Absence of BRAF V600E mutation in hematologic malignancies excluding hairy-cell leukemia

Abstract

Th e BRAF gene is a member of the Raf kinase family and encodes a serine/threonine-specifi c protein kinase that plays an important role in regulating the mitogen activated protein (MAP) kinase/ERK signaling pathway and aff ects cell division, diff erentiation and secretion. Somatic mutations in the BRAF gene occur in a wide variety of human tumors, most commonly in melanoma (43%) and thyroid (39%), colon (12%) and ovarian (12%) cancers. BRAF missense mutations are mainly localized in the glycine-rich loop and activation segment, encoded by exon 11 and exon 15, respectively. A missense mutation in DNA (1799T → A) resulting in an amino acid change from valine to glutamic acid at codon 600 (V600E) accounts for approximately 80% of BRAF mutations found in human cancers [1]. Recently, the BRAF V600E mutation was found in all 48 patients with hairy-cell leukemia (HCL) in one study [2]. Nevertheless, the BRAF V600E mutation has been reported to be rare in other myeloid and lymphoid neoplasms [2–15]. In order to confi rm the prevalence of the BRAF V600E mutation in hematologic malignancies other than HCL, we investigated the prevalence of this gene mutation in patients with a variety of hematologic malignancies. A total of 780 patients were enrolled in this study, including 288 patients with de novo acute myelogenous leukemia (AML), 84 with de novo myelodysplastic syndromes (MDS), 84 with myeloproliferative neoplasms (MPN), 71 with chronic myeloid leukemia (CML) in chronic phase, 51 with CML in blast crisis, 10 pediatric and 102 adult patients with precursor B-cell acute lymphoblastic leukemia (B-ALL), four pediatric and 13 adult patients with T-cell acute lymphoblastic leukemia (T-ALL), 47 with chronic lymphocytic leukemia (CLL) and 26 with multiple myeloma (MM) with bone marrow infi ltration ( 30%). Diagnoses in these patients were made according to the French–American–British (FAB) classifi cation and revised with application of the World Health Organization (WHO) 2008 classifi cation. Th e disease burden for all patients was above the sensitivity of the Sanger direct DNA sequencing technique. Th e median age of the case series was 42 years (range 5–88 years), and the majority were male (54%) and mostly ethnic (Han) Chinese. Th e main characteristics of patients in this study are summarized in Supplementary Table I to be found online at http:// informahealthcare.com/doi/abs/10.3109/10428194.2012. 695777. Th e study was approved by the Ethics Committee of the First Affi liated Hospital of Soochow University and was conducted according to the Declaration of Helsinki. We examined the BRAF V600E mutation by polymerase chain reaction (PCR) amplifi cation of exon 15 of the BRAF gene followed by direct bidirectional DNA sequencing in cells from 780 patients with hematologic malignancies. Genomic DNA was extracted from frozen bone marrow mononuclear cells (BMMCs) collected at diagnosis after Ficoll gradient centrifugation using standard procedures. For PCR amplifi cation of exon 15 of the BRAF gene, primers 5 ’ -TCATAATGCTTGCTCTGATAGGA-3 ’ (forward) and 5 ’ -GGCCAAAAATTTAATCAGTGGA-3 ’ (reverse) were used for both PCR and sequencing. PCR products were directly sequenced on both strands using an ABI 3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA). Th e aim of this study was to determine the occurrence of the somatic BRAF V600E mutation in patients with hematologic malignancies, excluding HCL, especially in patients with myeloid malignancies, ALL or MM. We sequenced exon 15 in all 780 patients enrolled in the study. However, no evidence of BRAF mutations invoving exon 15, including the most common mutation BRAF V600E, was found in these patients. By contrast, the BRAF V600E mutation was detected in BMMCs from fi ve patients with HCL. Our data confi rm that the BRAF V600E mutation is common in HCL but indicate it to be absent in MDS, AML, MPN, precursor B-ALL, T-ALL, MM and lymphoma, suggesting that it may not be widespread in hematologic malignancies, excluding HCL.