[Sensitive detection of K-ras oncogene codon 12 mutations by nested PCR using mismatched primers and selective digestion of non-mutated PCR fragments with restriction enzyme].

Sensitive detection of K-ras oncogene mutation at codon 12 using nested polymerase chain reactions (PCR) with mismatched primers combined with selective digestion of nonmutated DNA fragments after the first PCR amplification was reported by Levi S, et al. (Cancer Res 51:3497-3502, 1991). We examined the usefulness of this novel technique in cell lines carrying various K-ras codon 12 mutations and clinical samples such as colon carcinoma tissue, corresponding normal colonic mucosa and pancreatic juice obtained from the patients with pancreatic carcinoma undergoing endoscopic retrograde pancreatography (ERP). In analysis of tumor cell lines carrying mutated K-ras at codon 12, the fragment length of the amplified DNA was 135 bp after digestion using restriction enzyme BstN1, whereas those of nonmutated cell lines were 106 bp. This method was highly sensitive to detect mutant DNA diluted at a ratio of 2048 fold with normal DNA samples obtained from peripheral blood lymphocytes. In analysis of clinical samples, 4 out of 10 colorectal carcinoma tissues were positive for K-ras gene mutation at codon 12, however, no mutations were detected in corresponding normal colonic mucosa. In analysis of pancreatic juice taken from the patients with pancreatic carcinoma, 1 out of 3 samples were positive for K-ras mutation at codon 12. Thus, this novel approach was thought to be useful for detection of minimum number of cancer cells from compound samples containing larger amounts of normal cells.

Evidence for increased somatic cell mutations in patients with hepatocellular carcinoma.

The measurement of somatic cell mutation may assist in the assessment of human cancer risk. The glycophorin A (GPA) assay, which measures the frequency of variant erythrocytes in persons with blood type MN, was used to directly assess in vivo mutability in 30 patients with hepatocellular carcinoma (HCC). HCC patients showed significantly increased frequencies of both hemizygous (MO) and homozygous (MM) variants, due to somatic loss of expression of the N allele, when compared with 27 patients with chronic liver disease and 21 healthy controls. The mean elevations of the MO and MM variant frequencies (VF) in HCC patients were 2-3-fold greater than the comparable VF in the control groups. The mean MO and MM VF in the patients with chronic liver disease was slightly elevated compared to that in healthy controls, but the difference was not significant. In the 19 anti-hepatitis C virus (HCV)-positive patients with a history of blood transfusion, significant linear relations between VF and the duration of HCV infection were observed for MO and MM. These data indicate a high background frequency of somatic mutations in HCC patients. The GPA assay may prove to be a useful estimation of the individual's risk of development of HCC.

Methods in laboratory investigation. Rapid and simple detection of c-Ki-ras2 gene codon 12 mutations by nonradioisotopic single-strand conformation polymorphism analysis.

BACKGROUND: To achieve simple and rapid detection of codon 12 mutations of the c-Ki-ras2 gene, we developed a nonradioisotopic single-strand conformation polymorphism analysis using silver stain. EXPERIMENTAL DESIGN: After a conventional polymerase chain reaction, amplified DNA fragments were mixed with formamide, heated and subjected to electrophoresis for 1.5 hours using minigels of polyacrylamide. The gels were then silver stained and single-strand DNA fragments were visualized directly. RESULTS: Electrophoresis and subsequent silver staining were completed within 2.5 hours. Plasmids carrying various codon 12 mutations of the c-Ki-ras2 gene were used as controls of Nonradioisotopic single-strand confirmation polymorphism analysis, and all six mutations were successfully separated from the normal allele in a single electrophoretic run. In the analysis of tumor cell lines and tumor samples, the results were identical with those of conventional dot blot hybridization using 32P-labeled oligonucleotide probes. Of 10 colorectal carcinoma tissues examined, 4 tumors were shown to carry codon 12 mutations of the c-Ki-ras2 gene. CONCLUSIONS: We conclude that this novel technique is a rapid, simple and useful method that may replace conventional methods.

Intratumoral DNA heterogeneity of small hepatocellular carcinoma.

BACKGROUND: Intratumoral DNA heterogeneity provides important information regarding biologic and clinical behavior. The purpose of this study was to evaluate the incidence of DNA heterogeneity in small hepatocellular carcinoma (HCC) nodules. METHODS: The DNA content of 28 surgically resected small HCC nodules (< or = 3.0 cm) was measured using flow cytometry of fresh or frozen samples taken from different parts of each nodule with reference to the macroscopic features. RESULTS: Of the 28 small HCC nodules, 14 (50.0%) had only DNA diploid stemline characteristics. Five nodules (17.9%) manifested DNA diploid and DNA aneuploidy within the same tumor. Of the remaining nine nodules (32.1%) that showed only DNA aneuploidy, two contained tumor tissues with apparently different DNA content. Thus, DNA heterogeneity was found in 7 (25.0%) of 28 nodules. DNA heterogeneity correlated well with macroscopic histologic features. All four early HCC were composed of only DNA diploid cells, whereas three of six nodule-in-nodule lesions were composed of DNA heterogeneous cells, in which the inner obviously cancerous nodule showed DNA aneuploidy and the outer well differentiated HCC portion demonstrated DNA diploid. Four of 18 overt HCC nodules showed DNA heterogeneity; 2 of these 4 nodules showed both diploid and aneuploid peaks, and the other 2 two showed different aneuploid peaks within the same nodule. CONCLUSIONS: DNA heterogeneity correlating with macroscopic features is found frequently even in small HCC nodules. Therefore, multiple sampling based on macroscopic features is required for the accurate assessment of DNA ploidy, particularly when the information about DNA ploidy is used as a prognostic indicator.

Elevated serum interleukin-6 levels in patients with pancreatic cancer.

The vast majority of pancreatic cancer patients have advanced disease at the time of diagnosis and they eventually become so emaciated that death primarily occurs from cancer cachexia. Cancer cachexia may be mediated by certain cytokines such as interleukin-6. In this study, we measured serum interleukin-6 levels in 55 patients with histologically proven pancreatic cancer and investigated their relationships to the clinical status of pancreatic cancer. A control population of 20 normal healthy adults and 25 chronic pancreatitis patients with comparable gender and age distribution characteristics was also studied. Serum interleukin-6 levels were measured using a quantitative sandwich enzyme-linked immunosorbent assay. Thirty pancreatic cancer patients (54.5%) had detectable levels, although interleukin-6 levels were detectable in only one healthy control and in two chronic pancreatitis patients. The specificity of serum interleukin-6 in this population was 93.3%, resulting in high diagnostic accuracy (72.0%). Among the pancreatic cancer patients, the detection rates of serum interleukin-6 levels increased significantly with the disease extent (p < 0.01). Moreover, a significant difference was also found in the detection rates between the 30 pancreatic cancer patients with body weight loss (76.7%) and the remaining 25 patients without weight loss (28.0%, p < 0.01). These results may provide new insight into both diagnosis and treatment of pancreatic cancer, because the diagnostic accuracy of serum interleukin-6 was high and because anti-interleukin-6 therapeutics could improve symptoms in pancreatic cancer patients with high interleukin-6 levels.

Effect of heavy alcohol intake on long-term results after curative resection of hepatitis C virus-related hepatocellular carcinoma.

We studied the effect of heavy alcohol intake (ethanol intake > or = 80 g/day for > or = 5 yr) on long-term results in 53 patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) who had undergone curative hepatic resection. Cell proliferative activity in the tumor and non-tumorous liver was also assessed by counting argyrophilic nucleolar organizer region-associated proteins (Ag-NOR) in the resected specimens. Twenty patients (20 males, 0 females) were positive for heavy alcohol intake [AI(+)] and 33 (28 males, 5 females) were not [AI(-)]. All patients were positive for HCV antibody and negative for hepatitis B surface antigen. Carcinoma recurred within 3 to 51 postoperative months in 42 (79.2%) of the 53 patients. The median disease-free survival time was 12.6 mo in the AI(+) group and 25.4 mo in the AI(-) group (P < 0.01). The AI(+) group also had significantly poorer survival than the AI(-) group (P < 0.05, 3-year survival rate: 66.7% vs. 93.5%). HCC tumor in the AI(+) group showed significantly increased proliferative activity compared with that in the AI(-) group (P < 0.05, Ag-NOR number: 2.3 +/- 0.8 vs. 1.9 +/- 0.4). However, there was no significant difference between the numbers of Ag-NORs in non-tumorous liver from these two groups (1.5 +/- 0.2 vs. 1.5 +/- 0.2). Patients with heavy alcohol intake should be followed particularly closely, even if they have received curative surgery, since heavy alcohol intake is closely related to a poor postoperative prognosis.

Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma.

BACKGROUND: Mutations in the K-ras oncogene at codon 12 are detected at a remarkably high frequency in pancreatic carcinomas and are believed to be a critical event in oncogenesis. The authors attempted to detect K-ras mutations in DNA obtained from pure pancreatic juice collected endoscopically, as a novel diagnostic approach to pancreatic carcinoma. METHODS: K-ras mutations were examined using the two-step polymerase chain reaction (PCR) combined with restriction enzyme digestion, followed by nonradioisotopic single-strand conformation polymorphism (SSCP) analysis. RESULTS: Specific mutations of the K-ras gene at codon 12 were found in six of nine (67%) duct cell carcinomas, all of which were negative by cytodiagnosis of the same pure pancreatic juice. K-ras mutations were not detected in the pancreatic juice from 14 healthy control subjects, 10 patients with chronic pancreatitis, or 3 patients with islet cell tumors. CONCLUSIONS: Detection of K-ras mutation at codon 12 in pancreatic juice is highly specific for diagnosing pancreatic duct cell carcinoma and may be a valuable diagnostic modality for pancreatic carcinoma and for differentiating chronic pancreatitis from carcinoma.