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Pancreatic cancer is the most lethal of all solid tumors partially because of its chemoresistance. Although gemcitabine is widely used as a first selected agent for the treatment of this disease despite low response rate, molecular mechanisms of gemcitabine resistance in pancreatic cancer still remain obscure. The aim of this study is to elucidate the mechanisms of gemcitabine resistance. The 81-fold gemcitabine resistant variant MiaPaCa2-RG was selected from pancreatic cancer cell line MiaPaCa2. By microarray analysis between MiaPaCa2 and MiaPaCa2-RG, 43 genes (0.04%) were altered expression of more than 2-fold. The most upregulated gene in MiaPaCa2-RG was ribonucleotide reductase M1 subunit (RRM1) with 4.5-fold up-regulation. Transfection with RRM1-specific RNAi suppressed more than 90% of RRM1 mRNA and protein expression. After RRM1-specific RNAi transfection, gemcitabine chemoresistance of MiaPaCa2-RG was reduced to the same level of MiaPaCa2. The 18 recurrent pancreatic cancer patients treated by gemcitabine were divided into 2 groups by RRM1 levels. There was a significant association between gemcitabine response and RRM1 expression (p = 0.018). Patients with high RRM1 levels had poor survival after gemcitabine treatment than those with low RRM1 levels (p = 0.016). RRM1 should be a key molecule in gemcitabine resistance in human pancreatic cancer through both in vitro and clinical models. RRM1 may have the potential as predictor and modulator of gemcitabine treatment., ((c) 2006 Wiley-Liss, Inc.)

Background: Portal or splenic vein thrombosis (PSVT) is a common disorder after laparoscopic splenectomy (LS). Splenomegaly is a well-known risk factor for PSVT. However, no treatment strategy for PSVT has been established., Methods: Thirty-three consecutive patients who had undergone LS and postoperative imaging surveillance were examined. PSVT was classified according to the site of thrombosis. We evaluated patient background, operative factors, and clinical symptoms., Results: Spleen weight of patients with PSVT (n = 17, median 218 g) was greater than that of patients without PSVT (n = 16, median 101 g). Seven patients developed thrombosis involving the entire splenic vein (total splenic vein thrombosis), and 4 of them had clinical symptoms (fever >38 degrees C and/or abdominal pain). The incidence of clinical symptoms was significantly more frequent in patients with than without total SVT. Operation time, blood loss, and spleen weight were also significantly greater in patients with total SVT. Multiple logistic regression analysis demonstrated spleen weight was the strongest predictor of PSVT and total SVT., Conclusion: Patients with total SVT have greater risk factors for PSVT and frequently have clinical symptoms. They are candidates for anticoagulation therapy.

The major cause of death in colorectal cancer is related to liver metastasis. Although the metastatic process has been well studied, many aspects of the molecular genetic basis of metastasis remain unclear. Elucidation of the molecular nature of liver metastasis is urgent to improve the outcome of colorectal cancer. We analyzed the chronological gene expression profiles of 104 colorectal samples corresponding to oncogenic development including normal mucosa, localized and metastasized primary tumors, and liver metastatic lesions as fundamental samples using a custom cDNA microarray. The gene expression patterns in 104 samples were classified into four groups closely associated with their metastatic status, and the genes of each group appropriately reflected the metastatic process. To investigate the existence of metastatic potential in primary tumors using metastasis-related genes detected by chronological analysis, we performed a hierarchical cluster and supervised classification analysis of 28 independent primary tumors. Hierarchical cluster analysis segregated the tumors according to their final metastatic status, rather than their clinical stages, and the profile of metastasized primary tumors resembled one of a metastatic lesion apart from a primary lesion rather than one of a non-metastasized primary tumor. Using the supervised classification approach, the expression profile of these genes allowed the classification of tumors diagnosed as localized cancer into two classes, the localized and the metastasized class, according to their final metastatic status. The disease-free survival and overall survival were significantly longer in the localized class than the metastasized class. Chronological analysis of the gene expression profile provides a better understanding of the metastatic process. Our results suggest that the metastatic potential is already encoded in the primary tumor and is detectable by a gene expression profile, which allows the prediction of liver metastasis in patients diagnosed with localized tumors and also the design of new strategies for the treatment and diagnosis of colorectal cancer.

We investigated the wide variability of 18F-2-fluoro-2-deoxy-D: -glucose (FDG) uptake, semiquantified as standardized uptake value (SUV), in positron emission tomography (PET) scanning, in 20 patients with colorectal cancer (CRC), including 1 with synchronous hepatic metastasis. The sensitivity of PET in CRC diagnosis was 100%, with a mean SUV of 8.0 (3.1-11.9). Tumor size and depth of invasion were associated with higher SUVs (P=.0004, .042, respectively). Strong glucose transporter-1 (GLUT-1) expression had significantly positive correlation with the SUV (r=.619, P=.003). GLUT-1 expression revealed positive staining in 17 (85%) of the 20 primary lesions. The central part of the tumor, thought to be relatively hypoxic, had stronger GLUT-1 expression and a higher SUV than the periphery, in both the primary tumor and hepatic metastatic foci. Our data suggest that the SUVs of FDG uptake in PET may be a noninvasive biomarker for advanced CRC, indicative of a large hypoxic tumor with deep invasion.

Background: Surgical attempts for locally recurrent rectal cancer often fail due to local re-recurrence and distant metastasis. Preoperative chemoradiation may enhance better local control and survival. The aim of this study was to assess the safety of oral uracil and tegafur (UFT) plus leucovorin (LV), and irinotecan combined with radiation and determine the maximum-tolerated dose (MTD) and dose limiting toxicity (DLT) of the triple drug regimen., Patients and Methods: Patients with locally recurrent rectal cancer received escalating doses of irinotecan on days 1, 8, 15, and 22 (starting at 30 mg/m2, with 10 mg increments between consecutive cohorts) and fixed doses of UFT (300 mg/m2) plus LV (75 mg/day) on days 3 to 7, 10 to 14, 17 to 21, and 24 to 28. Radiation was given 5 days per week totaling 40 to 50 Gy (2Gy/day)., Results: Six patients were treated at the starting dose, and 2 received the full scheduled chemoradiotherapy. The other 4 patients had grade 3 diarrhea and diarrhea was the DLT. One patient had partial response and he had subsequently radical surgical resection. Median progression free survival for local recurrence was 320 days., Conclusion: Irinotecan plus UFT/LV with concomitant radiotherapy in patients with locally recurrent rectal cancer was not feasible due to diarrhea in this setting. Modification of the treatment is needed.

The aim of the study was to evaluate the usefulness of serial lung perfusion scintigraphy prospectively using single-photon emission computed tomographic image (SPECT) in screening for pulmonary embolism (PE) after elective surgery for gastrointestinal malignancy. PE was examined pre- and postoperatively with SPECT. Diagnosis of PE was based on segmental perfusion defect visualization in at least two of three planes on a SPECT image compared with preoperative SPECT images. Final diagnosis was determined by detection of embolus with multidetector helical CT (MDCT). No perioperative anticoagulant was used. Thirty-four patients were enrolled. One patient was excluded because of thrombophilia. In preoperative scans, nonsegmental defects were detected in 11 and a segmental defect in 1 patient, who was then diagnosed as PE preoperatively. Among 21 patients with normal preoperative SPECT, 2 had nonsegmental and 5 had segmental defects postoperatively. Among 11 patients with nonsegmental preoperative SPECT, 7 had nonsegmental and 4 had segmental defects postoperatively. Postoperative segmental defects were differentiated by their shape only and there was no need to compare pre- and postoperative SPECT. MDCT confirmed four patients with PE among nine with segmental defects postoperatively. Our results of screening for PE by visualization at least two planes of SPECT images suggest that postoperative SPECT scan is suitable for the diagnosis of postoperative PE.

Whether peroxisome proliferator-activated receptor (PPAR) delta is a good target for the chemoprevention and/or treatment of colorectal cancer (CRC) remains controversial. Our goal was to examine PPARdelta expression in multistage carcinogenesis of the colorectum and to assess the relevance of PPARdelta in CRC. Immunohistochemical analysis indicated that PPARdelta expression increased from normal mucosa to adenomatous polyps to CRC. In cancer tissues, the PPARdelta protein was accumulated only in those cancer cells with highly malignant morphology, as represented by a large-sized nucleus, round-shaped nucleus, and presence of clear nucleoli. Interestingly, the cancer tissue often contained both PPARdelta-positive and -negative areas, each retaining their respective specific morphological features. Moreover, this pattern persisted even when PPARdelta-positive and -negative cells were aligned next to each other within a single cancer nest or gland and was present in the majority of CRC cases. Immunohistochemistry for Ki-67 proliferation marker showed no significant correlation between Ki-67 and PPARdelta in CRC samples. Based on Western blot analysis and quantitative RT-PCR, high PPARdelta protein expression correlated with high PPARdelta mRNA levels. Peroxisome proliferator-activated receptor delta may have a supporting role in tumorigenesis, and the close association between PPARdelta expression and malignant morphology of CRC cells suggests a pivotal role in cancer tissue.

Poorly differentiated adenocarcinoma (Por) and signet-ring cell carcinoma (Sig) are rare but highly malignant types of colorectal cancer. To explore their genetic backgrounds we investigated TGF-beta type II receptor (TGF-beta RII) and SMAD4 in the TGF-beta signaling pathway, and to identify their mutator phenotype we examined microsatellite instability (MSI) status. Loss of SMAD4 expression was significantly more frequent in Por (12 of 38; 31%) and Sig (4 of 5; 80%) tumors than in well (Well) and moderately differentiated (Mod) carcinomas (p = 0.04, 0.003, respectively). Mutation of the SMAD4 gene was detected in 2 of 26 Por tumors. MSI was positive in 14 of the 38 Por tumors and in 1 of the 5 Sig tumors, but in none of the Well or Mod tumors examined. We also found mutation of TGF-beta RII, a putative target of MSI, in 10 of 35 Por tumors (28.6%), but in none of 3 Sig tumors. As a whole, about 50% of the Por tumors and 80% of the Sig tumors showed abnormalities of either TGF-beta RII or SMAD4 expression. This suggests that disruption of the TGF-beta signaling pathway may play a central role in the pathogenesis of Por and Sig tumors of the colorectum.

Peritoneal metastasis is the most common cause of tumour progression in advanced gastric cancer. Clinicopathological findings including cytologic examination of peritoneal lavage have been applied to assess the risk of peritoneal metastasis, but are sometimes inadequate for predicting peritoneal metastasis in individuals. Hence, we tried to construct a new prediction system for peritoneal metastasis by using a PCR-based high throughput array with 2304 genes. The prediction system, constructed from the learning set comprised of 30 patients with the most informative 18 genes, classified each case into a 'good signature group' or 'poor signature group'. Then, we confirmed the predictive performance in an additional validation set comprised of 24 patients, and the prediction accuracy for peritoneal metastasis was 75%. Kaplan-Meier analysis with peritoneal metastasis revealed significant difference between these two groups (P=0.0225). By combining our system with conventional clinicopathological factors, we can identify high risk cases for peritoneal metastasis more accurately.

Purpose: Our aim was to determine the effects of cyclin D1 inhibition on tumor-associated neovascularization and endothelial cell growth., Experimental Design: We have generated adenovirus system for antisense to cyclin D1 (AS CyD1) and evaluated in vitro and in vivo effects. Small interfering RNA against cyclin D1 was also used to analyze cyclin D1 inhibition-associated vascular endothelial growth factor (VEGF) regulation., Results: The xenografts treated with adenoviral AS CyD1 showed less vessel density and displayed smaller tumor size in colon cancer cell lines HCT116 and DLD1. In vitro studies indicated that AS CyD1 decreased VEGF protein expression in DLD1 but not in HCT116. Cyclin D1 small interfering RNA caused a decrease in VEGF expression at protein and RNA levels in DLD1. A modest decrease was noted in the VEGF promoter activity, with inactivation of the STAT3 transcription factor through dephosphorylation. On the hand, the cyclin D1 inhibition plus STAT3 inhibitor markedly decreased VEGF expression in HCT116, although VEGF did not change by the STAT3 inhibitor alone. In cultures of human umbilical vein endothelial cells (HUVEC), VEGF augmented cyclin D1 expression and cell growth. AS CyD1 significantly inhibited HUVEC growth even in the presence of VEGF. AS CyD1 also significantly suppressed in vitro tube formation in VEGF-treated HUVEC and in vivo macroaneurysm formation in VEGF-treated Matrigel plug., Conclusions: Our results suggest that cyclin D1 may play a role in the maintenance of VEGF expression and that AS CyD1 could be potentially useful for targeting both cancer cells and their microenvironment of tumor vessels.

The oncogenic beta-catenin/T-cell factor (TCF) signal is a common trigger inducing expressions of various cancer-related genes and is activated in various types of human malignancy. The aim of this study was to create an effective double-stranded DNA decoy that would interfere with endogenous TCF hyperactivity in tumor cells. We first established the TCF-activated model using nontumor human embryonic kidney 293 (HEK293) cells by introducing a beta-catenin cDNA. Based on a consensus TCF-binding sequence in the cyclin D1 and c-myc promoters, several double-stranded oligodeoxynucleotides were designed and tested for their ability to inhibit TCF activity in the HEK293 model. Among them, the 18-mer oligodeoxynucleotide stably formed double-stranded DNA and efficiently inhibited TCF activity. FITC-labeled oligodeoxynucleotide was efficiently incorporated into the nucleus at 6 hours and remained within cells for up to 72 to 96 hours. When compared with scrambled oligodeoxynucleotide, we found that the 18-mer TCF decoy significantly inhibited TCF activity and promoter activities of the downstream target genes, such as cyclin D1, c-myc, and matrix metalloproteinase 7 in HCT116 colon cancer cells. Reverse transcription-PCR assays indicated that mRNA expression of these genes decreased with treatment of the TCF decoy. Proliferation assay showed that the TCF decoy significantly inhibited growth of HCT116 tumor cells, but not of nontumor HEK293 cells. Our data provide evidence that the TCF decoy reduced both TCF activity and transcriptional activation of downstream target genes. Thus, this TCF decoy is potentially an efficient and nontoxic molecular targeting therapy for controlling malignant properties of cancer cells.

Angiogenesis is a compensatory mechanism that enables malignant tumors to survive in an oxygen-deficient environment. To test our hypothesis that hypoxia stimulates the production of angiopoietin-2 (Ang-2) in colorectal cancer (CRC), we investigated the expression of Ang-2 in three cultured CRC cell lines, and in specimens from 11 CRC metastatic liver tumors. Hypoxia-induced Ang-2 mRNA expression was clearly evident in HCT116 cells that did not express Ang-2 under normoxic conditions. Ang-2 mRNA was detected only after 48 h in hypoxic serum-deprived cultures in a LoVo cell line, and under both normoxic and hypoxic conditions without any noticeable difference in the HT29 cells. There was a stepwise increase in Ang-2 expression from the periphery to the central part of the liver metastatic foci, whereas an inverse result was noted in tumor blood vessels, with a gradual decrease in CD31-positive ECs from the edge to the central region of the metastatic lesion. An expression pattern similar to Ang-2 was found in glucose transporter 1 (Glut-1), a known hypoxia-induced factor. These findings suggest that hypoxia plays an important role in inducing the expression of Ang-2 in CRC.

Hepatocellular carcinoma (HCC) is a common malignancy, but the prognosis remains poor due to the lack of sensitive diagnostic markers. To gain insight into the central molecular features common to all types of HCC, and to identify novel diagnostic markers or therapeutic targets for HCC, we performed a gene expression profiling analysis using a high throughput RT-PCR system. After examining the mRNA expression of 3,072 genes in 204 (119 tumor and 85 non-tumor) liver samples, we identified differential gene expression between the HCV group (n=80), HBV group (n=19) and non-B, non-C group (n=20) with a principal component analysis and a correlation spectrum analysis. After selection of genes differentially expressed between tumor and non-tumor tissues (p<0.01) within each HCC group, a total of 51 differentially expressed genes (23 upregulated and 28 downregulated genes) were found to be common to the three HCC groups. Gene Ontology grouping analysis revealed that genes with functions related to cell proliferation or differentiation and genes encoding extracellular proteins were found to be significantly enriched in these 51 common genes. Using an atelocollagen-based cell transfection array for functional analysis of eight upregulated genes, five (CANX, FAM34A, PVRL2, LAMR1, and GBA) significantly inhibited cellular apoptosis by two independent assays. In conclusion, we identified 51 differentially expressed genes, common to all HCC types. Among these genes, there was a high incidence of anti-apoptotic activity. This combination approach with the advanced statistical methods and the bioinformatical analysis may be useful for finding novel molecular targets for diagnosis and therapy.

Background: The enhanced antitumor effect of paclitaxel when used with oxaliplatin in gastric cancer is reported, however the underlying biological mechanism is unknown., Methods: We tested the cytotoxic activity, apoptosis, and mitotic catastrophe of paclitaxel and oxaliplatin in MKN-28 and MKN-45 gastric cancer cell lines. The modulation of survivin expression was determined by Western blotting., Results: WST-1 assay indicated that paclitaxel plus oxaliplatin showed better cytotoxicity than paclitaxel alone, even when low concentrations of oxaliplatin were used. Flow cytometry analysis revealed significantly greater increases in apoptotic cells after treatment with paclitaxel followed by low-dose oxaliplatin (1 microM) than after any single-reagent regimen in the MKN-45 cell line. In MKN-28, a difference existed only between combination treatment and oxaliplatin treatment. Morphologic examination showed that the cells undergoing mitotic catastrophe were highest in the combination groups in the both cell lines. Downregulation of survivin expression was found by Western blotting with treatment by paclitaxel, oxaliplatin, or their combination., Conclusion: Our findings suggest that the mechanism of enhanced cytotoxicity might be through enhanced mitotic catastrophe and apoptosis, which is possibly due to chemotherapy-induced downregulation of surviving. The combination of paclitaxel and low-dose oxaliplatin should be incorporated into the design of a clinical trial.

Massive lymph node metastasis of the para-aortic region and supraclavicular lymph nodes, Virchow's lymph node metastasis due to colon cancer, is extremely rare. We herein report a case of such systemic lymph node metastasis that was successfully treated with a combination of irinotecan (CPT-11) and UFT, a combination drug of tegafur and uracil. The patient was a 57-year-old woman who had a tumor in the ascending colon, and massively swollen para-aortic and supraclavicular lymph node metastasis. She was treated with combination chemotherapy of CPT-11 and UFT. The main tumor was detected as a decompressed scar, and the supraclavicular and para-aortic lymph nodes had completely disappeared after the second cycle of treatment. A histopathological examination and immunohistochemistry with cytokeratin showed complete remission of adenocarcinoma in the tumor and para-aortic lymph nodes. She remains alive without recurrence 52 months after chemotherapy. Combination chemotherapy of CPT-11 and UFT may be of potential value in the treatment of advanced colorectal carcinoma, and both histopathological and immunohistochemical confirmation of a complete remission may indicate prolonged disease-free survival.

Clinical studies have shown that oxaliplatin, a novel platinum derivative, is a potent chemotherapeutic agent for colorectal cancer when combined with 5-fluorouracil and leucovorin. Although the toxic activity is based on covalent adducts between platinum and DNA, its actual biological behavior is mostly unknown. In an effort to explore the mechanism of tumor susceptibility to oxaliplatin, we examined the cytotoxic effects of oxaliplatin in colorectal cancer cell lines in reference to p53 gene status. Although p53 gene status did not clearly predict sensitivity to oxaliplatin, p53 wild-type cells including HCT116 were sensitive but HCT116 p53-/- were found to be resistant to oxaliplatin. Oxaliplatin caused strong p21waf1/cip1 induction and G0-G1 arrest in p53 wild-type cells, whereas cisplatin did not induce G0-G1 arrest. Assays using p53 wild but p21waf1/cip1 null HCT116 cells revealed that oxaliplatin did not show G0-G1 arrest and reduced growth-inhibitory effects, suggesting that p21waf1/cip1 may be a key element in oxaliplatin-treated p53 wild-type cells. Although HCT116 is DNA mismatch repair-deficient, a mismatch repair-proficient HCT116+ch3 cell line displayed similar responses with regard to p21waf1/cip1-mediated growth inhibition and G0-G1 arrest. In p53 mutant cells, on the other hand, oxaliplatin caused an abrupt transition from G1 to S phase and eventually resulted in G2-M arrest. This abrupt entry into S phase was associated with loss of the p21waf1/cip1 protein via proteasome-mediated degradation. These findings suggest that p21waf1/cip1 plays a role in oxaliplatin-mediated cell cycle and growth control in p53-dependent and -independent pathways.

Background: Clinical studies have demonstrated that oxaliplatin, a novel platinum derivative, is a potent chemotherapeutic agent, especially when combined with other reagents. The aim of the present study was to explore the mechanism of such action., Methods: Using colon cancer cell lines, we examined changes in cell cycle, apoptosis and mitotic catastrophe induced by oxaliplatin and/or paclitaxel., Results: Oxaliplatin at its IC(50) induced apoptosis and cell cycle arrest at G(2)-M phase. Western blot analyses indicated that oxaliplatin decreased mitosis-commencing protein cdc2 and anti-apoptotic proteins, phospho-Bcl(2) and Bcl-xl in the three colon cancer cells tested. Since cdc2 stabilizes survivin, a putative IAP (inhibitor of apoptosis) family member, through phosphorylation of Thr34, we examined the level of survivin and found a marked decrease due to oxaliplatin. This finding is of particular interest because survivin is a promising molecular target against various human cancers and a key molecule involved in both apoptosis and mitotic catastrophe. When used in combination with paclitaxel (taxol), a putative apoptosis-inducing reagent, the isobologram indicated that the taxol-oxaliplatin sequence or taxol plus oxaliplatin had synergic or additive effects, while the oxaliplatin-taxol sequence resulted in a prominent antagonism. The taxol-oxaliplatin sequence caused marked growth inhibition of DLD1 and SW480 cells, possibly due to upregulation of apoptotic and non-apoptotic pathways, respectively. Morphological surveys indicated that the non-apoptotic process could be mitotic catastrophe., Conclusion: Our results suggest that oxaliplatin that potently inhibited survivin may exert outstanding cytotoxic effects when combined with certain chemoreagents through enhancement of apoptosis and mitotic catastrophe.

The cyclin-dependent kinase inhibitor p21cip1/waf1 negatively regulates the progression of cell cycle and the potential usefulness of p21cip1/waf1 gene is proposed in gene therapy. However, studies have demonstrated a protective role of p21cip1/waf1 against apoptosis and little is known about effects of ectopic expression of p21cip1/waf1 on differentiation of colon cancer cells. In the present study, we found diffuse p21cip1/waf1 expression in only a few clinical samples of colorectal cancer with wild-type p53 gene. To explore the role of p21cip1/waf1 in cell growth, apoptosis and differentiation, we constitutively overexpressed p21cip1/waf1 in HT29 colon carcinoma cells. Ectopic overexpression of p21cip1/waf1 was associated with inhibition of CDK2-associated kinase activity, indicating the functionality of the introduced p21cip1/waf1 gene. Overexpression of p21cip1/waf1 caused an appreciable growth inhibition in monolayer and soft agar cultures and it significantly reduced sodium butyrate- but not 5-fluorouracil-induced apoptosis. p21cip1/waf1 overexpressing cells exhibited marked decrease of intestinal differentiation when assayed with intestinal alkaline phosphatase. Our findings suggest that introduction of p21cip1/waf1 gene into colon cancer cells may be useful for inhibiting cell growth but caution should be taken regarding the increased resistance to certain apoptosis-inducing agents and dysregulation of endogenous p21cip1/waf1-mediated differentiation process.

The prognosis of patients with advanced gastric cancer remains unfavorable. Even after curative resection, 40% of patients with advanced gastric cancer die of recurrence. Conventional clinicopathlogic findings are sometimes inadequate for predicting recurrence in individuals. Hence, we tried to construct a new diagnostic system, which predicts recurrence in patients with advanced gastric cancer after curative resection based on molecular analysis. Gastric cancer progression is a function of multiple genetic events that may affect the expression of large number of genes. We performed gene expression profiling with 2,304 genes in 60 advanced gastric cancer patients who underwent curative resection using a PCR array technique, a high-throughput quantitative RT-PCR technique. The diagnostic system, which was constructed from the learning set comprised of 40 patients with the most informative 29 genes, classified each case into a good-signature or poor-signature group. Then, we confirmed the predictive performance in an additional test set comprised of 20 patients, and the prediction accuracy for recurrence was 75%. Kaplan-Meier analysis revealed significant difference between the good-signature and the poor-signature group (p = 0.0125). Especially in patients with smaller tumor (< or = 5 cm), less developed LN metastasis (N(0,1)), or earlier stage (stages I and II), the prediction accuracy was high (88.9%, 84.6%, or 81.8%, respectively). Our diagnostic system based on systematic analysis of gene expression profiling can predict the recurrence at clinically meaningful level. By combining our system with conventional clinicopathologic factors, we can improve the prediction of recurrence in patients with advanced gastric cancer who underwent curative surgery.

Purpose: The prognosis of hepatocellular carcinoma (HCC) is very poor, particularly in patients with tumors that have invaded the major branches of the portal vein. Combination chemotherapy with intra-arterial 5-fluorouracil and subcutaneous interferon-alpha has shown promising results for such advanced HCC, but it is important to develop the ability to accurately predict chemotherapeutic responses., Experimental Design: We analyzed the expression of 3,080 genes using a polymerase chain reaction-based array in 20 HCC patients who were treated with combination chemotherapy after reduction surgery. After unsupervised analyses, a supervised classification method for predicting chemotherapeutic responses was constructed. To minimize the number of predictive genes, we used a random permutation test to select only significant (P < 0.01) genes. A leave-one-out cross-validation confirmed the gene selection. We also prepared an additional 11 cases for validation of predictive performance., Results: Hierarchical clustering analysis and principal component analysis with all 3,080 genes revealed distinct gene expression patterns in responders (those with complete response or partial response) and nonresponders (those with stable disease or progressive disease) to the combination chemotherapy. Using a weighted-voting classification method with either all genes or only significant genes as assessed by permutation testing, the objective responses to treatment were correctly predicted in 17 of 20 cases (accuracy, 85%; positive predictive value, 100%; negative predictive value, 80%). Moreover, patients in the validation dataset could be classified into two distinct prognostic groups using 63 predictive genes., Conclusions: Molecular analysis of 63 genes can predict the response of patients with advanced HCC and major portal vein tumor thrombi to combination chemotherapy with 5-fluorouracil and interferon-alpha.

To identify candidate genes involved in human colorectal carcinogenesis, we constructed the gene expression profiles of 50 colorectal cancers (CRCs) and 12 normal colorectal epithelia using a cDNA microarray specially constructed for CRC. Hierarchical clustering analysis and principal component analysis could clearly distinguish the gene profiles of cancer tissues from those of normal tissues. Our results confirm there are indeed differences in gene expression between cancer and normal mucosa. Our cDNA microarray identified 22 up-regulated genes and 32 down-regulated genes in CRC. Many of these genes have been previously identified in relation to human carcinogenesis, 68% and 78%, respectively. Subsequent validation of selected genes by serial analysis of gene expression and reverse transcription polymerase chain reaction, demonstrated expression patterns that were almost identical to our microarray analysis. Using a four-fold larger sample relative to that used in our previous study, candidate genes involved in human colorectal carcinogenesis were reproducibly identified. Further studies of comprehensive gene expression using our technique may elucidate the mechanism of CRC tumorigenesis.

Background/aims: Hepatocellular carcinoma (HCC) has a very poor prognosis, due to the high incidence of tumor recurrence. As the current morphological indicators are often insufficient for therapeutic decisions, we sought to identify additional biologic indicators for early recurrence., Methods: We analyzed gene expression using a PCR-based array of 3,072 genes in 100 HCC patients. Informative genes predicting early intrahepatic recurrence were selected by random permutation testing, and a weighted voting prediction method was constructed. Following estimation of prediction accuracy, a multivariate Cox analysis was performed., Results: By permutation testing, we selected 92 genes demonstrated distinct expression patterns differing significantly between recurrence cases and recurrence-free cases. Our prediction method, using the 20 top-ranked genes, correctly predicted the early intrahepatic recurrence for 29 of 40 cases within the validation group, and the odds ratio was 6.8 (95%CI 1.7-27.5, P = 0.010). The 2-year recurrence rates in the patients with the good signature and those with the poor signature were 29.4 and 73.9%, respectively. Multivariate Cox analysis revealed that molecular-signature was an independent indicator for recurrence (hazard ratio 3.82, 95%CI 1.44-10.10, P = 0.007)., Conclusions: Our molecular-based prediction method using 20 genes is clinically useful to predict early recurrence of HCC.

Many trials using DNA microarrays have been reported for various human malignancies, but an efficient molecular diagnostic system has yet to be established. Here, we adopted a high throughput quantitative PCR-array system based on adaptor-tagged competitive PCR (ATAC-PCR), as a novel technique for gene expression profiling of hepatocellular carcinoma (HCC). This PCR-array contained 3,072 genes derived from three different cDNA libraries, including 298 additional known genes suspected to be involved in hepatocarcinogenesis. Using this PCR-array with 20 pairs of liver tissues (20 HCC, 20 surrounding nontumor liver), we identified a total of 117 genes differing in expression levels in the two liver tissues. Hierarchical clustering analysis and principal component analysis with these genes revealed distinct gene expression patterns in the HBV-positive group and the HCV-positive groups. Among 117 genes, only 7 (GPAA1, TMEM9, FACL4, ADFP, MAWBP, PACE4, FOS) were common to both groups. In conclusion, this PCR-array analysis with an appropriate set of genes is considered useful for gene expression profiling of HCC, and we identified some genes which may play a common key role in hepatocarcinogenesis.

Background/aims: Hepatocellular carcinoma (HCC) usually develops following chronic liver inflammation caused by hepatitis C or B virus. Through expression profiling in a rare type of HCC, for which the causes are unknown, we sought to find key genes responsible for each step of hepatocarcinogenesis in the absence of viral influence., Methods: We used 68 non-B, non-C liver tissues (20 HCC, 17 non-tumor, 31 normal liver) for expression profiling with PCR-array carrying 3072 genes known to be expressed in liver tissues. To select the differentially expressed genes, we performed random permutation testing. A weighted voting classification algorithm was used to confirm the reliability of gene selection. We then compared these genes with the results of previous expression profiling studies., Results: A total of 220 differentially expressed genes were selected by random permutation tests. The classification accuracies using these genes were 91.8, 92.0 and 100.0% by a leave-one-out cross-validation, an additional PCR-array dataset and a Stanford DNA microarray dataset, respectively. By comparing our results with previous reports on virus-infected HCC, four genes (ALB, A2M, ECHS1 and IGFBP3) were commonly selected in some studies., Conclusions: The 220 differentially expressed genes selected by PCR-array are potentially responsible for hepatocarcinogenesis in the absence of viral influence.

Motivation: Gene expression profile analyses have been used in numerous studies covering a broad range of areas in biology. When unreliable measurements are excluded, missing values are introduced in gene expression profiles. Although existing multivariate analysis methods have difficulty with the treatment of missing values, this problem has received little attention. There are many options for dealing with missing values, each of which reaches drastically different results. Ignoring missing values is the simplest method and is frequently applied. This approach, however, has its flaws. In this article, we propose an estimation method for missing values, which is based on Bayesian principal component analysis (BPCA). Although the methodology that a probabilistic model and latent variables are estimated simultaneously within the framework of Bayes inference is not new in principle, actual BPCA implementation that makes it possible to estimate arbitrary missing variables is new in terms of statistical methodology., Results: When applied to DNA microarray data from various experimental conditions, the BPCA method exhibited markedly better estimation ability than other recently proposed methods, such as singular value decomposition and K-nearest neighbors. While the estimation performance of existing methods depends on model parameters whose determination is difficult, our BPCA method is free from this difficulty. Accordingly, the BPCA method provides accurate and convenient estimation for missing values., Availability: The software is available at http://hawaii.aist-nara.ac.jp/~shige-o/tools/.

Background: Individual human carcinomas have distinct biological and clinical properties: gene-expression profiling is expected to unveil the underlying molecular features. Particular interest has been focused on potential diagnostic and therapeutic applications. Solid tumors, such as colorectal carcinoma, present additional obstacles for experimental and data analysis., Results: We analyzed the expression levels of 1,536 genes in 100 colorectal cancer and 11 normal tissues using adaptor-tagged competitive PCR, a high-throughput reverse transcription-PCR technique. A parametric clustering method using the Gaussian mixture model and the Bayes inference revealed three groups of expressed genes. Two contained large numbers of genes. One of these groups correlated well with both the differences between tumor and normal tissues and the presence or absence of distant metastasis, whereas the other correlated only with the tumor/normal difference. The third group comprised a small number of genes. Approximately half showed an identical expression pattern, and cancer tissues were classified into two groups by their expression levels. The high-expression group had strong correlation with distant metastasis, and a poorer survival rate than the low-expression group, indicating possible clinical applications of these genes. In addition to c-yes, a homolog of a viral oncogene, prognostic indicators included genes specific to glial cells, which gives a new link between malignancy and ectopic gene expression., Conclusions: The malignancy of human colorectal carcinoma is correlated with a unique expression pattern of a specific group of genes, allowing the classification of tumor tissues into two clinically distinct groups.

cDNA microarray analyses can be used to identify candidate genes that play important roles in human carcinogenesis. To gain insight into the molecular sketch of colorectal cancer, we have constructed cDNA microarrays specialized for colorectal cancer, which we named "Colonochip" by selecting genes that are expressed in colorectal cancer, normal colonic mucosa, and liver metastatic cancer tissues. This microarray contained 4608 nonredundant cDNA clones from over 30,000 cDNA clones derived from the three types of human cDNA libraries, as well as clones from 170 additional conventional major genes suspected to be involved in colorectal carcinogenesis, according to literatures. Using this "Colonochip," we were able to identify 59 genes showing twofold or more differential expression between primary cancer and normal colonic mucosa, potent candidates for diagnosis, and therapy of colorectal cancer for further studies., (Copyright 2001 Academic Press.)

p57KIP2, the second class of KIP family protein, is one of the negative regulators of the cell cycle. To elucidate the role of p57KIP2 in colorectal normal mucosa and cancer, we examined the expression of p57KIP2 protein in 110 pairs of colorectal non-tumor and cancer tissues. Immunohistochemical analysis showed that p57KIP2 was weakly detected in the normal colonic epithelium and lymph follicles. A unique expression pattern of p57KIP2 was exclusively noted in the elastic fibers within the walls of relatively large blood vessels (diameter > 100 microm). In cancer tissues, p57KIP2 protein was localized mainly in nuclei. Using the mean percentage of nuclear p57KIP2 expression (25%) as the cut-off value, we divided our cases into those with high expression (n = 44, 40%) and low expression (n = 66, 60%) of p57KIP2 among 110 colorectal cancer cases tested. The clinical and pathological survey showed a significant correlation between low expression of p57KIP2 and large tumor size (p < 0.05) or the presence of tumors in females (p < 0.01). Survival analysis showed that p57KIP2 expression did not influence prognosis. RT-PCR analysis was also performed using RNA extracts from 6 colorectal cancer tissues. When the levels of p57KIP2 mRNAs were compared with expression of p57KIP2 protein, a clear correlation was found, suggesting that expression of the p57KIP2 protein may be regulated at the transcription level. The present study revealed p57KIP2 expression in colorectal cancer and suggests that p57KIP2 may not play a central role in the progression of colorectal cancer.

We here report a 53-year-old woman who had undergone resection of a choledochal cyst and hepaticojejunostomy three years before. She was readmitted because of intermittent fever, and abdominal computed tomography revealed a 4-cm tumor in the head of the pancreas. We performed pancreatoduodenectomy, and examination of the resected specimen showed well-differentiated papillary adenocarcinoma. Only 5 cases of carcinoma occurring after the resection of a choledochal cyst have been reported, and to our knowledge, this is the second case of carcinoma of the head of the pancreas.

There is evidence to suggest that CDC25B phosphatase is an oncogenic protein. To elucidate the role of CDC25B in colorectal carcinoma, we examined the expression of CDC25B at the mRNA and protein levels. Reverse transcription-PCR assay indicated that CDC25B was overexpressed in tumor tissues relative to normal mucosa in 6 of 10 cases. Using immunohistochemistry, we identified high expression of CDC25B in 77 of 181 colorectal cases (43%). Univariate analysis showed that high expression was a significant predictor for poor prognosis compared with low expression (5-year survival rate; 59% versus 82%, respectively; P < 0.0001). Multivariate analysis indicated that CDC25B was an independent prognostic marker (risk ratio for death, 3.7; P < 0.0001) even after controlling for various factors such as lymph node metastasis, tumor size, degree of differentiation, and depth of invasion. Furthermore, the level of CDC25B expression clearly predicted the outcome of patients with Dukes' B and Dukes' C tumors. On the other hand, CDC25A mRNA was overexpressed in 9 of 10 colorectal cancer cases, and immunohistochemistry for CDC25A showed high expression in 52 of 111 cases (47%), but no significant correlation with prognosis. Our findings suggest that CDC25B is a novel independent prognostic marker of colorectal carcinoma and that it may be clinically useful for selecting patients who could benefit from adjuvant therapy.

Background: There is now evidence from three randomized controlled trials (from Minnesota, Nottingham and Funen) that screening average-risk individuals for colorectal cancer (CRC) with fecal-occult-blood tests (FOBT) can reduce mortality from CRC. In Japan, mortality rates from CRC have increased and mass screening with FOBT has been performed since 1992. Although there is growing proportion of CRC with FOBT, there is no conclusive evidence that they reduce mortality from this cause. We evaluated the feature of CRC with FOBT, as one of the methods for evaluation of the efficacy of screening with this test., Methods: Between January, 1982 and December, 1996, 2071 cases with CRC resected in our hospital were considered. We evaluated the clinicopathological findings about age, sex, location of tumor, size depth, incidence of lymph node metastasis, stage and prognosis of CRC with FOBT (376) compared with CRC with no screening (controls; 1695)., Results: CRC with FOBT were earlier stage and smaller-sized cancers than controls. No significant difference was found in the incidence of lymph node metastasis at each depth and prognosis in each stage., Conclusions: By screening with FOBT, we can detect at an earlier stage and with a smaller cancer, with the same biological behavior as controls. Now, we must encourage colon screening, continuing to research ways to improve identification of high-risk subgroups and increase complicance.

Colorectal cancer (CRC) infiltrating the submucosa (sm cancer) and the proper muscle (mp cancer) represent an early and an intermediate stage in the development of CRC, and sm and mp cancer are therefore appropriate to discuss the natural history of CRC. A total of 337 sm cancers and 291 mp cancers resected in our series were evaluated. We divided sm and mp cancers into three categories, respectively, sm 1, sm 2, sm 3, mp 1, mp 2, and mp 3, according to the depth of infiltration of the submucosa or the proper muscle. The deeper the invasion, the larger of the tumor size, and the proportion of depressed type in their configuration increases. On the contrary, the proportion of tumors with adenomatous component decreases. A larger proportion of tumors in all categories of infiltration showed PG more of ten than that of NPG in their marginal structure. In this study, 17% of sm cancer and 23% of mp cancer might develop not via the adenoma-carcinoma sequence.

The significance of continuous hepatic arterial infusion (HAI) chemotherapy of 5-FU to prevent recurrence in the remnant liver following curative hepatectomy for colorectal metastases was analyzed in the prospective randomized study. Patients of HAI chemotherapy group received 6 weeks of 5-FU 500 mg/day. The one- and two-year overall survival rates of nine patients with HAI therapy were 87.5% and 87.5%, respectively, while the rates of ten cases without the regional chemotherapy were 100% and 66.7%, respectively, indicating no obvious difference. On the other hand, the disease-free survival rates of HAI Group were higher than those of control group. The disease-free survival rates of the former were 75.0% at one-year and 75.0% at two-year, and the rates of the latter were 47.6% and 47.6%, respectively. No serious complications such as severe liver dysfunction, sclerotic cholangitis or hepatic necrosis were observed. Although the follow-up period was not enough long to accurately evaluate the efficacy, local prophylactic chemotherapy by continuous infusion of 5-FU could be a promising method as an adjuvant chemotherapy after hepatic resection for colorectal metastases.