结直肠癌RAS、BRAF基因状态与临床病理特征的关系

目的分析中国人群结直肠癌患者中KRAS、NRAS及BRAF基因突变状态与临床病理特征之间的关系。方法回顾性分析2018年1月—2020年11月于福建省肿瘤医院接受治疗的370例结直肠癌患者的肿瘤组织标本,检测KRAS、NRAS、BRAF基因突变状态及MMR蛋白表达状态,分析基因突变状态与结直肠癌临床病理特征的关系。结果370例患者中存在KRAS基因突变155例(41.9%),NRAS基因突变19例(5.1%),BRAF基因突变40例(10.8%),其中1例患者突变类型为G469V,其他均为V600E突变。KRAS基因在女性、高中分化患者中突变率高(P<0.05)。NRAS基因突变多见于低分化、浸润型患者(P<0.05)。BRAF V600E突变在右半结肠癌、年龄小于50岁、低分化、浸润型、淋巴结及远处转移患者中的突变率高(P<0.05)。结论KRAS基因突变与性别、分化程度有关,NRAS基因突变与分化程度、肿瘤大体分类有关,BRAF基因V600E突变与肿瘤原发部位、年龄、分化程度、肿瘤大体分类、区域淋巴结及远处转移有关。对结直肠癌患者进行KRAS、NRAS及BRAF基因的检测,为了解中国患者热点基因突变分布情况和临床精准诊疗提供理论依据。     

TP53 Mutations in Myelodysplastic Syndrome

Mutations of the TP53 tumor suppressor gene, contributing to the development and progression of a wide variety of human malignancies, are found in some of the patients with myelodysplastic syndromes (MDS). Previous reports revealed that TP53 mutations were found in 0-25% of patients with MDS and are closely associated with a complex abnormal karyotype including such chromosomal losses as -5/5q-, -7/7q- and/or 17p-, which are known to be frequent in therapy-related leukemias. We have also detected TP53 mutation in 10 (14%) of 70 patients with MDS. All of the mutations were detected at the time of diagnosis, which suggest the TP53 mutation may play a role in the development of MDS. Those patients with a TP53 mutation had a poor prognosis regardless of leukemic transformation or not.

The reported mutational spectra of TP53 in MDS and ANLL differ from those of colon and lung cancers. Compared with other hematological disorders, the spectrum of TP53 mutations in MDS and ANLL is assumed to be associated with pathogenic exposure to known or unknown carcinogens, as suggested by the chromosomal findings. Further studies are required to clarify the pathogenesis of this heterogenous disease entity.     

Rapid detection of exon 1 NRAS gene mutations using universal heteroduplex generator technology

Specific NRAS oncogene missense mutations have been frequently found in some tumors and several hematological diseases, especially in those of myeloid origin. There is a wide range of PCR-based methods for screening and detection of NRAS exon 1 single-base substitutions. However, there are disadvantages and ambiguities associated with these techniques because all of them require either separate probes, separate PCR amplifications, or complicated post-PCR manipulations. This report describes a new approach for detection of NRAS gene mutations at codon 12 and 13 based on the DNA heteroduplex analysis method. The strategy relies upon differential electrophoretic behavior of induced heteroduplex molecules formed by cross-hybridization of two PCR-amplified species, the sample under analysis and the synthetic universal heteroduplex generator (UHG). The screening of a panel of all codon 12 and 13 NRAS mutant DNA variants indicated that this approach discriminates all 12 relevant mutations. The sensitivity of the method was estimated by a competitive assay where mutant alleles could be detected at a dilution level of 1 to 16 wild-type alleles. This UHG technology was tested on some clinical samples previously studied by PCR-ASO. This methodology is highly specific, sensitive, and achieves an appreciable reduction in workload and time because it requires one PCR amplification followed by polyacrylamide gel electrophoresis in standard conditions. We propose that this new approach may be applied as an alternative strategy for codon 12-13 NRAS mutations and it could be easily incorporated into the range of routine assays performed in oncology laboratories.     

Loss of heterozygosity of chromosome 12p does not correlate with KRAS mutation in non-small cell lung cancer

Activating mutations of RAS gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. However, several studies have shown a frequent loss of the wild-type KRAS allele in the tumors of murine models and an inhibition of oncogenic phenotype in tumor cell lines by transfection of wild-type RAS, indicating that wild-type RAS may have oncosuppressive properties. To determine whether loss of wild-type KRAS is involved in the development of human lung cancer, we investigated the mutations of KRAS, NRAS and BRAF in 154 primary non-small cell lung cancers (NSCLCs) as well as 10 NSCLC cell lines that have been shown to have KRAS mutations. We also determined the loss of heterozygosity status of KRAS alleles in these tumors. We detected point mutations of KRAS in 11 (7%) of 154 NSCLCs, with 10 cases at codon 12 and 1 at codon 61, but no mutations of NRAS or BRAF were found. Using the laser capture microdissection technique, we confirmed that 9 of the 11 tumors and 7 of the 10 NSCLC cell lines retained the wild-type KRAS allele. Among the cell lines with heterozygosity of mutant and wild-type KRAS, all of the cell lines tested for expression were shown to express more mutated KRAS than wild-type mRNA, with higher amounts of KRAS protein also being expressed compared to the cell lines with a loss of wild-type KRAS allele. In addition, among 148 specimens available for immunohistochemical analysis, 113 (76%) showed positive staining of KRAS, indicating that the vast majority of NSCLCs continue to express wild-type KRAS. Our findings indicate that the wild-type KRAS allele is occasionally lost in human lung cancer, and that the oncogenic activation of mutant KRAS is more frequently associated with an overexpression of the mutant allele than with a loss of the wild-type allele in human NSCLC development.     

Study of promoter hypomethylation profiles of RAS oncogenes in hepatocellular carcinoma derived from hepatitis C virus genotype 3a in Pakistani population

Epigenetic modifications such as DNA methylation contribute to progression of hepatitis C virus (HCV) infection to life‐threatening hepatocellular carcinoma (HCC) by promoting the silencing of tumor suppressor genes through DNA hypermethylation and by causing genomic instability through global hypomethylation. However few studies have addressed the promoter region hypomethylation status of the oncogenes involved in HCV derived HCC. In this study, we analyzed the promoter region methylation pattern of RAS oncogenes (HRAS, KRAS, and NRAS) using methylation‐specific PCR for 50 chronic HCV patients infected with genotype 3a (27 HCC patients and 23 control non‐HCC patients). Methylation‐specific polymerase chain reaction analysis revealed that the NRAS oncogene promoter (P = .0025) was significantly hypomethylated in HCC patients compared to the non‐HCC patients suggesting its contribution to the progression of HCV towards HCC. To identify the agent for alteration in the RAS oncogene expression, 7 HCV genes were expressed in the Huh‐7 cell line followed by measurement of the NRAS expression level in Huh‐7 by a quantitative real‐time polymerase chain reaction. An increase in the messenger RNA level of the NRAS gene was detected when Huh‐7 were transfected with Core, NS5a, and NS2 genes. Our findings suggest the involvement of NRAS oncogene in the pathogenesis of HCV3a derived HCC in Pakistani population and also identifies the HCV genes responsible for its enhanced expression. Our study raises the hypothesis that a single HCV gene may increase the chances of malignancy. Therefore, our study may have identified a useful epigenetic biomarker of HCC progression in HCV patients and may help to develop novel diagnostic tools.