We read with interest the study by Gustafsson et al.1 regarding BRAF mutations in childhood acute lymphoblastic leukemia (ALL). They investigated exons 11 and 15 of BRAF and exons 1 and 2 of NRAS in 29 cases (25 pre-B ALL, 3 T-cell ALL and 1 undifferentiated ALL), and identified six (21%) BRAF mutations and seven (24%) NRAS mutations. The frequencies of BRAF mutations seemed to be particularly common in T-cell ALL (two out of three; 67%); only four (16%) of the pre-B ALL harbored a BRAF mutation. All mutations were located in exon 15—three L597Q and one V590I among the pre-B ALL and one V600E and one G596S in the T-cell ALL. Hou et al.2 recently demonstrated that the L597Q mutation is a functional oncogene, at least in an in vitro experimental system. In the study by Gustafsson et al.,1 and as expected based on previous studies,3, 4 the BRAF and NRAS mutations were generally mutually exclusive. The high frequency of BRAF mutations was unexpected since such mutations otherwise have been demonstrated to be very rare in hematologic malignancies. In fact, previous analyses revealed no BRAF mutations in 53 cell lines from leukemias and lymphomas,3 65 multiple myelomas/plasma cell leukemias,5 21 multiple myelomas6 or in 149 acute myeloid leukemias (AML).7, 8 We know of only three additional studies reporting acquired BRAF mutations in malignant hematologic disorders. In a series of 164 B- and T-cell lymphomas, Lee et al.9 detected BRAF mutations in ∼2.5%. The same group10 also reported such mutations in 20% of B-lineage ALL, 9% of acute biphenotypic leukemia and 4% of AML; all the investigated patients were adults. Finally, Christiansen et al.,11 who analyzed 140 treatment-related myelodysplastic syndromes and AML, found three t-AML with a BRAF mutation; all these were adult acute monoblastic leukemias with t(9;11)(p21;q23). Interestingly, germ line mutations in the closely related RAF1 (previously CRAF) were recently reported in two patients who developed t-AML.8 Hence, there may be an association between mutations in RAF genes and t-AML. It may also be noteworthy that among the 17 BRAF-positive hematologic malignancies reported to date,1, 9, 10, 11 only four (24%)—one T-cell ALL1 and three t-AML11—have carried the highly activating V600E amino-acid substitution, which results in a 500-fold activation of the BRAF protein, inducing a pronounced RAS-RAF-MEK-ERK signaling.3
To date, the report by Gustafsson et al.1 is the only one focusing on BRAF mutations in pediatric ALL, except a recent case report of a germ line BRAF mutation (E501G) in a child with a congenital cardiofaciocutaneous syndrome who later developed pre-B ALL.12 Hence, the high incidence of such mutations in childhood ALL has as yet not been confirmed. Considering that BRAF mutations may be more prevalent in some cytogenetic subgroups, as indicated by the correlation between t(9;11)-positive t-AML and BRAF mutations,11 one may expect that different karyotypic ALL entities may be associated with different mutation frequencies. Unfortunately, Gustafsson et al.1 did not include cytogenetic data. However, as most of their BRAF-positive pre-B ALL occurred in children aged 1.5–4 years, one may expect that they were characterized by either high hyperdiploidy or t(12;21)(p13;q22)/ETV6–RUNX1 fusion, as these two cytogenetic subgroups constitute approximately 80% of all B-cell precursor (BCP) ALL in the 2–7 years frequency peak,13 or by dic(9;20)(p13;q11), also known to occur primarily in this age group.14
For the above-mentioned reasons, we screened a total of 92 childhood BCP ALL, comprising 67 cases with high hyperdiploidy (>50 chromosomes), 22 with ETV6–RUNX1 fusion and three with dic(9;20) as well as 17 pediatric T-ALL for mutations in exons 11 and 15 of BRAF. DNA was extracted from bone marrow and/or peripheral blood samples obtained at the time of diagnosis, using standard methods. A one-step PCR amplification of the exons was performed as reported,15 by the use of the primer combinations BRAFEx11For: IndexTermATCCCTCTCAGGCATAAGGTAATG and BRAFEx11Rev: IndexTermGCGAACAGTGAATATTTCCTTTGA for exon 11 and BRAFEx15For: IndexTermTGCTTGCTCTGATAGGAAAATGAG and BRAFEx15Rev: IndexTermTCTCAGGGCCAAAAATTTAATCA for exon 15, previously validated and shown to identify BRAF mutations in malignant melanomas.15 For this study, the primers were used successfully to identify a homozygous V600E in the cell line SK-MEL-28, again showing that the assay is capable of detecting BRAF mutations. Both the sense and antisense strands were directly sequenced, and the sequences were analyzed using the Seqscape software (PE Applied Biosystems, Foster City, CA, USA). The study was approved by the research ethics committee at Lund University and it was conducted in accordance with the Declaration of Helsinki.
None of the 92 BCP ALL or 17 T-cell ALL harbored any BRAF mutations in exons 11 and 15, a result which has several implications. First, we have very recently shown that approximately one-third of all high hyperdiploid pediatric ALL carry mutations in genes (FLT3, NRAS, KRAS and PTPN11) involved in the RTK–RAS pathway, indicating that activation of this signaling cascade is pathogenetically important in this cytogenetic subgroup.16 As BRAF also participates in this pathway one could have expected, considering that the aforementioned mutated genes are mutually exclusive in high hyperdiploid ALL, that the cases negative for FLT3, NRAS, KRAS and PTPN11 mutations frequently would harbor BRAF mutations. However, this is obviously not the case as none of the 67 cases investigated had a mutation. Second, the lack of BRAF mutations in ALL cases with high hyperdiploidy, t(12;21) or dic(9;20), which constitute the vast majority of all pediatric BCP ALL occurring in the characteristic age peak,13, 14 shows that activation of the BRAF protein does not play an important leukemogenic role in this patient cohort. Third, we could not confirm the high frequency of BRAF mutations in childhood T-ALL reported by Gustafsson et al.1 The reasons for the discrepant results between this study and the one by Gustafsson et al.1 are unclear, although one possibility is that their T-ALL and pre-B ALL cases belonged to other cytogenetic/molecular genetic subgroups than did the cases analyzed by us. This notwithstanding, based on the present large series, we conclude that BRAF mutations are very rare in B- and T-cell pediatric ALLs.
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Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. Mutations of the BRAF gene in human cancer. Nature 2002; 417: 949–954.
Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE . Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002; 418: 934.
Bonello L, Voena C, Ladetto M, Boccadoro M, Palestro G, Inghirami G et al. BRAF gene is not mutated in plasma cell leukemia and multiple myeloma. Leukemia 2003; 17: 2238–2240.
Ng MHL, Lau KM, Wong WS, To KW, Cheng SH, Tsang KS et al. Alterations of RAS signalling in Chinese multiple myeloma patients: absent BRAF and rare RAS mutations, but frequent inactivation of RASSF1A by transcriptional silencing or expression of a non-functional variant transcript. Br J Haematol 2003; 123: 637–645.
Smith ML, Snaddon J, Neat M, Cambal-Parrales M, Arch R, Lister TA et al. Mutation of BRAF is uncommon in AML FAB type M1 and M2. Leukemia 2003; 17: 274–275.
Zebisch A, Staber PB, Delavar A, Bodner C, Hiden K, Fischereder K et al. Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia. Cancer Res 2006; 66: 3401–3408.
Lee JW, Yoo NJ, Soung YH, Kim HS, Park WS, Kim SY et al. BRAF mutations in non-Hodgkin's lymphoma. Br J Cancer 2003; 89: 1958–1960.
Lee JW, Soung YH, Park WS, Kim SY, Nam SW, Min WS et al. BRAF mutations in acute leukemias. Leukemia 2004; 18: 170–172.
Christiansen DH, Andersen MK, Desta F, Pedersen-Bjergaard J . Mutations of genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal transduction pathway in therapy-related myelodysplasia and acute myeloid leukemia. Leukemia 2005; 19: 2232–2240.
Makita Y, Narumi Y, Yoshida M, Niihori T, Kure S, Fujieda K et al. Leukemia in Cardio-facio-cutaneous (CFC) syndrome: a patient with a germline mutation in BRAF proto-oncogene. J Pediatr Hematol Oncol 2007; 29: 287–290.
Forestier E, Schmiegelow K . The incidence peaks of the childhood acute leukemias reflect specific cytogenetic aberrations. J Pediatr Hematol Oncol 2006; 28: 486–495.
Forestier E, Gauffin F, Andersen MK, Autio K, Borgström G, Golovleva I et al. Clinical and cytogenetic features of pediatric dic(9;20)(p13.2;q11.2)-positive B-cell precursor acute lymphoblastic leukemias: a Nordic series of 24 cases and review of the literature. Genes Chromosomes Cancer 2008; 47: 149–158.
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Paulsson K, Horvat A, Strömbeck B, Nilsson F, Heldrup J, Behrendtz M et al. Mutations of FLT3, NRAS, KRAS, and PTPN11 are frequent and possibly mutually exclusive in high hyperdiploid childhood acute lymphoblastic leukemia. Genes Chromosomes Cancer 2008; 47: 26–33.
This study was supported by grants from the Swedish Children's Cancer Foundation, the Swedish Cancer Society and the Swedish Research Council.
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Davidsson, J., Lilljebjörn, H., Panagopoulos, I. et al. BRAF mutations are very rare in B- and T-cell pediatric acute lymphoblastic leukemias. Leukemia 22, 1619–1621 (2008). https://doi.org/10.1038/leu.2008.14
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