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.     

Analysis of K-ras, N-ras, H-ras, and p53 in lung neuroendocrine neoplasms.

This study screened 11 samples of typical carcinoid (TC), 4 samples of atypical carcinoid (AC), 1 sample of large cell neuroendocrine carcinoma (LCNEC), and four metastases for point mutations in exons 5 to 8 of the p53 gene, and exons 1 and 2 of the K-ras. H-ras, and N-ras genes using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) and direct sequencing and by immunohistochemistry for p53. Exon 1 of K-ras was mutated in two samples of low-grade AC and a metastasis from one of these tumors (GAT12 and AGT12, respectively). No mutations in N-ras or H-ras were found. Mutations in exons 5 and 8 of the p53 gene were identified in a high-grade AC and a LCNEC. Positive immunostaining for p53 was present in three samples, with only one genotypic mutation shown (LCNEC). In conclusion, point mutations of the p53 gene were infrequent in these pulmonary neuroendocrine tumors, did not correlate in all samples with immunostaining, and were associated with the higher-grade tumors. Second, the presence of K-ras mutations seems to be associated with the higher-grade carcinomas. Third, N-ras and H-ras mutations were not found with these pulmonary neuroendocrine tumors.     

Ras gene activation in malignant cells of human ovarian carcinoma peritoneal fluids

In this study, we showed, for the first time, the pattern of point mutations at codon 12 of the K-ras, H-ras and N-ras genes, using polymerase chain reaction and restriction fragment length polymorphism analysis in 47 malignant cytologic specimens of ovarian adenocarcinoma peritoneal fluids. Forty-seven % of the samples were found to carry a point mutation at codon 12 of K-ras gene. Also, 21 cystadenoma peritoneal fluids were used as control specimens for the detection of ras mutations. Fourteen % of these samples were found to carry a point mutation at codon 12 of the K-ras gene. The prevalence of K-ras gene mutations were statistically correlated with FIGO and surgical stage of the malignant specimens. Our data demonstrates that the K-ras gene mutations are mainly affected (47%) in the malignant cells of the peritoneal washings or ascites of women with ovarian adenocarcinomas and may have value for the early diagnosis and monitoring of these neoplasms.     

H-ras oncogene mutation in dedifferentiated liposarcoma. Polymerase chain reaction-restriction fragment length polymorphism analysis

Point mutations of the ras gene family (K-ras, H-ras, and N-ras) are thought to be involved in the development of a variety of human tumors. Dedifferentiated liposarcoma is characterized by the coexistence of well-differentiated (WD) and high-grade anaplastic (HG) components. The presence of point mutations at codons 12 and 13 of the H-ras gene was studied in 34 liposarcomas, comprising 15 well-differentiated liposarcomas and 19 dedifferentiated liposarcomas, and in 8 storiform-pleomorphic type malignant fibrous histiocytomas (MFHs) using polymerase chain reaction-restriction fragment length polymorphism and direct sequencing analysis. The 2 components of dedifferentiated liposarcoma were analyzed independently. H-ras mutations were seen only in dedifferentiated liposarcomas (4/19 [21%]), 1 in WD components and 3 in HG components. The mutation was not seen in any of 15 cases of well-differentiated liposarcoma. MFHs showed an H-ras mutation in 1 (12%) of 8 cases. Our results seem to suggest that the H-ras mutation is a relatively uncommon event in dedifferentiated liposarcoma, which may demonstrate an epiphenomenon of dedifferentiation in dedifferentiated liposarcoma.     

p53 and K-ras gene mutations in carcinoma of the rectum among Finnish women.

AIMS/BACKGROUND: The aim of this study was to identify p53 and K-ras gene mutations in carcinoma of the rectum among Finnish women. Mutation patterns might give clues to aetiological factors when comparisons are made with other human tumours. METHODS: Of 134 women with carcinoma of the rectum, paraffin wax embedded specimens of the tumour tissue were obtained from 118 patients. Genomic DNA was extracted, and exons 4-8 of the p53 gene and codons 12/13 and 61 of the K-ras gene were amplified, and analysed for mutations by single strand conformation polymorphism and direct sequencing. The production of p53 and K-ras proteins was studied by immunohistochemistry. RESULTS: The overall crude frequency for mutations in the p53 gene was 35% but the true frequency appears to be higher (up to 56%). In the K-ras gene, the mutation frequency (15%) was significantly lower than that reported for colon cancer. In the p53 gene, the mutation frequency increased significantly with patient age. In a high proportion of patients (14%) the rectal tumours contained small subclones of tumour cells that displayed extremely rare mutations at codons 110 and 232 of the p53 gene. Hot spot codon 175 mutations were significantly less common in rectal cancer than in cancer of the colon. CONCLUSIONS: Rectal cancer among Finnish women has characteristics in the mutations of the p53 and K-ras genes that are uncommon in other human tumours, including cancer of the colon. A biological explanation of these findings is not clear at present, but might be associated with an unidentified genetic factor in Finland.     

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     

Mutations in K-ras and epidermal growth factor receptor expression in Korean patients with stages III and IV colorectal cancer

K-Ras somatic mutations in advanced colorectal cancer (CRC) can predict resistance to mAbs that target the epidermal growth factor receptor (EGFR). The relationships between K-ras mutations and the EGFR status have not yet been examined, especially in Korean patients. A total of 82 colorectal tumors (stage III-IV) were analyzed. K-Ras mutations at codons 12 and 13 were detected by polymerase chain reaction-single strand conformational polymorphism. The EGFR expressions were examined by immunohistochemistry, and these were graded according to a modified EGFR expression scoring system. The relationships between the patients' characteristics and the survival time and the gene mutation status were analyzed. The EGFR expression was positive in 69 patients (84.1%) and negative in 13 patients (15.9%). The K-ras mutation rate was 35.4%. In all, 20 (68.9%) cases were mutated at codon 12 and 9 (31.1%) cases were mutated at codon 13. No relationship was observed between the EGFR status and K-ras mutation. The median overall survival (OS) was 68.1 months. There was no difference between the K-ras mutant group and the wild type group for overall survival (30.3% vs 21.0%, respectively, at 36 months, P = .777). K-ras mutation and the EGFR status were not independent prognostic factors for OS (P = .105 and P = .499, respectively). For the Korean patients with CRC, the rate of an EGFR protein expression was greater than that for the patients in Western countries, and the rate of K-ras mutations was lower than that for patients in Western countries. This study found no correlation between the EGFR status and K-ras mutations in colorectal tumors.     

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.     

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。     

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.     

High-degree tumor budding and podia-formation in sporadic colorectal carcinomas with K-ras gene mutations

In vitro ras activation enhances the epithelial-mesenchymal transition of colorectal carcinoma cells. But ras effects are known to be highly dependent on cell types and the tissue context. Therefore, this study was made to test the hypothesis that in clinical colorectal carcinoma specimens, aggressive invasion phenotypes, specifically tumor budding and podia formation, would correlate with K-ras gene mutations. In a series of 95 clinically sporadic primary colorectal carcinomas collected ad hoc, tumor budding and podia formation were counted using pan-cytokeratin immunohistochemistry, and K-ras gene mutations in codons 12 and 13 were determined. Consistent with the hypothesis, tumor budding and podia formation were observed to be significantly higher in the 32 (34.7%) of the tumors with K-ras gene mutations (29 mutations in codon 12, 3 in codon 13), and this correlation was observed independent of the patterns of invasion (expansive versus infiltrative). Microsatellite status, numbers of losses of heterozygosity, adenomatous polyposis coli and p53 gene mutations, and degree of promoter methylations (CIMP status) were not associated with K-ras gene mutations. Besides their effects on the tumor cell cycles, oncogenic K-ras gene mutations in colorectal carcinomas could be important for aggressive tumor invasion. This may be important in metastasizing disease and could provide a rationale for developing drugs that interrupt ras-signaling cascades.     

K-ras mutations in lung tumors and tumors from other organs are consistent with a common mechanism of ethylene oxide tumorigenesis in the B6C3F1 mouse

Ethylene oxide is a multisite carcinogen in rodents and classified as a human carcinogen by the National Toxicology Program. In 2-year mouse studies, ethylene oxide (EO) induced lung, Harderian gland (HG), and uterine neoplasms. We evaluated representative EO-induced and equivalent spontaneous neoplasms for K-ras mutations in codons 12, 13, and 61. K-ras mutations were identified in 100% (23/23) of the EO-induced lung neoplasms and 25% (27/108) of the spontaneous lung neoplasms. Codon 12 G to T transversions were common in EO-induced lung neoplasms (21/23) but infrequent in spontaneous lung neoplasms (1/108). K-ras mutations were found in 86% (18/21) of the EO-induced HG neoplasms and 7% (2/27) of the spontaneous HG neoplasms. Codon 13 G to C and codon 12 G to T transversions were predominant in the EO-induced HG neoplasms but absent in spontaneous HG neoplasms (0/27). K-ras mutations occurred in 83% (5/6) of the EO-induced uterine carcinomas and all were codon 13 C to T transitions. These data show a strong predilection for development of K-ras mutations in EO-induced lung, Harderian gland, and uterine neoplasms. This suggests that EO specifically targets the K-ras gene in multiple tissue types and that this event is a critical component of EO-induced tumorigenesis.     

膀胱癌组织中K－ras基因点突变及其基因产物P21的表达研究

为在分子生物学水平探讨膀胱癌的发生发展以及恶性程度的判别和预后评价的指标，作者应用聚合酶链反应（ＰＣＲ）结合单链构象多态性分析（ＳＳＣＰ）和免疫组织化学方法，研究了５４例膀胱癌组织和５例正常膀胱组织的Ｋ－ｒａｓ基因第１２位密码子点突变和其产物Ｐ２１的表达情况。结果表明：５４例膀胱癌组织中，仅有４例Ｋ－ｒａｓ基因第１２位密码子点突变和３４例Ｐ２１蛋白阳性表达，并且浅表性肿瘤Ｐ２１阳性表达率明显低于浸润性肿瘤。提示Ｋ－ｒａｓ基因点突变在膀胱癌中发生率较低，Ｐ２１蛋白的过量表达主要是膀胱癌晚期表现，它可能与Ｈ－ｒａｓ和Ｎ－ｒａｓ基因突变有关。为肿瘤病因学研究和临床研究提供了有用的资料。     

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.     

Single somatic ras gene point mutation in soft tissue malignant fibrous histiocytomas.

The frequency of ras gene mutations in human soft tissue malignant fibrous histiocytomas within and around the hot spot codons (12, 13, and 61) of all ras genes, (H-ras-1, K-ras-2, and N-ras) was studied by nested polymerase chain reaction and direct DNA sequencing from archival formalin-fixed, paraffin-embedded tissue. Light microscopy and immunohistochemistry served to define malignant fibrous histiocytoma. All of the four differentiation subtypes (storiform-pleomorphic, inflammatory, myxoid, and giant cell) were investigated. Nine of thirty-two malignant fibrous histiocytomas (28%) contained ras gene point mutations. The highest incidence was found in the myxoid subtype (four of nine). H-ras-1 gene codon 12.2 was the only codon affected and contained in all mutated cases a GGC-->GTC exchange. Seven of the nine mutations were homozygous and probably affected more than 80% of the tumor DNA. The flanking regions of all hotspot codons did not contain any point mutation. The presence of a single and often homozygous point mutation of the H-ras-1 gene, especially in myxoid malignant fibrous histiocytoma could serve as a basis for further genomic discrimination of myxoid sarcomas.     

Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer

Mutational analysis of KRAS codons 12 and 13 is standard for patients with metastatic colorectal cancer since mutations in these codons predict lack of response to anti-EGFR therapies. However, even among patients whose tumors are wildtype for KRAS codons 12 and 13, only a subset respond to therapy. Since additional activating mutations downstream of EGFR may also play a role in treatment resistance, we sought to establish the frequency of these mutations. We evaluated 2121 colorectal tumors for mutations in codons 12 and 13 of the KRAS gene. A subset of these samples, comprised of 513 samples wildtype for KRAS codons 12 and 13, were tested for mutations in codons 61 and 146 of KRAS, codon 600 of BRAF, and codons 12, 13, and 61 of NRAS. Mutation status was determined by targeted pyrosequencing. Mutations in KRAS codon 12 or 13 were identified in 900/2121 (42.4%) samples. Of the 513 wildtype samples tested for additional mutations, 78 samples were mutant for BRAF, 19 for KRAS codon 61, 17 for KRAS codon 146, and 26 for NRAS. In total, 140/513 (27.3%) tumors wildtype for KRAS codons 12 and 13 harbored a mutation in another of the RAS pathway genes. While further study is needed to determine the full therapeutic implications of mutations in these codons, mutational testing of these codons may be useful for identifying a significant proportion of patients who may also be resistant to anti-EGFR therapies.     

Immunoexpression of ultraviolet photoproducts and p53 mutation analysis in atypical fibroxanthoma and superficial malignant fibrous histiocytoma.

p53 mutation is one of the major results of ultraviolet (UV) radiation. UV photoproducts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (64PPs) also play an important role in skin cancer development. Atypical fibroxanthoma (AFX), which mimics malignant fibrous histiocytoma (MFH) histologically, occurs in the sun-exposed skin of the elderly, and therefore, an association with UV has long been suspected. Eighteen fibrohistiocytic skin lesions comprising AFX (n = 7), storiform-pleomorphic type MFH centered in the subcutis (superficial MFH; S-MFH; n = 4) and benign fibrous histiocytoma (BFH; n = 7) were used for immunohistochemical and molecular analysis. Eight cases of deep MFH (D-MFH) were also analyzed for UV photoproduct expression for the purposes of comparison. Immunohistochemically, the CPD scores of AFX (3.6 +/- 0.4) were significantly higher than those of S-MFH (1.3 +/- 0.8), D-MFH (0.8 +/- 0.5), or BHF (1.4 +/- 0.7); however, the 64PP scores were extremely low in all these tumors (AFX, 0.1 +/- 0.1; S-MFH, 0.0 +/- 0.0; D-MFH, 0.0 +/- 0.0; and BHF, 0.0 +/- 0.0). AFX, S-MFH, and BFH showed immunoexpression for p53 (2/7, 2/4, and 0/7), respectively. p53 mutations were detected in AFX (4/6; 67%) and S-MFH (1/4; 25%), but not in BFH (0/5; 0%) using polymerase chain reaction-single-strand conformation polymorphism, and all of the mutations in AFX were either C-T transitions or at dipyrimidine sites. In conclusion, AFX and S-MFH are both similar fibrohistocytic lesions; however, AFX has high immunoreactivity for CPDs compared with S-MFH, D-MFH, or BFH. These data suggest that CPDs may play an important role in the pathogenesis of AFX.     

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.     

Gene mutations in lymphoproliferative disorders of T and NK/T cell phenotypes developing in renal transplant patients

Post-transplantation lymphoproliferative disorder (PT-LPD) is characterized by lymphoid proliferation after organ or bone marrow transplantation. In Western countries, most cases of PT-LPD are B-cell-derived and Epstein-Barr virus-associated, in which alterations of c-myc, p53, and N-ras genes might play a role in disease progression. In Japan, PT-LPD of T- and NK/T-cell types are not uncommon in renal transplant patients. Mutations of p53 (exons 4 through 8), K-ras (exons 1 and 2), c-kit (exons 11 and 17), and beta-catenin genes (exon 3) in 12 cases of these diseases were analyzed by PCR single strand conformation polymorphism and then by direct sequencing. p53 gene mutations were detected in 5 of 5 cases of peripheral T-cell lymphoma, 3 (60%) of 5 cases of adult T-cell leukemia/lymphoma, and 1 of 2 cases of NK/T cell lymphoma. Twenty-five percent of T and NK/T cell lymphomas showed K-ras mutations. Mutations of c-kit and beta-catenin genes were found in 33% of cases. Among a total of 42 substitution mutations, 40 were transitions with involvement of CpG sites in 20 to 30% of cases. Most cases had at least one mutation that changed an amino acid, which might have provided the selection pressure for expansion. These findings suggested that p53 gene mutations might play a central role in development of peripheral T-cell lymphoma including adult T-cell leukemia/lymphoma in renal transplant patients.     

Frequent polymorphism of peroxisome proliferator activated receptor gamma gene in colorectal cancer containing wild-type K-ras gene

We analyzed the K-ras gene mutation and the polymorphism of peroxisome proliferator activated receptor gamma (PPARgamma) gene, which is known as a master gene for adipocyte differentiation and a tumor suppressor gene, in patients with colorectal cancer by polymerase chain reaction-restriction fragment length polymorphism. We detected a mutation of K-ras gene at codon 12 in 8 of 39 colorectal cancers. We also detected a polymorphism of PPARgamma gene at codon 12 in 10 of 39 patients with colorectal cancer. Homozygous polymorphism (Ala12Ala) in PPARgamma gene was found only in one patient, but heterozygous polymorphism (Pro12Ala) was found in 9 of 29 patients. Surprisingly, all 10 colorectal cancers developed in the patients with polymorphism of PPARgamma gene carrying the wild-type K-ras gene. These findings suggest that PPARgamma gene polymorphism may be implicated with the development of colorectal cancers, in which K-ras gene is not mutated.