Published papers

Background/Aims: The biological and clinicopathological significance of microsatellite instability in hepatocellular carcinoma still remains to be determined. The aim of this study was to assess the role of microsatellite instability in hepatocarcinogenesis. Methods: Genomic DNA extracted from 38 fresh samples of hepatocellular carcinoma was amplified by polymerase chain reaction using 29 fluorescence-labeled microsatellite markers and analyzed using a semi-automated laser scanning system. Associations between the incidence of replication error and the clinicopathological features of hepatocellular carcinoma were evaluated. Since reference DNA was extracted from corresponding fresh samples of non-cancerous liver tissue, the incidence of microsatellite instability in non-cancerous liver tissues was not assessed in this study. Results: Four (11%) hepatocellular carcinomas had a replication error in one or two microsatellite markers; they were all poorly differentiated hepatocellular carcinomas. The incidence of replication error correlated significantly with the histological differentiation of the tumor (p<0.05) and with portal vein involvement (p<0.05). All four hepatocellular carcinomas with replication errors showed loss of heterozygosity in one or more of the 29 markers we examined. No replication errors were detected in six markers in the coding regions of the BAX, insulin-like growth factor II receptor, transforming growth factor-beta type II receptor, E2F-4, hMSH3 and hMSH6 genes. Conclusions: The results of this study indicate that: (1) microsatellite instability is a rare event during hepatocarcinogenesis and may be specifically associated with progression of hepatocellular carcinoma; and (2) frame-shift mutation in the above six genes is not a common mechanism involved in progression of this cancer.

A 44-year-old woman presented with a right huge axillary mass. Both mammography and ultrasonography revealed a primary cancer of 2.8 cm maximum diameter in the right breast and metastases in the axillary lymph nodes, both being confirmed by aspiration cytology as ductal carcinoma. Right standard radical mastectomy with level III axillary lymph node dissection was carried out. Pathologically, the tumor was diagnosed as ductal carcinoma in situ with microinvasion (DCISM), histologic grade 3. The area of stromal invasion measured 1 mm at its widest point. Sixteen of the 17 resected axillary lymph nodes contained metastases, including six level III lymph nodes. Immunohistochemical studies of the tumor revealed overexpression of p53 protein, but not that of c-erbB-2 protein. The frequency of lymph node metastases from DCISM is reported to be very low. Therefore, the present case with extensive involvement of level III lymph nodes was unusual.

A 49-year-old premenopausal woman with stage I breast carcinoma underwent left quadrantectomy with axillary dissection in 1992. The tumor was 0.7×0.5 cm. Histopathologically, this was a pure tubular carcinoma without lymph node metastasis or lymphatic or vascular invasion. Although the surgical margin was pathologically negative, atypical ductal hyperplasia was present close to the cut margin's edge. Neither adjuvant chemotherapy nor radiotherapy had been given after the operation. Approximately 5 years after the first surgery, she had a local recurrence in the vicinity of the operative wound. There was no clinical evidence of distant metastasis. A salvage mastectomy was performed. Histopathological examination revealed that the second tumor was an invasive ductal carcinoma, histological grade 2, with extensive intraductal component. It was difficult to determine whether this was a true in-breast recurrence or a second primary cancer. Overexpression of p53 and c-erbB-2 was observed in the second tumor. Estrogen receptor and progesterone receptor were both negative. No postoperative chemotherapy was given. Multifocality and atypical ductal hyperplasia were observed in 7 (87.5%) and 6 [75%) of 8 patients, respectively, with tubular carcinoma between 1991 and 1997 at the National Cancer Center Hospital. Coexisting disease associated with tubular carcinoma suggests that radiotherapy may be an important component of breast conservation treatment to prevent local recurrence in this type of tumor.

Purpose: To determine the appearance of pancreatic adenocarcinoma on delayed contrast enhanced computed tomography (CT) and verify the diagnostic significance. Patients and Methods: Twenty-two surgically resected lesions of pancreatic adenocarcinoma were studied with dynamic CT and findings were compared with those on histopathology. Results: Ten 45% of 22 pancreatic adenocarcinoma demonstrated masses on unenhanced CT, while 21 (95%) demonstrated on early contrast-enhanced CT (early CT), and 15 (68%) demonstrated masses on delayed contrast-enhanced CT (late CT). Pancreatic mass was demonstrated as a low-density area on unenhanced and early CT, but mass density varied on late CT. Delayed enhancement of the mass was predominantly observed in small lesions; 3 (75%) of 4 lesions 2 cm or smaller were detected as high-density areas. Histologically, delayed enhanced lesions showed severe or moderate degrees of fibrosis. Conclusion: Late CT is useful for the detection of pancreatic adenocarcinoma with fibrosis as a high-density area, especially in small lesions.

Background: The causes and pathologic and prognostic phenotypes of late-onset familial breast cancers are still unknown. The purpose of this study was to document the clinicopathological features of late-onset familial breast cancers using genetic testing of BRCA1 and BRCA2. Methods: We analyzed 11 breast cancers from 10 patients from 8 Japanese late-onset Breast cancer families. Results: The average age of the patients was 55 years (range 43 to 89). Bilateral occurrence was noted in 2 patients (8%). All the tumors were invasive ductal carcinomas, except for 1 case of invasive lobular carcinoma. Tumor size ranged from 0.8 cm to 7.8 cm (median 2.3 cm) and lymph node metastasis occurred in 6 of the 11 patients (55%). Six (55%) of the 11 tumors were histologically grade 2 and 5 (45%) were histologically grade 3. Estrogen receptor (ER) positivity was 80% (8/10). Overexpression of c-erbB-2 and p53 protein was detected in 18% (2/11) and 9% (1/11) of the tumors, respectively. Five patients from 4 families received genetic testing but all were negative for BRCA 1 and BRCA2 germline mutations. All the patients were alive after a median follow-up period of 32 months, except for 1 patient. Conclusions: In this study, no germline mutations of BRCA1 or BRCA2 were detected. However, there was a tendency towards ER-positive tumors, but the positivity of p53 protein was considered to be lower then that of sporadic tumors.

Objective: To evaluate the use of helical computed tomography (CT) in diagnosing portal vein (PV) invasion by pancreatic head carcinoma and its usefulness in predicting outcome. Design: Validation cohort study. Setting: Tertiary care public hospital. Patients: Twenty-seven patients with carcinoma involving the pancreatic head were preoperatively studied with helical CT. All patients underwent resection. Main Outcome Measure: By quantifying the contact between the tumor and PV on helical CT, the relationship between them was classified into 1 of 4 types: type 1, visible fat layer between PV and the tumor; type 2, with the total PV circumference defined as 360°, contact between the tumor and PV was considered to be 90°or less; type 3, contact ranged between 91°and 180°; and type 4, contact greater than 180°. Helical CT results were compared with intraoperative observation, histological findings of the resected specimen, and postoperative course. Results: When helical CT showed type 3 or 4, the case was diagnosed as positive for PV invasion. Sensitivity, specificity, and overall accuracy were 83%, 100%, and 89% when compared with the intraoperative assessment, and 92%, 79%, and 85% with the histological assessment, respectively. One- and 2-year survival rates were 86% and 69% for type 1,100% and 75% for type 2, and 33% and 12% for type 3, respectively. The survival rates of patients with types 1 and 2 were significantly higher than that of those with type 3 (P<.05). All 3 patients with type 4 died within 9 months. Conclusions: Helical CT facilitates detection of PV invasion by pancreatic head carcinoma. The extent of PV involvement reflected the outcome after pancreatectomy.

The present study was designed to determine whether changes in DNA methyltransferase (DNA MTase) expression are involved in hepatocarcinogenesis. We examined DNA MTase expression in normal liver tissue (with no remarkable histological findings), liver tissue showing chronic hepatitis or cirrhosis, which are generally thought to be precancerous conditions, and hepatocellular carcinomas (HCCs) using the reverse-transcriptase polymerase chain reaction assay. DNA MTase mRNA levels were significantly higher in liver tissue showing chronic hepatitis and cirrhosis (DNA MTase mRNA/β-actin mRNA ratio = 0.30 ± 0.22, n = 24, P < 0.01) than in normal liver tissue either from patients with liver metastatic lesions of colonic cancer (0.14 ± 0.05, n = 6) or from patients with HCCs (0.16 ± 0.07, n = 3). DNA MTase mRNA levels were even higher in HCC tissue (0.34 ± 0.18, n = 29). These results suggest that increased DNA MTase expression may be an early event during hepatocarcinogenesis. DNA MTase is a potential target for HCC preventive therapy.

Clinicopathological features of 28 patients with intraductal papillary tumor (IDPT) and 10 patients with mucinous cystic tumor (MCT) of the pancreas were studied. Both IDPT and MCT showed papillary projections of the epithelium with abundant mucus secretion in the ectatic ducts or cystic spaces. The patients with IDPT comprised 19 men and 9 women with a mean age of 64.9 years. Three fourths of the IDPTs were located in the pancreatic head, and the mean tumor size was 9.5 cm. Local recurrence was observed in one patient, but none died of IDPT. In contrasts all patients with MCT were women, with a mean age of 49.4 years. None of the MCTs arose in the head, and the mean tumor size was 7.1 cm. One patient died of MCT, but all of the others survived without recurrence. Eight IDPTs and three MCTs showed invasion into the surrounding pancreatic tissue. Muconodular infiltration was mainly observed in five IDPTs and one MCTs and tubular infiltration in three IDPTs and two MCTs. A characteristic histological finding associated with the muconodular infiltration in IDPT was subepithelial 'mucin droplets' that appeared to represent a change in polarity of mucus secretion. The formation of such subepithelial 'mucin droplets' may he the initial step of muconodular infiltration in IDPT. Muconodular infiltration in IDPT appears different morphologically and biologically from the mucinous carcinoma subtype of conventional invasive ductal carcinorma.

We report a case of insulin-dependent diabetic fibrous mastopathy, which clinically simulated breast cancer. The patient is a 55-year-old woman with stony-hard breast lumps, who had been treated with insulin for diabetes mellitus. Mammmography revealed a highly-dense mass shadow in the left breast and ultrasonography detected hypoechoic lesions with acoustic shadows in both breasts, suggestive of malignancy. Excisional biopsy was performed for the left breast lump. On histopathological examination, this was fibrosis with dense lymphocytic infiltration around lobules, which is a characteristic feature of diabetic fibrous mastopathy.

A 7 year old boy with giant cell fibroblastoma (GCF) of the skin and subcutaneous tissue of the right chest wall is described. To date, the histogenesis of GCF has not been clarified. The reason for the diversity of immunohistochemical data among various authors may be because the specimens studied were from only part of the lesion, or reduction of antigenicity through the preparation process. However, our findings based on studies of many specimens from various parts of the tumor for accurate immunohistochemical evaluation suggest that GCF may be a myofibro histiocytic tumor. Recently, the suggestion that GCF is a juvenile form of dermatofibrosarcoma protuberans (DFSP) has been reported. In addition to the present case, we performed immunohistochemical examination of 10cases of definitely diagnosed DFSP for comparison. The immunohistochemical characteristics of these two neoplasms were concordant. However, from clinical and morphological viewpoints, it seems premature to recognize GCF as a juvenile form of DFSP. Acta Pathol Jpn 41: 552–560, 1991. Copyright © 1991, Wiley Blackwell. All rights reserved

To find new histopathological prognostic parameters for adenocarcinoma of the pancreatic head, 27 of these carcinomas, at stage IV according to the Japan Pancreas Society, which had been curatively resected, were histologically examined. In addition to previously recognized histological prognostic parameters, sections were examined for the following factors: fibrotic focus (FF), direct invasion of the tumor into the lymph node (DILN), and tumor necrosis (TN). Frequency of tumor recurrence or death was higher in patients with any one of the three new factors than in those without any of these factors. To develop an accurate system for predicting the outcome, the presence or absence of each factor and lymphatic permeation was given a score of 1 (present) or 0 (absent), and the total score was than calculated for each patient. All patients with a score of 0 were alive without tumor recurrence, at a mean follow-up of 20 months, whereas all seven patients with a score of 4 had experienced tumor recurrence, and six died subsequently. The results of this study demonstrated that FF, DILN, and TN are good histological parameters of ductal adenocarcinoma of the pancreatic head, and that the scoring system proposed in this paper is useful for prediction of the outcome of the disease. © Springer-Verlag 1997.

Regulation of cell adhesion systems is involved in both normal development and the invasive behavior of carcinomas. We examined alterations of cell morphology and adhesion molecules in response to phorbol ester treatment of the SW1116 colon cancer cell line, which forms well-organized dome-like tubular structures in culture. 12-O-Tetradecanoylphorbol-13 acetate (TPA) induced rapid spreading of cancer-cell colonies through formation of focal adhesion and disappearance of adherens junctions. Immunologic analyses demonstrated that tyrosine-phosphorylated proteins were concentrated at focal adhesions, and that tyrosine phosphorylation of two proteins, paxillin and an unidentified 130-kd protein, was significantly increased. Tyrosine phosphorylation of paxillin was detectable within 15 min after TPA treatment, when only lamellipodia had extended from the colony, and in cells treated with blocking antibodies against integrins β1 and β5, which strongly inhibited spreading and disorganization while preserving adherens junctions. The level of paxillin phosphorylation correlated well with the degree of morphologic change induced by low-dose TPA, and the dephosphorylation occurred before reversion of morphology upon removal of TPA. These findings suggest that the TPA signal was transduced to the tyrosine phosphorylation of paxillin strongly associated with formation of focal adhesion, and that this in turn induced dysfunction of the cadherin system and caused spreading and disorganization of the tubular structure. The mechanism responsible for disruption of the cadherin system at adherens junctions was not clear, but the transition of β-catenin into nuclei corresponded to the disappearance of its signal along areas of cell-cell contact. This SW1116 model provides a good system for studying the molecules involved in transient regulation and crosstalk between the cell-cell and cell-substratum adhesion systems, which may explain the mechanism of invasion of well-differentiated human carcinomas.

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.

Since the CD44 variant 6(v6) molecule has been noted as a marker for tumor metastasis and prognosis in several tumors, we examined whether or not v6 is a useful marker for evaluating the prognosis of pancreatic cancer patients. In addition, we attempted to assess the clinicopathological implications for pancreatic cancer of the variant 2 (v2) isoform using a recently developed monoclonal antibody against a v2 epitope. The expression of CD44 variants was evaluated immunohistochemically in paraffin-embedded pancreatic cancer tissues from 42 patients who were confirmed surgically and histologically to have received curative resection. An indirect immunoperoxidase method was used with monoclonal antibodies against epitopes of the standard (CD44s) portion, v6 and v2. Protein expression data were evaluated statistically for any correlations with the length of survival or with histological parameters. The expression of CD44v6 and v2 was observed only in tumor cells, if at all. On the other hand, expression of total CD44 (including CD44v, as well as CD44s) was observed in both tumors and adjacent normal sites. Tumor tissue from 21 (50%) and 16 (38%) patients showed positive immunoreactivity with mAb 2F10 (anti-CD44v6) and mAb M23.6.1 (anti-CD44v2), respectively. The expression of CD44v6 and v2 was correlated with decreased overall survival (P = 0.0160 and P = 0.0125, respectively). A significant correlation was obtained between CD44v2 peptide expression and vessel invasion (P = 0.026). These results suggest that CD44v2 and CD44v6 may be useful markers for poor prognosis in curatively resected primary pancreatic cancer.

E-cadherin, the major intercellular adhesion molecule of epithelial cells, is important in determining the architecture of sarcomas, especially those showing epithelioid features. In addition to its role in cell adhesion, β-catenin, a cadherin undercoat protein, has been shown to function as a downstream transcriptional activator of the Wnt/Wingless signaling pathway. In order to evaluate the significance of the cadherin cell adhesion system and the Wnt/Wingless signaling pathway in the morphogenesis and/or tumorigenesis of synovial sarcoma (a major type of sarcoma with epithelioid features), immunoreactivity for pan-cadherin, E-cadherin, and their undercoat proteins (α-, β-, and γ-catenins and p120) was evaluated in 15 synovial sarcomas. Immunoreactivity for pan-cadherin, E-cadherin, α-catenin, β-catenin, and p120 was observed in all 15 specimens. Immunoreactivity for pan-cadherin was stronger than that for E-cadherin. Expression of γ-catenin was detected in ten specimens. Although β-catenin was observed only at the cell-cell boundaries in four specimens, it was present in the nucleus and cytoplasm and at the cell-cell boundaries in the other 11, suggesting constitutional activation of the Wnt/Wingless signaling pathway in synovial sarcoma. Direct sequencing for exon 3 of the β-catenin gene, however, revealed no mutations in any of the 15 specimens. In conclusion, other types of cadherin besides E-cadherin, together with cadherin undercoat proteins, may play a role in cell adhesion in synovial sarcoma. Furthermore, mechanisms other than mutation of exon 3 of the β-catenin gene may activate the Wnt/Wingless signaling pathway in this type of tumor.

The aim of this study was to elucidate the pathogenetic backgrounds of early-onset gastric cancers. Mutations of the E-cadherin and β-catenin genes were analyzed by subjecting microdissected cancer cells and corresponding non-cancerous epithelial cells obtained from 9 gastric cancer patients under 35 years old to polymerase chain reaction-single strand conformation polymorphism analysis. Somatic, but no germline, E-cadherin gene mutations were detected in 6 (67%) of the patients. The cancer cells of 2 patients were exon 9-deleted E-cadherin molecule-immunoreactive. Neither somatic nor germline mutations in exon 3 of the β-catenin gene were observed in any patient. One patient lacked β-catenin immunoreactivity and the cancer cells of 6 others showed cytoplasmic β-catenin immunoreactivity. The E-cadherin-mediated cell adhesion system in the cancer cells of all the patients examined appeared to be disrupted, indicating that somatically acquired dysfunction of this system plays an important role in early-onset diffuse-type gastric cancers. Helicobacter pylori infection was observed in 6 (67%) of our 9 patients, an incidence higher than the average in young Japanese individuals. Thus, early-onset gastric cancers may be attributabIe to environmental factors such as Helicobacter pylori infection.

The aim of this study was to elucidate the significance of aberrant DNA methylation in gastric carcinogenesis. The DNA methylation status at the D17S5 locus, at which a candidate tumor suppressor gene, HIC-1, was identified, of gastric cancers and non-cancerous gastric mucosae from 42 gastric cancer patients was examined by Southern blotting using a methylation-sensitive restriction enzyme. DNA hypermethylation was observed in 15, 13, 25 and 45% of the tissues showing no remarkable histological findings, chronic gastritis without intestinal metaplasia, intestinal metaplasia and gastric cancer, respectively. The incidence of DNA hypermethylation was significantly higher in gastric cancers than in non-cancerous gastric mucosae (P < 0.05). DNA hypermethylation was often accompanied by allelic loss at the same locus in gastric cancers. DNA hypermethylation at the D17S5 locus, which was even detected in precancerous conditions, including intestinal metaplasia, may play a role in gastric carcinogenesis.

The aim of this study was to examine the association between DNA hypermethylation and clinicopathological features of non-small cell lung cancers (NSCLCs). The DNA methylation status at the D17S5 loci, at which a candidate tumor suppressor gene, HIC-1 (hypermethylated in cancer), was identified, of 51 paired tumor and nontumorous lung tissue specimens from NSCLC patients were examined by Southern blot analysis, using a methylation- sensitive restriction enzyme. DNA hypermethylation at this locus was found in 17 (33%) tumors and 16 (31%) nontumorous lung tissues. DNA in hypermethylation at this locus occurred more frequently in poorly than in well-differentiated tumors, especially in adenocarcinomas, and correlated significantly with the differentiation grade (P = 0.01). DNA hypermethylation at the D17S5 locus correlated significantly with the loss of heterozygosity at this locus in tumors (P = 0.01). The incidence of DNA hypermethylation was significantly higher in smokers than those who had never smoked in both tumors and nontumorous lung tissues (P = 0.03 and P = 0.01, respectively). These results suggest that DNA hypermethylation at the D17S5 locus may play a role in the development of NSCLCs in cigarette smokers.

Background: Hexamethylene bisacetamide (HMBA), a potent differentiating agent of transformed cell lines, has been reported to affect phenotypic modulation and suppress proliferation of vascular smooth muscle cells (VSMCs). The effects of HMBA on the growth of VSMCs were studied in the rat carotid injury model. Methods: HMBA was added to culture medium of VSMCs explanted from aorta of Sprague Dawley rats and the cell number was counted after 5 days of incubation. The carotid artery of Sprague Dawley rats was denuded with a balloon embolectomy catheter. The rats were treated with continuous infusion of HMBA (1 g/day) or saline for 7 days after injury. Results: HMBA inhibited VSMC proliferation in a dose-dependent manner (at a HMBA concentration of 1-5 mM). Plasma concentration of HMBA during continuous infusion was in the range 0.8-1.0 mM. Immunohistochemistry of proliferating cell nuclear antigan showed that HMBA inhibited VSMC proliferation after vascular injury. Cell number in the intima and neointimal thickening were significantly reduced in HMBA-treated rats at 14 days after injury. Conclusions: Systemic administration of HMBA inhibited VSMC proliferation in the rat carotid injury model.