Pre-conditioning modifies the TME to enhance solid tumor CAR T cell efficacy and endogenous protective immunity.

Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its effectiveness in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate and pancreas cancer, including those targeting prostate stem cell antigen (PSCA), are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human PSCA knockin (hPSCA-KI) immunocompetent mouse model, we evaluated the safety and therapeutic efficacy of PSCA-CAR T cells. We demonstrated that cyclophosphamide (Cy) pre-conditioning significantly modified the immunosuppressive TME and was required to uncover the efficacy of PSCA-CAR T cells in metastatic prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous expression of PSCA. This combination dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, as well as endogenous and adoptively transferred T cells, resulting in long-term anti-tumor immunity.

Experience with a Reduced Toxicity Allogeneic Transplant Regimen for Non-CGD Primary Immune Deficiencies Requiring Myeloablation.

PURPOSE: A need exists for reduced toxicity conditioning regimens that offer less toxicity while maintaining myeloablation, especially for primary immune deficiencies where myeloablation or high donor myeloid chimerism is required to achieve cure. We adapted a busulfan and fludarabine regimen by Gungor et al. for children and young adults undergoing allogeneic HCT for non-CGD primary immune deficiencies requiring myeloablation or high donor myeloid chimerism, and herein report our experience. METHODS: We retrospectively reviewed records of 41 consecutive patients who underwent allogeneic HCT for Wiskott-Aldrich syndrome (n = 12), primary HLH/XLP (n = 10), CD40L deficiency (n = 7), or other (n = 12) primary immune deficiencies with a conditioning regimen containing pharmacokinetic-guided busulfan dosing which achieved a cumulative AUC between 57 and 74 mg/L × h (65-80% of conventional myeloablative exposure), along with fludarabine and alemtuzumab or anti-thymocyte globulin at 3 transplant centers between 2014 and 2019. RESULTS: Forty-one patients underwent a first (n = 33) or second (n = 8) allogeneic HCT. Median age was 2.3 years (range, 0.3 years-19.8 years). All but one patient (97.5%) achieved neutrophil recovery at a median of 14 days (range, 11-34 days). One patient developed sinusoidal obstruction syndrome and two patients developed diffuse alveolar hemorrhage. Four patients developed grades II-IV acute GVHD. Three patients developed chronic GVHD. One-year overall survival was 90% (95% confidence interval [CI] 81-99%) and event-free survival was 83% (95% CI 71-94%). CONCLUSIONS: Our experience suggests that a reduced toxicity busulfan-fludarabine regimen offers low toxicity, low incidence of grades 2-4 GVHD, durable myeloid engraftment, and excellent survival, and may be considered for a variety of primary immune deficiencies where myeloablative HCT is desired.

Fludarabine/Melphalan 100 mg/m2 Conditioning Therapy Followed by Allogeneic Hematopoietic Cell Transplantation for Adult Patients with Secondary Hemophagocytic Lymphohistiocytosis.

Our previous research indicated that a reduced-intensity conditioning regimen (fludarabine and melphalan at 100 mg/m2) was useful in allogeneic hematopoietic cell transplantation (HCT) for patients with lymphoma. This retrospective study evaluated the reduced-intensity conditioning regimen in allogeneic HCT for adult patients with hemophagocytic lymphohistiocytosis (HLH). Sixteen patients with HLH were evaluated, including 6 patients who were enrolled in a prospective clinical trial (NCT00772811) and 10 patients who received the same conditioning regimen (fludarabine at 30 mg/m2/day on days -6 to -2 and melphalan at 100 mg/m2 on day -2). The median age was 42 years (range, 18 to 64), and 12 patients had Epstein-Barr virus (EBV)-associated HLH. Donors were an HLA matched sibling for 10 patients, an unrelated matched volunteer for 4 patients, and a mismatched family member for 2 patients. After excluding 3 patients who died soon after HCT, 12 patients achieved an engraftment (neutrophil median, day 12; platelet median, day 16). Five patients experienced acute graft-versus-host disease (GVHD), including 1 case of grade II and 4 cases of grades III to IV. Chronic GVHD occurred in 3 patients (moderate, 1 case; severe, 2 cases). After a median follow-up of 33.8 months 1 patient progressed, 3 patients relapsed, and 9 patients died. Five deaths were unrelated to relapse or progression and were caused by infection (n = 3), bleeding (n = 1), and GVHD (n = 1). No deaths or relapses were observed at >124 days post-transplant. The overall survival rate was 48.6%, and significant differences were observed according to pretransplant ferritin level (P = .007) and cytopenia lineage (P = .021). Before allogeneic HCT 10 of 12 patients still tested positive for EBV DNA: 6 patients tested negative for EBV DNA after HCT, 2 patients had persistent EBV DNA, and 2 patients were unassessable because of early death. Conditioning therapy using a lower dose of melphalan combined with fludarabine appears to be promising in allogeneic HCT for adults with HLH. However, strategies are needed to reduce the risk of early death.

Reduced-Toxicity Myeloablative Conditioning Consisting of Fludarabine/Busulfan/Low-Dose Total Body Irradiation/Granulocyte Colony-Stimulating Factor-Combined Cytarabine in Single Cord Blood Transplantation for Elderly Patients with Nonremission Myeloid Malignancies.

The optimal intensity of a conditioning regimen might be dependent on not only age and comorbidities but also disease activity and the type of graft source. We evaluated the outcome of unrelated single cord blood transplantation (CBT) using a conditioning regimen of fludarabine 180 mg/m2, i.v. busulfan 9.6 mg/kg, 4 Gy total body irradiation, granulocyte colony-stimulating factor-combined high-dose cytarabine (12 g/m2) in 23 elderly patients (median, 64 years) with nonremission myeloid malignancies between 2013 and 2018 in our institution. All but 1 patient achieved neutrophil engraftment at a median of 23.5 days (range, 18 to 50). With a median follow-up of 28 months, the probabilities of overall survival (OS), disease-free survival (DFS), and cumulative incidence of relapse at 2 years were 62%, 52%, and 26%, respectively. The cumulative incidences of nonrelapse mortality at 100 days and 2 years were 9% and 22%, respectively. In the univariable analysis a higher proportion of blasts in bone marrow and in peripheral blood and a monosomal or complex karyotype were significantly associated with inferior OS and DFS. Poor cytogenetics were significantly associated with inferior DFS and increased relapse incidence. These data demonstrate that this reduced-toxicity myeloablative conditioning regimen was tolerable and effective in terms of engraftment, relapse, and survival in single CBT for elderly patients with nonremission myeloid malignancies.

Effects of Prophylactic Foscarnet on Human Herpesvirus-6 Reactivation and Encephalitis in Cord Blood Transplant Recipients: A Prospective Multicenter Trial with an Historical Control Group.

Cord blood transplantation (CBT) is a distinct risk factor for human herpesvirus-6 (HHV-6) reactivation and HHV-6 encephalitis. In a prospective multicenter trial we investigated the effects of prophylactic foscarnet (90 mg/kg i.v. infusion from days 7 to 27 after CBT) on the occurrence of HHV-6 reactivation, HHV-6 encephalitis, and acute graft-versus-host disease (aGVHD) in CBT recipients. Between 2014 and 2016, 57 patients were included in a foscarnet-prophylaxis group. Outcomes were compared with an historical control group who received CBT between 2010 and 2014 (standard-treatment group, n = 63). The cumulative incidence of high-level HHV-6 reactivation, defined as plasma HHV-6 DNA ≥ 104 copies/mL, at 60 days after CBT was significantly lower in the foscarnet-prophylaxis group than in the standard-treatment group (18.3% versus 57.3%, P < .001). Multivariate analysis revealed that myeloablative preconditioning and standard treatment were significant risk factors for high-level HHV-6 reactivation. The cumulative incidence of HHV-6 encephalitis at 60 days after CBT was not different between the groups (foscarnet-prophylaxis group, 12.4%; standard-treatment group, 4.9%; P = .14). The cumulative incidences of grades II to IV and grades III to IV aGVHD at 60 days after CBT were not different between the groups (grades II to IV aGVHD: foscarnet-prophylaxis group, 42.0%; standard-treatment group, 40.5%; P = .96; grades III to IV aGVHD: foscarnet-prophylaxis group, 14.5%; standard-treatment group, 14.5%; P = 1.00). In the setting of this study foscarnet significantly suppressed systemic HHV-6 reactivation in CBT recipients but failed to prevent the development of HHV-6 encephalitis. Suppression of HHV-6 reactivation by foscarnet did not show any effects against the incidence of aGVHD.

Adiponectin Enhances Quiescence Exit of Murine Hematopoietic Stem Cells and Hematopoietic Recovery Through mTORC1 Potentiation.

Myelotoxic injury, such as chemotherapeutic agents and ionizing radiation, unlocks the vigorous power of hematopoietic stem cells (HSCs) to replenish the hematopoietic system, making quiescent HSCs enter the cell cycle. Considering that both HSC-intrinsic and -extrinsic mechanisms enforce quiescence of HSCs, the drastic change in bone marrow (BM) environment after injury, represented by massive expansion of BM adipocytes, might trigger HSC activation. BM adipocytes, the major cellular component in the ablated marrow, however, reportedly suppress proliferation of hematopoietic cells, which may indicate the BM adipocytogenesis is an irrational response of injured organism. Given that adipose tissue is an endocrine organ with pleiotropic functions, we hypothesized that adipocyte-derived factors, especially adiponectin, an anti-inflammatory adipokine involved in regulation of granulopoiesis, are implicated in HSC activation. Myeloablative intervention increased BM adiponectin by multiple mechanisms, including adipocyte expansion and increased diffusion from the blood. Adiponectin-null (Adipoq -/- ) mice showed delayed hematopoietic recovery after BM injury, with Adipoq-/- HSCs more quiescent and defective in mammalian target of rapamycin complex 1 (mTORC1) activation. Recombinant adiponectin promoted not only HSC activation in vivo but cytokine-induced activation in vitro, and shortened the time for exit from quiescence in an mTORC1-dependent manner. These data illustrate a scarcely-reported example of a cell-extrinsic factor, adiponectin, enhancing quiescence exit of HSCs, and subsequent hematopoietic recovery. Our findings also highlight adipocytes as a source of adiponectin to ensure the proliferative burst of hematopoietic cells in ablated marrow. Stem Cells 2017;35:1835-1848.

B-cell reconstitution for SCID: should a conditioning regimen be used in SCID treatment?

Bone marrow transplantation has resulted in life-saving sustained T-cell reconstitution in many infants with severe combined immunodeficiency (SCID), but correction of B-cell function has been more problematic. At the annual meeting of the Primary Immunodeficiency Treatment Consortium held in Boston, Massachusetts, on April 27, 2012, a debate was held regarding the use of pretransplantation conditioning versus no pretransplantation conditioning in an effort to address this problem. Reviews of the literature were made by both debaters, and there was agreement that there was a higher rate of B-cell chimerism and a lower number of patients who required ongoing immunoglobulin replacement therapy in centers that used pretransplantation conditioning. However, there were still patients who required immunoglobulin replacement in those centers, and therefore pretransplantation conditioning did not guarantee development of B-cell function. Dr Rebecca H. Buckley presented data on B-cell function according to the molecular defect of the patient, and showed that patients with IL-7 receptor α, ADA, and CD3 chain gene mutations can have normal B-cell function after transplantation with only host B cells. Dr Elie Haddad presented a statistical analysis of B-cell function in published reports and showed that only a conditioning regimen that contained busulfan was significantly associated with better B-cell function after transplantation. The question is whether the risk of immediate and long-term toxicity with use of busulfan is justified, particularly in patients with SCID with DNA repair defects and in very young newborns with SCID who will be detected by using newborn screening.

Role of NF-κB-dependent signaling and p38 MAPK signaling pathway in the control of hemopoiesis during cytostatic administration.

The study examines the role of signal pathways in triggering the compensatory reactions in the blood system in response to the cytostatic administration. In vitro experiments elucidated the involvement of protein kinase p38 in the synthesis of granulocytic CSF by cells of hemopoietic microenvironment. The important role of the transcriptional factor NF-κB and protein kinase p38 in limitation of the processes of maturation of the hemopoietic precursors was demonstrated.

Prediction of area under the cyclosporine concentration versus time curve in children undergoing hematopoietic stem cell transplantation.

This prospective study aimed to validate a previously developed first-dose limited sampling strategy (LSS) to predict the area under the cyclosporine concentration-versus-time curve (AUC) and to develop and then validate an LSS to predict cyclosporine AUC at steady state. This two-center Canadian study included children (ages .4 to 17.2 years) undergoing myeloablative allogeneic hematopoietic stem cell transplantation receiving cyclosporine for acute graft-versus-host disease prophylaxis. There were three cohorts, each incorporating 24 AUC determinations: first-dose LSS validation, steady-state LSS development, and steady-state LSS validation. Patients contributing data to either of the development cohorts were excluded from the corresponding validation group. Cyclosporine was given every 12 hours as a 2-hour infusion. Cyclosporine AUC was determined after administration of the first cyclosporine dose (8 samples) and then once weekly (9 samples) until engraftment. Steady-state LSSs were developed using stepwise multiple linear regression. An LSS was considered to provide an acceptable estimate of AUC if the lower limit of the 95% confidence limit (CL) of the intraclass coefficient was .8 or higher and both bias and precision were 15% or less. Fifty-three children age .4 to 18 years participated. Cyclosporine concentrations drawn up to 4 hours from the start of the infusion correlated most strongly with AUC. The previously developed first-dose LSSs and three steady-state LSSs met criteria for acceptability. The intraclass coefficients of the three-point first-dose LSS validation cohort, three-point steady-state LSS development cohort, and three-point steady-state LSS validation cohort were .974 (95% CL: .941 to .988), .984 (95% CL: .965 to .993), and .993 (95% CL: .984 to .997), respectively. The three-point first-dose (2, 6, and 8 hours) and steady-state (2, 2.5, and 8 hours) LSSs are valid measures of cyclosporine AUC after intravenous administration over 2 hours. Their use in a prospective evaluation of the relationship between cyclosporine AUC and hematopoietic stem cell transplantation clinical outcomes in children is suggested.

Adenovirus viremia and disease: comparison of T cell-depleted and conventional hematopoietic stem cell transplantation recipients from a single institution.

Adenovirus (ADV) is an important cause of viral mortality in hematopoietic stem cell transplantation (HSCT). Recipients of T cell-depleted (TCD) HSCT are at increased risk for viral infections. We compared the rates and outcomes of ADV viremia and disease between TCD and conventional (CONV) HSCT at our institution. This was an observational study of 624 adult and pediatric recipients of myeloablative HSCT at Memorial Sloan-Kettering Cancer Center between January 1, 2006, and March 11, 2011. Viral cultures and ADV PCR were ordered as clinically indicated. ADV viremia by quantitative PCR assay was defined as 1 or more positive values ≥1,000 copies/mL or 2 or more consecutive positive values. Competing-risk regression analyses were used to identify predictors for ADV viremia. ADV viremia at 1 year after HSCT occurred in 8% of TCD HSCT recipients and in 4.0% of CONV HSCT recipients (P = .041). Among the TCD recipients, ADV viremia was seen in 15% of children, compared with 5% of adults (P = .008). Young age (hazard ratio [HR], 3.0; P < .001) and acute graft-versus-host disease (GVHD) (HR, 3.2; P = .001) were identified as risk factors for ADV viremia. ADV viremia was predictive of mortality (HR, 6.0; P < .001). ADV disease developed in 3.5% of TCD HSCT recipients and in 0.4% of CONV HSCT recipients (P = .022), with an attributable mortality of 27%. Among TCD HSCY recipients, grade II to IV GVHD was a risk factor for ADV disease (HR, 13; P < .001), but age was not. More than 90% of the cases of ADV disease involved a viral load of ≥10,000 copies/mL. Rates of ADV disease were 10-fold greater in TCD HSCT recipients compared with CONV HSCT recipients, predominantly in patients who developed acute GVHD. The benefit of preemptive therapy for an ADV viral load ≥10,000 copies/mL for preventing ADV disease in TCD HSCT recipients should be evaluated in prospective clinical trials.

Dose intensification of busulfan in the preparative regimen is associated with improved survival: a phase I/II controlled, randomized study.

Dose intensity is important for disease control in patients undergoing allogeneic stem cell transplantation. We conducted a phase I/II controlled, adoptive, randomized study to determine the optimal dosing schedule of i.v. busulfan. Patients aged ≤75 years with advanced hematologic malignancies with human leukocyte antigen-compatible donor were eligible. All patients received fludarabine at 30 mg/m(2)/d for 4 days, and busulfan was administered in different doses in oral or i.v. formulations. As determined by the phase I trial, i.v. busulfan at a dose of 11.2 mg/kg/d was used for the phase II expansion cohort. Altogether, 80 patients with a median age of 56 years were enrolled. Forty percent had active disease at the time of transplantation. Engraftment occurred in 91%, and a complete response was achieved in 79% of patients posttransplantation. At a median follow-up of 91 months in the surviving patients, the outcomes for i.v. busulfan dose of 11.2 mg/kg/d versus other doses were as follows: nonrelapse mortality, 34% versus 23% (P = .4); cumulative incidence of relapse, 43% versus 68% (P = .02); relapse-free survival, 25% versus 9% (P = .017); and overall survival, 27% versus 9% (P = .02). We conclude that optimizing i.v. busulfan dose intensity in the preparative regimen may overcome disease-associated poor prognostic factors.

Feasibility of frequent patient-reported outcome surveillance in patients undergoing hematopoietic cell transplantation.

Patient-reported outcomes (PROs), including symptoms and health-related quality of life (HRQOL), provide a patient-centered description of hematopoietic cell transplantation (HCT)-related toxicity. These data characterize the patient experience after HCT and may have prognostic usefulness for long-term outcomes after HCT. We conducted a study of 32 patients after HCT (10 autologous HCT recipients, 11 full-intensity conditioning allogeneic HCT recipients, and 11 reduced-intensity conditioning allogeneic HCT recipients) to determine the feasibility of weekly electronic PRO collection from HCT until day (D) +100. We used questions from the PRO version of the Common Terminology Criteria for Adverse Events to capture symptoms, and the Patient-Reported Outcomes Measurement Information System Global Health scale to measure physical and mental HRQOL. The vast majority (94%) of patients used the electronic PRO system, with only 6% opting for paper-and-pencil only. The median weekly percentage of participants who completed the surveys was 100% in all cohorts through hospital discharge, and remained 100% for the autologous HCT and reduced-intensity allogeneic HCT cohorts through D+100. Patients were satisfied with the electronic system, giving high marks for readability, comfort, and questionnaire length. Symptom severity varied by absolute level and type of symptom across the 3 cohorts, with the full-intensity allogeneic HCT cohort exhibiting the greatest median overall symptom severity, peaking at D+7. Median physical health HRQOL scores decreased with time in the 3 cohorts, and HRQOL was generally correlated with overall symptom severity. Our results demonstrate the feasibility of frequent electronic PROs in the early post-HCT period. Future studies in larger populations to explore predictive models using frequent PRO data for outcomes, including long-term HRQOL and survival, are warranted.

Fractal organization of the human T cell repertoire in health and after stem cell transplantation.

T cell repertoire diversity is generated in part by recombination of variable (V), diversity (D), and joining (J) segments in the T cell receptor ß (TCR) locus. T cell clonal frequency distribution determined by high-throughput sequencing of TCR ß in 10 stem cell transplantation (SCT) donors revealed a fractal, self-similar frequency distribution of unique TCR bearing clones with respect to V, D, and J segment usage in the T cell repertoire of these individuals. Further, ranking of T cell clones by frequency of gene segment usage in the observed sequences revealed an ordered distribution of dominant clones conforming to a power law, with a fractal dimension of 1.6 and 1.8 in TCR ß DJ and VDJ containing clones in healthy stem cell donors. This self-similar distribution was perturbed in the recipients after SCT, with patients demonstrating a lower level of complexity in their TCR repertoire at day 100 followed by a modest improvement by 1 year post-SCT. A large shift was observed in the frequency distribution of the dominant T cell clones compared to the donor, with fewer than one third of the VDJ-containing clones shared in the top 4 ranks. In conclusion, the normal T cell repertoire is highly ordered with a TCR gene segment usage that results in a fractal self-similar motif of pattern repetition across levels of organization. Fractal analysis of high-throughput TCR ß sequencing data provides a comprehensive measure of immune reconstitution after SCT.

Fifty years of melphalan use in hematopoietic stem cell transplantation.

Melphalan remains the most widely used agent in preparative regimens for hematopoietic stem cell transplantation (SCT). From its initial discovery more than 50 years ago, it has been gradually incorporated in the conditioning regimens for both autologous and allogeneic transplantations because of its myeloablative properties and broad antitumor effects as a DNA alkylating agent. Melphalan remains the mainstay conditioning for multiple myeloma and lymphomas, and it has been used successfully in preparative regimens of a variety of other hematological and nonhematological malignancies. The addition of newer agents to conditioning, such as bortezomib or lenalidomide for myeloma or clofarabine for myeloid malignancies, may improve antitumor effects for transplantation, whereas melphalan in combination with alemtuzumab may represent a backbone for future cellular therapy because of reliable engraftment and low toxicity profile. This review summarizes the development and the current use of this remarkable drug in hematopoietic SCT.

Impact of high-risk cytogenetics and achievement of molecular remission on long-term freedom from disease after autologous-allogeneic tandem transplantation in patients with multiple myeloma.

Within a prospective protocol, the incidence and impact of achievement of molecular remission (mCR) and high-risk cytogenetics was investigated in 73 patients with multiple myeloma (MM) after autologous (auto)-allogeneic (allo) tandem stem cell transplantation (SCT). After induction chemotherapy, patients received melphalan 200 mg/m(2) before undergoing auto-SCT, followed 3 months later by melphalan 140 mg/m(2) and fludarabine 180 mg/m(2) before allo-SCT. Sixteen patients had high-risk cytogenetic features, defined by positive FISH for del(17p13) and/or t(4;14). Overall, 66% of the patients achieved CR or near-CR, and 41% achieved mCR, which was sustained negative (at least 4 consecutive samples negative) in 15 patients (21%), with no significant difference in incidence between the patients with high-risk cytogenetics and others (P = .70). After a median follow-up of 6 years, overall 5-year progression-free survival was 29%, with no significant difference between del 17p13/t(4;14)-harboring patients and others (24% versus 30%; P = .70). The 5-year progression-free survival differed substantially according to the achieved remission: 17% for partial remission, 41% for CR, 57% for mCR, and 85% for sustained mCR. These results suggest that auto-allo tandem SCT may overcome the negative prognostic effect of del(17p13) and/or t(4;14) and that achievement of molecular remission resulted in long-term freedom from disease.

Prognostic impact of posttransplantation iron overload after allogeneic stem cell transplantation.

In patients referred for allogeneic hematopoietic stem cell transplantation (HSCT), iron overload is frequent and associated with increased morbidity and mortality. Both the evolution of iron overload after transplantation and its correlation with late posttransplantation events are unknown. We studied 290 patients undergoing myeloablative allogeneic HSCT between 2000 and 2009. Serum ferritin, transferrin saturation, transferrin, iron, and soluble transferrin receptor were determined regularly between 1 and 60 months after HSCT, and values were correlated with transplantation outcome. Ferritin levels peaked in the first 3 months posttransplantation and then decreased to normal values at 5 years. Transferrin saturation and iron behaved analogously, whereas transferrin and soluble transferrin receptor increased after an early nadir. Landmark survival analysis showed that hyperferritinemia had a detrimental effect on survival in all periods analyzed (0 to 6 months P < .001; 6 to 12 months P < .001; 1 to 2 years P = .02; 2 to 5 years P = .002). This effect was independent of red blood cell transfusion dependency and graft-versus-host disease. Similar trends were seen for other iron parameters. These data show the natural dynamics of iron parameters in the setting of allogeneic HSCT and provide evidence for a prognostic role of iron overload extending beyond the immediate posttransplantation period. Interventions to reduce excessive body iron might therefore be beneficial both before and after HSCT.

Reconstitution of interleukin-17-producing T helper cells after allogeneic hematopoietic cell transplantation.

Interleukin 17A (IL-17)-producing CD4(+) T helper type 17 (Th17) cells have recently drawn attention as possible effector cells of acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) in murine models. Their role after allogeneic HCT in humans is unknown. In this prospective study, Th17, Th1/17, and Th1 cells were quantified in the peripheral blood of 80 patients within the first 3 months after allogeneic HCT using intracellular cytokine staining and flow cytometry. Within the observation period, Th1, Th1/17, and Th17 cells did not reconstitute to levels of healthy control subjects. In contrast to Th1 cells, no further expansion of Th1/17 and Th17 cells was observed during the first month after HCT. Antithymocyte globulin during conditioning significantly reduced the frequency of Th1/17 and Th17 cells but not of Th1 cells. Acute GVHD was not associated with significant changes in the size of the Th1, Th1/17, or Th17 cell subsets. Cytomegalovirus reactivation triggered the expansion of all T helper subsets, and Th1 cells showed the strongest increase. In contrast, no significant changes were found in the T helper cell compartment of patients with bacterial infections compared with time-matched control subjects. In conclusion, quantitative reconstitution of Th1, Th1/17, and Th17 cells is impaired within the first 3 months after HCT, especially when antithymocyte globulin is administered during conditioning. Cytomegalovirus reactivation, but not acute GVHD or bacterial infection, triggered the absolute expansion of these T cell subsets.

Outcome of poor response paediatric AML using early SCT.

BACKGROUND: Children with poor response acute myeloid leukaemia (AML) generally have a very poor outcome. Allogeneic stem cell transplantation (SCT) is often recommended for these children but the benefit is unclear. The aim of this study was to investigate survival for poor response AML patients treated with SCT. MATERIAL AND METHODS: Treatment was given according to the NOPHO-AML 2004 protocol. All patients received AIET (Cytarabine, Idarubicin, Etoposide, Thioguanine) and AM (Cytarabine, Mitoxantrone) as induction. We included poor response defined as > 15% blasts on day 15 after AIET (n = 17) or > 5% blasts after AM (n = 14, refractory disease). Poor response patients received intensively timed induction and proceeded to SCT when a donor was available. RESULTS: Thirty-one of 267 evaluable patients (12%) had a poor response. SCT was performed in 25; using matched unrelated donors in 13, matched sibling donors in 6, cord blood donor in 4, and haploidentical donor in two. The median follow-up for the 31 poor responding patients was 2.6 years (range 0.4 - 8.1 years) and 3-year probability of survival 70% (95% CI 59-77%). CONCLUSIONS: The poor responders in the NOPHO-AML 2004 protocol had a favourable prognosis treated with time-intensive induction followed by SCT.

Prognostic impact of immune status and hematopoietic recovery before and after fludarabine, IV busulfan, and antithymocyte globulins (FB2 regimen) reduced-intensity conditioning regimen (RIC) allogeneic stem cell transplantation (allo-SCT).

This retrospective analysis aimed to assess hematopoietic and immune recovery in a cohort of 53 patients [males: n = 33; median age: 59 yr (range: 22-70)] who received a FB2 (fludarabine 120-150 mg/m² + IV busulfan 6.4 mg/kg + antithymocyte globulin thymoglobulin 5 mg/kg) reduced-intensity conditioning (RIC) allo-stem cells transplantations (SCT). With a median follow-up of 19 months (range: 2-53), the 2-yr overall survival, disease-free survival (DFS), relapse incidence, and non-relapse mortality were 63%, 59.5%, 35%, and 6%, respectively. In univariate analysis, the factors correlated with a significantly higher 2-yr OS and DFS were a higher total circulating lymphocytes count at transplant (>730/mm(3) ; OS: 81% vs. 43%, P = 0.02; DFS: 73% vs. 45.5%, P = 0.03) and a higher recovery of leukocytes (>5300/mm(3) ) (2-yr OS: 81% vs. 44%, P = 0.007; 2-yr DFS: 72% vs. 46%, P = 0.08), neutrophils (>3200/mm(3) ) (2-yr OS: 76% vs. 50%, P = 0.03; 2-yr DFS: 67% vs. 52.0%, P = 0.1), and monocytes (>590/mm(3) ; 2-yr OS: 80% vs. 45%, P = 0.004; 2-yr DFS: 76% vs. 42%, P = 0.01) at day +30 post-transplant. In multivariate analysis, the only independent factors associated with a significantly higher OS and DFS were a better immune status at transplant (lymphocytes count >730/mm(3) ) and a higher monocytes count (>590/mm(3) ) at day +30 post-transplant. These results suggest that immune status and hematopoietic recovery before and after FB2 RIC allo-SCT can be significant predictors of outcome. This paves the way for future studies aiming to closely monitor the kinetics of immune recovery after RIC allo-SCT and to evaluate the impact of growth factors and other immunostimulatory cytokines in the setting of RIC allo-SCT.

Perivascular, but not parenchymal, cerebral engraftment of donor cells after non-myeloablative bone marrow transplantation.

Myeloablative (MyA) bone marrow transplantation (BMT) results in robust engraftment of BMT-derived cells in the central nervous system (CNS) and is neuroprotective in diverse experimental models of neurodegenerative diseases of the brain and retina. However, MyA irradiation is associated with significant morbidity and mortality and does not represent a viable therapeutic option for the elderly. Non-myeloablative (NMyA) BMT is less toxic, but it is not known if the therapeutic efficacy observed with MyA BMT is preserved. As a first step to address this important gap in knowledge, we evaluated and compared engraftment characteristics of BMT-derived monocytes/microglia using several clinically relevant NMyA pretransplant conditioning regimens in C57BL/6 mice. These included chemotherapy (fludarabine and cyclophosphamide) with or without 2 Gy irradiation, and 5.5 Gy irradiation alone. Each regimen was followed by transplantation of whole bone marrow from green fluorescent protein-expressing wild type (wt) mice. While stable hematopoietic engraftment occurred, to varying degrees, in all NMyA regimens, only 5.5 Gy irradiation resulted in significant engraftment of BMT-derived cells in the brain, where these cells were exclusively localized to perivascular, leptomeningeal, and related anatomic regions. Engraftment in retina under 5.5 Gy NMyA conditions was significantly reduced compared to MyA, but robust engraftment was identified in the optic nerve. Advancing the therapeutic applications of BMT to neurodegenerative diseases will require identification of the barrier mechanisms that MyA, but not NMyA, BMT is able to overcome.