Minoru Tada

Clinical application of ras gene mutation for diagnosis of pancreatic adenocarcinoma

Most pancreatic adenocarcinomas are known to have ras gene (oncogene) mutations. The site of the mutations is localized in codon 12 of K-ras gene. Such high incidence and localization of the ras gene mutations have not been observed in any other human malignancies. Polymerase chain reaction and direct sequencing method enabled us to analyze DNA sequence around codon 12 of K-ras gene in small quantities of specimens obtained from needle biopsies and aspirate samples for pathological diagnosis. All the materials obtained from 12 patients with pancreatic adenocarcinoma showed the mutations, whereas those obtained from 6 patients with chronic pancreatitis showed no mutations. In several cases using the mutations of K-ras gene as a marker, this analysis supplemented conventional pathology and cytology in making the diagnosis of pancreatic adenocarcinoma. The analysis of ras-gene mutations was useful for the clinical diagnosis of pancreatic adenocarcinoma.

Analysis of ras gene mutations in biliary and pancreatic tumors by polymerase chain reaction and direct sequencing.

Ras gene is one of the oncegenes most commonly detected in human cancers and consists of three families (H‐ras, K‐ras, N‐ras) that are converted to active oncogenes by point mutations occurring in codon 12, 13, or 61. The authors analyzed mutations of these codons in 12 extrahepatic bile duct carcinomas, nine gallbladder carcinomas, and 20 pancreatic tumors (18 pancreatic adenocarcinomas and two islet cell tumors) by a method to directly sequence nucleotides, using polymerase chain reaction and a direct sequencing method. Point mutations at K‐ras codon 12 were found in all of 18 pancreatic adenocarcinomas and in one bile duct carcinoma, but there were no mutations in the remaining 11 bile duct carcinomas, in all of 9 gallbladder carcinomas, or in two islet cell tumors. A very high incidence of ras gene mutations may be used clinically for the diagnosis of debatable cases of pancreatic adenocarcinoma.

Ras gene mutations in intraductal papillary neoplasms of the pancreas. Analysis in five cases

Five intraductal papillary neoplasms of the pancreas were analyzed for the presence of the Ras gene mutations. Three (60%) of the five neoplasms showed point mutations at K‐ras codon 12. This incidence of the mutations was rather low compared with that found with ordinary pancreatic adenocarcinoma. The presence of the mutations did not correlate with the severity of cellular atypia, but was apparently related to the size of the tumor. The two neoplasms that had no mutations were smaller than the others that had mutations. The analysis suggested that Ras gene mutation is not the first genetic alteration of tumor progression, but that it occurs during the development of neoplasms of the pancreas.

Randomized, double-blind, placebo-controlled trial of eight-week course of recombinant α-interferon for chronic non-A, non-B hepatitis

Forty-nine Japanese patients were enrolled in a randomized, placebo-controlled, doubleblind trial of α-interferon for chronic non-A, non-B hepatitis: 24 patients received 3 million units of recombinant human alpha α-interferon (α-2a) thrice weekly for eight weeks, and 25 patients received placebo in a similar schedule. The mean serum alanine aminotransferase (ALT) dropped from 155±91 (sd) to 69±72 during interferon treatment, but remained unchanged (158±140 to 147±130) during placebo treatment (P<0.001). Serum ALT level fell to the normal range in 29% of interferon-treated patients, but in only 4% of placebo-treated patients. Pre- and posttreatment liver biopsies were obtained in all but one case. Average histological activity indices (HAI) were markedly improved in the interferon-treated group (9.5±3.7 to 7.0±4.3), but were unchanged in the placebo group (8.5±4.3 to 8.5±4.9). In addition, we compared the efficacy of interferon treatment between anti-hepatitis C virus (HCV) antibody positive and negative groups. Biochemical and histological improvements were similar and statistically significant in patients with and without antibody to hepatitis C virus. These data indicate that a eight-week course of α-interferon induces biochemical and histological improvement in more than half the patients with chronic non-A, non-B hepatitis.

Detection and direct sequencing of hepatitis B virus genome by DNA amplification method

Hepatitis B virus (HBV) DNA was detected with amplification by the polymerase chain reaction method. Cloned HBV DNA equivalent to one virus genome (3 x 10(-6) pg) was detectable by ethidium bromide staining after 50 cycles of polymerase chain reaction. By applying this method, presence of HBV DNA was studied in 23 hepatitis B surface antigen (HBsAg)-positive and 11 HBsAg-negative sera from patients with chronic liver disease. Hepatitis B virus DNA was positive in 8 of 8 hepatitis B e antigen (HBeAg)-positive, in 2 of 2 HBeAg- and anti-HBe-negative, and in 12 of 13 anti-HBe-positive sera. Hepatitis B virus DNA was undetectable in all HBsAg-negative sera even with amplification. To confirm specificity, the amplified product was directly sequenced. Sequences around 122nd and 160th codon of HBs gene, which determines subtypes d/y and w/r, respectively, were analyzed. The results were compatible with recent reports regarding the relation between HBV subtypes and HBV DNA sequence at those codons. Hepatitis B virus DNA could be detected at the level of one virion by gene amplification method, and its sequence could be determined by direct sequencing in a few days.