Hisanao Ohkura

Production of alpha‐fetoprotein, normal serum proteins, and human chorionic gonadotropin in stomach cancer: Histologic and immunohistochemical analyses of 35 cases

By immunoperoxidase histochemical staining of formalin‐fixed paraffin‐embedded sections, the production of alpha‐fetoprotein(AFP), albumin(ALB), transferrin(TF), alpha‐1‐antitrypsin(AAT), and human chorionic gonadotropin(HCG) was examined in 35 operatively resected stomach cancers with elevated serum AFP levels (higher than 20 ng/ml as determined by radioimmunoassay). Cells positive for AFP were found in 19 cases (54%). In 29 cases (83%), some tumor cells contained normal serum proteins (ALB, TF, or AAT). All 19 tumors with AFP‐positive cells also stained positively for two or three kinds of normal serum proteins. In some cases, AFP and normal serum proteins were localized in the same cells. There were two cases in which metastatic tumors produced AFP, whereas the primary sites did not. In nine cases (26%), HCG was present in tumor cells and HCG‐ and AFP‐positive cells were coexistent in six tumors. Histologic examination of AFP‐producing stomach tumors revealed medullary or papillotubular arrangements with marked nuclear atypia and eosinophilic granular or clear cytoplasms containing no glycogen or mucin. Some tumors with medullary patterns resembled liver cell carcinomas. Concordant phenotypic expression of AFP and normal serum protein production appears to be a general feature of AFP‐producing tumors such as liver cell carcinoma, yolk sac tumor, and stomach cancer.

The usefulness of CEA and/or CA19-9 in monitoring for recurrence in gastric cancer patients: a prospective clinical study

AbstractBackground. Many studies on postoperative carcinoembryonic antigen (CEA) and/or carbohydrate antigen (CA)19-9 monitoring after operation for gastric cancer have been reported, but most have been retrospective. Methods. A nationwide observational study was implemented in 135 leading institutions in Japan to evaluate the significance of CEA and/or CA19-9 in postoperative monitoring for recurrence in patients with advanced gastric cancer. Three hundred and twenty-one patients examined in this analysis underwent radical gastrectomy at one of Japans leading institutions between November 1993 and March 1996 and had been followed up for at least 5 years. Serum levels of CEA and CA19-9 were examined preoperatively and every 3 months postoperatively, with diagnostic imagings, such as chest X-ray, computed tomography (CT), and ultrasonography also being performed every 3 months. Results. Recurrence was observed in 120 patients (peritoneum, 48; liver 16; lymph node, 16; multiple sites, 25; and others, 12). Sensitivities of CEA and either CEA or CA19-9, or both, for recurrence were 65.8% and 85.0%, respectively, both of which values were significantly higher than the preoperative positivities (28.3% and 45.0%, respectively). In most patients with high preoperative levels CEA and/or CA19-9, these tumor markers increased again at recurrence. Recurrent diseases were detected between 5 months after detection by diagnostic imagings and 12 months before detection by diagnostic imagings (mean of 3.1 ± 3.6 months before detection by diagnostic imagings) and between 10 months after detection by diagnostic imagings and 13 months before detection by diagnostic imagings (mean of 2.2 ± 3.9 months before detection by diagnostic imagings) by CEA and CA19-9 monitorings, respectively. Conclusion. These results suggest that CEA and/or CA19-9 monitoring after operation was useful to predict the recurrence of gastric cancer, especially in almost all the patients with high preoperative levels of these markers.

Clinical significance of monitoring serum levels of 5-fluorouracil by continuous infusion in patients with advanced colonic cancer.

SummarySerum concentrations of 5-fluorouracil (5-FU) given by continuous infusion to 19 patients with advanced colonic cancer were measured by an HPLC method, and steady-state concentration (SSc), area under the curve (AUC72) and total body clearance (Cl) were calculated as pharmacokinetic parameters. The serum level of 5-FU rapidly increased, reaching a plateau within 2 h after the start of administration. There were positive correlations between the dose and both SSc (r = 0.578,P <0.01) and AUC72 (r = 0.558,P <0.05). When the patients were divided into toxic and non-toxic groups according to the degree of toxicity, the values for SSc and AUC72 in the toxic group were significantly higher than those in non-toxic patients. The Cl value in the toxic group was also significantly different from that in the non-toxic group when data were calculated on a log scale. Furthermore, no differences in these parameters between effective and non-effective groups were detected when the patients were divided into two groups according to anti-neoplastic responses. These results indicate that increased serum concentration does not always provide therapeutic benefits to patients receiving continuous infusions of 5-FU.

Detection of Hepatitis C Virus RNA in Sera and Liver Tissues of Non-A, Non-B Hepatitis Patients Using the Polymerase Chain Reaction

Sera obtained from patients with non‐A, non‐B hepatitis were examined for the presence of hepatitis C virus (HCV) genome by using the reverse transcription‐polymerase chain reaction assay, as well as for antibody to HCV (anti‐HCV) by using an enzyme‐linked immunosorbent assay (ELISA). We also examined the presence of HCV RNA in liver tissue obtained by surgical resection of hepatocellular carcinoma. Among 33 patients, HCV RNA was detectable in 21 (64%), and the antibody was also positive in 21 (64%). Eighteen (55%) patients were positive for both assays. The two assays gave inconsistent results in 3 patients who were positive for HCV RNA but negative for anti‐HCV, and in 3 other patients who were negative for HCV RNA and positive for anti‐HCV. HCV RNA was also detected in 6 out of 10 non‐cancerous liver tissue specimens and in 3 out of 7 tumor tissue specimens. Using the polymerase chain reaction, the HCV genome was detected directly in many specimens obtained from patients with non‐A, non‐B hepatitis, suggesting the presence of replicating virus in patients positive for anti‐HCV. In addition, the differing results of the two assay systems suggest that the application of both is important for evaluation of the status of HCV infection.

Combined measurement of the c-erbb-2 protein in breast carcinoma tissues and sera is useful as a sensitive tumor marker for monitoring tumor relapse

c‐erbB‐2 protein levels in tissue extracts and sera were determined in a retrospective analysis of 158 patients who underwent surgical resection of breast carcinoma by means of a sandwich enzyme immunometric assay (EIA) using monoclonal antibodies (MAbs) directed to the extracellular domain of the c‐erbB‐2 oncogene protein (ErbB‐2). In the analysis of tissue extracts, 48 samples (30.3%) showed ErbB‐2 levels exceeding 18.0 ng/mg protein (group A), while in 110 samples these levels were below 18.0 ng/mg protein (group B). Immunohistochemical examination of resected tissues using anti‐c‐erbB‐2 antibody revealed positive staining in 93.8% (45/48) in group A and 13.6% (15/110) in group B (p < 0.00001). The proportion of patients who preoperatively showed a serum ErbB‐2 value above 5.4 ng/ml was 52.1% (25/48) in group A and 10.0% (11/110) in group B (p < 0.00001). Thus, the level of ErbB‐2 in tissue extracts was significantly associated with immunohistochemistry and ErbB‐2 levels in preoperative sera. During follow‐up, 48 patients (30.3%) developed recurrent disease: 17 in group A (35.4%) and 31 in group B (28.2%). From an ROC analysis based on the postoperative serum ErbB‐2 levels in patients either with or without relapse, the cutoff value of serum ErbB‐2 for tumor relapse was determined to be 6.5 ng/ml. The sensitivity of serum ErbB‐2 in patients with relapsed breast cancer was 58.3% (21/36) overall, 84.6% (11/13) in group A and 43.5% (10/23) in group B. In the analysis of serum samples taken before relapse, 90.9% (10/11) of the subjects in group A and 26.7% (4/15) of those in group B were shown to be positive for serum ErbB‐2. Serum ErbB‐2 in group A was a more sensitive marker than other tumor markers such as CEA, CA15‐3, and NCC‐ST‐439. Thus, the determination of ErbB‐2 in tissue extracts of breast carcinoma may be useful for assessing c‐erbB‐2 protein expression in the primary tissue and indicates that serum ErbB‐2 may be a sensitive marker for monitoring tumor relapse. Int. J. Cancer 89:329–336, 2000.

Detection of aberrant crypt foci by magnifying colonoscopy

Aberrant crypt foci (ACF), first described by Bird et al., 1 have been considered an early event in colorectal carcinogenesis because they are associated with a carcinogen-induced model of colorectal tumorigenesis in rodents TM and are present at a greater frequency in the colons of patients with familial adenomatous polyposis or sporadic colorectal cancer than in patients with benign colonic diseases, a, ~ These foci consist of abnormally large, darkly stained, slightly bulged mucosal crypts. 1 Histopathologically, they resemble the minute (microscopic) adenomatous polyps that have been studied extensively in familial adenomatous polyposis 7, s but have been observed only rarely in the mucosa of other patients?, lo Furthermore, a recent study 11 suggested that ACF arise as a result of clonal genetic alterations. However, until now, these studies were investigated only in specimens taken from animals or surgically resected colons from patients with colorecta~ neoplasms. To gain a better understanding of colorectal carcinogenesis, the further analysis of ACF in a large number of patients with a wide variety of diseases and in healthy individuals is needed. In this study, the value of magnifying colonoscopy in the detection of ACF in the human colorectum was evaluated.

Sensitive detection of loss of heterozygosity in the TP53 gene in pancreatic adenocarcinoma by fluorescence‐based single‐strand conformation polymorphism analysis using blunt‐end DNA fragments

We have developed a fluorescence‐based single‐strand conformation polymorphism analysis to detect HaeIII‐sensitive polymorphic sites in intron I of the TP53 gene. It is important to treat the PCR products with Klenow fragment to remove a 3′‐protruding nucleotide from the amplified DNA fragments added during the reaction in order to obtain a single peak for each allele. A comparison of the signal profiles of two alleles with those of normal heterozygotes by data processing using computer software has enabled sensitive detection of loss of heterozygosity (LOH) from clinical materials with a fraction of tumor cells below 10%. In analysis of 14 pancreatic carcinomas in which the proportion of the tumor cells is usually low due to the abundance of the stromal component, 7 samples (50%) were informative and 5 of the 7 (71.4%) were positive for LOH at the TP53 locus. This approach would be useful for allelotyping tumors with low cellularity, as well as other clinical samples such as biopsied specimens and paraffin embedded tissues. Genes Chromosom Cancer 15:157–164 (1996).

Diagnosis of bladder cancer by analysis of the allelic loss of the p53 gene in urine samples using blunt‐end single‐strand conformation polymorphism

The novel approach of blunt‐end single‐strand conformation polymorphism (SSCP) has been applied in the analysis of urine samples from bladder‐cancer patients for detecting loss of heterozygosity (LOH) of 3 polymorphic markers in the p53 gene. Of the 28 urine samples examined by SSCP analysis of blunt‐ended DNA fragments using a fluorescence‐based automated sequencer, 16 were informative in more than 1 of the 3 polymorphic markers at the p53 locus and 8 (50.0%) showed allelic loss of the p53 gene. In analysis of resected tumor tissues, LOH of the p53 gene was detected in 8 of 8 informative samples (100%) with T1 and higher stages and/or Grade 2 and Grade 3 tumors, while it was detected in 6 (75.0%) urine samples obtained from these 8 patients. This new diagnostic modality enables sensitive detection of tumor cells in urine samples and would be applicable for diagnostic bladder cancer with invasive character. Int. J. Cancer 74:403–406, 1997.

Sandwich Radioimmunometric Assay with Murine Monoclonal Antibody, NCC-ST-439, for Serological Diagnosis of Human Cancers

Sandwich radioimmunometric assay (RIA) for a new tumor‐associated carbohydrate antigen defined by a monoclonal antibody (MoAb), NCC‐ST‐439, was developed and the antigen levels were determined in sera of normal donors, and patients with various malignant and non‐malignant disorders. In normal donors, 97.0% (226/233) of sera were antigen‐negative (less than 12 units/ml) except for 7 serum samples from young females. In patients with malignant disorders, 34.2% (82/240) were antigen‐positive, in particular 64.0% (16/25) of patients with pancreatic carcinoma, 66.7% (16/24) of patients with recurrent colorectal carcinoma and 54,5% (6/11) of patients with recurrent breast carcinoma. In patients with non‐malignant disorders, 6.0% (7/116) were antigen‐positive. The positive rate in benign hepatobiliary disorders, including gallstones, hepatitis and liver cirrhosis, was especially low at 4.3% (1/23). We concluded that determination of serum NCC‐ST‐439 antigen would be useful in serodiagnosis of carcinoma patients.

Identification of mycobacteria by nonradioisotopic single-strand conformation polymorphism analysis

Clinical isolates of mycobacteria were identified to species levels using nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis of 16S rRNA gene fragments amplified by polymerase chain reaction with primers common to all of mycobacterial species. The method is based on a hypervariable region within the 16S rRNA in mycobacteria, which is characterized by species-specific nucleotide sequences. A total of 92 mycobacterial strains (Mycobacterium tuberculosis, M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. nonchromogenicum, M. xenopi, and unidentified strain) were studied. They were classified into nine types of pattern showing single-strand DNA bands having different mobilities. Each strain was shown in the species-specific mobility by non-RI SSCP analysis. The results of non-RI SSCP analysis were identical to those of standard biochemical methods and 16S rRNA sequencing.