Detection of circulating gastric cancer cells in peripheral blood using real time quantitative RT-PCR.

BACKGROUND/AIMS: Detection of occult cancer cells in peripheral blood or bone marrow has recently received a great deal of attention regarding the prediction of postoperative recurrence of the cancer, and for novel strategies of adjuvant therapy. This study addresses the detection of circulating tumor cells in peripheral blood in patients with gastric cancer using Quantitative RT-PCR. METHODOLOGY: Common mRNA targets for RT-PCR for detection of small numbers of cancer cells in gastric cancer are CK18, CK19, CK20, and CEA. Ten milliliter of peripheral venous blood was taken from 14 healthy Japanese volunteers and 101 patients with gastric cancer. Samples were analyzed using real-time TaqMan technology and a Model 7700-sequence system. The group of gastric cancer patients included 69 individuals with curative disease on preoperative diagnosis and 32 individuals with a non-curative operation or recurrence of the disease. RESULTS: The number of CK19 and CK20 mRNA copies was significantly increased in patients with a non-curative operation or recurrence of gastric cancer (CK19; p=0.0087, CK20; p=0.0022) compared with healthy volunteers. Cut-off levels of CK19 or CK20 copy numbers were determined by the maximum value of healthy volunteers. For CK19, there were 61 (88.4%) negative cases and 8 (11.6%) positive cases in 69 individuals with curative gastric cancer. There was a significant difference in tumor stages between CK19 positive and negative patients with curative disease on preoperative diagnosis. For CK20, there were 59 (85.5%) negative cases and 10 (15.5%) positive cases. There was no statistical difference between CK20 positive and negative cases for all clinicopathological factors. On postoperative day 14, there was a significant difference between positive and negative cases regarding tumor size, tumor stage, and lymph node metastasis for CK19, and tumor stage and lymph node metastasis for CK20. Five-year survival rates of patients with CK19 positive or negative cases were 50.0% or 79.0%, respectively (p=0.0347). While, for CK20, 5-year survival rates for positive cases was 51.9%, and for negative cases 78.9% (p=0.0490). CONCLUSIONS: Micrometastases of gastric cancer can be detected in circulating peripheral blood using quantitative real-time RT-PCR. CK19 is a better marker than CK18, CK20 and CEA, and could be clinically useful to estimate prognosis or to make a postoperative strategy of adjuvant treatment.

Overexpression of hypoxia-inducible factor 1alpha and p53 is a marker for an unfavorable prognosis in gastric cancer.

PURPOSE: To investigate the effects of the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and p53 on the prognosis of human gastric cancer, the clinicopathologic characteristics of the tumors and the clinical outcome were analyzed. EXPERIMENTAL DESIGN: The expressions of HIF-1alpha and p53 proteins were studied by immunohistochemistry in 216 specimens of primary gastric cancer. RESULTS: HIF-1alpha(+)/p53(+) tumors more frequently showed an undifferentiated type, an infiltrative growth appearance, and an invasive lymphatic involvement compared with HIF-1alpha(-)/p53(-) tumors. HIF-1alpha(+)/p53(+) tumors also had more lymph node metastasis compared with HIF-1alpha(-)/p53(-) tumors. When stratified for HIF-1alpha and p53 positivity, the patients who were p53-negative and HIF-1alpha-negative had the most favorable prognosis, whereas patients who were p53-positive and HIF-1alpha-positive had the worst prognosis (P=0.0018). Using a multivariate Cox regression analysis, the depth of invasion, lymph node metastasis, and HIF-1alpha positivity were all found to be independent prognostic factors in patients with gastric cancer. CONCLUSION: Thus, HIF-1alpha is considered to be a useful independent prognostic factor in gastric cancer, and the combination of a HIF-1alpha protein overexpression with nonfunctional p53 tends to indicate a dismal prognosis.

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 < or =6 bp. Type B changes are more drastic and involve modifications of > or =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.

Pharmacokinetics of gamma-hydroxybutylic acid (GHB) and gamma-butyrolactone (GBL), the anti-angiogenic metabolites of oral fluoropyrimidine UFT, in patients with gastric cancer.

Gamma-hydroxybutylic acid (GHB) and gamma-butyrolactone (GBL), the metabolites of UFT, which is an oral fluoropyrimidine, have been reported to inhibit angiogenesis with IC50 values of 25.8 ng/ml. The pharmacokinetics of GHB and GBL were examined after the administration of UFT in patients with gastric cancer. The patients received 200 mg of UFT orally twice a day. Peripheral blood samples were collected at 0, 0.5, 1, 2 and 4 hr after the time of dosing on day 5. The baseline and endogenous GBL concentrations in plasma were 20.2 +/- 7.5 ng/ml for patients and 16.8 +/- 4.0 ng/ml for volunteers (P = 0.221). The values of C(max) for tegafur, uracil, 5-FU and GBL were 14.7 +/- 5.2 and 4.0 +/- 2.8 microg/ml, 191.2 +/- 115.3 and 147.5 +/- 57.3 ng/ml, respectively, and the values of Tmax were 1.0 +/- 0.6, 1.1 +/- 0.6, 0.9 +/- 0.6 and 1.2 +/- 0. 6 hr, respectively. The concentration of GBL was much higher than its IC50 value for angiogenesis. GBL is thus suggested to contribute to the anticancer effects of UFT in addition to that of 5-FU, which is continuously metabolized from UFT.

Microsatellite instability in cancer: what problems remain unanswered?

Analyses of microsatellite instability have been prevalent, particularly in the field of oncology. However, the literature on this subject is diverse. The discrepancies may derive from methodological problems in the conventional techniques used for analysis. Problems include low quantitativity in the detection systems, inaccurate migration in electrophoresis, and Taq polymerase-mediated modifications of polymerase chain reaction products. Indeed, use of a new fluorescent technique where these problems have been overcome has elucidated various intriguing and previously unrecognized aspects of microsatellite instability in human cancers. Patterns of microsatellite changes observed in various human cancers can be classified into 2 subtypes, those showing relatively small changes within 6 base pairs (type A) and those exhibiting drastic changes over 8 base pairs (type B). Although type A microsatellite instability has been connected to defective mismatch repair phenotype, the relationship between type B microsatellite instability and defective mismatch repair phenotype remains unclear. Nevertheless, as symbolized in cases of hereditary nonpolyposis colorectal cancer, connections between type B microsatellite instability and familial predisposition have been suggested in some cancers. The molecular background of type B microsatellite changes warrants particular attention.

Hyperthermia for rectal cancer.

BACKGROUND: In order to improve the clinical results of rectal cancer, hyperthermia has been prescribed in combination with chemotherapy and radiotherapy. The techniques of hyperthermia and their clinical applications to rectal cancer were reviewed. METHODS: The development of heating devices has been intensively investigated, including external heating devices, intraluminal heating devices, circulation of warmed saline solution, and whole body hyperthermia. RESULTS: Nonrandomized and randomized trials for rectal cancer have demonstrated an improved local response with the combined use of hyperthermia and conventional treatments. A preoperative therapy with hyperthermia increased resectability and decreased local recurrence, resulting in the improvement of the postoperative prognosis. There were no major postoperative complications related to the preoperative treatment. A lower incidence of local recurrence was observed in groups that underwent intra- or postoperative hyperthermia treatment, as compared with control groups. In cases with unresectable or local recurrent rectal cancer, hyperthermia achieved a local tumor regression and prolonged pain relief. CONCLUSIONS: These clinical data suggest that hyperthermia combined with radiation or chemotherapy demonstrates great promise for the treatment of patients with carcinoma of the rectum.

Angiogenesis as a target for gastric cancer.

BACKGROUND: The growth of solid tumors and the formation of metastases depend on angiogenesis. Both tumor cells and host cells secrete a variety of factors to stimulate angiogenesis. METHODS: We investigated the expression of angiogenic factors in gastric cancer in vitro and in vivo. RESULTS: The expression of one of the angiogenic factors, vascular endothelial growth factor antigen, in gastric cancer cells can thus serve as a pertinent predictive factor for hematogenous invasion or metastasis, in addition to having prognostic value. The presence of micrometastasis in bone marrow was closely related to vascular endothelial growth factor positivity and microvessel density in the primary gastric cancer. In in vivo experiments antiangiogenic agents with cytotoxic anticancer drugs formed a highly effective modulator combination for the treatment of the Lewis lung carcinoma against primary and metastatic disease. CONCLUSIONS: Antiangiogenic agents may thus be valuable for long-term administration to maintain tumor dormancy because drug resistance does not develop, and these agents have a sustained effect. As a target, antiangiogenic therapy may therefore be potentially able to prolong survival time of patients with gastric cancer.

Estimation of angiogenesis with anti-CD105 immunostaining in the process of colorectal cancer development.

BACKGROUND: The expression of panendothelial markers (eg, CD34, CD31, and factor VIII) is not always observed in angiogenic vessels, and such markers are not useful for measuring angiogenesis. In contrast, CD105 is preferentially expressed in angiogenic vessels and thus may be valuable for measuring angiogenesis. We hypothesized that microvessel quantification by means of CD105 might be useful for measuring angiogenesis in the colorectal adenoma-carcinoma sequence. METHODS: We immunohistochemically investigated 54 cases of colorectal adenomas and 20 cases of carcinomas using monoclonal antibodies CD34 and CD105, and microvessel density (MVD) was counted at x200 magnification. RESULTS: Microvessels positive for CD34 were distributed almost uniformly in adenomas. In contrast, microvessels positive for CD105 were preferentially observed in the surface area of adenomas. In carcinomas, CD34 stained only a proportion of blood vessels that were positive for CD105. No significant difference of MVD for CD34 was observed in the colorectal adenoma-carcinoma sequence. In contrast, an increment of MVD for CD105 from low-grade to high-grade dysplasia (P <.0001) and that from high-grade dysplasia to carcinomas (P <.05) was statistically significant. CONCLUSIONS: Assessing neovascularization with CD105 in the process of colorectal cancer development may thus be a valuable marker for predicting the risk of colorectal cancer development.

Overexpression of cyclooxygenase-2 and tumor angiogenesis in human gastric cancer.

BACKGROUND/AIMS: Cyclooxygenase-2 (COX-2) protein is overexpressed in various cancers, including esophageal, gastric, colon, and pancreatic. To better comprehend the role of COX-2 in gastric cancer, especially with regard to angiogenesis, we investigated COX-2 and vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in 108 patients with gastric cancer. METHODOLOGY: We used immunohistochemical analysis of formalin-fixed tissues of gastric cancer. RESULTS: Expression of COX-2 showed diffuse staining in the cytoplasm of tumor cells, however, no staining in normal epithelial cells. Of the 108 tumors examined, 71 (65%) were positive for COX-2 expression, the VEGF-positive cases numbered 43 of 108 cases (39.8%). The intensity of COX-2 expression did not correlate with any clinicopathological characteristics. The positive rate of VEGF expression in COX-2-positive cases was significantly higher than in COX-2-negative ones (47.9% vs. 24.3%, P<0.05). MVD in COX-2-positive cases was significantly higher than in COX-2-negative ones (22.0+/-7.8 vs. 18.5+/-7.5/1 mm2; P<0.05). CONCLUSIONS: Our study provides evidence that COX-2 is closely related with angiogenesis.

Role of surgery in the patients with stage I and II primary gastric lymphoma.

BACKGROUND/AIMS: Primary gastric lymphoma is a relatively rare disease. As the concept of MALT (mucosa-associated lymphoid tissue) lymphoma has been acknowledged, a relation between primary gastric lymphoma and Helicobacter pylori infection seems apparent, appropriate treatment, including surgical resection, and/or chemotherapy, and/or radiotherapy has remained controversial. METHODOLOGY: Between 1974 and 1996, we treated 85 Japanese patients with histologically proven primary gastric lymphomas (stage I and II according to modified Ann Arbor staging) with surgery, and/or adjuvant chemotherapy. The clinicopathological factors, especially regarding surgical curability and survival were evaluated retrospectively. RESULTS: Of the 85 patients (44 men, and 41 women), average age was 60.5 years. The lesion was frequently located at the middle third of the stomach, and multiple lesions were noted in 19 patients. Microscopic lymph node metastasis was positive in 37.6% (32/85), and the rate of positive metastasis increased in proportion to depth of invasion. There was a discrepancy between macro- and microscopic findings regarding lymph node metastasis. Total gastrectomy was done for 50 patients, distal gastrectomy for 31, pancreatoduodenectomy for 3, and gastrojejunostomy for 1 patient. Lymph node dissection was done at the D1,2 level for 73, and at the D3 level for 12 patients. There were no major complications except in 3 cases (subphrenic abscess), and there was 1 operative death. The overall 5-year survival and 10-year survival was 82.3% and 74.0%, respectively. In the stage I patients, the 5-year and 10-year survival rate was 97.5%, respectively. All cases of surgery were curative, and surgery alone resulted in the same survival time as the surgery plus chemotherapy group (10-year survival; 100% vs. 96.6%, no statistical difference). In the stage II patients, 5-year and 10-year survival rates were 58.6% and 44.2%, respectively. Surgery was curative in 59.3% (19/32), and palliative in 40.7% (13/32). Palliative surgery was done because of extensive lymph node metastasis in 6 patients, invasion to adjacent organ in 6, and both in 1 patient. Chemotherapy was prescribed in 71.9% of the patients (23/32), and when compared with the patients who were on chemotherapy, the survival rate showed no statistical difference regardless of palliative or curative surgery. CONCLUSIONS: Surgery for primary gastric lymphoma had low complication rate, and led to good survival rate in stage I disease. In stage II disease, surgical curability did not affect the survival, implying the necessity of the evaluation about the treatment strategy by randomized study. However, considering the stage migration before and after and possible inaccuracy of preoperative staging, the application of non-surgical treatment must be prudent.