Seiji Akiyama

Altered mRNA expression of specific molecular species of fucosyl- and sialyl-transferases in human colorectal cancer tissues

Human colorectal cancers express various cancer‐associated carbohydrate determinants such as Lewis Y or sialyl Lewis A, suggesting a considerable alteration in glycosyltransferase activities occurring upon malignant transformation. We investigated the mRNA amounts of fucosyltransferase (Fuc‐T) and sialyltransferase (ST) isoenzymes, including Fuc‐T III, IV, V, VI and VII and ST‐3N, ST‐30 and ST‐4, in human colorectal cancer tissues by Northern blotting and RT‐PCR. Regarding fucosyltransferases, mRNA of Fuc‐T III and VI was not significantly altered, and only Fuc‐T IV mRNA showed a moderate increase in cancer tissues when compared with adjacent non‐malignant colonic epithelia taken from the same patient (273 ± 96%; p < 0.001). The moderate increase of Fuc‐T IV message may be related to an enhanced expression of Lewis Y in colon cancer tissues. In the ST isoenzymes, mRNA for ST‐3N remained unchanged, whereas that for ST‐4 decreased significantly in cancer tissues, to 32 ± 29%, (p < 0.005). The most remarkable finding was that the message of ST‐30 was prominently increased in cancer tissues compared with non‐malignant colorectal mucosa. When further investigated by quantitative RT‐PCR assays on a larger series of patients with colorectal cancers, the average increase in mRNA for ST‐30 was 459 ± 200% compared with that in adjacent non‐malignant epithelium (significant at p < 0.0001). The increase of ST‐30 message was more prominent in the cancer tissues strongly expressing sialyl Lewis A than in the cancer tissues expressing sialyl Lewis A only weakly or moderately (significant at p < 0.05). The marked increase in the message of ST‐30 is suggested to be related to an enhanced expression of sialylated carbohydrate determinants in colon cancer tissues including sialyl Lewis A, since the enzyme exhibited a significant activity against the type I chain carbohydrate substrate and produced the precursors for sialyl Lewis A synthesis, when its cDNA was expressed in Cos‐7 cells. Int. J. Cancer 71:556‐564, 1997

Laparoscopy-assisted distal gastrectomy with systemic lymph node dissection for early gastric carcinoma: a review of 43 cases☆

BACKGROUND Recently, laparoscopy and laparoscopy-assisted surgery have been used increasingly as less-invasive alternatives to conventional open surgery. But the use of this approach in gastric carcinoma has received little attention, possibly from the low incidence of early-stage disease in the West and the relative complexity of the surgical procedure. STUDY DESIGN A prospective feasibility study of laparoscopy-assisted distal gastrectomy was performed in patients with histologically confirmed gastric carcinoma located in the lower or middle third of the stomach. Patients whose preoperative evaluations, including endoscopic ultrasonography and computerized tomography, led to a diagnosis of T1 N0 stage disease, and who had no advanced disease discovered during laparoscopy, were eligible. Intraoperative blood loss, time of operation, mortality, and morbidity were assessed, along with the number of lymph nodes retrieved and shortterm survival. RESULTS Between 1998 and 1999, 43 patients were enrolled. Laparoscopy-assisted distal gastrectomy was converted to an open procedure in one patient. There were no operative or in-hospital deaths, but the incidence of anastomotic leakage was 14% (6 of 43). The mean blood loss was 239 mL, the time of operation was 225 minutes, and lymph node retrieval was 20.2 nodes. These results are comparable with a series of conventional open operations. One patient died of recurrent disease, and all other patients remain disease-free to date. Port-site recurrence was not observed. CONCLUSIONS Although laparoscopy-assisted distal gastrectomy was equivalent to open surgery in several clinical parameters, the relatively high morbidity was a drawback. Its appropriateness to gastric cancer surgery must be verified by further studies.

Molecular detection of p16 promoter methylation in the serum of colorectal cancer patients

Assays based on the molecular detection of genetic changes in serum have been shown as potential diagnostic tools for colorectal cancer. We examined the methylation status of p16 in colorectal cancers using methylation-specific PCR (MSP). Forty-four of 94 (47%) cancer DNA exhibited abnormal promoter methylation of p16 gene while no corresponding normal DNA exhibited such methylation. Subsequently, we examined whether aberrant methylation could be detected in corresponding serum DNA, and found that 13 of 44 (30%) patients with p16 promoter methylation in tumor DNA demonstrated abnormal methylation in their serum DNA. Moreover, abnormal methylation was found in the serum of patients in all clinical stages, suggesting that early colorectal cancer could be detected using the MSP method.

Expression of midkine in the early stage of carcinogenesis in human colorectal cancer

SummaryIt has been suggested that a heparin-binding growth factor, midkine (MK), plays an important role in carcinogenesis because of its frequent overexpression in various malignant tumours. To clarify whether or not MK contributes to the early stage of carcinogenesis, we examined the status of MK mRNA in 20 adenomas with moderate- and severe-grade dysplasia, 28 carcinomas and 28 corresponding normal tissues, by means of Northern blotting. The MK expression level was significantly more elevated in adenomas than in normal tissues (P< 0.001, unpaired Students t -test). A difference was also observed between carcinomas and the corresponding normal tissues (P< 0.04, paired Students t-test). Moreover, MK immunostaining was positive in the adenomas with moderate- and severe-grade dysplasia and in the carcinomas,but not in mild-grade dysplasia or in normal tissues. These findings were in line with those on Western blotting. In three patients with both adenomas with moderate- or severe-grade dysplasia and carcinomas, elevated MK expression was observed in the neoplasticlesions. This is the first report of the association of elevated MK expression with the early stage of carcinogenesis in humans.

Detection of mitochondrial DNA alterations in primary tumors and corresponding serum of colorectal cancer patients.

We previously examined colorectal cancer patients using mutation‐specific mismatch ligation assay for genetic alterations in primary tumors and paired serum samples and proved that genetic alterations present in the tumors of cancer patients can be detected in the serum of those same patients. Recent evidence has proved that various cancers frequently have mutations in the D‐loop region of mitochondrial DNA (mtDNA). Therefore, we thought that mutations in the mitochondrial genome might also become a genetic marker of colorectal cancer to detect tumor DNA in the serum of patients. We first sequenced the D‐loop region of mtDNA in colorectal cancers. We then proceeded with a sensitive method, i.e., mismatch ligation assay to examine the possibility that mtDNA alterations can be found in the serum DNA. We analyzed the D‐loop region of mtDNA in 77 primary colorectal cancers, 7 of which (9%) contained true somatic mutations in this region. We then examined whether mtDNA alterations can be found in the serum DNA using mismatch ligation assay. Of 7 alterations that were examined, 1 (14%) could be detected in the serum. This result suggested that the mtDNA alteration could also be used as a tumor marker to detect tumor DNA in the serum.

Real-time observation of micrometastasis formation in the living mouse liver using a green fluorescent protein gene-tagged rat tongue carcinoma cell line

Initial arrest, attachment, extravasation and subsequent extravascular growth of tumor cells in the secondary organs are believed to be crucial events for hematogenous metastasis, but the actual processes in living animals remain unclear. For the present study, we established green fluorescent protein (GFP)–expressing rat tongue carcinoma cell lines (RSC3) that permit real‐time analysis of micrometastasis formation in combination with intravital video microscopy (IVVM). With this system, GFP‐expressing metastatic (LM‐EGFP) and non‐metastatic (E2‐EGFP) cell lines could be visualized at the cellular level in live mice for more than 1 month. Real‐time IVVM analysis of liver metastases after intraportal injection of cells via a mesenteric vein revealed that both LM‐EGFP and E2‐EGFP tumor cells arrest similarly in sinusoidal vessels near terminal portal venules within 0.4 sec, during which time no evidence of a “rolling”‐like movement along endothelial cell surfaces was observed. Quantitative analysis of GFP‐positive foci showed that E2‐EGFP cells were completely sheared from the liver sinusoid within 3 days, with no solitary dormant cells, whereas a substantial number of LM‐EGFP cells remained in the liver, probably due to stable attachment to the sinusoidal wall. Confocal laser scanning microscopic study in combination with laminin immunohistochemistry revealed that only LM‐EGFP cells started growth at 3 to 4 days after inoculation and that most of the growing foci were surrounded by subsinusoidal basement membrane. Our results suggest that micrometastasis formation by LM‐EGFP cells consists of initial tumor cell arrest due to size constraints of the vessel, stable attachment to subsinusoidal basement membrane and subsequent intravascular growth before extravasation. The difference in metastatic potential between the 2 lines may reside in their capacity to attach stably to the vessel wall rather than their potential for initial cell arrest or subsequent growth. The system used in the present study may be a powerful tool for analyzing targets for various anti‐metastatic agents in the sequential process of metastasis.

Quantitative detection of CEA expressing free tumor cells in the peripheral blood of colorectal cancer patients during surgery with real-time RT-PCR on a LightCycler

We applied novel real-time reverse transcriptase-polymerase chain reaction (RT-PCR) with a LightCycler for quantitative detection of carcinoembryonic antigen (CEA) mRNA expressing tumor cells in the peripheral blood of colorectal cancer patients. Analysis of peripheral blood samples from 99 potentially curative colorectal cancer patients revealed a significantly higher mean CEA mRNA value in post-operative bloods (18.71) than in pre-operative blood (1.03) (P=0.003). Kaplan-Meier analysis demonstrated disease free survival of patients with positive CEA mRNA in post-operative blood to be significantly shorter than in cases negative for CEA mRNA (P=0.03). These results suggest that tumor cells could be shed into the bloodstream during surgical procedures, and these free tumor cells are accompanied by a poor patient outcome. Real-time quantitative RT-PCR is a useful technique for quantitative assessment of free tumor cells in the peripheral blood of colorectal cancer patients.

Follow-up surveillance for recurrence after curative gastric cancer surgery lacks survival benefit.

AbstractBackground: Although routine follow-up to detect asymptomatic recurrence after surgery for gastric cancer is recommended, the effect of such reassessment on survival has not been evaluated. Methods: Clinical records of patients developing recurrent disease after potentially curative resection between 1985 and 1996 were retrieved. Among these patients, 197 were in our follow-up program. We analyzed survival in these patients according to the presence or absence of cancer-related symptoms when recurrent disease was diagnosed. Results: Of all patients with recurrent disease, 50% were diagnosed within 1 year and 75% within 2 years of surgery. Asymptomatic recurrence, detected in 88 patients (45%), frequently represented distant metastasis. Although early detection significantly improved survival after detection of recurrent disease, disease-free survival for this subset was shorter. Thus, no significant difference in overall survival was observed. Conclusions: Early detection of asymptomatic gastric cancer recurrence did not improve overall survival of patients with recurrence after curative resection. Until development of more effective treatment for this disease, close follow-up may offer no survival benefit.

Mitochondrial DNA alteration in esophageal cancer.

It has been reported that various cancers frequently have mutations in the D‐loop region of mitochondrial DNA (mtDNA). We examined the genetic alterations in this region of esophageal cancer using direct sequencing. Of 68 sequence variants, 15 have not been reported to date. Tumor mtDNA with these variants were compared with mtDNA from corresponding normal esophageal mucosa. Two of 37 primary esophageal cancers (5%) contained somatic mutations in the D‐loop region of mtDNA. Although the mutation rate of mitochondrial tumor DNA within the D‐loop was not high, this result suggested that mtDNA mutations play a role in the development of esophageal cancer.

TNF-α promotes progression of peritoneal metastasis as demonstrated using a green fluorescence protein (GFP)-tagged human gastric cancer cell line

The mechanisms underlying progression of peritoneal metastasis by gastric cancer after micrometastasis formation remain unclear. In the present study, we investigated metastasis to the abdominal wall peritoneum, one of the major features of peritoneal spread, using a human gastric cancer cell line (GCIY-EGFP) tagged with the green fluorescence protein gene (GFP). This model allows sensitive, specific and sequential observation of metastasis development from the initial deposits to peritoneal carcinomatosis at the end stage. In the initial phase, GCIY-EGFP cells could form micrometastasis selectively on the omentum and mesenterium in a milky spot-dependent manner, but not on abdominal wall peritoneum lacking milky spots until the late stages. In vitro analysis using primary mesothelial cells revealed addition of TNF-α to decrease their stress fibers, leading to morphological change followed by exposure of the submesothelial extracellular matrix (ECM) in intercellular gaps. Such TNF-α pretreatment was found to enhance attachment of tumor cells to the mesothelial monolayer. When tumor cells were injected into the peritoneal cavity of TNF-α pretreated mice, they could metastasize to the abdominal wall peritoneum from the very early stages, resulting in accelerated accumulation of ascites than in TNF-α non-pretreatment controls. RT-PCR analysis revealed that tumor cells express cytokines and chemokines, including TNF-α. Furthermore, TNF-α treatment results in up-regulation of expression of monocyte chemoattractant protein-1(MCP-1) and IL-8 by mesothelial cells and of TNF-α itself by inflammatory leukocytes in the peritoneal cavity. These results suggest that metastasis to the abdominal wall peritoneum occurs as a second step from the first omental metastasis in a milky spot-independent manner and that TNF-α derived from tumor cells, mesothelial cells and inflammatory leukocytes in the peritoneal cavity may be involved in the progression of peritoneal metastasis.