Recommendations on hematopoietic stem cell transplantation for inherited bone marrow failure syndromes.

Allogeneic hematopoietic stem cell transplantation (HSCT) offers the potential to cure patients with an inherited bone marrow failure syndrome (IBMFS). However, the procedure involves the risk of treatment-related mortality and may be associated with significant early and late morbidity. For these reasons, the benefits should be carefully weighed against the risks. IBMFS are rare, whereas case reports and small series in the literature illustrate highly heterogeneous practices in terms of indications for HSCT, timing, stem cell source and conditioning regimens. A consensus meeting was therefore held in Vienna in September 2012 on behalf of the European Group for Blood and Marrow Transplantation to discuss HSCT in the setting of IBMFS. This report summarizes the recommendations from this expert panel, including indications for HSCT, timing, stem cell source and conditioning regimen.

Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia.

To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59-100%) and a specificity of 87% (CI 70-96%). Positive and negative predictive values were 71% (CI 42-92%) and 96% (CI 82-100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria.

Mesenchymal stem cells from Shwachman-Diamond syndrome patients display normal functions and do not contribute to hematological defects.

Shwachman-Diamond syndrome (SDS) is a rare inherited disorder characterized by bone marrow (BM) dysfunction and exocrine pancreatic insufficiency. SDS patients have an increased risk for myelodisplastic syndrome and acute myeloid leukemia. Mesenchymal stem cells (MSCs) are the key component of the hematopoietic microenvironment and are relevant in inducing genetic mutations leading to leukemia. However, their role in SDS is still unexplored. We demonstrated that morphology, growth kinetics and expression of surface markers of MSCs from SDS patients (SDS-MSCs) were similar to normal MSCs. Moreover, SDS-MSCs were able to differentiate into mesengenic lineages and to inhibit the proliferation of mitogen-activated lymphocytes. We demonstrated in an in vitro coculture system that SDS-MSCs, significantly inhibited neutrophil apoptosis probably through interleukin-6 production. In a long-term coculture with CD34(+)-sorted cells, SDS-MSCs were able to sustain CD34(+) cells survival and to preserve their stemness. Finally, SDS-MSCs had normal karyotype and did not show any chromosomal abnormality observed in the hematological components of the BM of SDS patients. Despite their pivotal role in the hematopoietic stem cell niche, our data suggest that MSC themselves do not seem to be responsible for the hematological defects typical of SDS patients.

The isochromosome i(7)(q10) carrying c.258+2t>c mutation of the SBDS gene does not promote development of myeloid malignancies in patients with Shwachman syndrome.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder, characterized by exocrine pancreatic insufficiency, skeletal abnormalities and bone marrow (BM) dysfunction with an increased risk to develop myelodysplastic syndrome and/or acute myeloid leukaemia (MDS/AML). SDS is caused, in nearly 90% of cases, by two common mutations (that is, c.183_184TA>CT and c.258+2T>C) in exon 2 of the SBDS gene, localized on chromosome 7. Clonal chromosome anomalies are often found in the BM of SDS patients; the most frequent is an isochromosome for long arms of chromosome 7, i(7)(q10). We studied eight patients with SDS carrying the i(7)(q10) who were compound heterozygotes for SBDS mutations. By assessing the parental origin of the i(7)(q10) using microsatellite analysis, we inferred from the results which mutation was present in double dose in the isochromosome. We demonstrate that in all cases the i(7)(q10) carries a double dose of the c.258+2T>C, and we suggest that, as the c.258+2T>C mutation still allows the production of some amount of normal protein, this may contribute to the low incidence of MDS/AML in this subset of SDS patients.

The polymorphisms -318C>T in the promoter and 49A>G in exon 1 of CTLA4 and the risk of aplastic anemia in a Caucasian population.

Aplastic anemia (AA) is a rare disease with a major autoimmune pathogenetic component. CTLA4 is a T-lymphocyte surface molecule involved in the maintenance of immune tolerance. Some polymorphisms associated with a reduced expression of CTLA4, and thus presumably with increased tendency to autoimmunity, have been associated with various autoimmune diseases. In this study, we evaluated the distribution of the low expression polymorphisms -318C > T and 49A > G of CTLA4 in a population of 67 patients with acquired AA and in 100 normal controls. There was no difference in the distribution of the tested polymorphism between patients and controls and, within the patient group, between those who responded to immunosuppression vs those who did not respond. This study indicates that the polymorphisms -318C > T and 49A > G of CTLA4 do not affect the risk of developing AA and do not influence the response to immunosuppression.

Allogeneic bone marrow stem cell transplantation following CD34+ immunomagnetic enrichment in patients with inherited metabolic storage diseases.

T-cell depletion is an essential step in reducing the risk of graft-versus-host disease (GVHD) in patients with inherited metabolic storage diseases (IMSD) undergoing hematopoietic stem cell transplantation. This goal can be achieved either by selective removal of T cells or by positive selection of CD34+ cells. Large-scale preparations of purified CD34+ cells from bone marrow products have not been extensively described. We report our results with bone marrow CD34+ cell enrichment using the CliniMACS system in eight children with IMSD. The median recovery of positively selected CD34+ cells was 46.2% with a purity of 97.5%, and a residual T cell content of 0.04 x 10(6). A median of 5.5 x 10(6)/kg of CD34+ cells was infused. All patients engrafted at a median time of 12 days and none of the patients developed GVHD. This method is technically feasible and can be successfully used to transplant children with IMSD.

Leukaemic transformation of donor cells in a patient receiving a second allogeneic bone marrow transplant for severe aplastic anaemia.

Allogeneic blood or bone marrow transplantation is a successful treatment for leukaemia and severe aplastic anaemia (SAA). Graft rejection following transplantation for leukaemia is a rare event but leukaemic relapse may occur at varying rates, depending upon the stage of leukaemia at which the transplant was undertaken and the type of leukaemia. Relapse is generally assumed to occur in residual host cells, which are refractory to, or escape from the myeloablative conditioning therapy. Rare cases have been described, however, in which the leukaemia recurs in cells of donor origin. Lack of a successful outcome of blood or bone marrow transplantation for severe aplastic anaemia (SAA), however, is due to late graft rejection or graft-versus-host disease. Leukaemia in cells of donor origin has rarely been reported in patients following allogeneic bone marrow transplantation for SAA. This report describes leukaemic transformation in donor cells following a second allogeneic BMT for severe aplastic anaemia. PCR of short tandem repeats in bone marrow aspirates and in colonies derived from BFUE and CFU-GM indicated the donor origin of leukaemia. Donor leukaemia is a rare event following transplantation for severe aplastic anaemia but may represent the persistence or perturbation of a stromal defect in these patients inducing leukaemic change in donor haemopoietic stem cells.

Purified autologous grafting in childhood acute lymphoblastic leukemia in second remission: evidence for long-term clinical and molecular remissions.

Autologous transplantation is a treatment option for relapsed childhood acute lymphoblastic leukemia (ALL) in second complete remission (CR2) when a suitable donor is not available. In an attempt to prevent relapses originating from graft leukemic contamination, the experimental protocol of in vitro purification of leukapheretic products with monoclonal antibodies (MoAbs), previously reported for adults, was adopted in 11 of 12 consecutive patients (median age, 9 years) with B cell precursor ALL in CR2 after late relapse (median, 37; range, 31-51 months after the onset) enrolled between July 1997 and July 1999 at a single pediatric center. At a median of 12 days after the mobilizing chemotherapy followed by G-CSF, a median of 13.9 (range, 5.9-18.7) x 10(6) CD34+ cells/kg were collected from each patient and a median of 7.5 (range, 4.1-12.6) x 10(6) CD34+ cells/kg underwent the purification procedure. The first step of immunorosetting allowed a one-log reduction of the total cell count, by eliminating more than 90% of the CD11b+ cells; the second step, performed after incubation with anti-CD19 MoAbs, allowed the depletion of 99% (range, 93-100) of the CD19+ cells, kept within the magnetic field of the immunodepletion column, with a median recovery of 73% (range, 55-87) of the collected CD34+ cells. Molecular analysis assessed the in vitro eradication of detectable leukemic cells. A median reinfusion of 5.2 (range, 3.2-9.1) x 10(6) CD34+ cells/kg for each patient (median viability, 90%), after conditioning with the 'TBI-VP16-CY' regimen, allowed prompt engraftment and immunological reconstitution; no patients experienced severe transplant-related toxicity or major infections. One patient relapsed 7 months after transplantation, while 10 patients are alive in clinical and molecular remission, at a median follow-up of 29 months (range, 15-40) (2-year EFS, 89%, s.e. 9). In conclusion, the procedure proved to be reproducible for pediatric purified autografting, highly efficient concerning stem cell recovery and depletion of leukemia-lineage specific cells, and promising in terms of final outcome.

Bone marrow transplantation for childhood hematological disorders: a global pediatric approach in a twelve year single center experience.

One hundred and 43 consecutive pediatric patients (June 1985-December 1996) with at least 18 months of follow-up, were considered: most of the patients (111/143, 77.6%) underwent allogeneic BMT. The median follow-up was 5.7 years. Overall survival and 5 years EFS were 48.6% and 46.9%, respectively. For patients who underwent allogeneic BMT from HLA-identical siblings, the 5 years EFS for ALL was 75% in 1st CR, 60.4% in 2nd CR, 22.3% in > 2nd CR and 86.7% for AML in 1st CR. The EFS for Allo-BMT in "good" and "poor" prognosis patients was 68.6% and 21.8%, respectively (p value = 0.001). Early mortality in Allo-BMT patients was 17.7% between 1985-1990 and 10.3% between 1991-1996. Early treatment-related organ complications occurred mostly in patients who underwent BMT from an unrelated or a mismatched family donor. Late toxicity was evaluated in 57 patients (median follow-up of 82 months): none of the patients complained of significant late cardiac or respiratory dysfunction. With regards to growth, 18/57 patients (31.6%) lost more than two height centile channels. Three cases of thyroid neoplasms were observed. Evaluation of psychosocial functioning, studied in 39 patients who had at least 2 years of follow-up in CR, did not reveal any evident quality of life impairment. The possibility of curing childhood hematological malignancies is based on a global pediatric and multidisciplinary approach. A continuous need to improve results in terms of EFS and quality of life suggests that further multicenter prospective studies should be carried out.

Mild pre-transplant cardiomyopathy may not impair long-term quality of life after bone marrow transplantation.

An 8-year-old child with acute myeloid leukemia (AML), underwent an allogeneic bone marrow transplant (BMT) from his HLA matched sister in spite of having a mild cardiomyopathy. We followed the patient with periodic electrocardiograms (ECG) and echocardiograms which have not worsened, and the patient's quality of life is not compromised 14 years after BMT. Bone Marrow Transplantation (2000) 25, 335-336.

Interleukin-10 increases mannose receptor expression and endocytic activity in monocyte-derived dendritic cells.

Human monocyte-derived dendritic cells were differentiated in vitro for 7 days with granulocyte macrophage-colony stimulating factor and interleukin-13. These cultured dendritic cells are at an immature stage of differentiation and exhert high endocytic activity via surface mannose receptor and via fluid-phase macropinocytosis. We have investigated the modulation of endocytosis by interleukin-10 in these cells. When added during the last 24 h of the 7-day culture, interleukin-10 significantly stimulated the uptake of fluorescein-labelled dextran (39 +/- 16% increase, mean +/- SD of 6 experiments), a sugar binding to the mannose receptor. This effect was dose dependent and correlated with the length of exposure to interleukin-10, with a maximal effect (more than seven-fold increase) when the cytokine was added at the beginning of the culture (day 0). The interleukin-10-increased fluorescein-labelled-dextran endocytosis was mostly mediated via the mannose receptor, as unlabelled mannose and specific antimannose receptor monoclonal antibody inhibited most of the uptake. Moreover, interleukin-10-treated cells expressed increased levels (up to four-fold) of mannose receptor. Interleukin-10 also increased, although to a lesser extent, the fluid-phase endocytosis (macropinocytosis) of fluorescein-labelled albumin. Interleukin-10 had the opposite effect on the differentiation and functional activity of monocyte-derived dendritic cells; cells having a very low stimulatory capacity and reduced expression of MHC class II and CD1a after a 7-day exposure. Thus interleukin-10 had a strong immunosuppressive effect on the differentiation and functional activity of monocyte-derived dendritic cells and yet strongly stimulated endocytosis in these cells. We speculate that an increased endocytic activity would eventually result in a decreased availability of antigens in the external milieu, thus contributing to the immunosuppressive and tolerogenic activity of interleukin-10.