Tsuyoshi Akiyoshi

Molecular detection of circulating solid carcinoma cells in the peripheral blood: the concept of early systemic disease

Detection of the mRNA of selected genes by reverse transcriptase‐polymerase chain reaction (RT‐PCR) is a sensitive and powerful tool for detecting cancer cells in bone‐marrow or peripheral‐blood samples. In this study, we determined whether carcinoembryonic antigen (CEA) mRNA is detectable in the peripheral blood of patients with gastrointestinal or breast cancer. In addition, we studied selected patients undergoing surgical procedures to assess whether tumor manipulation during operation enhances cancer‐cell dissemination. Peripheral blood from 55 patients with gastrointestinal or breast cancer and from 22 control cases was analysed for CEA mRNA using RT‐PCR. For 15 selected cases undergoing curative surgery for cancer, samples were also obtained during and after surgery. The lower limit of detection was 1 to 10 CEA‐positive cells diluted among 1 × 107 blood mononuclear cells. The test was positive for 20 of the 55 patients with cancer (36%). None of the 22 control samples were positive. An increase in positivity was observed with increasing stage of disease; however, even some patients with early‐stage cancer showed positive results. In addition, CEA mRNA could be detected in the peripheral blood during operation in 3 of 13 patients whose pre‐operative CEA mRNA in the peripheral blood had been negative. These findings suggest that, (1) RT‐PCR amplification of CEA mRNA is an efficient means of detecting circulating solid cancer cells in the peripheral blood, although long‐term clinical studies should be done to evaluate its usefulness; (2) not only breast cancer but also gastrointestinal cancer might be better regarded as a systemic disease even in early stages of carcinoma; and (3) surgical manipulation can provoke cancer‐cell dissemination. ©1996 Wiley‐Liss, Inc.

Clinical significance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcription-polymerase chain reaction in patients with gastrointestinal or breast carcinomas.

PURPOSE This study evaluates the clinical significance of detection of carcinoembryonic antigen (CEA) mRNA in the dissected lymph nodes and peripheral blood samples of patients with gastrointestinal or breast carcinomas. PATIENTS AND METHODS A total of 406 lymph nodes obtained from 65 patients were analyzed by both histologic and molecular examination of CEA-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Peripheral blood samples from another 102 patients were also analyzed by CEA-specific RT-PCR. Patients were followed up prospectively for 24 +/- 12 months. RESULTS Of 406 lymph nodes, the positive detection rate increased from 20% by histologic examination to 60% by RT-PCR examination. The recurrence rate was 40% in 15 cases showing positive results in both examinations, 14% in 29 cases showing histologically negative but RT-PCR positive results, and none in 21 cases showing negative results in both examinations. The positive detection rate for CEA mRNA in peripheral blood samples increased with advancing stage of disease. With respect to 62 curatively operated cases, CEA mRNA was detected in 12 cases. Four of these 12 cases developed metastatic disease after surgery whereas none of 50 cases negative by RT-PCR developed metastasis. CONCLUSION It has been shown that RT-PCR is a powerful tool to detect CEA mRNA in the lymph nodes or the peripheral blood. This is potentially very useful to determine high-risk patients for metastasis. Serial analysis is warranted to assess the long-term significance of this method and its therapeutic and prognostic implications.

Matrix metalloproteinase-7 expression in gastric carcinoma.

BACKGROUND/AIMS: Matrix metalloproteinase-7 (MMP-7) belongs to the same family as matrix degrading metalloproteinase (MMPs) that may play an important part in cancer cell invasion and metastasis. This study reports on the MMP-7 mRNA expression level both in human gastric carcinomas and the normal gastric mucosa. METHODS: From fresh specimens of 47 surgical pairs of primary gastric carcinomas and corresponding normal tissue specimens, cDNA was obtained by reverse transcription (RT) and thereafter MMP-7 mRNAs were detected by means of a polymerase chain reaction. The tumour/normal (T/N) ratio of MMP-7 expression was calculated after correcting for glyceraldehyde-3-phosphate dehydrogenase as an internal control. RESULTS: The expression corrected levels of MMP-7 mRNA of the tumour was greater than that of the normal mucosa in 41 of 47 cases (87%). The 13 cases whose T/N ratio was more than 2.1 showed a deeper invasion of the gastric wall, and more frequent lymphatic or vascular permeations than the 34 cases whose T/N ratio was less than 2.0. An immunohistochemical study showed that MMP-7 was predominantly expressed in the cancer cells, weakly expressed in normal epithelial cells, and not expressed in the surrounding stromal cells. CONCLUSIONS: These findings suggest that the overexpression of MMP-7 may thus play an important part in tumour invasion in gastric carcinomas while, in addition, MMP-7 may also prove to be a useful marker for determining the biological aggressiveness of gastric carcinoma.

Analysis of MT1‐MMP and MMP2 expression in human gastric cancers

Membrane‐type 1 matrix metalloproteinase (MT1‐MMP) is a presumed activator of MMP2, which is one of the major proteinases in tumor cell invasion. In this study, we determined the clinico‐pathologic significance of MT1‐MMP expression in 68 human gastric carcinomas. The tumor‐normal ratio (T/N ratio) of MT1‐MMP expression was determined by reverse transcription‐polymerase chain reaction analysis. To visualize the localization of MT1‐MMP, an immunohistochemical study was performed. In addition, a gelatin zymography was done to examine the activation ratio of MMP2, and a correlation between MT1‐MMP expression and activation of MMP2 was studied. The expression of MT1‐MMP mRNA was higher in tumor tissue than in corresponding normal tissue in most cases. The mean value of the T/N ratio was 4.8. Twenty cases with T/N ≥ 4.8 showed significantly deeper invasion and higher frequency of lymph node metastasis than 48 cases with T/N < 4.8. MT1‐MMP expression was an independent factor influencing both tumor invasion of the gastric wall and lymph node metastasis. Although MT1‐MMP expression was not an independent prognostic factor, the patients with T/N ≥ 4.8 showed a significantly worse prognosis than those with T/N < 4.8. An immunohistochemical study demonstrated that MT1‐MMP expression was mainly recognized in the tumor cells. There was a significant correlation between MT1‐MMP expression and activation of MMP2. Our findings suggest that: 1) the expression of MT1‐MMP may influence prognosis via tumor invasion of the gastric wall and lymph node metastasis, and 2) MT1‐MMP activation of MMP2 may be clinically relevant in gastric carcinoma tumors. Int. J. Cancer 74:316‐321, 1997.

A MAGE‐1‐encoded HLA‐A24‐binding synthetic peptide induces specific anti‐tumor cytotoxic T lymphocytes

Although several MAGE‐1 peptides have already been identified, the MAGE‐1‐encoded peptide presented by HLA‐A24, which is the most common allele in Japanese population and is also frequently present in Caucasians, might have a wide applicability for immunotherapy using these peptides. To identify this potential peptide, we examined the induction of specific cytotoxic T lymphocytes (CTL) from the peripheral‐blood mononuclear cells (PBMC) in HLA‐A24 healthy donors by in vitro stimulation with MAGE‐1‐encoded synthetic peptides with a binding affinity for HLA‐A24, by a simplified method. Of the 5 peptides tested, the highest HLA binder (NYKHCFPEI) was able to elicit CTL from unseparated PBMC by stimulation with freshly isolated, peptide‐pulsed PMBC as antigen‐presenting cells (APC) and by also using interleukin 7 and keyhole‐limpet hemocyanin for a primary culture. The induced CTL could thus lyse HLA‐A24 tumor cells expressing MAGE‐1, as well as the peptide‐pulsed target cells, in an HLA‐class‐I‐restricted manner. By using the MAGE‐1/HLA‐A24 peptide, NYKHCFPEI, we found it possible to immunize many more patients, especially Japanese patients, by means of such peptide‐based immunotherapeutic approaches to MAGE‐1‐positive malignant tumors. Int. J. Cancer 80:169–172, 1999.

The expression of tumor-rejection antigen “MAGE” genes in human gastric carcinoma

BACKGROUND & AIMS The genes MAGE-1 and MAGE-3 both encode melanoma peptide antigens recognized by major histocompatibility complex-restricted cytotoxic T lymphocytes. The antigens may be a target for immunotherapy. There is, however, little information on the expression of these genes in gastric carcinomas. Therefore, the expression of MAGE genes in gastric carcinomas was evaluated. METHODS The expression of MAGE-1, MAGE-2, and MAGE-3 genes in tumors and corresponding normal tissue specimens was studied using a reverse-transcription polymerase chain reaction. The results were analyzed according to clinicopathologic factors of the tumor. RESULTS In the 68 gastric carcinomas studied, MAGE-1, MAGE-2, and MAGE-3 messenger RNA were detected in 41%, 31%, and 38%, respectively. Fifty percent of the gastric carcinomas expressed at least one of the MAGE genes. Messenger RNA for the three MAGE proteins was not detected in normal gastric tissue. MAGE gene expression in gastric carcinomas was not associated with a significant clincopathology of the tumor. However, gene expression was lower in mucinous carcinomas (3 of 10). CONCLUSIONS MAGE-1, MAGE-2, and MAGE-3 are expressed in a high percentage of gastric carcinomas. These tumor rejection antigens may provide tumor-specific targets for immunotherapy.

A novel variant of human Grb7 is associated with invasive esophageal carcinoma.

The cDNAs of a putative growth factor-bound (Grb) 7 signal transduction molecule and Grb7V novel splice variant were isolated from an invasive human esophageal carcinoma. Although both Grb7 isoforms share homology with the Mig-10 cell migration gene, the Grb7V isoform lacks 88 base pairs in the C terminus; the resultant frame shift leads to substitution of an SH2 domain with a short hydrophobic sequence. The wild-type Grb7 protein, but not the Grb7V isoform, is rapidly tyrosyl phosphorylated in response to EGF stimulation in esophageal carcinoma cells. Analysis of human esophageal tumor tissues and regional lymph nodes with metastases revealed that Grb7V was expressed in 40% of Grb7-positive esophageal carcinomas. More importantly, Grb7V expression was enhanced after metastatic spread to lymph nodes as compared to the original tumor tissues. Finally, transfection of an antisense Grb7 RNA expression construct lowered endogenous Grb7 protein levels and suppressed the invasive phenotype exhibited by esophageal carcinoma cells. These findings suggest that Grb7 isoforms are involved in cell invasion and metastatic progression of human esophageal carcinomas.

Minimal increase in serum interleukin-6 levels during laparoscopic cholecystectomy.

The chronologic changes in the serum levels of interleukin-6 (IL-6), a mediator for acute-phase inflammation, were compared between laparoscopic cholecystectomy (LC) and open cholecystectomy (OC), since these two types of operations were considered to be a unique model for examining the role of local tissue injury in postoperative inflammatory reactions. The increase in the serum IL-6 level during LC was found to be significantly smaller than that during OC and resulted in a smaller extent of postoperative elevations for C-reactive protein. These results suggest that laparoscopic surgery associated with minimal tissue injury can help limit an increase in the serum IL-6 level during surgery, thus contributing to a reduction in surgical stress.

Microsatellite instability in Japanese gastric cancer

Background. Recent studies have shown that micro‐satellites are unstable in various types of cancers, and such genetic instability at the microsatellite loci (micro‐satellite instability) has been considered to play an important role in the development of cancer. However, the clinicopathologic significance of microsatellite instability in gastric cancer has not been clarified.

Elongation factor 1 gamma mRNA expression in oesophageal carcinoma.

Elongation factor 1 gamma (EF1 gamma) is known to be a subunit of EF1, one of the G proteins that mediate the transport of aminoacyl tRNA to 80S ribosomes during translation. As little is known regarding the expression of EF1 gamma in human oesophageal carcinoma, this study looked at its expression using a northern blot analysis. Thirty six cases of oesophageal carcinoma and 15 oesophageal carcinoma cell lines were studied. The EF1 gamma mRNA overexpression at a level of twofold or more was seen in five (14%) of 36 carcinomatous tissues compared with the normal counterparts. All five overexpressed cases showed severe lymph node metastases compared with the non-overexpressed cases, and the difference was significant (p = 0.028). The stage of the disease of these five cases was far advanced compared with the nonoverexpressed cases (p = 0.012). All 15 oesophageal carcinoma cells expressed EF1 gamma mRNA relatively lower than the gastric or pancreatic carcinoma cell lines, in which EF1 gamma was originally isolated. As the expression of EF1 gamma mRNA could be detected even in the biopsy specimens, its overexpression in tumour tissue may provide preoperative useful information for predicting the aggressiveness of tumours.