K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung

BACKGROUND. The capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas. METHODS. We studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes. RESULTS. Nineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001). CONCLUSIONS. The presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.     

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.     

RET oncogene activation in papillary thyroid carcinoma.

The RET proto-oncogene encodes a cell membrane tyrosine-kinase receptor protein whose ligands belong to the glial cell line-derived neurotrophic factor. RET functions as a multicompetent receptor complex that includes alphaGFRs and RET. Somatic rearrangements of RET designated as RET/PTC (from papillary thyroid carcinoma) were identified in papillary thyroid carcinoma before RET was recognized as the susceptibility gene for MEN2. There are now at least at least 15 types of RET/PTC rearrangements involving RET and 10 different genes. RET/PTC1 and RET/PTC3 are by far the most common rearrangements. All of the rearrangements are due to DNA damage and result in the fusion of the RET tyrosine-kinase (RET-TK) domain to the 5'-terminal region of heterologous genes. RET/PTC rearrangements are very common in radiation-induced tumors but have been detected in variable proportions of sporadic (i.e., non-radiation associated) papillary carcinomas. It is estimated that up to approximately half the papillary thyroid carcinomas in the United States and Canada harbor RET/PTC rearrangements, most commonly RET/PTC-1, followed by RET/PTC-3 and occasionally RET/PTC-2. The cause of these rearrangements in sporadic papillary carcinomas is not known, but the close association between their presence and the papillary carcinoma phenotype indicates that they play a causative role in tumor development. The proposed mechanisms of RET/PTC-induced tumorigenesis and the clinical and pathologic implications of RET/PTC activation are discussed.     

Possible role of K-ras oncogene in mutagenic activity of ethylnitrosourea on lymphocyte hyperproliferation in rat colon.

N-ethyl-N-nitrosourea (ENU) is a potential carcinogenic agent that is commonly used in industry. Therefore the present study aimed to find out the possible effects of this agent on the rat digestive tract especially on the colon. We have studied the complementary mutation activity of the exon 2 of K-ras oncogene by ENU treatment in rat colonal tissue. While the two experimental group rats were injected once a week with 20 mg/kg and 300 mg/kg body weight-body weight with ENU (i.p.), the last experimental group was administered only with PEG and the control group animals received no treatment. Following 45 weeks, all animals were sacrificed and colonal tissues were obtained. Tissues were processed for light and electron microscopy and also for molecular biological analyses. While no colonal tumour development was observed in the control and in the PEG treated group, an extensive tumour development was seen in a wide range of tissues in the high dose ENU treated group. The light and electron microscopical examination of the rat colonal tissue revealed a lymphocyte hyperproliferation in the submucosal region, an increased number of polymorphonuclear leukocytes (PMLs) and occasional epithelial lesions. The mutation analyses of exon 2 of K-ras by HpaII demonstrated more than one recognition sites in the ENU treated group whereas there was only one enzyme cognate site in the control group.     

Evaluation of K-ras 12 oncogene mutations in Venezuelan patients with Helicobacter pylori infection

This work evaluated the infection of H. pylori in the different gastric pathologies and its association with the oncogen K-ras 12. Endoscopy was performed in 62 patients and 3 biopsies from the antral region were taked and used for the histological diagnostic, PCR, and point mutations determination. The results showed a high incidence of H. pylori infection in patients with active chronic gastritis (AcCG) 90%, chronic atrophic gastritis (AtCG) 70%, intestinal metaplasia (IM) 67%, dysplasia (D) 83%, and decrease in in gastric cancer (GC) 33%. Evaluation of the oncogen K-ras 12 showed that 68% of the patients presented mutations in the different analyzed amino acids. In the 12 codon of the K-ras gene, we observed simple point mutations and combination in the same sample in different gastric pathologies. In AcCG samples were detected the greater number of mutations. A decrease of the point mutations were observed in the progression stages to gastric cancer. The presence of these specific mutations would be tumor markers and it determine the possible development of gastric tumors.     

K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.

We examined 82 surgically resected or biopsied, formalin-fixed, paraffin-embedded primary adenocarcinomas of the pancreas for the presence of activating point mutations in codon 12 of the K-ras oncogene. Mutations were detected using primer-mediated, mutant-enriched, polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. This combination of mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and allele-specific oligonucleotide hybridization results in a rapid and sensitive characterization of the mutations in codon 12 of K-ras. Sixty-eight (83%) of the 82 carcinomas examined harbored a point mutation. Of the 68 mutations, 33 (49%) were guanine to adenine transitions, 27 (39%) were guanine to thymine transversions, and eight (12%) were guanine to cytosine transversions. Mutations were found in carcinomas of the head (61 of 75, 81%) as well as in carcinomas of the body or tail (seven of seven, 100%) of the pancreas. The overall prevalence of K-ras point mutations in adenocarcinomas of the pancreas obtained from patients who smoked cigarettes at some point during their lives (88%; 86% in current smokers and 89% in ex-smokers) was greater than that seen in pancreatic adenocarcinomas from patients who never smoked cigarettes (68%, P = 0.046). The presence of K-ras point mutations did not correlate with tumor ploidy, tumor proliferating index, or patient survival. These results demonstrate that primer-mediated, mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis combined with allele-specific oligonucleotide hybridization can be used to detect and characterize mutations in codon 12 of the K-ras oncogene in formalin-fixed, paraffin-embedded tissues, and the results confirm that activating point mutations in codon 12 of the K-ras oncogene occur frequently in adenocarcinomas of the pancreas.     

Some neuronal properties of PC12 cells differentiated by the K-ras oncogene

Summary When infected with a virus containing the Kirsten-ras oncogene, rat phaeochromocytoma or PC12 cells elaborated neurites and ceased mitosis, that is, they underwent neuronal differentiation. Such differentiated cells could be replated and maintained up to 20 weeksin vitro without the need of an exogenous, continuous supply of nerve growth factor (NGF). The neurites of K-ras infected PC12 cells, filled with microtubules and actin which was concentrated within the growth cones, resembled those of primary neuronsin vitro. As in the NGF-primed PC12 cells, two types of secretory vesicles were present in the K-ras-infected PC12 neurites: large (100 nm), dense core granules, and small (45 nm), clear vesicles.Compared to naive PC12 cells, K-ras infected PC12 cells had (a) higher activities of acetylcholinesterase and choline acetyltransferase, two enzymes involved in acetylcholine metabolism; (b) enhanced activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis; (c) a higher, evoked norepinephrine release; and (d) similar levels of sodium-dependent uptake of both choline and norepinephrine.Although the total content of catecholamines in K-ras-differentiated PC12 cells was less than that of naïve cells, both norepinephrine and dopamine were present in substantial amounts and norepinephrine was released after stimulation. According to their enzymatic activity, these cells can also synthesize acetylcholine and thus have potential as donors for the intracerebral replacement of either catecholaminergic or cholinergic neurotransmitters.     

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.     

Clinical significance of depressed-type colorectal neoplasms based on growth and development

A route of colorectal cancer development other than the adenoma-carcinoma sequence has recently become an issue due to the discovery of depressed-type early colorectal cancers. Moreover, the fact that some polyp-like cancers actually originate from depressed-type lesions has become obvious. Despite the protruding shapes of depressed-type early colorectal cancers, they probably have biological characteristics, which are different from those of the usual polyp lesions. We undertook this study to evaluate the clinical significance of depressed-type colorectal neoplasms. The authors recently experienced 87 cases of depressed-type colorectal neoplasms. Using Kudo's classification, we classified these 87 cases into three types based on their growth patterns, type IIc, type IIa + IIc, and type Is + IIc, and then analyzed these types on the basis of size, type, and submucosal invasion rate. The submucosal invasion rate of cancers of type IIa + IIc was significantly higher than that of type IIc (p < 0.05), and the rate for cancers of types IIa + IIc and Is + IIc together was significantly higher than that of type IIc (p < 0.05). However, no significant difference was found between the rates of types IIa + IIc and Is + IIc. In conclusion, the IIa + IIc and Is + IIc sub-types of depressed-type colorectal neoplasms, individually and together, have higher rates of submucosal invasion than type IIc lesions. Accordingly, type IIa + IIc and type Is + IIc must be differentiated from the usual polyps and should be managed cautiously, despite their protruding shapes.     

应用间期荧光原位杂交技术检测原发性肝细胞癌c-myc基因扩增的临床意义

目的：探讨c-myc基因在原发性肝细胞癌中的扩增及其与临床及预后的关系。方法：应用间期核荧光在位杂交技术对100例原发性肝癌和6例带卫星结节的肝癌组织标本进行检测。结果：92％（92例）原发性肝癌组织存在c-myc基因扩增,其中高拷贝数扩增达37％（37例）,低拷贝数扩增55％（55例）,只有8％（8例）,无扩增,结合临床资料分析,c-myc扩增与患者临床分期,肿瘤大小和病理类型无明显相关（P＞0．05）,原发肿瘤和相应卫星结节肿瘤组织具有相似的c-myc基因扩增水平和相同病理类型的特点,在可随诊两年半的90例病例中,91．1％（82例）病例有c-myc基因扩增,其中高拷贝数扩增34例,低拷贝数扩增48例,无扩增8例,分析c-myc扩增程度与患者术后复发的时间及生存期关系,发现c-myc高拷贝数扩增的病例术后1年内复发率（70．6％,24／34）高于c-myc低拷贝数扩增（39．6％,19／48）和无扩增者（12．5％,1／8）,三者这产差异有显著性意义（P＜0．05）,而在术后＞12个月的复发率则与c-myc不同扩增程度间差异无显著性意义（P＞05）,另外,c-myc高拷贝数扩增者术后2年存活率明显低于低拷数扩增和无扩增者（P＝0．01）,结论：原发性肝细胞癌存在高频率的C-myc基因扩增,基因扩增与患者临床分期,肿瘤大小和病理类型无明显相关性,但与患者预后有关,高拷贝数扩增者复发时间是,复发率高,生存期短。     

Clinical significance.

This article initiates the special section on clinical significance. Within a brief précis and overview, the 4 methodological articles and the integrative commentary of the special section are introduced. A call for the inclusion of the assessment of clinical significance in treatment evaluations is extended.     

An analysis of oncogene activation in ovarian carcinoma]

Evidence is increasing that oncogenes are involved in the development and/or progression of gynecological malignancies including ovarian carcinoma. While histopathologic examination remains an indispensable tool in the diagnosis and evaluation of patients with ovarian carcinoma, the advancement of technology and the development of new knowledge regarding neoplastic transformation are providing a basis for new opportunities to improve patients care. In this review, a variety of techniques to study the abnormalities of oncogenes, especially of c-myc oncogenes in clinical specimens of human ovarian malignancies are reviewed. Emphasis in placed on whether the techniques are feasible in routine clinical laboratories and have potential values to the care of patients with ovarian carcinoma. At this juncture, an examination of c-myc oncogene abnormalities at the DNA level appears to have a greater potentials in the field described above than those at mRNA and protein levels.     

原癌基因RET与甲状腺乳头状癌关系的研究进展

许多肿瘤有其特异性的基因改变。RET是与甲状腺乳头状癌(papillary thyroid carcinoma)有着特殊关系的原癌基因。RET／PTC重排和甲状腺滤泡性癌中PAX8．PPAR7的重排,以及发生于乳头状肾细胞癌的基因重排,是迄今在人类上皮性癌中所确定的为数不多的融合基因。现已证实在甲状腺乳头状癌中存在着RET原癌基因的多种重排形式。     

C-myc oncogene product P62c-myc in ovarian mucinous neoplasms: immunohistochemical study correlated with malignancy.

The monoclonal antibody Myc 1-6E10 was used to determine the cellular distribution of the c-myc oncogene product p62c-myc in 60 mucinous ovarian tumours. Three patterns of immunostaining were apparent: (i) nuclear staining alone; (ii) staining of the nucleus and basal cytoplasm; and (iii) staining of the entire cell. Of the 21 cases of mucinous cystadenoma, 11 showed nuclear staining alone, and a further case showed additional weak staining of the basal cytoplasm. Nuclear staining alone was not present in any of the 17 borderline mucinous tumours examined. Strong staining of the nucleus and basal cytoplasm was seen in 16 of these borderline cases, six of which also showed focal staining of the apical cytoplasm. All 22 cases of mucinous cystadenocarcinoma showed staining of the cell nucleus and entire cell cytoplasm. Focal staining of the apical cytoplasm in six of 17 borderline mucinous tumours produced a pattern of c-myc immunostaining similar to that of cystadenocarcinoma. Retrospective analysis of the clinical data showed that no significant differences between patients with borderline tumours of these two categories could be defined. Although immunostaining with Myc 1-6E10 can be used in the categorisation of mucinous ovarian tumours, it is concluded that standard histological criteria are more accurate indicators of tumour behaviour than is an assessment of c-myc expression.     

Quantitative enriched PCR (QEPCR), a highly sensitive method for detection of K-ras oncogene mutation

We have developed a rapid and highly sensitive method for the detection of mutant K-ras codon 12 allele in the presence of 10⁵ copies of the wild-type alleles. This sensitivity is achieved by selective amplification of mutant K-ras sequences, using a two-stage procedure with modified primers. In the first stage, primers consist of K-ras sequences in the 3′ portion and polyomavirus sequence (to minimize homology with human genome) on the 5′ portion. The 3′ portion also consists of mismatch sequence that generates an MvaI site in normal, but not mutant, K-ras codon 12 alleles. Thus, following the first round of 20 cycles, restriction enzyme cleavage is carried out to selectively digest normal K-ras codon 12 alleles. To enrich mutant alleles, a second amplification is performed using tail primers that recognize the polyoma, but not human sequences. This design ensures that in the second amplification only mutant alleles that were pre-amplified in the first round would serve as template for this reaction. Ethidium bromide-stained polyacrylamide gel electrophoresis (PAGE) of second–stage PCR product that has been digested with MvaI is used to monitor the presence of mutant alleles, detected at sensitivity of 1/10⁵. This technique offers high sensitive detection of mutant K-ras alleles using a new concept of tail-primer design and is likely to assist in identifying patients at risk to develop pancreatic, colon, or lung cancer, which harbor high incidence of mutant ras alleles. Hum Mutat 10:322–325, 1997. © 1997 Wiley-Liss, Inc.     

Diagnosis of duodenal and ampullary epithelial neoplasms by endoscopic biopsy: a clinicopathologic and immunohistochemical study

A series of 19 duodenal and 16 ampullary neoplasms was studied to determine their pathologic features on endoscopic biopsy, to evaluate the diagnostic accuracy of this procedure, and to assess the usefulness of immunohistochemical staining for carcinoembryonic antigen (CEA) in these neoplasms. The 11 benign neoplasms (31 per cent) were adenomas, five of which had focal hyperplastic features; the 24 malignant neoplasms (69 per cent) included ten intestinal-type carcinomas (resembling colonic carcinoma), seven anaplastic carcinomas (resembling diffuse gastric carcinoma), two adenocarcinomas in situ, and five lesions of unoriented, cytologically malignant epithelium. Malignancy was suspected endoscopically in 19 of 24 carcinomas, and the majority of the benign neoplasms were described as polyps or plaques. Resections (performed in 20 cases) demonstrated the accuracy of the biopsy diagnoses in 17 cases (85 per cent). In the three discordant cases, diagnosed by biopsy as adenoma in two cases and carcinoma in situ in one, coexistent in situ or infiltrating carcinomas were identified in the resected specimens. Carcinoembryonic antigen (20 cases) was identified mostly along glycocalyceal borders in normal and adenomatous tissues, whereas the carcinomas also showed strong cytoplasmic staining for CEA. Endoscopic biopsy is a valuable procedure in the diagnosis of duodenal and ampullary neoplasms. Correlation of the pathologic features of biopsy specimens with endoscopic appearances may result in more accurate diagnoses.     

Claudin-18 in biliary neoplasms. Its significance in the classification of intrahepatic cholangiocarcinoma

Claudin-18 (CLDN18), a tight junction protein specific to stomach and lung, is aberrantly expressed in preinvasive and invasive neoplasms of the pancreas. To investigate the significance of CLDN18 expression in biliary neoplasms, immunohistochemical analysis was performed. CLDN18 expression was frequently observed in the epithelial cells of extrahepatic bile duct carcinomas (90%, n = 99), intrahepatic intraductal papillary neoplasms of the bile duct (IPNBs, 100%, n = 11), and extrahepatic IPNBs (89%, n = 9), while it was less frequent in intrahepatic cholangiocarcinomas (ICCs, 43%, n = 83). Interestingly, CLDN18 expression was also frequently observed in precancerous lesions such as biliary intraepithelial neoplasias (78%, n = 18). Among ICCs, CLDN18-positive cases showed higher frequencies of periductal infiltrative growth, perineural invasion, and lymph node metastasis. Multivariable analysis demonstrated that positive CLDN18 expression was an independent risk factor for lymph node metastasis in ICCs. Furthermore, CLDN18 expression was associated with poor overall survival by univariable analysis, as well as lymph node metastasis. These results suggest that CLDN18 may play an important role in biliary carcinogenesis, and especially in ICCs, it is associated with aggressive behavior and serves as a useful marker for the classification of ICC.