Sarkar F H, Valdivieso M, Borders J, Yao K L, Raval M M, Madan S K, Sreepathi P, Shimoyama R, Steiger Z, Visscher D W
Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Diagn Mol Pathol. 1995 Dec;4(4):266-73. doi: 10.1097/00019606-199512000-00007.
The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.
人们发现，几乎在所有人类实体瘤中，p53肿瘤抑制基因都发生了改变，而K-ras基因突变仅在少数人类癌症（结肠癌、胰腺癌和肺癌腺癌）中被观察到。对同一非小细胞肺癌患者样本中的这两个基因进行突变失活研究的报道较少。为了进行此类分析，我们开发并比较了一些方法（用于p53和K-ras基因的突变检测），这些方法在DNA提取、聚合酶链反应（PCR）扩增以及非放射性PCR-单链构象多态性（PCR-SSCP）分析方面代表了一种经过改进的通用方案，该方案可轻松应用于福尔马林固定、石蜡包埋组织或冷冻肿瘤标本。我们将此方法应用于55例非小细胞肺癌患者p53（外显子5 - 8）和K-ras（密码子12和13）基因突变的评估。通过放射性PCR-SSCP评估p53基因的突变状态，并与使用我们标准化非放射性检测系统、利用单个6微米组织切片的PCR-SSCP进行比较。通过PCR-SSCP观察到的突变模式随后通过PCR-DNA测序得到证实。K-ras基因的突变状态同样通过PCR-SSCP进行评估，特定突变通过使用针对密码子12和13的32P标记的序列特异性寡核苷酸探针进行Southern斑点杂交来确认。在55例非小细胞肺癌患者中，有10例（18%）发现了K-ras（密码子12）的突变。腺癌在密码子12处的K-ras突变率为33%，而在密码子13处仅发现1例突变。正如预期的那样，鳞状细胞癌样本（25例）未显示K-ras突变。在55例患者中有19例（34.5%）记录到p53基因外显子5 - 8的突变。19例突变中有10例为单核苷酸点突变，导致氨基酸替换。6例显示插入突变，3例显示缺失突变。只有3个样本同时显示K-ras和p53基因的突变。我们得出结论，尽管K-ras和p53基因突变在非小细胞肺癌中很常见，但同一患者样本中两个基因同时发生突变并不常见。我们还得出结论，这种通用的非放射性方法优于其他类似方法，并且易于应用于对大量福尔马林固定、石蜡包埋或冷冻样本进行多个基因的突变分析的快速筛查。
