Mutation analysis of the BRAF oncogene in juvenile myelomonocytic leukemia

Abstract

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative/myelodysplastic disorder associated with mutations in the Ras-Raf-MEK-ERK-signaling pathway. B-Raf plays a central role in this pathway. In 65 screened JMML patients we identified no BRAF mutations and we conclude that this gene is unlikely to play a role in the pathogenesis of JMML.

JMML is a rare myelodysplastic/myeloproliferative disease of early childhood, accounting for less than 3% of all hematologic malignancies in children. JMML is characterized by prominent hepatosplenomegaly, absolute monocytosis, presence of myeloid precursors in peripheral blood, low platelet count, frequent skin involvement and in vitro granulocyte-macrophage colony stimulating factor (GM-CSF) hypersensitivity.1 GM-CSF hypersensitivity in JMML results from continuous activation of the GM-CSF-receptor-Ras-Raf-MEK-ERK signal transduction pathway due to activating mutations of RAS2 or PTPN11.3 These mutations occur in 25% and 35% of JMML cases respectively. Furthemore, approximately 11% of cases of JMML cases occur in individuals with neurofibromatosis type 1 (NF1) and JMML cells show bi-allelic inactivation of the NF1 tumor suppressor gene encoding for neurofi-bromin, a GTPase activating protein for Ras. Loss of NF1 leads to activation of RAS signaling.4 The remaining cases presumably carry somatic mutations of other yet undefined genes of the Ras signaling pathway.

The Ras effector B-Raf plays a central role in transmitting growth signals from Ras to downstream molecules including MEK and ERK. Somatic mutations of BRAF occur at high frequency in numerous human cancers.5 The BRAF mutation results in increased kinase activity and accounts for more than 90% of the mutations.5 Germline mutations of BRAF cause cardio-faciocutaneous syndrome (CFC), a dominant disorder characterized by short stature, cardiac defects, distinct facial features, developmental delay, and ectodermal abnormalities.6 CFC shares many features with other dominant syndromes including Costello syndrome (CS) and Noonan syndrome (NS).7 CS and NS are caused by germline mutations in genes of the Ras pathway, including HRAS (mutated in CS) and PTPN11, KRAS, or SOS1 (mutated in NS).7 Interestingly, infants with NS are predisposed to develop a myeloproliferative disease resembling JMML. So far, a BRAF mutational screen of JMML specimens has not been conducted.

In this study, we screened 65 JMML specimens for somatic mutations of BRAF. The diagnosis of JMML was based on criteria described by Hasle et al.8 and specimens were treated according to the guidelines for JMML of the European Working Group on Childhood MDS (EWOG- MDS).

DNA was extracted from mononuclear cells derived from cryopreserved primary bone marrow, peripheral blood, or spleen at diagnosis. Sequence analysis was performed using forward and reverse primers for BRAF exon 15: forward 5′-AGC CCC AAA AAT CTT AAA AG- 3’ and reverse 5′-CTC AGG GCC AAA AAT TTA AT- 3’. In a subset of patients the entire coding sequence of BRAF was analyzed. Details on the used primers are available on request. Of the 65 patients with JMML included in this study, 26 had no clinical diagnosis of NF1 and lacked mutations of KRAS, NRAS or PTPN11. Six patients were diagnosed with NF1, 12 had a somatic mutation of NRAS or KRAS, and 12 patients had a somatic mutation of PTPN11. No data on the molecular background were available for nine patients. No V600E mutation of the BRAF gene was found in any of the 65 patients. Furthermore, in a subset of 15 patients without a clinical diagnosis of NF1 and without mutations of PTPN11 or RAS, the entire coding region of BRAF was analyzed. No mutations were identified in this group of patients.

JMML is regarded as a model disease that is associated with hyperactive Ras signaling. Mutant proteins of the Ras-Raf-MEK-ERK pathway play an important role in the pathogenesis of JMML and explain the GM-CSF hypersensitivity observed ex vivo. In the majority of cases of JMML, these mutations are mutually exclusive and affect NRAS, KRAS, NF1, or PTPN11 genes. Additionally, mutations in this pathway function as an important diagnostic tool. They are a potential marker of minimal residual disease9 and an attractive new potential therapeutic target.

Our hypothesis was that B-Raf might play an important role in JMML as it is an important downstream effector of Ras. Somatic BRAF mutations occur frequently in other types of human cancer and recently, BRAF mutations were found in leukemia. We excluded the occurrence of BRAF mutations in a large cohort of JMML patients. Therefore, additional screening of genes of the Ras pathway will be necessary to identify genetic aberrations in cases without known mutations.

Acknowledgments

we would like to acknowledge the contribution of O.A. Haas, E.R. van Wering, C.M. Zwaan, F. Locatelli, M. Zecca, H. Hasle and J. Stary