Takachika Ozawa

Characterization of various antibodies against tissue plasminogen activator using highly sensitive enzyme immunoassay

Polyclonal and three monoclonal anti-tissue plasminogen activator (a-t-PA) antibodies (1:3 C5, 1:3 G5 and 2:2 B10) were characterized by using enzyme immunoassay (EIA) in which beta-D-galactosidase was coupled to a-t-PA antibody. Monoclonal antibodies called 2:2 B10 and 1:3 G5, specific for both one-chain and two-chain t-PA, strongly bound to one-chain t-PA purified from cultured melanoma cell lines, but 1:3 C5 antibody bound weakly to such t-PA. When polyclonal t-PA antibody was used as the first reaction antibody immobilized on silicone pieces, few determinants were available for monoclonal antibody used in the second reaction due to previous interaction of these determinants in the first reaction. When t-PA levels in the plasma were determined, the presence of EDTA enhanced the sensitivity of t-PA determination by the present EIA technique. Plasma concentrations of t-PA were measured to be higher with 2:2 B10 monoclonal antibody than with polyclonal antibody as the first antibody. Tissue-PA was mainly detected in the endothelial cells, but not in the muscular layer of the inferior mesenteric artery when immunochemical technique was used with polyclonal t-PA antibody.

Genetic Alterations of Mixed Hyperplastic Adenomatous Polyps in the Colon and Rectum

Some mixed hyperplastic adenomatous polyps (MHAPs) contain dysplastic lesions or even carcinomas. These polyps are considered to be different from ordinary hyperplastic polyps and may have a preneoplastic potential. We investigated APC and K‐ras mutations in MHAPs of the colon and rectum, and also in colorectal adenomas and hyperplastic polyps to identify molecular differences between MHAPs, adenomas and hyperplastic polyps, using direct sequencing of mutation cluster regions (MCR) in APC and K‐ras. No APC mutations were identified in 12 MHAPs and 8 hyperplastic polyps, whereas 10 of 27 (37.0%) adenomas showed somatic mutations. K‐ras mutations were identified in one of 12 (8.3%) MHAPs, one of 8 (12.5%) hyperplastic polyps, and 10 of 27 (37.0%) adenomas. p53 mutation was found in a carcinoma arising in an MHAP. Mutations other than APC mutations may play a role in the development of MHAPs.

Positron emission tomography with 18F-fluoro-2-deoxyglucose for the detection of recurrent ovarian cancer.

BackgroundRecurrent ovarian cancer is refractory and resistant to treatment in most patients, and no effective treatment for it has been established. Starting a treatment when tumors still consist of micro foci may contribute to improvement of prognosis. Therefore, the early diagnosis of relapse is important.MethodsAmong patients with epithelial ovarian cancer in whom initial treatment achieved remission between April 1998 and December 2003, those patients in whom the cancer-related antigen (CA)125 level was increased during the subsequent follow-up period, or those who showed abnormal computed tomography (CT)/magnetic resonance imaging (MRI) findings despite normal CA125 levels, were examined by 18F-fluoro-2-deoxyglucose – positron emission tomography (FDG-PET). We compared the rates of accurate diagnosis of recurrence achieved using CT/MRI, CA125, and FDG-PET in patients with a definitive diagnosis of relapse.ResultsWe investigated 29 patients with epithelial ovarian cancer. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of FDG-PET were 84.6% (22/26), 100% (3/3), 100% (22/22), 42.9% (3/7), and 86.2% (25/29), respectively. These values were higher than the corresponding values obtained using CT/MRI or CA125 levels.ConclusionFDG-PET may be very useful for identifying sites of recurrent ovarian cancer, although this procedure had a low NPV because of the high rate of false-negative findings for micro or cystic lesions.

Germline alterations in the CDH1 gene in familial gastric cancer in the Japanese population

Germline point or small frameshift mutations of the CDH1 (E‐cadherin) gene are known to cause familial gastric cancer (FGC), but the frequency of CDH1 mutations is low in Japanese patients with FGC. Because recent studies have reported germline large genomic deletions of CDH1 in European and Canadian patients with FGC, in the present study we examined DNA samples from 13 Japanese patients with FGC to determine whether similar germline changes were present in CDH1 in this population. Using a sequencing analysis, a 1‐bp deletion (c.1212delC), leading to the production of a truncated protein (p.Asn405IlefsX12), was found in an FGC family; immunohistochemical analysis revealed the loss of CDH1 protein expression in the tumors in this family. Using a combination of multiplex ligation‐dependent probe amplification (MLPA) and RT‐PCR analyses, we also found a large genomic deletion (c.164‐?_387+?del), leading to the loss of exon 3 and the production of a truncated protein (p.Val55GlyfsX38), in another FGC family. The functional effects of the detected mutations were examined using a slow aggregation assay. Significant impairment of cell–cell adhesion was detected in CHO‐K1 cells expressing Ile405fsX12‐ and Gly55fsX38‐type CDH1 compared with cells expressing wild‐type CDH1. Our results suggest that the p.Asn405IlefsX12 and p.Val55GlyfsX38 mutations of the CDH1 gene contribute to carcinogenesis in patients with FGC. This is the first report of CDH1 germline truncating mutations in Japanese patients with FGC. Screening for large germline rearrangements should be included in CDH1 genetic testing for FGC. (Cancer Sci 2011; 102: 1782–1788)

Solitary splenic metastasis from gastric cancer: Report of a case

Abstract.We report a rare case of metachronous and solitary metastasis to the spleen from gastric cancer. A 69-year-old man who had undergone a distal gastrectomy for gastric cancer 48 months earlier was found to have a solitary lesion in the spleen, and an increased serum carcinoembryonic antigen (CEA) level. The patient underwent a laparotomy for suspected metastasis to the spleen. At laparotomy, a tumor was found in the upper pole of the spleen without involvement of other organs, and a splenectomy was performed. Histological examination confirmed that the splenic tumor was a well-differentiated adenocarcinoma similar to the primary gastric cancer. The postoperative course was uneventful and his serum CEA decreased to within normal levels. The patient died of multiple metastases to the liver and peritoneal dissemination 40 months after the splenectomy.

Evaluation of the Interobserver Agreement in the Number of Mitotic Figures of Breast Carcinoma as Simulation of Quality Monitoring in the Japan National Surgical Adjuvant Study of Breast Cancer (NSAS-BC) Protocol

In the National Surgical Adjuvant Study for Breast Cancer (NSAS‐BC), node‐negative breast cancers were divided into higher‐ and lower‐risk groups according to the histopathological nuclear grade given at individual collaborating hospitals, and the higher‐risk group was entered into a randomized protocol of adjuvant therapy. Because the nuclear grade was the composite of nuclear atypia and mitotic counts, maintenance of interobserver agreement in mitotic counts was indispensable for the success of the protocol study. Fourteen pathologists participating in the protocol judged whether or not 20 photomicrographs suspected of showing mitotic cancer‐cell figures truly showed mitoses. After standardizing the counting method, these pathologists counted the number of mitotic figures per 10 high‐power fields of hematoxylin‐eosin‐stained main‐tissue sections of 20 tumors. Areas where mitotic counts were considered to be the most frequent by each pathologist were compared for these tumors. For the judgment of whether the photomicrograph indicated mitosis, the level of interobserver agreement was moderate (k=0.569). In the observations of 20 tumors, interobserver agreement level of mitotic counts was moderate (k=0.506), that of nuclear atypia scoring was fair (k=0.265), and that of nuclear grading was substantial (k=0.633). The counted area was almost the same among the observers in 9 tumors, split into two areas in 6, and dispersed in 5. Concordance in judgment was achieved in 7 of the first 9 and in all of the third 5, but only in one of the second 6. The cause of discordance was mostly derived from tumor heterogeneity and the difference in the site where mitoses were counted. Interobserver agreement level was considered to be satisfactory, and it was expected that the case entry would be performed appropriately in the protocol study. The selection of the counting area was confirmed to be important for the acquisition of high‐level agreement level in mitotic counts.

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin‐fixed, paraffin‐embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.

Chronic Gastric Anisakiasis Presenting as Pneumoperitoneum

Typically, the diagnosis of gastric anisakiasis is made at endoscopy with the identification of anisakis larvae. We report a case of gastric anisakiasis presenting as pneumoperitoneum. A 70-year-old Japanese woman was admitted to our hospital with abdominal fullness and pain. Plain chest X-ray in the upright position showed the presence of free gas below the diaphragm. A tentative diagnosis of perforation peritonitis was made and an emergency laparotomy was performed. At laparotomy, a 4 cm, circumscribed red mass was noted on the anterior wall of the upper body of the stomach near the lesser curvature and a partial gastrectomy was carried out. The histological diagnosis showed a foreign body, assumed to be a part of anisakis larvae, seen in the centre of the granuloma. On the serosal aspect, there was histological evidence of peritonitis with fibrin and neutrophils. In addition, an antianisakis larvae immunoglobulin G antibody test was positive. Chronic gastric anisakiasis was suspected because of the presence of eosinophilic granuloma in the resected area and denatured anisakis larvae. Thus, we interpret this case as gastric perforation acutely based on chronic gastric anisakiasis.

CASE REPORT: Extrahepatic Biliary Schwannoma

Schwannoma (neurilemoma or neurinoma) rarely develops in the biliary tract. We report here a case of extrahepatic biliary schwannoma found in a 47-yr-old Japanese woman presenting with obstructive jaundice. The radiological imaging studies were suggestive of nonepithelial tumor involving the common bile duct. The patient underwent tumor resection. The tumor extended inward and outward from the wall of the common bile duct in the shape of a dumbbell. The extraductal tumor was solid with microcystic changes, while the intraductal lesion presented cystic changes. Microscopically, the tumor was predominantly composed of spindle-shaped cells with nuclear palisading, and it contained lymphoid aggregates. Immunohistochemically, the tumor cells were positive for S-100 protein. The final diagnosis was benign schwannoma of the common bile duct. The tumor differed from usual soft tissue schwannoma and closely resembled gastrointestinal schwannoma.

CD5^+ Diffuse Large B-Cell Lymphoma with c-myc/IgH Rearrangement Presenting as Primary Effusion Lymphoma

We report an instructive case of diffuse large B-cell lymphoma presenting as acute heart failure. A 69-year-old human immunodeficiency virus-negative man was admitted to our hospital for general fatigue. A computed tomographic scan of the chest and abdomen showed pericardial effusion, but there was no evidence of tumor masses, lymph node enlargement, or hepatosplenomegaly. During the chemotherapy, increased lactate dehydrogenase and pleural effusion appeared. The tumor cells in the effusion showed positivity for CD5, CD19, CD20, κ chain, and Bcl-2 and negativity for CD10 and CD23.The chromosomes showed t(8;14)(q24;q32) with c-myc/immunoglobulin (Ig)H rearrangement, and the MIB-1 index was not high (60%). Neither human herpes virus 8 nor Epstein-Barr virus DNA was detected in the cells by polymerase chain reaction. The response to chemotherapy was very poor, and the patient died 4 months after the diagnosis. A spectrum of the symptoms of CD5+ lymphoma encompasses pericardial effusion and also can accompany c-myc/IgH rearrangement.