Markedly High Plasma Thrombopoietin (TPO) Level is a Predictor of Poor Response to Immunosuppressive Therapy in Children With Acquired Severe Aplastic Anemia.

BACKGROUND: Immunosuppressive therapy (IST) is commonly used for patients with acquired severe aplastic anemia (SAA). Because the clinical response rate and therapeutic outcome for individual patients to IST varies, an in vitro test that identifies potential responders would be desirable. METHODS: We evaluated the relationship between thrombopoietin (TPO) levels at the time of diagnosis and the response to IST at 6 months in 85 children (median age, 9.0 years; range, 1.0-15.5 years) with acquired SAA using enzyme-linked immunosorbent assay. Thirty-one age-matched healthy individuals were used as controls. All patients received antithymocyte globulin and cyclosporine. RESULTS: Overall, 39 patients (45.9%) responded to IST at 6 months. TPO plasma levels were significantly higher in nonresponders than in responders (1,555.8 vs. 1,284.7 pg/ml, respectively; P = 0.031). Multivariate analysis identified the TPO levels of >1,796.7 pg/ml (TPO-high group, 20 patients; odds ratio (OR), 8.285; 95% confidence interval (CI), 2.114-32.904; P = 0.002) as independent poor predictors of IST response at 6 months. Moreover, the TPO-high group was associated with lower 5-year failure-free survival rates (30% vs. 68%, P = 0.012) compared with the TPO-low group. CONCLUSION: The measurement of TPO levels at diagnosis is useful for predicting the response to IST in children with SAA and may help in decision making.

Correlation of rabbit antithymocyte globulin serum levels and clinical outcomes in children who received hematopoietic stem cell transplantation from an alternative donor.

We analyzed the correlation between rabbit ATG (rATG) serum levels and clinical outcomes in 37 children who received rATG at a total dose of 10 or 15 mg/kg during HSCT conditioning from an alternative donor. Fourteen patients had advanced malignant diseases, 13 had severe AA, and 10 had inherited disorders. Complete engraftment was achieved in all patients, and no rejection occurred. The cumulative incidence of grades II-IV acute GVHD and extensive chronic GVHD was 27% (95% CI, 12.5-39.6%) and 8.1% (95% CI, 0-23.1%), respectively. Multivariate analysis identified lower rATG levels at week 4 as an independent risk factor in the development of grades II-IV acute GVHD (p = 0.037). Serious infections were not observed in any patient following HSCT. No correlation was found between EBV reactivation and rATG levels at week 2 and week 4 after HSCT. Furthermore, no correlation was found between relapse and rATG levels two and four wk post-transplantation. The probability of five-yr OS among patients was 70.3% (95% CI, 59.8-79.2%). Our results suggest that targeted rATG administration may protect patients from severe acute GVHD without increasing the risk of EBV reactivation or relapse.

A Cytokine-Based Diagnostic Program in Pediatric Aplastic Anemia and Hypocellular Refractory Cytopenia of Childhood.

BACKGROUND: Distinguishing hypocellular refractory cytopenia of childhood (RCC) from aplastic anemia (AA) is challenging. Thus far, no studies have compared the cytokine profiles in patients with AA to those with hypocellular RCC. In the present study, we addressed whether thrombopoietin (TPO) and interleukin 17 (IL-17) plasma levels are useful for differentiating between the two diseases. METHODS: We measured the endogenous plasma concentrations of TPO and IL-17 in 29 patients with AA, 34 patients with hypocellular RCC, and 31 healthy controls using sensitive enzyme-linked immunosorbent assays. RESULTS: The TPO and IL-17 plasma levels were significantly lower in patients with hypocellular RCC than in patients with AA (P < 0.001 and P = 0.007, respectively). The multivariate logistic regression analysis identified moderate disease severity, TPO levels of <1,369.8 pg/ml (TPO-low group, n = 32; odds ratio (OR), 13.40; 95% confidence intervals (CI), 3.001-51.254; P < 0.001), and IL-17 levels of <22.2 pg/ml (IL-17-low group, n = 33; OR, 4.11; 95% CI, 1.033-19.404; P = 0.031) as independent factors discriminating hypocellular RCC from AA. Importantly, 25 (78.1%) of 32 patients in the TPO-low group and 25 (75.8%) of 33 patients in the IL-17-low group were diagnosed as having hypocellular RCC. Moreover, 22 (71%) of 31 patients in the TPO-high group and 21 (70%) of 30 patients in the IL-17-high group were diagnosed as having AA. CONCLUSIONS: TPO and IL-17 levels are useful for differentiating hypocellular RCC from AA. Prospective studies are required to confirm our findings.

RUNX1 mutation associated with clonal evolution in relapsed pediatric acute myeloid leukemia with t(16;21)(p11;q22).

TLS/FUS-ERG chimeric fusion transcript resulting from translocation changes involving chromosomes 16 and 21 is a rare genetic event associated with acute myeloid leukemia (AML). The distinct t(16;21) AML subtype exhibits unique clinical and morphological features and is associated with poor prognosis and a high relapse rate; however, the underlying mechanism remains to be clarified. Recently, whole-genome sequencing revealed a large set of genetic alterations that may be relevant for the dynamic clonal evolution and relapse pathogenesis of AML. Here, we report three pediatric AML patients with t(16;21) (p11; q22). The TLS/FUS-ERG fusion transcript was detected in all diagnostic and relapsed samples, with the exception of one relapsed sample. We searched for several genetic lesions, such as RUNX1, FLT3, c-KIT, NRAS, KRAS, TP53, CBL, ASXL1, IDH1/2, and DNMT3A, in primary and relapsed AML samples. Interestingly, we found RUNX1 mutation in relapsed sample of one patient in whom cytogenetic analysis showed the emergence of a new additional clone. Otherwise, there were no genetic alterations in FLT3, c-KIT, NRAS, KRAS, TP53, CBL, ASXL1, IDH1/2, or DNMT3A. Our results suggest that precedent genetic alterations may be essential to drive the progression and relapse of t(16;21)-AML patients.

Somatic mosaicism for oncogenic NRAS mutations in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. Somatic mutations in genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and CBL, have been identified in more than 70% of children with JMML. In the present study, we report 2 patients with somatic mosaicism for oncogenic NRAS mutations (G12D and G12S) associated with the development of JMML. The mutated allele frequencies quantified by pyrosequencing were various and ranged from 3%-50% in BM and other somatic cells (ie, buccal smear cells, hair bulbs, or nails). Both patients experienced spontaneous improvement of clinical symptoms and leukocytosis due to JMML without hematopoietic stem cell transplantation. These patients are the first reported to have somatic mosaicism for oncogenic NRAS mutations. The clinical course of these patients suggests that NRAS mosaicism may be associated with a mild disease phenotype in JMML.

De novo childhood myelodysplastic/myeloproliferative disease with unique molecular characteristics.

Myelodysplastic/myeloproliferative uclassifiable (MDS/MPN-U) is a rare myeloid neoplasm characterized by myelodysplasia and myeloproliferation at the time of initial presentation, which is usually a diagnosis of exclusion. The molecular pathogenesis of MDS/MPN-U patients remains to be elucidated. Among five patients diagnosed with MDS/MPN-U, three patients harboured RUNX1 (AML1) mutations; one carried somatic mosaicism of RUNX1 mutation with JAK2(V617F) mutation and one had dual RUNX1 and FLT3-internal tandem duplication mutations with progression to acute myeloid leukaemia (AML). Germline mutation of TP53 was detected as a sole genetic lesion in one patient. JAK2(V617F) and somatic mosaicism of KRAS and TET2 mutations co-existed in one patient. Otherwise, no alterations were detected in PTPN11, NRAS, CBL and ASXL1 genes. ETV6-PDGFRB fusion transcript was not detected in all patients. Four patients recieved haematopoietic stem cell transplantation (HSCT); three patients relapsed and one achieved complete remission after three donor lymphocyte infusions. Our findings suggest that the mutational spectrum observed in childhood MDS/MPN-U is quite different from that seen in juvenile myelomonocytic leukaemia and, to some extent, resemble chronic myelomonocytic leukaemia. Moreover, two patients had constitutional alterations of genes frequently found in AML. Further investigations are required to define the roles of these genetic alterations in the pathogenesis of childhood MDS/MPN-U.

Excellent outcome of allogeneic bone marrow transplantation for Fanconi anemia using fludarabine-based reduced-intensity conditioning regimen.

Fanconi anemia (FA) is a disorder characterized by developmental anomalies, bone marrow failure and a predisposition to malignancy. It has recently been shown that hematopoietic stem cell transplantation using fludarabine (FLU)-based reduced-intensity conditioning is an efficient and quite safe therapeutic modality. We retrospectively analyzed the outcome of bone marrow transplantation (BMT) in eight patients with FA performed in two institutes between 2001 and 2011. There were seven females and one male with a median age at diagnosis = 4.5 years (range 2-12 years). The constitutional characteristics associated with FA, such as developmental anomalies, short stature and skin pigmentation, were absent in three of the patients. One patient showed myelodysplastic features at the time of BMT. All patients received BMT using FLU, cyclophosphamide (CY) and rabbit anti-thymocyte globulin (ATG) either from a related donor (n = 4) or an unrelated donor (n = 4). Acute graft-versus-host disease (GVHD) of grade I developed in one patient, while chronic GVHD was not observed in any patient. All patients are alive and achieved hematopoietic recovery at a median follow-up of 72 months (range 4-117 months). BMT using FLU/low-dose CY/ATG -based regimens regardless to the donor is a beneficial therapeutic approach for FA patients.

Mutations profile of polycythemia vera and essential thrombocythemia among Japanese children.

BACKGROUND: Acquired somatic mutations of JAK2 have been reported to play a pivotal role in the pathogenesis of BCR-ABL1-negative myeloproliferative neoplasm (MPN). However, the molecular characteristics of childhood MPN remain to be elucidated. PATIENT AND METHODS: We investigated a group of pediatric patients diagnosed either with essential thrombocythemia (ET; N = 9) or polycythemia vera (PV; N = 4) according to WHO criteria (median age = 10 years; range 1.5-15 years) in whom direct sequencing was performed for the existence of genetic alterations in JAK2, MPL, TET2, ASXL1, CBL, IDH1, and IDH2. More sensitive allele specific polymerase chain reaction was used for JAK2(V617F) genotyping. RESULTS: We found three patients harbor JAK2(V617F) mutation (2/9 ET and 1/4 PV). Bone marrow examination showed small and large megakaryocytes with dysplastic features in JAK2(V617F)-positive ET patients compared to those without JAK2(V617F). We identified a previously unrecognized missense mutation at codon 1230 in exon 12 of ASXL1 gene in ET and PV patients (1/9 ET and 1/4 PV). Otherwise, no genetic alterations could be detected in JAK2 exon 12, MPL, TET2, CBL, IDH1, and IDH2 in all ET and PV patients. CONCLUSION: Although JAK2 mutations in childhood ET and PV are not as frequent as reported in adult patients, JAK2 is the most frequently mutated gene in childhood MPN known so far. Owing to the presence of childhood MPN without any genetic alterations in JAK2, MPL, TET2, ASXL1, CBL, IDH1, and IDH2, new biological markers have to be found.