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Polish Journal of Pathology
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1/2010
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BRAF mutations in sporadic colorectal carcinoma from polish patients

Piotr Wójcik
,
Krzysztof Okoń
,
Czesław Osuch
,
Agnieszka Klimkowska
,
Romana Tomaszewska

Pol J Pathol 2010; 1: 23-26
Online publish date: 2010/05/12
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Introduction
The BRAF gene encodes serine/threonine kinase activated by somatic point mutations in human cancers. K-Ras and B-Raf are involved in the transduction of mitogenic signals from the cell membrane to the nucleus. Recent clinical trials indicate that BRAF mutations are associated with resistance to anti-EGFR antibodies in colorectal cancers with wild-type KRAS [1-3]. Moreover, several drugs targeting B-Raf are in development or currently in clinical trials, including selective V600E-mutant inhibitors [4, 5]. Depending on therapeutic approach, BRAF mutations may serve as an exclusion or inclusion criterion; thus they constitute an important predictive factor. Allele-specific PCR is frequently in use for V600E mutation detection based on an assumption that this mutation is predominant; however, rare mutations in other codons are omitted.
Here we present the results of SSCP and sequencing analysis of exons 11 and 15 of the BRAF gene in routinely resected sporadic colorectal cancers previously screened for KRAS mutations

.
Materials and methods

Samples

One hundred and sixty-three unselected sporadic colorectal carcinoma cases were studied. All the patients (91 men and 72 women, aged 34-87 years) underwent surgery at the 1st Department of Surgery, Collegium Medicum, Jagiellonian University, Kraków, Poland. Carcinomas were histologically staged according to the TNM classification. The tumour and normal mucosa samples were cut into small pieces, snap frozen and stored at –80°C for further investigation. DNA was extracted from fresh-frozen tumour and corresponding non-neoplastic tissues according to the manufacturer’s instructions (QIAamp DNA Mini Kit, Qiagen).

Mutation detection
Mutational analysis of BRAF was performed by SSCP and sequencing. PCR products were amplified with RedTaq polymerase (Sigma-Aldrich) using the primers:


5'-TTCTGTTTGGCTTGACTTGACTT
5'-ACTTGTCACAATGTCACCACATT
for BRAF exon 11 [6] and
5'-TCATAATGCTTGCTCTGATAGGA
5'-GGCCAAAAATTTAATCAGTGGA for BRAF exon 15 [7] (annealing temp. 57°C).
Electrophoresis was carried out in TBE buffer (MP Biomedicals) using 80% MDE gels (Cambrex Bio Science). Thermostatically controlled circulating water was used to maintain a constant temperature of 8°C. Gels were run for 18 h with constant 170 V and developed by silver staining. Mutations were confirmed by direct sequencing. The sequence data were collected and analysed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). KRAS mutations were detected by SSCP and sequencing as described in detail previously [8].

MSI analysis
PCR was performed according to the previously reported protocol using a panel of 5 microsatellite markers located near APC, p53, BATRII, BAT26, and BAX [9]. All the cases that demonstrated MSI or LOH in at least 1 microsatellite marker were examined with a panel of 9 microsatellite markers (D2S123, D5S346, D3S1611, D18S35, NM-23, D7S501, D1S2883, TP53-pentanucleotide, TP53-dinucleotide) using a set of primers labelled with fluorescent dyes. The PCR products were separated on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). According to the MSI/LOH results, the cancers were classified into 3 groups: MSS (microsatellite stable cancers, without detectable instability), MSI-high (at least 40% unstable markers), and MSI-low [10].

Results
Mutational analysis performed on 163 sporadic colorectal adenocarcinoma cases revealed BRAF mutations in 6 tumours (3.7%). All of these mutations were missense. Five mutations were localized in exon 15: D594G, G596R, K601N and twice V600E. Only 1 mutation was detected in exon 11 (G469A).
Tumours with mutation in exon 15 were locally advanced (T3 or T4) at the time of resection. The only tumour with mutation in exon 11 was invading a muscular tissue (T2). All were metastatic to lymph nodes without diagnosed distant metastases. A BRAF mutation was detected in only 2 of 14 MSI-H cancers (Fisher’s test MSI-H vs. MSS/MSI-L; p = 0.08). These 2 mutated tumours with a high level of microsatellite instability had typical right-sided location and moderate or poor differentiation. Four others were situated distally to the splenic flexure with stable microsatellites (MSS) or a low level of instability (MSI-L) and moderate to poor differentiation. No mutation was detected in well differentiated tumours, which made up 25% (41/163) of the specimens. Tumours with V600E as well as exon 11 mutations were poorly differentiated. Mucus production characterized 19% (31/163) of collected adenocarcinomas and none of them carried a BRAF mutation whereas 45% (14/31) had a KRAS mutation. Mutated KRAS was detected in 36% (58/163) of the specimens [8]. The BRAF and KRAS mutations were mutually exclusive.

Discussion
Oncogenes BRAF and KRAS play a role in increased activity of the MAPK pathway, which is involved in cancer cell proliferation and migration. Due to mutations, altered B-Raf kinase may influence therapy outcome as it does in EGFR inhibition refractory colorectal cancers. BRAF mutation testing can provide useful information for planning therapy for individual patients. The reported frequency of mutations varies in sporadic colorectal cancers between 3 and 21% (Table II). In our study, the incidence was one of the lowest in unselected samples. A mutation was observed only in 3.7% of the collected tumours.

Allele-specific PCR is usually employed for detection of the most common mutation in codon 600. Interestingly, V600E mutation was not predominant in this sample set: only 2 out of 6 mutations (33%). Yuen et al. reported a similar proportion (36%) [15]. Some authors have analysed 2 exons of the BRAF gene. The percentage of V600E mutations varied between 36% and 95% of the BRAF mutations in such studies (Table III). In our study, exon involvement resembled already published data. Five mutations in exon 15 (codons 594–601) and only 1 in exon 11 (codon 469) were observed. Mutations in exon 11 occurs rarely. Depending on the cohort studied, the percentage of mutations in exon 11 fluctuated between 2% and 18% of all BRAF mutations (Table III).
Many authors have reported a high incidence of BRAF mutations in MSI-H cancers: Maestro et al. 18.4% [12], Lubomierski et al. 27% [27], Rajagopalan et al. 30.6% [20], Asaka et al. 32% [14], Nagasaka et al. 45% [18], Samowitz et al. 52% [19], Ahlquist et al. 62% [28], Barault et al. 63% [23], Goel et al. 71% [26]. The association with a high level of microsatellite instability is well established. However, in the present study, BRAF mutations were relatively rare in MSI-H cancers (14%). The trend towards higher incidence in this group is statistically insignificant in comparison with MSS. No BRAF mutation has been detected in well differentiated carcinomas. An association of mutated BRAF with poor differentiation is sometimes reported [18, 19]. Contrary to published data, none of the 6 tumours with mutation had mucinous histology [19, 30, 31].
Oncogenic mutation in either KRAS or BRAF is usually sufficient for dysregulation of the MAPK pathway in a cancer cell. Coexisting mutations in both genes are rare findings [15, 19]. Our results support this observation. No tumour with mutated BRAF had concomitant mutated KRAS.


Acknowledgement
The work was supported by grant No. PBZ-KBN-091/PO5/2003/24 of the Ministry of Science and Informatics, formerly State Committee of Scientific Research (KBN).

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Address for correspondence
Piotr Wójcik

Chair and Department of Pathomorphology
ul. Grzegórzecka 16
31-531 Kraków
e-mail: piotr.wojcik@uj.edu.pl
Copyright: © 2010 Polish Association of Pathologists and the Polish Branch of the International Academy of Pathology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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