Shinya Oda

Clinical significance of occult micrometastasis in lymph nodes from patients with early gastric cancer who died of recurrence

BACKGROUND Even after curative resection of an early gastric cancer, some patients die of a recurrence. It is our view that patients with early gastric cancer who died of their disease had occult micrometastases in perigastric lymph nodes at the time of the original diagnosis. In an attempt to identify these micrometastases, lymph nodes dissected from early gastric cancer lesions were stained after operation with monoclonal antibody against cytokeratin, an essential constituent of the cytoskeleton of epithelial cells. METHODS The 420 dissected lymph nodes from 34 patients with node-negative early gastric cancer who died of a recurrence were examined for the presence of tumor cells. We used immunocytochemical techniques and an antiserum to epithelial membrane antigen. The monoclonal antibody CAM 5.2 recognizes cytokeratin polypeptides (human cytokeratin numbers 8 and 18) commonly present in epithelial cells. Clinicopathologic characteristics and prognosis were determined for patients with cytokeratin-positive cells in the lymph nodes. RESULTS. Of 420 lymph nodes, 15 (3.6%) nodes and 23.5% (8 of 34) of the patients presented with cytokeratin-positive cells at the time of primary operation. The presence of cytokeratin positivity was not related to various clinicopathologic factors. The histologic stage of eight cytokeratin-positive cases was upstaged by the group of cytokeratin-positive lymph nodes from stage I to three of stage II, four of stage III, and one of stage IV, hematogenous recurrences were common, and the prognosis was poorer. CONCLUSIONS Immunohistochemical techniques aid in identifying micrometastatic disease in lymph nodes missed in routine hematoxylin-eosin staining. Cytokeratin staining of the dissected lymph nodes is recommended to precisely determine tumor stage and prognosis for patients with early gastric cancer.

Prognostic value of epidermal growth factor receptor (EGFR) and its relationship to the estrogen receptor status in 1029 patients with breast cancer

An epidermal growth factor receptor (EGFR) has been reported to be associated with a poor clinical outcome in breast cancer, while its prognostic value remains controversial. Immunohistochemical staining for EGFR was performed on frozen sections of primary breast cancer from 1029 patients with a mean follow-up duration of 46 months. EGFR was positive in 277 (26.9%) of 1029 cases, which inversely correlated with the estrogen receptor (ER) status. A univariated analysis indicated that EGFR had a significant prognostic value in both the disease free survival (DFS) and the overall survival (OS), while the same effect was also found in node negative as well as node positive breast cancer. A multivariate analysis indicated that EGFR was an independently significant prognostic factor for DFS (p = 0.0174) and OS (p = 0.0105) in all patients, but that EGFR demonstrated a prognostic significance only for DFS (p =0.0241) in node negative and only for OS (p = 0.0333) in node positive breast cancer. When all patients were stratified for EGFR and ER, a multivariate analysis indicated that the combination of EGFR(+)/ER(−) was an independently significant factor for both DFS and OS in node negative as well as node positive breast cancer. In conclusion, the prognostic value of EGFR was demonstrated by a multivariate analysis in a large series of breast cancer patients, but the value of EGFR was somewhat insufficient to achieve statistical significance for both DFS and OS in the subgroups divided by nodal status. On the other hand, the prognostic value of combination of EGFR and ER was sufficient to achieve statistical significance based on a multivariate analysis for both DFS and OS in the subgroups of node negative as well as node positive breast cancer patients.

Time trends of surgical treatment and the prognosis for Japanese patients with gastric cancer.

The incidence of gastric cancer is much higher in Japan than in other countries even though diagnostics and treatments of such patients have improved. The objective of this study was to present an overview of the past, present and future of surgical treatment for our patients with gastric cancer. We analysed data on 2152 Japanese men and women with gastric cancer who underwent surgical resection from 1965 to 1995 at Kyushu University in Fukuoka, Japan, based on a univariate and the multivariate analysis. We focused on time trends of surgical treatment and the postoperative outcome. Over the years, there have been favourable changes in the numbers of patients with early gastric cancer. In all cases of gastric cancer, the rate of 18% in the first six year period (group 1) was 57% in the last 5 year period (group 6). Size of the tumour was smaller, well-differentiated tumour tissue was more common, and lymphatic involvement was less frequent. Lymph node metastasis, liver metastasis and peritoneal dissemination all decreased. Extensive lymph node dissection was more frequently done and the rate of curative resection (curability A and B) increased. With increases in identifying the early stage of cancer and better perioperative care, mortality rates 30 days after the surgery greatly decreased. Multivariate analysis revealed that the 10 factors of depth of invasion, lymph node metastasis, lymph node dissection, tumour size, liver metastasis, peritoneal dissemination, lymphatic invasion, vascular invasion, lesion in the whole stomach and lesion in the middle stomach were independent factors for determining the prognosis. Detection of the tumour in an early stage, standardized surgical treatment, including routine lymph node dissection, close follow-up schedules and better perioperative management are expected to increase survival time for patients with this malignancy.

Proliferative activation of quiescent Rat-1A cells by delta FosB.

Fos and Jun transcription factors are induced during the normal course of the proliferative response of quiescent cells to serum or to growth factors. We have shown that delta FosB, an alternatively spliced form of FosB, is formed as rapidly as FosB in serum-stimulated Rat-1A cells. Although delta FosB lacks the C-terminal region of FosB carrying the transactivation function, constitutive expression of delta FosB transforms Rat-1A cells as does expression of FosB. The transforming ability of delta FosB suggests that delta FosB may lead to proliferative activation of quiescent cells without activating AP-1-responsive genes. To address this question, FosB or delta FosB was expressed as a fusion protein with the ligand binding domain of the human estrogen receptor (ER) in Rat-1A cells. After estrogen treatment, the fusion protein accumulates in nuclei and forms stable complexes with Jun proteins. We have shown that ER-delta FosB or to a lesser extent ER-FosB triggers quiescent Rat-1A cells to transit G1, initiate DNA replication, and ultimately undergo cell division at least once. Since ER-FosB, but not ER-delta FosB, induced expression of the AP-1-responsive transin/stromelysin gene, we concluded that the N-terminal region and the DNA binding domain of FosB or delta FosB itself have the potential to regulate cell proliferation and that the transactivation function carried by the C-terminal region of FosB is not essential for the proliferative activation of quiescent cells.

Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of ≤6 bp. Type B changes are more drastic and involve modifications of ≥8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Mutated gene-specific phenotypes of dinucleotide repeat instability in human colorectal carcinoma cell lines deficient in DNA mismatch repair

Mutations in DNA mismatch repair (MMR) genes in hereditary non-polyposis colon cancer (HNPCC) patients revealed the importance of MMR deficiency as a risk for carcinogenesis. Since diverse mutations occur in several MMR genes, the instability of repeat sequences dispersed in the genome, which are also governed by the MMR system, is a well used marker. However, the relationship between repeat sequence instability and MMR gene mutation in human cells has not been well defined mainly because precise systems to analyse repeat sequences have not been available. Using our newly developed system, we analysed alteration of dinucleotide repeats in human cell lines which harbour mutations in MMR genes. Among 24 subclones of DLD-1 cells (hMSH6−) only one had a dinucleotide repeat alteration in only one microsatellite locus, while LoVo cells (hMSH2−/hMSH6−) exhibited marked dinucleotide repeat instability (DRI). HCT116 cells, a hMLH1-mutant, showed an ultimate DRI phenotype. Interestingly, SW48 cells lacking hMLH1 expression also demonstrated DRI, albeit the extent of diversity being significantly lower than HCT116. These data suggest that the DRI phenotype in human cells is highly dependent on mutated MMR genes and on forms of mutation. The results of DRI analyses used to detect MMR-deficiency should be interpreted with caution.

Cytokeratin-positive cells in bone marrow for identifying distant micrometastasis of gastric cancer.

Direct evidence of tumour seeding in distant organs at the time of surgery for gastric cancer is not available. An immunocytochemical assay for epithelial cytokeratin protein may fill this gap since it is a feature of epithelial cells that would not normally be present in bone marrow. The bone marrow of 46 patients with primary gastric cancer was examined for tumour cells, using immunocytochemical techniques and antibody reacting with cytokeratin, a component of the intracytoplasmic network of intermediate filaments. The monoclonal antibody CK2 recognises a single cytokeratin polypeptide (human cytokeratin no. 18) commonly present in epithelial cells. The expression of tumour-suppressor genes p53 and RB for the primary lesion was also determined using the monoclonal antibodies PAb 1801 and 3H9 respectively, and the proliferating activity was determined by the Ki-67 antigen labelling index for MIB-1 antibody staining. Of these 46 patients, 15 (32.6%) presented with cytokeratin-positive cells at the time of primary surgery. The positive findings were related to the undifferentiated tissue type and to the prominent depth of invasion, but not to other clinicopathological factors. In 2 of 15 (13.3%) patients, the depth of invasion was limited to the mucosa. The metastatic potential to bone marrow did not relate to expressions of p53 and RB genes, or to the proliferating activity of MIB-1 staining for the primary lesion of gastric cancer. As tumour cells in bone marrow are indicative of the general disseminative capability of an individual tumour, this technique may be useful for identifying patients at high risk of metastasis from a gastric tumour.

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

Genomic sequences encoding the 3′ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms.

Concurrent overexpression of ETS-1 and C-met correlates with a phenotype of high cellular motility in human esophageal cancer

Hepatocyte growth factor (HGF) stimulates cell motility as well as mitotic activity of cells. High concentrations of HGF or overexpression of its cellular receptor c‐Met in cancer have been reported. We analyzed the expression status of c‐Met immunohistochemically in 76 cases of human esophageal cancer. Overexpression of c‐Met was noted at a considerably high frequency. Intriguingly, c‐Met overexpression was frequent in a specific type of cell nest formation in tumors, i.e., the small nest type, in which tumors form small, dispersed cell nests. Further immunohistochemical analyses using serial sections revealed a striking coincidence between overexpression of c‐Met and its transcriptional factor, Ets‐1. Overexpression of c‐Met and Ets‐1 was statistically more frequent in small nest type tumors. The close correlation in expression status between Ets‐1 and c‐Met was also confirmed using 6 established human esophageal cancer cell lines. In addition, cells that expressed high levels of Ets‐1 and c‐Met exhibited an extremely motile phenotype by HGF stimulation in vitro. The presence of HGF in tissue sections was confirmed using similar immunohistochemical approaches. These observations suggest that in human esophageal cancer cells the transcriptional factor Ets‐1 upregulates the expression of c‐Met and, consequently, confers on cells a highly motile phenotype leading to a specific form of tumor development.

Frequency of Microsatellite Instability inBreast Cancer Determined by High-Resolution Fluorescent Microsatellite Analysis

In breast cancer, the rates of positivity for microsatellite instability (MSI), vary greatly in the literature. Using high-resolution fluorescent microsatellite analysis (HFRMA), we studied microsatellite alterations in 75 patients with sporadic breast cancer. In this system, several devices were prepared to improve reproducibility of polymerase chain reaction products, migration accuracy of electrophoresis, and characteristics of the detection system. Precise and objective analyses of microsatellite alterations are made feasible using HRFMA. Seven of the 75 cases were judged to be positive for MSI, the rate of positivity being 9.3%. This rate is relatively low compared to the data in the literature. All the microsatellite changes observed in this system can be classified into two types: type A with relatively small changes in microsatellite sequences observed in limited loci and type B with drastic and widely dispersed changes. The former was thought to be connected to abnormal activity in DNA mismatch repair (MMR). Among the 7 cases, 6 (8.0%) had type A alterations, which means that the tumors may have an abnormal MMR activity. Application of precise and objective systems for microsatellite analysis is expected to be clinically useful to detect patients at high risk for cancers.