Masao Ohto

Mutations in the Precore Region of Hepatitis B Virus DNA in Patients with Fulminant and Severe Hepatitis

BACKGROUND The presence of the hepatitis B e antigen (HBeAg) in serum is known to be a marker of a high degree of viral infectivity. However, fulminant hepatitis may occur in persons who are negative for HBeAg. A single point mutation has been reported to produce a stop codon in the precore region of hepatitis B virus DNA and prevent the formation of the precore protein required to make HBeAg. To determine whether a precore-mutant virus is causally related to severe liver injury, we analyzed the entire precore region in viral strains isolated from patients with fatal cases and uncomplicated cases of hepatitis B. METHODS Serum was obtained from 9 patients with fatal hepatitis B (5 with fulminant and 4 with severe exacerbations of chronic hepatitis) and 10 patients with acute, self-limited hepatitis B. Serum samples from a sex partner implicated as the source of the virus in one case of fulminant hepatitis were also studied. The 87 nucleotides in the precore region of the hepatitis B virus were amplified by the polymerase chain reaction and then directly sequenced. RESULTS Of the nine patients with fatal hepatitis, seven had retrievable hepatitis B DNA: In all seven there was a point mutation from G to A at nucleotide 1896 of the precore region, converting tryptophan (TGG) to a stop codon (TAG). In contrast, this mutation was not found in the 10 patients with acute, self-limited hepatitis B. The hepatitis B DNA from the implicated source contained a sequence with the stop-codon mutation that was identical to the sequence in her partner, who had fulminant hepatitis. CONCLUSIONS The presence of a mutant viral strain is associated with and may be involved in the pathogenesis of fulminant hepatitis B and severe exacerbations of chronic hepatitis B.

Natural History of Minute Hepatocellular Carcinoma Smaller Than Three Centimeters Complicating Cirrhosis

Twenty-two patients with cirrhosis and minute hepatocellular carcinoma less than 3 cm in diameter were followed for periods of 6-37 mo without specific treatment. The survival curve drawn by the Kaplan-Meier method showed a 1-yr survival of 90.7%, a 2-yr survival of 55.0%, and a 3-yr survival of 12.8%. The ultrasonic patterns of these masses in the liver were correlated with the size and showed a tendency to change from a low echo pattern to a low periphery and, finally, to a massive pattern. The growth speed calculated from the doubling time for tumor volume varied considerably from case to case with an average of 6.5 +/- 5.7 mo; it also changed in some cases during the observation period. Serum alpha-fetoprotein levels were generally low, rarely assisted in diagnosis, but tended to increase when the mass attained a diameter of greater than 3 cm; sudden acceleration in the rate of increase in alpha-fetoprotein level often coincided with a change of ultrasonic pattern to the massive one.

Percutaneous ethanol injection for the treatment of small hepatocellular carcinoma : study of 95 patients

Abstract Percutaneous ethanol injection (PEI) was applied to 120 lesions in 95 patients with hepatocellular carcinomas (HCC) smaller than 3 cm in the past 6 years. All main target tumours, in 67 patients who had been followed by sonography for more than 6 months after PEI, decreased in size; 28 tumours (41.8%) became undetectable and have remained so until now. The 1‐, 2‐, 3‐, 4‐ and 5‐year survival rates calculated by the Kaplan‐Meier method were 93%, 81%, 65%, 52% and 28% respectively. These survival rates were better than those of patients with HCC smaller than 3 cm who did not receive anticancer treatment (P <0.01). The survival of patients of the Childs A or Childs B status was better than that of those with Childs C disease. Recurrence occurred in areas within the liver different from the original lesion in 34% in one year, 61% in two years and 66% in three years after PEI. PEI was then repeated in 61% of such patients.

Abdominal wall fat index, estimated by ultrasonography, for assessment of the ratio of visceral fat to subcutaneous fat in the abdomen

PURPOSE To establish a new index of regional fat distribution using ultrasonography for assessment of the ratio of the visceral fat area (V) to the subcutaneous fat area (S) (V/S ratio). SUBJECTS AND METHODS The subjects examined were 62 patients (23 males and 39 females); 51 patients had hyperlipidemia and 11 patients had glucose intolerance. The mean body mass indices ranged from 20.3 to 42.9. The mean age of the patients was 44 +/- 13 years. The thicknesses of the preperitoneal fat layer (P) and subcutaneous fat layer (S) in the abdomen were measured by ultrasonography and the P/S ratio was calculated. The V/S ratio was obtained with radiographic computed tomography. RESULTS Of the various P/S ratios examined, the ratio of the maximum thickness of preperitoneal fat to the minimum thickness of subcutaneous fat was most closely correlated with the V/S ratio (r = 0.746, p < 0.0001). This ratio was termed the abdominal wall fat index (AFI). AFI was positively correlated with serum triglyceride levels and negatively correlated with high-density lipoprotein cholesterol (r = -0.312, p < 0.05), whereas the V/S ratio was correlated with triglyceride levels. AFI was positively correlated with basal insulin levels in both men and women. CONCLUSION These results suggest that AFI measured by ultrasonography may be a new indicator of visceral fat deposition, and may reflect metabolic disorders such as lipid metabolism and glucose metabolism disorders.

Association of gallbladder carcinoma and anomalous pancreaticobiliary ductal union

A total of 96 patients with gallbladder carcinoma in whom direct cholangiography clearly opacified the pancreaticobiliary ductal union and the common channel, and 65 patients with an anomalous union of these two duct systems at a distance greater than 15 mm from the papilla of Vater (normally less than 4.6 +/- 2.2 mm, mean +/- SD) were studied. It was found that this anomalous ductal union occurred in 16.7% of the patients with gallbladder carcinoma in comparison with an incidence of 2.8% among 641 consecutive patients with various hepatobiliary and pancreatic diseases studied by endoscopic retrograde cholangiopancreatography who did not have gallbladder carcinoma. It was also found that gallbladder carcinoma occurred in 24.6% of the 65 cases of anomalous ductal union in comparison with a 1.9% incidence of this cancer among 635 consecutive patients similarly studied and found to have normal ductal union (p less than 0.001). Thus, a close etiologic association was suggested between this anomaly in the terminal segment of the biliary tract and gallbladder carcinoma. Of the 65 patients with anomalous ductal union, 50 had the so-called congenital cystic dilatation of the common bile duct and 15 did not. Five of the 50 (10%) and 11 of the 15 (73.3%) had gallbladder carcinoma (p less than 0.01), and this carcinoma seems to be related to anomalous ductal union rather than to cystic dilatation of the common bile duct. As a tumorigenic factor in this anomaly, regurgitation of pancreatic juice has been stressed.

Resolution of acute hepatitis C after therapy with natural beta interferon

To test whether interferon can prevent acute non-A, non-B hepatitis from becoming chronic, a prospective controlled trial was conducted in 25 patients; 11 were treated for an average of 30 days with a mean of 52 megaunits of interferon and 14 acted as controls. 4 patients in the treatment group who continued to have raised serum aminotransferase concentrations after a years follow-up were given a second course of interferon. Follow-up at 3 years has revealed that all but 1 of those treated showed normal serum aminotransferase, whereas only 3 controls showed such change (p less than 0.02). Serum hepatitis C virus RNA became undetectable in 10 of 11 treated and in only 1 of 12 control patients, which suggests that interferon prevents the progression of acute non-A, non-B hepatitis to chronicity by eradicating HCV.

Variations in codons 84-101 in the core nucleotide sequence correlate with hepatocellular injury in chronic hepatitis B virus infection.

Individuals with chronic hepatitis B virus (HBV) infection are generally divided into asymptomatic healthy carriers and patients with chronic liver disease. Several studies have suggested that the hepatitis B core antigen could be an immunological target of cytotoxic T lymphocytes (CTL). To investigate the possible pressure site from CTL, the entire core region of HBV DNA was sequenced in 30 subjects (10 asymptomatic healthy carriers and 20 patients with chronic liver disease). No significant changes in the nucleotide sequence and deduced amino acid residue were noted in the 10 healthy carriers. In contrast, a cluster of changes in a small segment of 18 amino acids (codons 84-101 from the start of the core gene) was found in 15 of the 20 chronic liver disease patients. All these 15 patients had advanced liver diseases (chronic active hepatitis and cirrhosis), whereas only mild liver disease (chronic persistent hepatitis) was found in the five patients without mutations. These data suggest that the region with mutation clustering is the major target of CTL, and that the mutations evolve under the pressure of immune selection.

Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction.

Hepatitis C virus (HCV) RNA was detected in the sera of patients with non-A, non-B chronic liver disease by polymerase chain reaction (PCR). RNA was extracted from the serum, reverse transcribed to cDNA, and amplified by PCR. With this method, 30 patients with non-A, non-B chronic liver disease and 10 healthy subjects were tested. HCV RNA was detected in 13 of 16 (81%) anti-HCV-positive patients and also in 7 of 14 (50%) anti-HCV-negative patients, but in none of 10 anti-HCV-negative healthy subjects. Specificity of this method was confirmed by direct sequencing of amplified cDNA segment. The nucleotide sequences (37 nucleotides) obtained from 15 patients showed only 68-78% homology compared with the prototype HCV nucleotide sequence. In addition, of 15 nucleotide sequences, there were 12 different types. But the translated amino acid sequences (12 amino acids) showed 83-100% homology compared with the prototype HCV amino acid sequence. These data suggest the majority of anti-HCV-positive patients are carriers of HCV. But to detect all the viremic patients, the anti-HCV antibody testing may be insufficient. Direct detection of HCV RNA may be useful in the study of virus replication and its association with various liver diseases.

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.

Mutations in core nucleotide sequence of hepatitis B virus correlate with fulminant and severe hepatitis.

Infection with hepatitis B virus leads to a wide spectrum of liver injury, including self-limited acute hepatitis, fulminant hepatitis, and chronic hepatitis with progression to cirrhosis or acute exacerbation to liver failure, as well as an asymptomatic chronic carrier state. Several studies have suggested that the hepatitis B core antigen could be an immunological target of cytotoxic T lymphocytes. To investigate the reason why the extreme immunological attack occurred in fulminant hepatitis and severe exacerbation patients, the entire precore and core region of hepatitis B virus DNA was sequenced in 24 subjects (5 fulminant, 10 severe fatal exacerbation, and 9 self-limited acute hepatitis patients). No significant change in the nucleotide sequence and deduced amino acid residue was noted in the nine self-limited acute hepatitis patients. In contrast, clustering changes in a small segment of 16 amino acids (codon 84-99 from the start of the core gene) in all seven adr subtype infected fulminant and severe exacerbation patients was found. A different segment with clustering substitutions (codon 48-60) was also found in seven of eight adw subtype infected fulminant and severe exacerbation patients. Of the 15 patients, 2 lacked precore stop mutation which was previously reported to be associated with fulminant hepatitis. These data suggest that these core regions with mutations may play an important role in the pathogenesis of hepatitis B viral disease, and such mutations are related to severe liver damage.