BRAF and KRAS mutations in prostatic adenocarcinoma Nam-Yun Cho 1 , Minhee Choi 1 , Baek-Hee Kim 2 , Yong-Mee Cho 3 , Kyung Chul Moon 2 and Gyeong Hoon Kang 1,2 * 1 Laboratory of Epigenetics, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea 2 Department of Pathology, Seoul National University College of Medicine, Seoul, Korea 3 Department of Diagnostic Pathology, Asan Medical Center, Seoul, Korea Constitutive activation of the kinase cascade involving RAS, RAF, MEK and ERK is common to human cancers, and mutations of KRAS and BRAF are mutually exclusive and serve as alternatives to activate the RAS/RAF/ERK signaling pathway. RAS mutations are known to occur in prostate adenocarcinomas, but little is known about BRAF mutations in these tumors. In the present study, BRAF and KRAS mutations were characterized in 206 prostate adenocar- cinomas by enhanced PCR-RFLP and direct sequencing. The iden- tified KRAS and BRAF mutations were then analyzed with respect to preoperative serum PSA levels, Gleason scores and tumor stages. Mutations in codon 600 of BRAF were identified in 21 (10.2%) of 206 prostate adenocarcinomas. KRAS mutations in codons 12 or 13 were found in 15 (7.3%) of 206 prostate adenocarcinomas. How- ever, no tumor specimen contained both BRAF and KRAS muta- tions. Prostate adenocarcinomas with a BRAF mutation tended to show higher preoperative serum PSA levels, Gleason scores and tu- mor stages than prostate adenocarcinomas with a KRAS mutation. The results obtained show that BRAF mutations are as uncommon as KRAS mutations in prostate adenocarcinoma. Although BRAF and KRAS are members of the same RAS/ERK signaling pathway, prostate adenocarcinomas with a BRAF mutation showed clinico- pathologic features that differed from those of prostate adenocarci- noma with a KRAS mutation. ' 2006 Wiley-Liss, Inc. Key words: BRAF; KRAS; prostate adenocarcinoma Prostate adenocarcinoma is one of the most commonly diag- nosed carcinomas in Western men and a leading cause of carci- noma-related death. 1 Although the incidence of prostate adenocar- cinoma is relatively low (the 6th leading cause of cancer death in Korean men), 2 there has been a regular and marked increase in the incidence of prostate adenocarcinoma in Korea. For the most part this increase is believed to be related to a westernization of life style, as reflected by increasing obesity and an increase in dietary fat, although some of this increase is evidently due to enhanced detection. Many molecular studies have indicated that genetic alterations are important for prostate carcinogenesis. However, few oncogenes or tumor suppressor genes have been consistently linked to prostate adenocarcinoma, and the molecular mechanisms underlying its development and progression remain poorly under- stood. Constitutive activation of the kinase cascade involving RAS, RAF, mitogen/extracellular signal-regulated kinase (MEK), ex- tracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) is common to numerous cancers. Approxi- mately 15% of human cancers have activating RAS mutations, 3 and prostate adenocarcinomas have been reported to show KRAS mutation frequencies of up to 13%, although a difference in KRAS mutation rates was found between Japanese and American men. 4–6 BRAF, a member of the RAF family of serine/threonine kinases, has been recently reported to be activated by somatic mutation in many human cancers, and these mutations occurred at high fre- quencies in malignant melanomas and thyroid papillary carcino- mas and at lower frequencies in a wide range of other human can- cers. 7 BRAF is known to have a mutation hot-spot at codon 600 (originally designated as codon 599 based on NCBI gene bank sequence NM_004333 which was missing one codon after the region coding for the amino acids 30 and the recently corrected sequence (GI33188458) is one codon longer, and therefore all numbering of subsequent numbering of codons has to be shifted by one position), which has been reported to account for 91% of BRAF mutations in human cancers. 7 Cohen et al. reported no BRAF mutations in prostatic adenocarcinomas, but the sample size was too small (n 5 17). 8 In the view of the fact that BRAF mutations tend to occur in many of the same cancer tissue types as KRAS mutations, 9–11 the possibility cannot be excluded that BRAF mutations may occur in prostate adenocarcinoma. In the present study, we examined 206 samples of prostate adenocarcinomas for BRAF codon 600 mutation and KRAS codon 12 and 13 mutations by using enriched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. We then correlated the results obtained with the clinicopathologic features of prostate adenocarcinomas. Material and methods A total of 206 prostate samples were collected from patients undergoing radical prostatectomy for prostate adenocarcinoma at the Seoul National University Hospital and the Asan Medical Cen- ter, Seoul, between 1999 and 2004. Tissue samples were formalin- fixed and paraffin wax-embedded. Clinicopathologic characteris- tics are summarized in Table I. Each tumor was graded and staged according to the Gleason system 12 and the tumor-node-metastasis staging system, 13 respectively. Prostate adenocarcinoma was identified by microscopic ex- aminations of hematoxylin and eosin-stained slides, and tumor areas were marked with red pen, on tissue blocks. Marked foci were dissected out using a scalpel, placed in microtubes, de- waxed with xylene, and then treated with alcohol. The dried tissue samples obtained were lysed with proteinase K, and DNA was extracted using the classical method of phenol/chloroform/ isoamyl alcohol. Detection of mutation at BRAF codon 600 and KRAS codons 12 and 13 The presences of mutations at codons 12 and 13 of KRAS were determined by enriched PCR-RFLP analysis, according to the method originally described by Kahn et al. 14 and modified by Nagasaka et al. 15 Sequences of the respective sets of primers tar- geting codons 12 and 13 are described in Table II. Briefly, genomic DNA was subjected to PCR amplification utilizing mis- matched oligonucleotide primers targeting exon 2 of KRAS. Amplifications were carried out in a 25-ll reaction volume con- taining genomic DNA (30–50 ng), ‘‘K12 & 13’’ and ‘‘Kwt-R’’ pri- mers (10 pmol each), dNTPs (each at 200 lM), 13 PCR buffer and 0.75 units of Taq polymerase (Intron Corporation. Seoul, Korea). Samples were subjected to 30 PCR cycles with primer Grant sponsor: Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea; Grant number: A050042; Grant sponsor: SNUH Research Fund; Grant number: 03-2005-005; Grant sponsor: BK21 project for Medicine, Seoul, Korea. *Correspondence to: Department of Pathology, Seoul National Univer- sity College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744 South Korea. Fax: 182-2-743-5530. E-mail: ghkang@snu.ac.kr Received 6 January 2006; Accepted 28 March 2006 DOI 10.1002/ijc.22071 Published online 23 May 2006 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 119, 1858–1862 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer