) were AYAs (15-30 years of age). We identified no significant differences in 5-year overall survival (48% vs 50%, P=0.89), disease-free (47% vs 47%, P=0.89), relapse (24% vs 33%, P=0.30) or TRM (27% vs 16%, P=0.10) between the two groups. However, AYA patients had a greater incidence of grade II-IV acute (48% vs 31%, P=0.01) and chronic GVHD (22% vs 7%, P<0.01). Based on this analysis we identified no differences in survival, relapse or TRM between AYAs and children with AML receiving allo-HCT.

Corticosterone levels of Atlantic puffins vary with breeding stage and sex but are not elevated in poor foraging years.

Corticosterone (CORT) levels in seabirds fluctuate across breeding stages and in different foraging conditions. Here we use a ten-year data set to examine whether CORT levels in Atlantic puffins differ in years with high or low availability of capelin, the preferred forage species. Female puffins had higher CORT levels than males, possibly related to cumulative costs of egg production and higher parental investment. Puffins had higher CORT levels and body mass during pre-breeding than during chick rearing. Yearly mean chick growth rates were higher in years when adults had higher body mass and in years where adults brought chicks a lower percentage of non-fish (invertebrates/larval fish) food. Unlike most results from seabird species with shorter chick-rearing periods, higher CORT levels in puffins were not associated with lower capelin abundance. Puffins may suppress CORT levels to conserve energy in case foraging conditions improve later in the prolonged chick-rearing period. Alternatively, CORT levels may be lowest both when food is very abundant (years not in our sample) or very scarce (e.g., 2009 in this study), and increase when extra foraging effort will increase foraging efficiency (most years in this study). If these data primarily represent years with medium to poor foraging, it is possible that CORT responses to variation in foraging conditions are similar for puffins and other seabirds.

Lower leukemia relapse in pediatric patients with pulmonary cytolytic thrombi following allogeneic transplant.

Pulmonary cytolytic thrombi (PCT) is an uncommon complication after hematopoietic cell transplantation. Although the pathogenesis is unknown, patients typically respond to systemic corticosteroid treatment. Considering corticosteroids may impair GVL reactions, we reviewed the records of 324 pediatric patients who received a transplant for leukemia and compared the outcomes of those with PCT (n=14) to those without PCT (n=310). PCT patients had a significantly more acute GVHD (aGVHD) and chronic GVHD (cGVHD). Though 3-year non-relapse mortality and OS were similar, there was significantly less relapse in patients with PCT compared to those without PCT (0 vs 28%, P=0.02), regardless of the presence or absence of aGVHD. In multivariate analysis, grade II-IV aGVHD (P=0.02), cGVHD (P=0.01) and development of PCT (P<0.01) were independently associated with less relapse. These data suggest that patients with PCT are at greater risk for GVHD, but at lower risk of leukemia relapse.

Transplantation of ex-vivo culture-expanded parental haploidentical mesenchymal stem cells to promote engraftment in pediatric recipients of unrelated donor umbilical cord blood: results of a phase I-II clinical trial.

Suboptimal neutrophil and platelet recovery after unrelated donor umbilical cord blood transplantation (UCBT) may be due in part to an impaired microenvironment after intensive chemoradiotherapy. In an attempt to speed hematopoietic recovery, 15 pediatric patients with high-risk acute leukemia were enrolled on a single-institution phase I-II clinical trial in which ex-vivo culture-expanded MSCs from haploidentical parental donors were infused at the time of UCBT. Eight patients received MSCs on day 0, with three patients having a second dose infused on day 21. No serious adverse events were observed with any MSC infusion. All eight evaluable patients achieved neutrophil engraftment at a median of 19 days. Probability of platelet engraftment was 75%, at a median of 53 days. With a median follow-up of 6.8 years, five patients remain alive and disease free. The results of this pilot study show that infusion of ex-vivo culture-expanded haploidentical MSCs into unrelated pediatric UCBT recipients can be performed safely. This encouraging safety profile with haploidentical MSCs supports the investigation of unrelated 'off the shelf' allogeneic HLA-mismatched MSC products.

Fludarabine vs cladribine plus busulfan and low-dose TBI as reduced intensity conditioning for allogeneic hematopoietic stem cell transplantation: a prospective randomized trial.

Purine analogs are often used for conditioning preceding allogeneic hematopoietic stem cell transplantation (HCT). We prospectively tested fludarabine (Flu) 40 mg/m(2)/day x 5 days vs cladribine (Clad) 10 mg/m(2)/day x 5 days plus oral busulfan (1 mg/kg q6 h x 2 days) and total body irradiation 200 cGy in 32 recipients of matched sibling and unrelated donor (URD) HCT. Patients were similar in age (median 52 years), diagnosis, extensive pre-HCT therapy (56 vs 63%), and high-risk disease status (81 vs 93%). Neutrophil engraftment was prompt (median 11 vs 12 days), but early graft failure using Clad halted randomization. Platelet recovery was prompt (median Flu 18 vs Clad 24 days). Graft-versus-host disease (GVHD) after Flu vs Clad was similar; (acute grade II/IV 56 vs 69%, P=0.26; chronic 50 vs 31%, P=0.27). Nonrelapse mortality (Flu 25 vs Clad 38%, P=0.47) and progression-free survival at 3 years were similar as well. Multivariate analyses showed slightly, but not significantly lower relative risk (RR) of neutrophil engraftment with Clad (RR 0.6 (95% CI 0.2-1.3) P=0.16) and with URD RR 0.4 (0.2-1.0) P=0.04). Older patients with advanced hematologic malignancies achieve satisfactory outcomes using either of these reduced intensity conditioning regimens.

Involvement of human thalamic neurons in internally and externally generated movements.

Several anatomical studies support the existence of recurrent neural pathways from cortical motor areas to the thalamus via basal ganglia and back to the cortex. Neuronal responses to internally and externally generated sequential movements have been studied in the motor and premotor cortex of monkeys, but the involvement of subcortical motor structures such as the thalamus have not been studied in monkeys or humans. We examined the activity of neurons during a sequential button press task in motor thalamus of parkinsonian as well as chronic pain patients undergoing implantation of deep brain stimulating electrodes. Single and dual microelectrode recordings were carried out during an internally generated task with a memorized sequence (MEM) and an externally driven task with the sequence given during task performance (follow). Average histograms of neuronal firing were constructed for each task and aligned with respect to visual cues (ready, go) or button presses (P1, P2, P3). Sequential movements were monitored with surface electromyography and hand accelerometry, and cell responses were divided into movement-defined epochs for ANOVA and post hoc means testing. Of 52 neurons tested, 31 were found to have task-related responses and 10 were task-selective with 4 responding preferentially to MEM and 7 responding preferentially to follow (1 was both). Complex responses were found including preparatory, delay period, and phase- and task-specific activity. These kinds of responses suggest a role of the thalamus in both internally and externally cued arms movement and provide some evidence for a role in sequential movements.

Randomized clinical trial of ganciclovir vs acyclovir for prevention of cytomegalovirus antigenemia after allogeneic transplantation.

Cytomegalovirus (CMV) disease remains a major cause of morbidity following allogeneic stem cell transplantation (SCT). In a prospective randomized trial, we tested prophylactic therapy with ganciclovir or acyclovir for patients at high risk of disease. Ninety-one CMV seropositive recipients of related (n = 53) and unrelated (n = 38) donor transplants were enrolled. All patients received intravenous (i.v.) ganciclovir 5 mg/kg every 12 h days -7 to -2, followed by acyclovir 10 mg/kg i.v. every 8 h from day -1 until neutrophil engraftment. Patients were then randomly assigned to either ganciclovir (n = 45) or acyclovir (n = 46) until day 100 post transplant. Any degree of antigenemia was treated with ganciclovir 5 mg/kg i.v. twice a day for 2 weeks, followed by 5 mg/kg i.v. each weekday for 6 weeks. At day 100, the cumulative incidence of antigenemia was 31% (95% CI 17-45%) for ganciclovir and 41% (95% CI 26-56%) (P = 0.22) for acyclovir prophylaxis, respectively. The assigned prophylaxis cohort did not predict for CMV antigenemia. The cumulative incidence of CMV disease at 12 months was 13% (95% CI 3-23%) and 17% (95% CI 6-28%) (P = 0.59) for the ganciclovir- and acyclovir-treated groups, respectively. An absolute neutrophil count (ANC)

Haematopoietic cell transplantation in patients with Fanconi anaemia using alternate donors: results of a total body irradiation dose escalation trial.

Allogeneic haematopoietic cell transplantation (HCT) is the only therapeutic modality capable of correcting the haematologic manifestations of Fanconi anaemia (FA). However, HCT from alternative donors has been associated with poor survival. Between June 1993 and July 1998, 29 FA patients (median age 12.1 years; range 3.7-48.5 years) were enrolled in a prospective phase I-II dose escalation study. All patients were treated with cyclophosphamide 40 mg/kg, total body irradiation (TBI) 450 cGy or 600 cGy and antithymocyte globulin (ATG), followed by HCT from an alternative donor. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A for 6 months, short course methylprednisolone (2 mg/kg/day) between days +5 and +19 and marrow T-cell depletion by counterflow elutriation. The probability of developing grade III-IV toxicity was 17% (95% CI 3-31%). For the 25 marrow recipients, the probability of neutrophil engraftment (ANC 0.5 x 109/l by day 45) was 63% (95% CI 42-82%). Probabilities of grade II-IV acute GVHD and chronic GVHD were 32% (95%CI 10-54%) and 0% respectively. With a median follow-up of 18 months, the probability of survival for the entire cohort at 1 year was 34% (95% CI 17-51%). The presence of lymphocyte somatic mosaicism [i.e. the presence of diepoxybutane (DEB)-insensitive cells] was associated with a significantly increased risk of graft failure. Disappointingly, the use of higher dose TBI and post-transplant ATG did not improve engraftment. More effective peritransplant immunosuppression, especially in FA patients with somatic mosaicism, was required to overcome the barrier of graft rejection. New conditioning regimens adapted to each individual's alkylator sensitivity are needed to improve the outcome of alternative donor HCT for FA.

Haemopoietic cell transplantation in children with juvenile myelomonocytic leukaemia.

Seven children, 11 months to 5.9 years of age, with juvenile myelomonocytic leukaemia (JMML) underwent allogeneic haemopoietic cell transplantation (HCT) using related or unrelated donor bone marrow or umbilical cord blood. All patients had active disease at time of transplant despite chemotherapy in five patients and chemotherapy and splenectomy in one patient prior to HCT conditioning. All patients received cyclophosphamide and TBI, with the addition of busulphan in two cases. Engraftment was documented in all cases. Notably, six of seven patients relapsed after allogeneic HCT with one achieving a return to full donor chimaerism after cyclosporin A (CSA) withdrawal. Two patients are alive in remission, 23+ and 30+ months after transplant. The role of allogeneic HCT in patients with JMML is discussed. A cooperative multicentre trial is needed to establish the optimal therapy for these patients.

Engraftment of unrelated donor stem cells in children with familial amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia is an extremely rare disorder for which bone marrow transplantation offers the only possibility for cure. The pathophysiology is unclear. Two children with familial amegakaryocytic thrombocytopenia underwent unrelated donor transplantation (bone marrow in one, umbilical cord blood in the second) after a preparative regimen of cyclophosphamide and total body irradiation. Both patients failed initial engraftment, required further donor stem cell infusions and are currently well with sustained engraftment, 16 months and 7 months after transplantation, respectively. The difficulty in achieving engraftment of unrelated donor stem cells in these children suggests that in future cases additional measures to achieve engraftment may be necessary, for example, a more aggressive preparative regimen or an increased stem cell dose.