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...

Using the data obtained in the present study, we describe situations faced in the terminal care of children. It is important to address the problems revealed by this analysis in order to achieve improvements in both the physical and psychological care of children with terminal cancer.

It is vital to develop effective therapy for children with acute lymphoblastic leukemia (ALL), in whom no remission occurs or who suffer relapse with current protocols. Cellular drug resistance is thought to be an important cause of induction failure and relapse. We performed in vitro tests of bone marrow samples in 196 children with newly diagnosed ALL with a 4-day culture and a methyl-thiazol-tetrazolium assay. We tested 16 drugs and calculated the 70% lethal dose (LD70) for 14 drugs and the leukemic cell survival (LCS) rate for dexamethasone and prednisolone. For each single drug, patients were classified into two groups, sensitive or resistant, by median concentration of LD70 or LCS. When patients were classified into three groups by sensitivity to four drugs of DPAV (dexamethasone, prednisolone, L-asparaginase, and vincristine), 3-year event-free survival (EFS; 95% confidence intervals) of the super sensitive group (SS; sensitive to all 4 drugs) was 0.833 (0.690 to 0.976), that of the intermediate sensitive group (IS; sensitive to 2 or 3 drugs) was 0.735 (0.609 to 0.863), and that of the relatively resistant group (RR; sensitive to no drugs or to 1 drug) was 0.541 (0.411 to 0.670; P = .0008). We then investigated the relationship between the above four-drug sensitivity and the time of relapse. The SS and IS patients tended to maintain continuous complete remission, and RR patients tended to undergo induction failure and early and late relapse (P = .004). Initial white blood cell count, immunologic classification, and age were also predictive factors, but the patient numbers showed no statistical correlation between these factors and the four-drug sensitivity groups (SS, IS, and RR). When we took three groups SS/IS/RR and investigated the EFS for various clinical groups, DPAV sensitivity strongly influenced EFS in the standard-risk ALL (P = .016). In vitro drug sensitivity testing provides additional prognostic information about childhood ALL, and early detection of drug resistance at the time chemotherapy commences may provide a successful strategy for individualizing treatment, as the results indicate de novo resistance to front-line drugs and suggest alternative, second-line drugs.