Ferritin concentrations correlate to outcome of hematopoietic stem cell transplantation but do not serve as biomarker of graft-versus-host disease.

Clinical presentation and laboratory data are often too unspecific to distinguish the onset or activity of graft-versus-host disease (GvHD) from infections or toxicity. Antigen-presenting cells such as monocytes/macrophages and dendritic cells are involved in GvHD pathogenesis after allogeneic hematopoietic stem cell transplantation (HSCT). To test whether ferritin, an iron storage marker and macrophage activation-linked acute-phase protein, represents a candidate biomarker for acute or chronic GvHD in pediatric HSCT, we retrospectively evaluated a 2-year follow-up data from 131 eligible consecutive patients with different malignant and nonmalignant diseases who underwent allogeneic HSCT. Thirteen patients (10 %) suffered from acute GvHD II-IV°, 18 (14 %) had limited, and 14 (11 %) had extensive chronic GvHD. In extension of previous studies in adults investigating pre-transplant ferritin, our data show that post-HSCT hyperferritinemia (analyzed on days 0, +30, +60, +100, +180, +360, and +720) was significantly associated with decreased long-term survival (p < 0.001-0.03) in children and adolescents. Increased ferritin concentrations were associated with number and timing of red blood cell transfusions and toxic or infectious multi-organ failure but did not show significant differences between patients without GvHD and with acute grades II-IV, limited, or extensive chronic GvHD. Thus, our data do not identify ferritin as specifically GvHD-linked biomarker; however, they support the prognostic value of ferritin levels for outcome after HSCT in children.

European guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ECIL-4 (2011).

Human adenovirus (HAdV) infections are increasingly recognized as important pathogens in immunocompromised hosts, especially in patients with severely suppressed T-cell function. The 4th European Conference of Infections in Leukemia (ECIL-4) has developed evidence-based guidelines for diagnosis and management of HAdV infections. The risk for HAdV-associated disease is increased in children, and risk factors for HAdV disease are T-cell depletion, unrelated and cord blood hematopoietic stem cell transplantation, graft-versus-host disease grades III-IV, and lymphopenia. The recommended technique for monitoring of high-risk patients is quantitative polymerase chain reaction. Cidofovir is the most used antiviral therapy, although no controlled study has been performed. HAdV-specific T-cell therapy is in development.

Importance of allogeneic T-cells for disease control after stem cell transplantation for high-risk Langerhans cell histiocytosis.

Reduced intensity conditioning followed by allogeneic SCT (RIC-SCT) has recently emerged as promising new salvage option for children suffering from Langerhans cell histiocytosis (LCH) with risk organ involvement and failure to conventional therapy. We report on the posttransplant course of female toddler with high-risk LCH, who achieved complete remission after RIC-SCT, despite a posttransplant chimerism constellation, in which only the T-cell subset proved to be of donor origin in the long-term. We therefore suggest that allogeneic T-cells have played a crucial role in controlling disease activity in this patient and may exert the major curative effect after RIC-SCT for LCH.

Donor leukocyte infusion after hematopoietic stem cell transplantation in patients with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a clonal myeloproliferative disorder of early childhood. In all, 21 patients with JMML who received donor leukocyte infusion (DLI) after allogeneic hematopoietic stem cell transplantation (HSCT) for either mixed chimerism (MC, n=7) or relapse (n=14) were studied. Six patients had been transplanted from an HLA-matched sibling and 15 from other donors. Six of the 21 patients (MC: 3/7 patients; relapse: 3/14 patients) responded to DLI. Response rate was significantly higher in patients receiving a higher total T-cell dose (> or =1 x 10(7)/kg) and in patients with an abnormal karyotype. None of the six patients receiving DLI from a matched sibling responded. Response was observed in five of six patients who did and in one of 15 children who did not develop acute graft-versus-host disease following DLI (P=0.01). The overall outcome was poor even for the responders. Only one of the responders is alive in remission, two relapsed, and three died of complications. In conclusion, this study shows that some cases of JMML may be sensitive to DLI, this providing evidence for a graft-versus-leukemia effect in JMML. Infusion of a high number of T cells, strategies to reduce toxicity, and cytoreduction prior to DLI may improve the results.

Multi-color immune-phenotyping of CD34 subsets reveals unexpected differences between various stem cell sources.

Flow cytometric routine CD34 analysis enumerates hematopoietic stem and progenitor cells irrespective of their subpopulations although this might predict engraftment dynamics and immune reconstitution. We established a multi-color CD34 assay containing CD133, CD45RA, CD10, CD38 and CD33. We examined PBSC, donor bone marrow (BMd) and BM of patients 1 year after allografting (BM1y) regarding their CD34 subset composition, which differed significantly amongst those materials: the early CD45RA(-)CD133(+)CD38(low) subpopulations were significantly more frequent in PBSC than in BMd, and very low in BM1y. Vice versa, clearly more committed CD34 stages prevailed in BM, particularly in BM1y where the proportion of multi-lymphoid and CD38(++) B-lymphoid precursors was highest (mean 59%). CD33 was expressed at different intensity on CD45RA(±)CD133(±) subsets allowing discrimination of earlier from more committed myeloid precursors. Compared with conventional CD34(+) cell enumeration, the presented multi-color phenotyping is a qualitative approach defining different CD34 subtypes in any CD34 source. Its potential impact to predict engraftment kinetics and immune reconstitution has to be evaluated in future studies.

Improved outcome of treatment-resistant high-risk Langerhans cell histiocytosis after allogeneic stem cell transplantation with reduced-intensity conditioning.

Children with multisystem Langerhans cell histiocytosis (LCH) and risk organ involvement who fail to respond to conventional chemotherapy have an extremely poor prognosis. Myeloablative stem cell transplantation (SCT) as a possible salvage approach for these patients has been associated with a high risk of transplant-related mortality. Therefore, allogeneic stem cell transplantation following a reduced-intensity conditioning regimen (RIC-SCT) has recently been performed as an alternative salvage approach. We report on the experience with allogeneic RIC-SCT in nine pediatric high-risk LCH patients. Conditioning regimen included fludarabine in all patients, melphalan in eight patients, total lymphoid irradiation in six patients, total body irradiation in two, antithymocyte globulin in five, and Campath in four patients. RIC-SCT was well tolerated with regard to common procedure-related complications. Two patients died 50 and 69 days after RIC-SCT, respectively. Seven out of the nine patients survived and showed no signs of disease activity (including one with nonengraftment and full autologous hematopoietic recovery) after median follow-up of 390 days post-SCT. Based on this observation, we conclude that RIC-SCT is a feasible procedure with low transplant-related morbidity and mortality and a promising new salvage approach for high-risk LCH patients with resistant risk organ involvement.

Transplantation of highly purified peripheral blood CD34+ cells from HLA-mismatched parental donors in 14 children: evaluation of early monitoring of engraftment.

HLA-mismatched family members may represent an important cell source for patients that require stem cell transplantation but lack both a matched sibling donor and a closely matched unrelated donor. We report the outcome of 19 transplantations from HLA two- or three- loci mismatched parental donors in which 14 pediatric patients with hematological malignancies or other disorders, received a median of 21.5 x 106 (range, 5.4-58) highly purified CD34+peripheral blood stem cells (PBSC), as well as 4.7 x 104 (range, 0.4-12) donor T cells per kg body weight. T cell depletion was performed using a two-step CD34-positive selection on two different magnetic beads devices. Ten of 14 patients presented with rapid myeloid engraftment. The four patients who presented with graft failure (two non-engraftments, two rejections) received a second stem cell graft and one a third. Graft rejection was detected early by polymerase chain reaction (PCR) analysis of FACS-sorted T cells. Eight of the 14 patients are still alive after a median observation period of 15. 6 months (range, 3-31.3) with full donor chimerism in all hematopoietic cell lineages. No acute organ graft-versus-host disease (GVHD) and no chronic GVHD have occurred. One patient experienced relapse of leukemia. We conclude that transplantation of allogeneic PBSC from haploidentical donors will open new perspectives for pediatric patients for whom an HLA-matched stem cell graft is not available. Close monitoring of recipient and donor hematopoiesis might be of clinical value, to recognize early engraftment or rejection.

Lineage-specific chimaerism after stem cell transplantation in children following reduced intensity conditioning: potential predictive value of NK cell chimaerism for late graft rejection.

Chimaerism of FACS-sorted leucocyte subsets (CD14+, CD15+, CD3-/56+, CD3+/4+, CD3+/8+, CD19+) was monitored prospectively between days +14 and +100 in 39 children undergoing allogeneic stem cell transplantation with reduced intensity-conditioning regimens. Cell subsets exceeding 1% of nucleated cells were subject to cell sorting. Chimaerism was analysed by dual-colour FISH and/or by short tandem repeat-polymerase chain reaction. The chimaerism pattern on day +28 was evaluated with regard to its correlation with graft rejection. Of 39 patients, nine patients had donor chimaerism (DC) in all subsets. Mixed/recipient chimaerism (MC/RC) was detectable within T cells in 62%, within NK cells in 39% and within monocytes and granulocytes in 38% of the patients. The correlation of secondary graft rejection with the chimaerism pattern on day +28 revealed the strongest association between RC in NK-cells (P<0.0001), followed by T cells (P=0.001), and granulocytes and monocytes (P=0.034). Notably, patients with RC in T cells rejected their graft only if MC or RC was also present in the NK-cell subset. By contrast, none of the children with DC in NK cells experienced a graft rejection. These observations suggest that, in the presence of recipient T-cell chimaerism, the chimaerism status in NK-cells on day +28 might be able to identify patients at high risk for late graft rejection.

Psychosocial adjustment of pediatric patients after allogeneic stem cell transplantation.

The purpose of this study was to assess the psychosocial adjustment of patients who had been treated with allogeneic stem cell transplantation (SCT) in our clinic. Selection criteria for patients were to be aged 14-30 years at the time of the follow-up, to be at least 2 years post-SCT and to have a very good knowledge of German. Among 31 eligible patients, 26 participated (84% response rate). The patients were between 15 and 27 years old and were on average 7 years (range 2-13) post-SCT. Research instruments consisted of a demographic questionnaire and various subscales of established psychological measures for which data from a sample of bone cancer survivors and population norms were available. About 35% of patients showed high levels of anxiety, 62% appeared to be extremely sensitive and vulnerable, and 35% showed strong, unfulfilled needs in their love lives. In the other domains tested (self-esteem, family and peer relationships, school/vocational performance, etc), no noticeable differences were found between the subjects and comparable populations. There was no significant association between psychosocial outcome and demographic features or clinical data. Our results suggest that patients who underwent SCT in their childhood or adolescence are at risk of developing long-term emotional or social problems. Due to the retrospective design of our study and the small sample size, no predictive factors for psychosocial distress could be identified.

Resolution of early cytomegalovirus (CMV) infection after leukocyte transfusion therapy from a CMV seropositive donor.

A 2 year and 8 month old CMV-negative boy suffering from stage III neuroblastoma underwent ABMT in first very good partial remission. He acquired early CMV infection on day +5, followed by consecutive graft failure and severe sepsis, and the clinical course deteriorated. Between days +16 and +21, he received seven leukocyte concentrates (LC) collected from a healthy, but CMV-IgG-seropositive relative stimulated with G-CSF (filgastrim, 5 microg/kg/day). A median of 5.7 x 10(10) neutrophils/m2/day (range, 1.2-8.3) were transfused, corresponding to a T cell number of roughly 4 x 10(8) CD3+ cells/kg/day. After infusion of the LCs, PCR analysis became negative for CMV and the patient received his rescue bone marrow. One year after ABMT, he is in complete remission and in good clinical condition. Our results suggest that the T cells infused together with the irradiated leukocytes played a major role in eradicating the CMV infection in this patient.

Allogeneic bone marrow transplantation-mediated transfer of specific immunity against Toxocara canis associated with excessive IgE.

A girl with myelodysplastic syndrome (RAEB-T) received HLA-identical bone marrow from her younger brother after myeloablative treatment with busulfan and cyclophosphamide. After bone marrow transplantation, fever, exanthema, pruritus, and a pulmonary infiltrate were treated symptomatically. Bacterial cultures remained negative. Leukocyte engraftment began on day 10, and all blood cell populations proved to be of donor origin on FISH analysis. Increasing IgE levels (21 000 U/ml) on day 14 after BMT, positive RAST, specific IgG-antibodies, and missing Toxocara (T.) canis antigens in the recipient indicated donor-derived seroconversion. Before BMT, the recipient had been negative for T. canis in routine parasitological screening, and the donor proved to be positive for T. canis antibody by ELISA. This report suggests that the transfer of IgE immunity in the absence of detectable antigens may be responsible for IgE-mediated symptoms consistent with toxocara infection and confirms the need for parasite screening in donor medical examinations.

Allogeneic bone marrow transplantation for juvenile myelomonocytic leukaemia: a single centre experience and review of the literature.

Juvenile myelomonocytic leukaemia (JMML) is a rare paediatric disease and allogeneic stem cell transplantation is the only curative approach. The roles of pretransplant treatment, conditioning regimen and graft-versus-host disease (GVHD) are still unclear. Eleven children with JMML underwent allogeneic BMT in our institution. Donors were matched unrelated (n = 6) matched siblings (n = 4) and one mismatch family donor. Transplant-related mortality (TRM) was 36%. Three patients relapsed after transplantation. Two of three patients with relapse are in continuous remission after donor lymphocyte infusion or second BMT, respectively. To evaluate the role of pretransplant treatment, conditioning regimen and GVHD, we have summarised our series with other published single centre reports and give an overview on a total of 65 patients with JMML who underwent allogeneic BMT. No significant correlation between pretransplant treatment, conditioning regimen and TRM could be observed. Overall relapse rate is high (47%). TBI is associated with a significantly higher relapse rate (P = 0.012). Other conditioning modalities, intensive chemotherapy and splenectomy prior to stem cell transplantation do not seem to have a significant impact on relapse rate. Patients with or without GVHD showed no significant difference in relapse rate (58% vs 45%). In the event of relapse after transplantation withdrawal of immunosuppression, donor lymphocyte infusion or second transplant was successful in 6/11 patients. Graft-versus-leukaemia effect seems to play an essential role in bone marrow transplantation for JMML.

Cidofovir for adenovirus infections after allogeneic hematopoietic stem cell transplantation: a survey by the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation.

Adenovirus is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation and there is no established therapy. Cidofovir has in vitro efficacy against adenovirus. We performed a retrospective analysis of 45 patients treated with cidofovir for adenovirus from 10 centers. In total, 16 patients had definite adenovirus disease, 13 probable disease, and 16 asymptomatic infections. A total of 31 (69%) patients were successfully treated with cidofovir, 10 failed, and four were not evaluable owing to early death from other causes. Cidofovir therapy was successful in 10 patients with adenovirus disease, 10 patients with probable disease, and in 10 patients with asymptomatic infections. The overall survival at 28 days and 6 months after initiation of cidofovir therapy was 76 and 46%, respectively. Of the patients, 18 developed toxicity associated with cidofovir: 14 developed renal toxicity and four other types of toxicities. We conclude that cidofovir may be useful against adenovirus after allogeneic hematopoietic stem cell transplantation but additional studies are needed.

Pre-emptive treatment of CMV DNAemia in paediatric stem cell transplantation: the impact of recipient and donor CMV serostatus on the incidence of CMV disease and CMV-related mortality.

Cytomegalovirus (CMV) DNAemia was detected by PCR in 30/125 (24%) consecutive paediatric patients undergoing allogeneic stem cell transplantation. All patients with CMV DNAemia received pre-emptive ganciclovir until two consecutive negative results were obtained. CMV-IgG-positive patients (R+) had a significantly increased risk of DNAemia as compared to CMV-IgG-negative (R-) patients (62% vs 8%) P<0.0001. The incidence of DNAemia was 71% (10/14) in R+ transplanted from seronegative donors (D-) compared to 54% (13/32) in those transplanted from seropositive donors (D+). Of 30 (40%) children with DNAemia, 12 developed CMV disease despite pre-emptive treatment. The overall incidence of disease was 0% (0/59) for R-/D-, 9% (3/23) for R+/D+, 7% (2/29) for R-/D+ and 57% (8/14) for R+/D-. In patients with DNAemia, 4/20 (20%) patients with D+ and 8/10 (80%) with D- became symptomatic. In the multivariate analysis of both groups, patients at risk (R+ and/or D+) and patients with DNAemia, a negative donor serostatus was the only factor associated with a significantly increased incidence of disease. Seven of 9 patients with lethal CMV disease had received CMV-IgG-negative grafts. The data suggest that in CMV seropositive recipients donor CMV seropositivity is associated with a reduced incidence of CMV disease and a favourable outcome following pre-emptive treatment.

Organ toxicity and quality of life after allogeneic bone marrow transplantation in pediatric patients: a single centre retrospective analysis.

One hundred and fifty five pediatric patients underwent allogeneic bone marrow transplantation between 1980 and 1996 in the St Anna Children's Hospital in Vienna with an overall survival of 52.3% (81 patients). Seventy-three patients with a minimum observation time of 1 year (1-13 years, median: 4.6) were analyzed retrospectively for chronic GVHD, organ toxicity (WHO score), growth and pubertal development. Chronic GVHD was diagnosed in 20 patients (27.3%), being extensive in 17 cases. Maximum organ toxicity was WHO III in two patients (3%) and WHO II in 11 patients (15%) 1 year after BMT and WHO III in one patient (2%) and WHO II in five patients (11%) 3 years after BMT. Impaired growth and pubertal development were detected in more than 30% 3 years after BMT. As all patients presented with a Karnofsky or Lansky score of more than 80%, they were asked to complete a questionnaire comprising 12 questions concerning physical state of health and psychosocial state of health. Restricted contacts were classified as imposing a severe handicap by six patients (8%), restriction in mobility and 'normal life activities' by three patients (4%) and two patients classified themselves as severely physically handicapped. Most patients (75%) reported no physical or psychical impairment.

CMV-viraemia during allogenic bone marrow transplantation in paediatric patients: association with survival and graft-versus-host disease.

Sixty-one consecutive paediatric patients undergoing allogenic bone marrow transplantation (BMT) were screened prospectively for cytomegalovirus (CMV)-viraemia by PCR. Sixteen patients (26%) presented with single or recurrent CMV-viraemia between day -7 and + 100. Although only four of them had evidence of CMV-disease, there was a significant difference in the incidence of acute Graft versus Host Disease (GVHD) grade III-IV (75% vs 15.5%), liver-involvement (68% vs 13%) and the incidence of chronic GVHD (83% vs 13.8%) between CMV-PCR-positive and CMV-PCR-negative patients. Transplant-related mortality (TRM) was 43.7% in the CMV-PCR-positive group versus 13% in patients which had no evidence for CMV-viraemia. In all but one cases mortality in CMV-PCR positive patients was GVHD-associated. Pre-emptive therapy with gancyclovir in case of CMV-viraemia seemed to have no impact on incidence and severity of GVHD.

Influence of transplantation regimen on prognostic significance of high-level minimal residual disease before allogeneic stem cell transplantation in children with ALL.

High MRD levels before transplantation in children receiving T cell-depleted unrelated grafts for relapsed ALL were associated with a 100% relapse risk. We report on two children with relapsed ALL who underwent non T cell-depleted BMT from unrelated donors. Despite a high residual tumor load pre- transplant and the occurrence of only aGVHD grade I, they are still in second complete remission 2.7 and 5.2 years after transplantation, respectively. Thus, we propose that the transplant regimens influence the prognostic significance of high MRD levels pre-BMT.

Early recipient chimerism testing in the T- and NK-cell lineages for risk assessment of graft rejection in pediatric patients undergoing allogeneic stem cell transplantation.

Timely diagnosis of impending graft rejection is crucial for effective therapeutic intervention after allogeneic hematopoietic stem cell transplantation (SCT). We have investigated the predictive potential of early leukocyte subset-specific chimerism for graft loss in children undergoing SCT. In total, 192 pediatric patients transplanted for the treatment of malignant and non-malignant diseases after reduced-intensity or myeloablative conditioning were investigated. Surveillance of lineage-specific chimerism was initiated upon first appearance of leukocyte counts amenable to cell sorting. Graft rejection occurred in 23 patients between 24 and 492 days post-transplant (median 63 days). The first chimerism analysis of T and NK cells performed at a median of 20 days after SCT identified three different risk groups that were independent from the conditioning regimen: recipient chimerism (RC) levels in T cells below 50% indicated a very low risk of rejection (1.4%), whereas high levels of RC (>90%) both in T and NK cells heralded graft loss in the majority of patients (90%) despite therapeutic interventions. RC >50% in T cells and ≤90% in NK cells defined an intermediate-risk group in which timely immunotherapy frequently prevented rejection. Early assessment of T- and NK-cell chimerism can therefore be instrumental in the risk assessment and therapeutic management of imminent graft rejection.