Velho Sérgia, Moutinho Cátia, Cirnes Luís, Albuquerque Cristina, Hamelin Richard, Schmitt Fernando, Carneiro Fátima, Oliveira Carla, Seruca Raquel
Institute of Molecular Pathology and Immunology, University of Porto, Portugal.
BMC Cancer. 2008 Sep 9;8:255. doi: 10.1186/1471-2407-8-255.
BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied.
Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies.
KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191).
Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated polyps and MSI CRC show a similar frequency of BRAF mutations. These results support that BRAF mutations harbour a mild oncogenic effect in comparison to KRAS and suggest that BRAF mutant colorectal cells need to accumulate extra epigenetic alterations in order to acquire full transformation and evolve to MSI CRC.
BRAF、KRAS和PIK3CA突变在散发性结直肠癌（CRC）中经常被发现。与KRAS和PIK3CA突变不同，BRAF突变与具有CpG岛甲基化表型（CIMP）、MLH1甲基化和微卫星不稳定性（MSI）的肿瘤相关。我们旨在通过一系列结直肠息肉和癌来确定KRAS、BRAF和PIK3CA突变在结直肠肿瘤发生过程中的频率。在该系列息肉中，还研究了CIMP、MLH1甲基化和MSI。
通过PCR/测序进行突变分析。经亚硫酸氢盐处理的DNA用于研究CIMP和MLH1甲基化。通过五重PCR和准单态单核苷酸重复序列的基因扫描分析检测MSI。使用卡方检验和Fisher精确检验进行关联研究。
KRAS、PIK3CA或BRAF突变出现在71%的息肉中，且相互排斥。KRAS突变出现在35%的息肉中。在其中一个息肉中发现了PIK3CA突变。V600E BRAF突变出现在29%的病例中，所有这些病例均被分类为锯齿状腺瘤。CIMP表型出现在25%的息肉中，且所有息肉均有BRAF突变。未检测到MLH1甲基化，所有息肉的微卫星均稳定。息肉和CRC（MSI和MSS）中致癌突变频率的比较使我们证明KRAS和PIK3CA可能先于这两种类型的CRC出现。BRAF突变可能先于MSI癌出现，因为在锯齿状息肉中发现的频率与在MSI CRC中发现的频率相似（P = 0.9112），但与在微卫星稳定（MSS）肿瘤中发现的频率有统计学差异（P = 0.0191）。
我们的结果表明，BRAF、KRAS和PIK3CA突变在恶性转化之前就已发生，这表明这些致癌改变是结直肠癌发生过程中的主要遗传事件。此外，我们表明BRAF突变与结直肠锯齿状息肉中的CIMP表型相关，并证实结直肠锯齿状息肉和MSI CRC中BRAF突变的频率相似。这些结果支持与KRAS相比，BRAF突变具有较轻的致癌作用，并表明BRAF突变的结直肠癌细胞需要积累额外的表观遗传改变才能实现完全转化并演变为MSI CRC。
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