结直肠粘膜隐窝异常病灶K-ras、APC基因突变的研究

目的　分析隐窝异常病灶（aberrant crypt foci,ACF）的基因水平变化特点及其作为结直肠癌最早期形态变化的分子依据,探讨ACF与腺瘤之间的关系。方法　经微解剖分离提取15例正常粘膜腺体、34例ACF、15例腺瘤和35例结直肠癌组织进行DNA测序,检测K-ras第12、13、61密码子和APC第15外显子“突变密集区”的突变。结果　正常粘膜无K-ras和APC基因突变,ACF、腺瘤和癌组织K-ras突变率分别为17.6%（6/34）、13.3%（2/15）和14.3%（5/35）,3者突变率相近。4例ACF、腺瘤和癌组织的突变位于12密码子的第2碱基（GGT→GAT）,2例ACF突变发生在13密码子的第2碱基（GGC→GAC）。ACF中K-ras的突变特点与同来源的癌组织保持一致,即患者年龄较大,大体以隆起型为主,所有组织未发现有61密码子的突变。癌和腺瘤APC基因突变率相近,癌为22.9%（8/35）,腺瘤为26.7%（4/15）明显高于ACF的2.9%（1/34,P<0.05),癌组织APC与患者的年龄、肿瘤位置、大体类型、组织分化程度无关。结论　ACF可能是结直肠癌最早期的形态改变,ACF的形态学变化、部分基因的改变均有别于腺瘤,提示（1）ACF有可能是腺瘤前的形态改变;（2）结直肠癌的发生可能存在“正常上皮→ACF→癌变”这一途径。     

Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung

To define the role of cellular oncogenes in human cancers, we studied the prevalence of mutational activation of ras oncogenes in untreated non-small-cell lung cancer. Genomic DNA was extracted from 39 tumor specimens obtained by thoracotomy and was examined for activating point mutations in codons 12, 13, and 61 of the H-ras, K-ras, and N-ras genes. A novel, highly sensitive assay based on oligonucleotide hybridization following an in vitro amplification step was employed. The K-ras gene was found to be activated by point mutations in codon 12 in 5 of 10 adenocarcinomas. Two of these tumors were less than 2 cm in size and had not metastasized. No ras gene mutations were observed in 15 squamous-cell carcinomas, 10 large-cell carcinomas, 1 carcinoid, 2 metastatic adenocarcinomas from primary tumors outside the lung, and 1 small-cell carcinoma. An approximately 20-fold amplification of the unmutated K-ras gene was observed in a tumor that proved to be a solitary lung metastasis of a rectal carcinoma. We conclude that mutational K-ras activation may be an important early event in the pathogenesis of adenocarcinoma of the lung but that amplification of ras genes or mutational activation of H-ras or N-ras does not play a major part in non-small-cell lung cancer.     

The role of p21ras in pancreatic neoplasia and chronic pancreatitis.

K-ras mutations have been detected in both ductal cell carcinoma and intraductal papillary mucinous tumor (IPMT) of pancreas. The frequency of this mutation in ductal cell carcinoma is high, whereas in IPMT, it is variable. It has been suggested that the relatively high frequency of this mutation in ductal cell carcinomas compared with IPMT may account for the differences in biological behavior between these tumor types. More recently, the significance of K-ras mutations in pancreatic tissue has been questioned with the demonstration of this mutation in nonneoplastic pancreata. The current study aims to estimate the relative frequency and evaluate the biological significance of K-ras gene mutations in these neoplasms by performing polymerase chain reaction (PCR) assays of microdissected areas of IPMT, ductal cell carcinomas, and resected chronic pancreatitis. The study also investigates whether alterations of p21ras occur in K-ras mutation-negative cases by using immunohistochemical staining for K-, N- and H-ras. K-ras codon 12 mutations were found almost as frequently in IPMT (71%) as in ductal cell carcinomas (78%). They were also associated with the earliest morphological lesion, flat mucinous change. This mutation also was detected in 42% of cases of chronic pancreatitis. Expression of p21ras was found to correlate closely with K-ras mutation status in IPMT and ductal cell carcinoma. Negative staining for pan-ras, H-ras, and N-ras in cases with wild-type K-ras genes suggests that alternative routes of ras gene alteration are not operative in IPMT or ductal carcinoma. The findings suggest that K-ras activation is frequently associated with both IPMT and ductal cell carcinoma. Its high prevalence in nonneoplastic pancreata suggests that it is also associated with self-limited morphological lesions of the pancreas that do not progress to malignancy.     

ras gene mutations in salivary gland tumors

OBJECTIVE: To assess the prevalence of activating mutations in K-ras and H-ras genes in salivary gland tumors with ductal or acinar differentiation and to evaluate their potential correlation with clinical parameters. DESIGN: Paraffin-embedded tissue samples of salivary gland carcinomas were investigated by the application of a direct sequence analysis procedure with automated DNA sequencing of polymerase chain reaction-amplified ras sequences. SETTING: Tertiary care teaching hospital. PATIENTS: Twenty-four patients with salivary gland carcinoma were surgically treated. Nine had adenocarcinoma, 1 had adenosquamous carcinoma, 11 had mucoepidermoid carcinoma, and 3 had acinic cell carcinoma. RESULTS: Point mutations were detected in 7 (29%) of the 24 carcinomas examined. The K-ras gene was mutated in only 2 samples (8%): a GGC-to-ATC mutation at codon 13 in an adenocarcinoma and a GGC-to-GTC transversion mutation at codon 13 in a mucoepidermoid carcinoma. Five (21%) harbored H-ras mutations: 4 contained a GGC-to-GTC transversion mutation at codon 12 and 1 had 2 distinct mutations, the same G-to-T at codon 12 as was shown in the other cases and a GGT-to-GGA heterozygous mutation at codon 13. All the H-ras mutations were in the group of mucoepidermoid carcinoma lesions (45%; 5/11). CONCLUSION: Our data suggest that K-ras gene alteration is probably not an important factor in the oncogenesis of human salivary gland tumors. However, mutational activation of the H-ras gene appears to play a role in the development and/or progression of salivary gland mucoepidermoid carcinomas.     

K-ras基因突变在内蒙古地区结直肠癌患者中研究

目的检测内蒙地区结直肠癌K-ras基因突变情况,并结合临床病理资料加以分析。方法提取15例结直肠癌患者结直肠癌手术切除标本组织的DNA,对产物进行基因序列癌组织的DNA聚合酶链反应（PCR）扩增、DNA直接测序分析。结果 K-ras基因突变率为0%,几种分化型的结直肠癌均未发现K-ras基因突变类型,包括12密码子（GGT）、13密码子（GGC）。结论我院结直肠癌患者k-ras基因突变率为0%,转移性结直肠癌患者原发肿瘤与转移灶肿瘤k-ras基因型均相同;结直肠癌患者k-ras基因突变与否与年龄、性别、肿瘤浸润深度、肿瘤组织学类型无关。     

K-ras mutation detection in colorectal cancer using the Pyrosequencing technique

The identification of gene mutations is a critical goal for the assessment of diagnosis and prognosis in cancer disease, particularly by direct sequencing. Pyrosequencing is a straightforward, non-electrophoretic DNA sequencing method using the luciferase-luciferin light release as a signal for nucleotide incorporation into a PCR template DNA. In this study, we aimed to investigate mutations in the K-ras gene using Pyrosequencing technology, because its reliable chemistry and robust detection mechanism allow for rapid, real-time detection of sequencing events. For the simultaneous detection of the predominant K-ras codons 12 and 13 mutations, we established a sequencing protocol based on the design of a single PCR primer pair and a single sequencing primer. The assay has been validated with DNA from 65 colorectal carcinomas. Furthermore, analysis of the rare K-ras codon 61 mutation was included. In 29% (19/65) of the patients, the K-ras gene was found to be mutated, whereas codons 12 and 13 were most frequently affected (18/65, 27.7%). Mutations with the highest frequency were G-->A transitions (12/19, 63%), followed by G-->T transversions (5/19, 26%). Overall survival was significantly shorter in patients with a tumor containing K-ras codon 12 mutations than in those without K-ras codon 12 mutations (p=0.024). In conclusion, we found Pyrosequencing to be a suitable technology for fast detection of hot-spot mutations in the K-ras oncogene. We demonstrated an important relationship between K-ras codon 12 mutations and overall survival in colorectal cancer patients.     

K-ras mutations are correlated to lymph node metastasis and tumor stage, but not to the growth pattern of colon carcinoma

In colorectal carcinoma, pathological assessment of tumors is essential for determining therapy and prognosis of the disease. Molecular associations of tumor complexity index and genetic alternations can be helpful to understand the tumor progression mechanism. Oncogenic K-ras is one of the major colorectal cancer associated genes, and is mutated in up to 50% of colorectal cancers. In this current study, we correlated tumor complexity index with mutations in K-ras codon 12, 13, and 61 in association with different clinicopathological parameters such as TNM stage, localization, sex, and age. Formalin-fixed paraffin embedded tissue blocks from colon cancer samples was selected from 88 patients diagnosed with adenocarcinoma. Mutations in the K-ras gene were detected using pyrosequencing technique. Tumor complexity index was calculated using immunohistochemically stained images of the tumor outline of the specimens and then analyzing these pictures using Photoshop CS, Fovea Pro, and Image J computer programs. Statistical analysis was performed with SPSS. K-ras mutations were detected in 17 (19.3%) colon cancer samples. Most of the samples were at a lower complexity index. No correlation was observed between K-ras mutations and complexity index. However, K-ras mutations were correlated with regional lymph node metastasis and tumor stages and complexity index with tumor wall penetration. In conclusion, complexity index and K-ras mutations are independent events; however, both correlate with tumor progression and are important in the biologic development of colon carcinoma.     

H-ras and K-ras gene mutations in primary human soft tissue sarcoma: concomitant mutations of the ras genes.

ras gene mutations have been described with varying frequency in several types of human malignancies. To determine the incidence and type of ras mutations in human soft tissue tumors, we studied 45 sarcomas, including 27 malignant fibrous histiocytomas (MFHs), 10 liposarcomas, 2 rhabdomyosarcomas, and 6 leiomyosarcomas. Al of the tumors were investigated by direct sequence analysis with the automated DNA sequencing of polymerase chain reaction-amplified ras sequences. Twenty (44%) of the sarcomas examined harbored K-ras mutations, 18 (90%) of which were MFHs. All of the K-ras mutations were G-to-A transition mutations in the second position of codon 13 (glycine --> aspartic acid). Of the samples with K-ras activation, 7 (16% of the total of 45 tumors), including 6 MFHs and 1 leiomyosarcoma, also contained H-ras mutation. All of the tumors that showed H-ras alteration had G-to-T transversion mutations in the second base of codon 12 (glycine --> valine). These data possibly implicate that ras gene activation may be a relatively uncommon event in soft tissue tumors, with the exception of MFH. It is suggested that the oncogenic process underlying the development of tumors between these groups may be different and that ras gene mutations may play a role in the etiology and/or progression of MFH. It is noteworthy that when ras gene activation occurs in sarcoma, it predominantly affects the K-ras gene, particularly codon 13. Moreover, H-ras mutations in our samples were detected only in association with tumors that also displayed K-ras-mutated genes. This study demonstrates for the first time concomitant mutations in two different members of the ras gene family in sarcoma     

APC and p53 mutations in de novo colorectal adenocarcinomas

To investigate genetic features in small and flat colorectal carcinomas that arise de novo, we searched for genetic alterations in six sporadic tumors by examining their APC, K-ras, and p53 genes. Two of the six tumors carried detectable mutations within the mutation cluster region (MCR) of the APC gene; both mutations were predicted to cause truncation of the gene product. Four tumors carried mutations of the p53 gene; three were missense mutations in exon 5, and the other was a 3-bp deletion in exon 6. However, neither codon 12 nor codon 13 of K-ras contained detectable mutation in any tumors. Hence, as “adenoma-carcinoma sequence” model of development of colorectal carcinoma, inactivation of the APC and p53 genes appear to be Involved in development of the de novo type of colorectal carcinoma even though the adenoma stage is not observed. © 1994 Wiley-Liss, Inc.     

K-ras基因突变与结直肠癌生物学行为的关系

目的: 观察结直肠癌组织中k-ras基因突变情况,探讨k-ras基因突变与结直肠癌生物学行为的关系。方法: 采用实时荧光定量PCR法检测123例结直肠癌组织中k-ras基因1号外显子12、13密码子突变情况,结合其临床病理资料分析。结果: 123例结直肠癌组织中k-ras基因突变者53例(40.8%),其中12密码子突变42例(34.1%),13密码子突变者11例(8.9%)。基因突变率与肿瘤大小、肿瘤侵润深度、分化程度无明显相关性,与淋巴结转移、肝脏转移及TNM分期有相关性（P<0.05）。淋巴结转移多者k-ras基因突变率高,有肝脏转移者基因突变率高,TNM分期越晚基因突变率越高。结论: K-ras基因突变可能在结直肠癌的发生、发展中起重要作用,而且与淋巴结转移和肝脏转移有密切相关,可作为判断结直肠癌恶性程度的一个分子生物学指标。     

Lack of ras mutations and prediction of long-term survival in carcinoma of the colon.

Ras oncogene mutations are found in a significant number of human colonic carcinomas. Correlation between patient survival and ras mutations was explored and compared with other clinical parameters. Colonic carcinoma embedded in paraffin was subjected to the polymerase chain reaction using primers to amplify codon 12 of the K-ras oncogene. Oligonucleotide probes to six mutations were used to identify mutated genes. A total of 71 cases were successfully amplified. Some 54% of the cases had mutations. There was no correlation between presence of a mutation and age. Patients in Stage D, patients with a family history of carcinoma, and males have a greater incidence of ras mutations. Patients in Stage C had a lower incidence of mutations. Presence of the mutation did not correlate with decreased survival except in Stage D. Some 61% of long-term survivors with colon carcinoma living for more than 6 yr had ras mutations. The absence of K-ras mutations did not identify long-term survivors.     

K-ras mutations and p53 alterations in neoplastic and nonneoplastic lesions associated with longstanding ulcerative colitis.

We have analyzed K-ras mutations and p53 alterations in 39 tumor and nontumor samples taken from nine patients with longstanding ulcerative colitis and colorectal carcinoma. Two of nine invasive carcinomas contained a K-ras mutation. By a combination of immunohistochemistry and single-strand conformation polymorphism analysis, p53 alterations were found in three of nine carcinomas. Five of 13 dysplastic lesions harbored a mutated K-ras gene, even in the absence of detectable changes in associated invasive tumors. One single focus of dysplastic mucosa harbored concomitant K-ras and p53 gene alterations. In two patients, a K-ras mutation was detected in epithelial lesions considered to be devoid of malignant potential (villous regeneration, active colitis). Our results indicate that: 1) the prevalence of K-ras and p53 genetic alterations found in ulcerative colitis-associated colonic carcinomas appears to be lower than in sporadic carcinomas; 2) K-ras mutations can be detected in dysplasia, villous regeneration, and active colitis and affect a subpopulation of the cells composing the lesions; 3) diverse genetic alterations can be detected in the same patient and the dysplastic lesions can exhibit a different genotype than the carcinomas; and 4) at least part of active colitis and villous regeneration lesions should be considered as preneoplastic in ulcerative colitis.     

Polymerase chain reaction/sequencing analysis of ras mutations in paraffin-embedded tissues as compared with 3T3 transfection and polymerase chain reaction/sequencing of frozen tumor deoxyribonucleic acids.

The efficiency of detection of H- and K-ras mutations in 27 CD-1 mouse liver tumors by direct sequencing of polymerase chain reaction (PCR)-amplified DNA isolated from formalin-fixed and paraffin-embedded tissues was compared with that after assay by both NIH 3T3 transfection (followed by sequencing of amplified transformant DNA) and direct sequencing of PCR-amplified DNA isolated from frozen tumors. Some tumor samples were chosen for comparison because they contained ras mutations that were detected by either NIH 3T3 transfection or sequencing of PCR-amplified DNA derived from frozen tumors, but were not detected by both techniques. The efficiency of detecting K-ras mutations was similar for sequencing of amplified fragments derived from both paraffin-embedded tissues and from frozen tumors. However, these two techniques differed in their efficacy for detection of H-ras codon 61 mutations. Furthermore, this difference appeared to be mutation-specific: the sequencing of amplified products from paraffin-embedded tumor tissues allowed increased detection of CAA to AAA mutations but decreased detection of CAA to CTA mutations relative to sequencing of amplified fragments derived from frozen tumor DNA. Direct sequencing of PCR products from paraffin-embedded sections was more sensitive than NIH 3T3 transfection for detection of activated K-ras genes containing codon 13 mutations but less sensitive for detection of activated H-ras genes containing codon 61 mutations. In summary, direct sequencing of amplified DNA from either frozen tumors or formalin-fixed, paraffin-embedded tissues can be more sensitive than NIH 3T3 transfection for detection of codon 13-activated K-ras genes. However, it appears to be less sensitive than NIH 3T3 transfection for detection of certain activating H-ras mutations. Depending upon the questions being asked of the data, each of the methods can provide useful information about ras gene mutations in tumor samples. The apparent differences in sensitivities between the methods is not yet understood, but such differences should be considered in the analysis of data obtained when only one method is used.     

Concurrent mutations of coding and regulatory sequences of the Ha-ras gene in urinary bladder carcinomas.

This report concerns the study of Ha-ras gene mutations and ras p21 expression in primary tumors of the urinary bladder. Polymerase chain reaction-based techniques and computerized image analysis were used. The data obtained were related to tumor grade, DNA ploidy, and tumor invasion. A point mutation (G-->T) at Ha-ras codon 12 was found in 30 of 67 tumors. The mutation frequency was greater in grade III (65%) than in grade II (44%) tumors; no mutations were observed in grade I tumors. The mutation was observed more often in aneuploid (58%) than in diploid (28%) tumors. No other substitution at codon 12 was seen and no codon 61 mutation was detected. The tumors were also tested for the A-->G mutation at position 2719 of Ha-ras intron D. Concurrent codon 12 and intron D mutations were identified in seven high-grade aneuploid tumors; six were invasive. The levels of the ras gene product p21 were approximately 10 times higher in tumors with intron D mutation than in those without. These findings confirm on human bladder tumors the observations of the effect of synchronous exon-intron mutations reported on the bladder cancer cell line T24. Our results are the first demonstration of Ha-ras intron D alterations in human tumor tissues and suggest that concurrent mutations at codon 12 and intron D of this gene within the same tumor may contribute to the aggressive behavior of human bladder carcinomas.     

Clinical significance of K-ras oncogene activation in ampullary neoplasms.

AIMS: To investigate the prevalence of K-ras codon 12 point mutations in ampullary neoplasms, to explore their clinical usefulness, and to test whether the detection of these mutations could be used to identify ampullary malignancies at an early stage. METHODS: Forty one tumour specimens from 28 patients with ampullary neoplasms were analysed for activating point mutations in K-ras codon 12 using a sensitive polymerase chain reaction (PCR) based assay. RESULTS: Eleven (39%) of the 28 primary tumours harboured point mutations in K-ras. Mutations were identified in seven (41%) of the 17 carcinomas and four (36%) of the 11 adenomas. Four of the possible six permutations in codon 12 were found in these 11 samples. This spectrum of mutations is different from pancreatic carcinoma but resembles that of colorectal neoplasms. Cytological brush specimens were available in 11 cases, and in all of these specimens, the K-ras status in the primary tumour and brush specimens was identical. CONCLUSIONS: K-ras codon 12 point mutations occur in about 40% of ampullary neoplasms at a relatively early stage in tumorigenesis. The pattern of mutations in these tumours resembles that of the adenoma-carcinoma sequence in the colorectum. These results indicate that ampullary neoplasms can be detected at an early stage by searching for genetic alterations in the K-ras oncogene in cytological brush specimens.     

Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years

1,3-Butadiene is a multisite carcinogen in rodents. Incidences of cardiac hemangiosarcomas were significantly increased in male and female B6C3F1 mice that inhaled 1,3-butadiene (BD) for 2 years. Eleven BD-induced cardiac hemangiosarcomas were examined for genetic alterations in ras protooncogenes and in the p53 tumor suppressor gene. Nine of 11 (82%) BD-induced hemangiosarcomas had K-ras mutations and 5 of 11 (46%) had H-ras mutations. All of the K-ras mutations were G-->C transversions (GGC-->CGC) at codon 13; this pattern is consistent with reported results in BD-induced lung neoplasms and lymphomas. Both K-ras codon 13 CGC mutations and H-ras codon 61 CGA mutations were detected in 5 of 9 (56%) hemangiosarcomas. The 11 hemangiosarcomas stained positive for p53 protein by immunohistochemistry and were analyzed for p53 mutations using cycle sequencing of polymerase chain reaction (PCR) amplified DNA isolated from paraffin-embedded sections. Mutations in exons 5 to 8 of the p53 gene were identified in 5 of 11 (46%) hemangiosarcomas, and all of these were from the 200- or 625-ppm exposure groups that also had K-ras codon 13 CGC mutations. Our data indicate that K-ras, H-ras, and p53 mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of BD and that these mutations may play a role in the pathogenesis of BD-induced cardiac hemangiosarcomas in the B6C3F1 mouse.     

大肠腺瘤与腺癌P21,P53,P185蛋白表达及ras,p53基因突变的检测

应用免疫组化及ＰＣＲ－ＲＦＬＰ法检测３０例大肠腺瘤、７４例大肠腺癌及癌旁粘膜Ｐ２１、Ｐ５３、Ｐ１８５蛋白表达及ｒａｓ、ｐ５３基因突变。结果表明，大肠腺瘤Ｐ２１、Ｐ５３蛋白阳性率（５３．３％，２７．６％）高于癌旁粘膜（Ｐ＜０．０１），二者过度表达与腺瘤恶性倾向有关。大肠腺癌Ｐ２１、Ｐ５３及Ｐ１８５蛋白阳性率（７２．９％，３７．８％，４７．２％）皆高于腺瘤Ｉ级不典型增生（Ｐ＜０．０１，Ｐ＜０．０５及Ｐ＜０．０５）。９例腺瘤，４０例腺癌存在两种以上蛋白表达，共同表达与腺瘤恶性倾向及腺癌预后有关。大肠腺瘤及腺癌ｒａｓ基因突变率分别为２６．７％及４１．９％，ｒａｓ基因突变与腺瘤恶性倾向有关。大肠腺瘤及腺癌ｐ５３基因２４８位点突变率分别为３．３％及１４．９％。６例腺癌存在两种基因突变，两种基因协同突变与大肠癌预后有关。提示ｒａｓ、ｐ５３及ｃ－ｅｒｂＢ－２基因改变均参与了大肠癌的发生、发展过程。     

Role of ras mutation in the progression of thyroid carcinoma of follicular epithelial origin

The histological differentiation of thyroid carcinoma is known to correlate with prognosis. Ras oncogene mutations, which have been identified in various human cancers, have been suspected playing an important role in carcinogenesis and tumor progression. The purpose of this study was to clarify the mechanism of thyroid tumor progression, focusing on ras oncogenes. We examined ras mutations using nested polymerase chain reaction (PCR) and direct sequencing methods. The ras oncogene product was also examined immunohistochemically. Our results indicated that the incidence of ras mutations correlated with the histological differentiation of thyroid cancer. Three poorly differentiated carcinomas showed a higher rate of ras mutations than did 17 well-differentiated counterparts. Hot spots were not identified except for a relative accumulation of the N-ras gene at codon 61. There was a correlation between the immunoreactivity of the ras oncogene product and ras mutation, although the immunoreactivity of ras-p21 did not correlate with the histological differentiation. Mutation of the ras gene seemed to be one of the important events in the progression from well-differentiated carcinoma to poorly differentiated thyroid carcinoma.     

卵巢浆液性交界及恶性肿瘤K-ras基因突变分析

目的探讨K-ras基因在卵巢浆液性交界及恶性肿瘤发生发展过程中的作用。方法收集51例卵巢浆液性肿瘤标本,包括经典型交界性浆液性肿瘤18例,微乳头型交界性浆液性肿瘤11例,浸润性微乳头型浆液性癌12例,经典型浆液性癌10例。采用显微切割技术获取肿瘤细胞后,提取基因组DNA、PCR技术扩增K—ras基因第一外显子,通过直接测序的方法鉴定K—ras基因第12、13密码子的突变情况。结果1例经典型交界性浆液性肿瘤K—ras基因第12密码子发生突变,突变类型为GGT→GTT即甘氨酸→缬氨酸,余50例标本未见突变;所有标本K—ras基因第13密码子均为野生型。结论K—ras基因第12、13密码子在被检患者中卵巢浆液性交界及恶性肿瘤中的突变频率很低,其在该肿瘤发生发展过程中可能不起主要作用。     

特异引物双扩增即时PCR与传统测序法检测肠癌、肺癌患者K-ras基因突变的比较

目的 以特异引物双扩增即时PCR技术K-ras检测试剂盒(下称ADx-K-ras即时PCR试剂盒)和Sanger DNA测序法同时检测结直肠癌和肺癌患者K-ras基因,以了解K-ras基因突变频率和突变类型,比较ADx-K-ras即时PCR试剂盒和Sanger DNA测序法用于肿瘤K-ras基因体细胞突变检测的临床价值.方法 收集临床肿瘤病理石蜡切片样品827例,其中肠癌样品583例,肺癌样品244例,提取DNA后,对K-ras基因第12和13密码子进行PCR扩增后,使用ADx-K-ras即时PCR试剂盒进行检测,与此同时,将PCR扩增后的产物进行Sanger DNA测序.两种方法对K-ras基因第12和13密码子的检测结果进一步进行各个突变类型的数目和突变率的统计对比.结果 ADx-K-ras即时PCR试剂盒对827例样品检测都得到了明确的结果,检测成功率为100%,Sanger DNA测序成功检测了677例,成功率为81.9%.583例肠癌样品中ADx-K-ras即时PCR试剂盒检测出突变192例,突变检出率为32.9%,Sanger DNA测序成功的样品533例,检出突变160例,突变检出率为30.0%.244例肺癌样品中ADx-K-ras即时PCR试剂盒检出突变26例,突变检出率为10.7%,Sanger DNA测序成功的样品144例,检出突变12例,突变检出率为8.3%.肠癌中第12密码子第2位的GGT→GAT最常见,占全部突变的35.1%(66/188),其次是第13密码子第2位的GGC→GAC,26.6%(50/188),第12密码子第2位的GGT→GTT,18.6%(35/188),第12密码子第1位的GGT→GCT最少见,1.6%(3/188).肺癌中第12密码子第1位的GGT→GTT最常见,占全部突变的40.9%(9/22),同样第12密码子第1位的GGT→GCT最少见,占全部突变的4.5%(1/22).结论 肠癌中K-ras突变率明显高于肺癌.对于甲醛固定石蜡包埋样品而言,ADx-K-ras即时PCR试剂盒对样品的DNA质量的耐受性较好,检测成功率高于Sanger DNA测序,可替代Sanger DNA测序法成为临床上对肿瘤K-ras基因体细胞突变检测的实用方法.