Juvenile myelomonocytic leukemia (JMML) is an intractable pediatric leukemia with poor prognosis whose molecular pathogenesis is poorly understood, except for somatic or germline mutations of RAS pathway genes, including PTPN11, NF1, NRAS, KRAS and CBL, in the majority of cases. To obtain a complete registry of gene mutations in JMML, whole-exome sequencing was performed for paired tumor-normal DNA from 13 individuals with JMML (cases), which was followed by deep sequencing of 8 target genes in 92 tumor samples. JMML was characterized by a paucity of gene mutations (0.85 non-silent mutations per sample) with somatic or germline RAS pathway involvement in 82 cases (89%). The SETBP1 and JAK3 genes were among common targets for secondary mutations. Mutations in the latter were often subclonal and may be involved in the progression rather than the initiation of leukemia, and these mutations associated with poor clinical outcome. Our findings provide new insights into the pathogenesis and progression of JMML.

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

Of 11 children with juvenile myelomonocytic leukemia (JMML) carrying RAS mutations (8 with NRAS mutations, 3 with KRAS2 mutations), 5 had a profound elevation in either or both the white blood cells and spleen size at diagnosis. Three patients had no or modest hepatosplenomegaly and mild leukocytosis at presentation but subsequently showed a marked increase in spleen size with or without hematologic exacerbation, for which nonintensive chemotherapy was initiated. The other three patients with NRAS or KRAS2 glycine to serine substitution received no chemotherapy, but hematologic improvement has been observed during a 2-to 4-year follow up. In the third group, all hematopoietic cell lineages analyzed had the RAS mutations at the time of hematologic improvement, whereas DNA ob- IntroductionSomatic point mutations of the RAS genes at codons 12, 13, and 61 (NRAS and KRAS2) are found in approximately 20% of patients with juvenile myelomonocytic leukemia (JMML). 1,2 Other patients show inactivation of NF1 or PTPN11 mutations. [3][4][5] Although most patients with JMML die from progressive disease unless treated with hematopoietic stem cell transplantation, there are a few patients who have been reported to spontaneously recover without intervention. 6,7 Some of these children have JMML associated with Noonan syndrome, but others do not. So far, the individual prognosis in JMML-carrying specific genetic aberrations remains unclear. We report the clinical course in 11 patients with RAS mutations. Materials and methodsThis study was approved by the Institutional Review Board of Shinshu University. Informed consent was obtained from the guardians of the patients following institutional guidelines. Cell preparationWe used peripheral blood (PB) or bone marrow (BM) mononuclear cells (MNCs) that had been frozen with liquid nitrogen. CD3-and CD56-positive PB cells were separated immunomagnetically. 8 Ninety-nine percent of the isolated cells were CD3-or CD56-positive according to a flow cytometric analysis. Clonal cell cultureTwenty thousand PB or BM MNCs were plated in a dish containing methylcellulose medium supplemented with or without 0.01 to 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF). 9 To examine the clonal derivation of myeloid and erythroid lineages, 2000 CD34 ϩ PB cells harvested immunomagnetically were cultured in methylcellulose medium supplemented with GM-CSF, stem cell factor, interleukin 3, and erythropoietin. Twelve days after incubation in 5% CO 2 , GM colonies, erythroid colonies, and mixed colonies were individually lifted and prepared as single cell suspensions. Sequence analyses were then performed on individual colonyconstituent cells. Detection of NRAS and KRAS2 mutationsDNA was extracted from PB or BM MNCs and nails. Exon 1 (codons 12 and 13) and exon 2 (codon 61) of NRAS and KRAS2 genes were amplified by polymerase chain reaction (PCR) using primer pairs described previously. 10,11 The PCR products were subjected to direct sequencing from both directions on an automatic DNA se...

ABSTRACThad a high risk of bias due to their study design and were conducted more than 10 years ago and may not be applicable to the standard of care of today.11 Updated evidence to aid treatment decisions in pediatric SAA is, therefore, required.In children, the choice of an appropriate treatment is particularly influenced by the long-term sequelae of the disease and its therapy. Thus, failure-free survival is much more important than survival alone when analyzing the long-term outcomes of children with aplastic anemia. Lack of response, relapse, and clonal evolution are problematic in the IST setting, whereas graft failure, acute and chronic graft-versus-host disease (GVHD), and infectious complications limit the success of BMT. In the present study, we compared the outcomes of children with SAA who received IST or BMT from an MFD as first-line treatment using data from nationwide IST and BMT registries. Methods PatientsBetween 1992 and 2009, a total of 599 consecutive children (younger than 17 years) with acquired SAA underwent BMT from an MFD or received IST as first-line treatment in Japan; 213 patients with an MFD underwent BMT and were registered in the Transplant Registry Unified Management Program (TRUMP) conducted by the Japanese Society for Hematopoietic Cell Transplantation, and 386 patients without an MFD were enrolled in two consecutive prospective multicenter trials (AA-92/97) conducted by the Japanese Childhood Aplastic Anemia Study Group and were initially treated with IST (Table 1). The disease severities were defined as previously reported.12,13 Underlying inherited marrow failure disorders were excluded clinically and by chromosome fragility testing. Marrow cytogenetic studies were performed for all patients, and patients with clonal cytogenetic abnormalities were excluded from this study. Patients with paroxysmal nocturnal hemoglobinuria with clinical symptoms and positive findings on the Ham test/sucrose test were also excluded from this analysis. All treatments were performed after obtaining written informed consent from patients or their parents in accordance with the Declaration of Helsinki. Immunosuppressive therapy and bone marrow transplantation proceduresThe characteristics of the treatment procedures are detailed in Table 2. Three hundred and eighty-six patients were enrolled in the AA-92 (n=84) and AA-97 (n=302) trials, and all the patients were initially treated with a combination of antithymocyte globulin and cyclosporine A. Response to IST and disease relapse were evaluated as previously reported.12 Transplantation data were collected with the use of standardized forms provided by the TRUMP. A total of 213 patients underwent BMT from an MFD as first-line treatment following the local protocols for conditioning regimens and GVHD prophylaxis. Patients who did not reach neutrophil counts >0.5×10 9 /L for 3 consecutive days after transplantation were considered to have had primary graft failure. Patients with initial engraftment in whom absolute neutrophil counts subsequently declin...

A novel MEF2D-BCL9 fusion we identified characterizes a novel subset of pediatric ALL, predicts poor prognosis, and may be a candidate for novel molecular targeting.