Membrane fluidity correlates with liver cancer cell proliferation and infiltration potential.

We developed a method to measure membrane fluidity of living cancer cells in two- and three-dimensional cultures, and found that there was a close relationship between the membrane fluidity of cancer cells and their proliferative and infiltrative ability. Membrane fluidity is thus a promising indicator of the probability of cancer recurrence.

Perioperative quantitative analysis of cytokeratin 20 mRNA in peripheral venous blood of patients with colorectal adenocarcinoma.

Hematogenous dissemination is a significant short-coming of colorectal carcinoma treatment. To screen patients with high risk for such blood-borne metastasis, we previously developed a highly sensitive system for the detection of cytokeratin 20 (CK-20) mRNA in blood. For a more practical application, we improved this system by making it quantitative and capable of analyzing peripheral venous blood for the detection of perioperative changes in CK-20 mRNA. CK-20 mRNA was not always detected in the preoperative blood, even in patients in an advanced stage, but it was identified without fail in intra- and post-operative blood. In addition, more copies of CK-20 mRNA were observed in the intra-operative blood than in pre- and post-operative blood. This study suggests that analysis of perioperative changes may provide important information for the precise evaluation of hematogenous dissemination and of the effect of surgical maneuvers on recurrence.

Quantitative analysis of alpha-fetoprotein mRNA in circulating peripheral blood of patients with hepatocellular and alpha-fetoprotein-producing gastric carcinomas.

In conjunction with strategies introduced in recent years to identify cancer micrometastasis through amplification of cancer-associated mRNA, we developed a highly sensitive system to detect alpha-fetoprotein mRNA in circulating peripheral blood of hepatocellular carcinoma patients. The aim of the present study was to make our original system quantitative. Peripheral venous blood from patients with hepatocellular carcinoma and alpha-fetoprotein-producing gastric carcinoma was subjected to reverse transcription followed by our original three-step polymerase chain reaction co-amplifying both the original sequence and our synthetic competitor. We succeeded in modifying our system for quantitative analysis, and investigated the perioperative change, the postoperative change and the change after chemotherapy in order to illustrate the possible application of this method. The quantitative analysis of alpha-fetoprotein mRNA present in the peripheral blood represents a useful tool for analyzing the relationship of surgery to recurrence, the effect of chemotherapy, and to predict impending recurrence in patients with hepatocellular and alpha-fetoprotein-producing gastric carcinomas.

Cytokeratin 20 mRNA in peripheral venous blood of colorectal carcinoma patients.

A highly sensitive system was previously developed by us to detect the presence of colorectal carcinoma cells in blood in the form of cytokeratin 20 (CK20) mRNA. In the present study, we used an improved version of this system to analyse the peripheral blood of 28 patients with colorectal carcinoma, five patients with non-cancerous intestinal diseases and six normal controls for the presence or absence of CK20 mRNA and to investigate the relationship between the mRNA results and prognosis. All eight patients with recurrence were positive for CK20 mRNA, as were four patients in the Dukes' C stage with either distant metastasis or dissemination. Five of the nine patients in the Dukes' C stage with neither distant metastasis nor dissemination were positive, and three of these developed recurrence within 11 months after the analysis. Only one of the seven patients in the Dukes' A or B stage was positive, and none showed recurrence during the 1-19 months of observation. None of the five patients without carcinomas or of the six normal controls was positive. Although the follow-up period is limited and the recurrences were all local at present, these results suggest that the presence of CK20 mRNA in circulation may be a useful indicator for the screening of advanced colorectal carcinoma patients with a high risk of recurrence.

Quantitative analysis of carcinoembryonic antigen messenger RNA in peripheral venous blood and portal blood of patients with pancreatic ductal adenocarcinoma.

One major therapeutic failure of pancreatic adenocarcinoma treatment is metastasis to the liver. To screen patients with high risk for such hematogenous dissemination, we previously developed a very sensitive system to detect carcinoembryonic antigen (CEA) in blood. For a more practical application, we improved this system by making it quantitative and capable of analyzing both preoperative peripheral blood and intraoperative portal blood for the presence of CEA mRNA. CEA mRNA was not detected in the peripheral venous blood of any of the three patients examined, but it was identified in the portal blood without fail. In addition, the quantities of CEA mRNA identified in the portal blood before and after pancreatectomy were different. This study suggests that analysis of the portal blood seems to be important for the precise evaluation of hematogenous dissemination and of the pathophysiology of pancreatic ductal adenocarcinoma.

Hematogenous spreading of hepatocellular carcinoma cells: possible participation in recurrence in the liver.

To detect hepatocellular carcinoma (HCC) cells in the circulating peripheral blood, we previously designed a highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) method targeting alpha-fetoprotein (AFP) messenger RNA (mRNA). Using this method, we analyzed peripheral blood of in- and out-patients bearing HCC for 2 months consecutively and examined the outcome thereafter. All 11 patients with recurrence either in the liver alone or in both the liver and the lung were positive for AFP mRNA. Among 10 recurrence-free patients, 2 patients were AFP mRNA-negative and remained recurrence-free during a period of 22 months observed. Four of 8 AFP mRNA-positive, recurrence-free patients developed a clinically evident recurrence in the liver after 2 to 16 months. Seven of 8 preoperative patients were already positive for AFP mRNA and the remaining negative patient became positive during surgery. Four of 6 preoperatively positive and still-alive patients had recurrence in the liver after 2 to 9 months. None of the HCC-negative patients were positive for AFP mRNA. We actually found an AFP protein-positive cell in peripheral blood obtained from one of the AFP mRNA-positive patients by immunostaining. The present results suggest that circulating HCC cells may have some relationship with the recurrence of HCC in the liver.

Detection of colorectal carcinoma cells in circulating peripheral blood by reverse transcription-polymerase chain reaction targeting cytokeratin-20 mRNA.

For the detection of circulating colorectal carcinoma cells, we investigated the presence of cytokeratin 20 (CK 20) mRNA in the peripheral blood of colorectal carcinoma patients. Application of our published technique resulted in analysis by reverse transcription followed by three-step nested polymerase chain reaction. This analysis could detect a single Colo 205 colon cancer cell mixed with 1 ml of blood. Our system also successfully detected the presence of CK 20 mRNA in actual patients' peripheral blood samples. Our highly sensitive and specific system for the detection of CK-20 mRNA from patients' peripheral blood thus seems to be useful for screening for circulating colorectal carcinoma cells.

Identification of carcinoembryonic antigen mRNA in circulating peripheral blood of pancreatic carcinoma and gastric carcinoma patients.

To detect adenocarcinoma cells in the circulating peripheral blood, we "analyzed the presence of carcinoembryonic antigen (CEA) mRNA in the peripheral blood obtained from patients with pancreatic carcinoma (PC) or with gastric carcinoma (GC) and also, as controls, from pancreatitis or gastritis patients without carcinomas, a gastric lymphoma patient and four healthy volunteers. Because of the small number of carcinoma cells expected in the peripheral blood, the analysis was performed by the reverse transcription followed by an original two-step polymerase chain reaction. By this sensitive method, 3 of 9 PC patients and 2 of 9 GC patients were positive for CEA mRNA. Except for 1 highly advanced PC patient, 3 of 4 CEA mRNA-positive patients developed recurrence after curative resection or liver metastasis after palliative operation within 9 months after the analysis. None of the control patients was positive for CEA mRNA in the peripheral blood. The results suggest that our sensitive RT-PCR method for detecting CEA mRNA in the peripheral blood is practically useful to find the hematogenous spreading of adenocarcinoma cells.