Spontaneous improvement of hematologic abnormalities in patients having juvenile myelomonocytic leukemia with specific RAS mutations

Abstract

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...

Acquisition of loss of the wild-type NRAS locus with aggressive disease progression in a patient with juvenile myelomonocytic leukemia and a heterozygous NRAS mutation

“…However, our recent study revealed hematologic improvement despite no chemotherapy during a 2-to 4-year follow-up in JMML patients with a NRAS or KRAS2 glycine to serine substitution, 9 implying that all types of RAS mutations are not always sufficient to confer the disease progression. Given the results of a 7,12-dimethylbenz[a]anthracene-induced rat leukemia model, 10 the two stages of genetic change in our case suggest that oncogenic heterozygous RAS mutations are an C. FISH analysis using probes for the region containing the NRAS gene on chromosome 1p13 and for the region containing D1S468 on chromosome 1p36 was performed on post-BC GM colony-constituent cells generated with GM-CSF and SCF.…”

Childhood Myelodysplastic Syndrome: Focus on the Approach to Diagnosis and Treatment of Juvenile Myelomonocytic Leukemia

“…31 Taken together, these data would suggest that the current spectrum of commonly occurring mutations in NF1, RAS, PTPN11, and CBL converge on the Ras/MAPK pathway in a highly specific way that remains to be fully elucidated. 32 Flotho et al were unable to confirm in the EWOG-MDS database that RAS G12S was associated with long-term survival in the absence of HSCT. 33 However, it was noted that among 12 patients observed without HSCT for more than 3 years from diagnosis, there were 5 patients experiencing long-term survival who harbored various RAS mutations.…”

Juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia

“…[52][53][54] Spontaneous improvement of other JMML patients with RAS mutations has also been noted. 85 Unfortunately, the majority of JMML patients will show disease progression, although conversion to a blast-type crisis is noted in o20%.…”