Takamitsu Kanazawa

Prediction of sensitivity of rectal cancer cells in response to preoperative radiotherapy by DNA microarray analysis of gene expression profiles.

Preoperative radiotherapy has been widely used to improve local control of disease and to improve survival in the treatment of rectal cancer. However, the response to radiotherapy differs among individual tumors. Our objective here was to identify a set of discriminating genes that can be used for characterization and prediction of response to radiotherapy in rectal cancer. Fifty-two rectal cancer patients who underwent preoperative radiotherapy were studied. Biopsy specimens were obtained from rectal cancer before preoperative radiotherapy. Response to radiotherapy was determined by histopathologic examination of surgically resected specimens and classified as responders or nonresponders. By determining gene expression profiles using human U95Av2 Gene Chip, we identified 33 novel discriminating genes of which the expression differed significantly between responders and nonresponders. Using this gene set, we were able to establish a new model to predict response to radiotherapy in rectal cancer with an accuracy of 82.4%. The list of discriminating genes included growth factor, apoptosis, cell proliferation, signal transduction, or cell adhesion-related genes. Among 33 discriminating genes, apoptosis inducers (lumican, thrombospondin 2, and galectin-1) showed higher expression in responders whereas apoptosis inhibitors (cyclophilin 40 and glutathione peroxidase) showed higher expression in nonresponders. The present study suggested the possibility that gene expression profiling may be useful in predicting response to radiotherapy to establish an individualized tailored therapy for rectal cancer. Global expression profiles of responders and nonresponders may provide insights into the development of novel therapeutic targets.

Distal Colorectal Cancers with Microsatellite Instability (MSI) Display Distinct Gene Expression Profiles that Are Different from Proximal MSI Cancers

Promoter methylation of the mismatch repair gene plays a key role in sporadic microsatellite instability (MSI) colorectal cancers. However, promoter methylation often occurs in proximal colon cancers, and molecular phenotypes underlying MSI cancers in distal colon have not been fully clarified. Our goal was to clarify the difference between MSI and microsatellite stability (MSS) cancers and, furthermore, to determine distinct characteristics of proximal and distal MSI cancers. By DNA microarray analysis of 84 cancers (33 MSI and 51 MSS), we identified discriminating genes (177 probe sets), which predicted MSI status with a high accuracy rate (97.6%). These genes were related to phenotypic characteristics of MSI cancers. Next, we identified 24 probe sets that were differentially expressed in proximal and distal MSI cancers. These genes included promoter methylation-mediated genes, whose expression was significantly down-regulated in proximal MSI cancers. Among discriminating genes between MSI and MSS, nine methylation-mediated genes showed down-regulation in MSI cancers. Of these, 7 (77.8%) showed down-regulation in proximal MSI cancers. Furthermore, methylation-specific PCR confirmed that frequency of hMLH1 promoter methylation was significantly higher in proximal MSI cancers (P = 0.0317). These results suggested that there is a difference between proximal and distal MSI cancers in methylation-mediated influence on gene silencing. In conclusion, using DNA microarray, we could distinguish MSI and MSS cancers. We also showed distinct characteristics of proximal and distal MSI cancers. The inactivation form of hMLH, per se, differed between proximal and distal MSI cancers. These results suggested that distal MSI cancers constitute a distinct subgroup of sporadic MSI cancers.

Tumour budding at the deepest invasive margin correlates with lymph node metastasis in submucosal colorectal cancer detected by anticytokeratin antibody CAM5.2

In the past few years, tumour budding at the invasive margin has been reported as a new risk factor for lymph node metastasis in advanced colorectal cancers, but it is sometimes difficult to detect tumour budding in submucosal colorectal cancer by haematoxylin and eosin staining. We immunohistochemically examined tumour budding at the deepest invasive margin of 56 surgically resected submucosal colorectal carcinomas using anticytokeratin antibody CAM5.2, furthermore checked by AE1/AE3, and determined the relation between tumour budding and clinicopathological factors. Moreover, we used the monoclonal antibody D2-40 for immunohistochemistry to detect lymphatic involvement. Tumour budding was detected in 42 cases (75.0%), and the budding-positive group showed a significantly higher rate of lymph node metastasis (including isolated tumour cells) (16/42 vs 0/14; P=0.004) than the budding-negative group. The sensitivity and negative predictive value of tumour budding alone for lymph node metastasis were superior to those of lymphatic invasion alone. Furthermore, the specificity and positive predictive value of the combination of either lymphatic invasion or tumour budding were superior to those of lymphatic invasion alone. Tumour budding detected immunohistochemically by using CAM5.2 is a newly found risk factor for lymph node metastasis and may help to avoid oversurgery in the future.

Poorly differentiated adenocarcinoma and mucinous carcinoma of the colon and rectum show higher rates of loss of heterozygosity and loss of E-cadherin expression due to methylation of promoter region.

Previous studies have reported that poorly differentiated adenocarcinoma and mucinous carcinoma of the colon and rectum (Por & Muc) show a low incidence and poor prognosis. However, genetic alterations and tumourigenesis of Por & Muc remain unclear. In our study, we analyzed the genetic and epigenetic alterations of Por & Muc to clarify the difference from those of well‐differentiated adenocarcinoma (WD). First, we evaluated the loss of heterozygosity (LOH) on 4 chromosomes (2p, 5q, 17p, 18q) frequently observed in colorectal cancer. Second, to determine large‐scale allelic losses, we performed wide‐ranging allelotyping study. Using 27 microsatellite markers spanning every 10 cM on chromosome 17 and 18, we defined the LOH ratio as the proportion of markers that exhibit LOH out of 27 markers. Third, we evaluated the methylation of E‐cadherin. With respect to LOH of 4 loci, higher rates of LOH were observed in Por & Muc and a statistical significance was found on the markers adjacent to MSH2 and SMAD4/DCC. Moreover, as the evidence of large‐scale allelic imbalance, the average LOH ratio was higher in Por & Muc (0.70) than in WD (0.24) (p = 0.01). Methylation analysis showed that 54.5% of Por & Muc demonstrated hypermethylation of E‐cadherin. In immunohistochemistry, 77.8% of Por & Muc exhibited abnormal expression of E‐cadherin. Hypermethylation of E‐cadherin correlated significantly with abnormal expression (p = 0.047). These results suggest that the higher rates of LOH contribute to Por & Muc tumourigenesis and most of Por & Muc lose normal E‐cadherin expression, partly because of the methylation of promoter region.

Gene Expression Signature and the Prediction of Ulcerative Colitis–Associated Colorectal Cancer by DNA Microarray

Purpose: Ulcerative colitis (UC) is associated with a high risk of colorectal cancer. To identify genes that could predict the development of cancer in UC, we conducted a DNA microarray analysis using nonneoplastic rectal mucosa of UC patients. Experimental Design: Gene expression in nonneoplastic mucosa of 53 UC patients were examined. Gene expression profiles were examined using human Genome U133 Plus 2.0 gene chip array (Affymetrix). Among 53 UC patients, 10 had UC-associated cancer (UC-Ca group) whereas 43 did not (UC-NonCa group). Results: By comparing gene expression profiles of nonneoplastic rectal mucosae between the UC-Ca and UC-NonCa groups, we could identify 40 genes that were differentially expressed between two groups. The list of discriminating genes included low-density lipoprotein receptor–related protein (LRP5 and LRP6). Previous studies suggested that LRP5 and LRP6 expression promotes cancer cell proliferation and tumorigenesis and are considered as candidate oncogenes. In the present study, both LRP5 and LRP6 showed significantly higher expression in the UC-Ca group, which suggests the importance of these genes in the development of UC-associated colorectal cancers. With the 40 selected discriminating genes, we did class prediction of the development of colorectal neoplasms in UC patients. Using the k-nearest neighbor method and the support vector machine, we could predict the development of UC-associated neoplasms with an accuracy of 86.8% and 98.1%, respectively. Conclusions: These findings have important implications for the early detection of malignant lesions in UC and may provide directions for future research into the molecular mechanisms of UC-associated cancer.

Expression of platelet-derived endothelial cell growth factor in inflammatory bowel disease

Background: Platelet-derived endothelial cell growth factor (PD-ECGF) is reported to be highly expressed in tumors and inflammatory tissues, but its expression and role in inflammatory bowel disease (IBD) are still unclear. In this study we examined the location and tissue density of cells immunoreactive for PD-ECGF in the colonic mucosa of IBD. Methods: Paraffin-embedded sections of colonic tissue from patients with ulcerative colitis (UC) or Crohns disease (CD) were immunostained for PD-ECGF. As controls, noninflamed mucosa of IBD, as well as normal colonic mucosa from patients with colorectal cancer, were used. Also, cancer tissues were evaluated. In addition, changes in the expression of PD-ECGF in human umbilical vein endothelial cells (HUVEC) after treatment with inflammatory cytokines and angiogenic factors, as well as after coculture with colon cancer cell lines, were evaluated by flow cytometry. Results: In normal colonic mucosa and noninflamed mucosa of IBD, PD-ECGF expression was negligible. In inflamed colonic mucosa, strong expression was observed, predominantly in macrophages and fibroblasts. Vascular endothelial cells of the inflamed colonic mucosa, but not of normal colonic mucosa or of neoplastic tissues, stained for PD-ECGF, and the microvessel density was significantly increased in the severely inflamed mucosa. Flow cytometry demonstrated that PD-ECGF was constitutively expressed in HUVEC. Inflammatory cytokines and vascular endothelial growth factor (VEGF) increased its expression, whereas basic fibroblast growth factor (bFGF) decreased it. Coculture with colon cancer cell lines in direct contact, but not in those without contact, also resulted in an important decrease in the expression of PD-ECGF in HUVEC. Conclusions: Autocrine production of PD-ECGF by endothelial cells may be a mechanism of inflammatory angiogenesis, but not tumor angiogenesis, and may be particularly important for the maintenance of damaged vasculature in IBD.

Chromosome 18q deletion and Smad4 protein inactivation correlate with liver metastasis: a study matched for T- and N- classification

Smad4 protein, whose gene is coded at chromosome 18q21.1, is an important tumour suppressor that mediates transforming growth factor-beta. It has been reported that inactivation of the Smad4 gene and allelic loss of chromosome 18q correlate with liver metastasis and poorer prognosis in colorectal cancers. Utilising a recently developed method of immunohistochemical staining for Smad4 protein, we focused on the specific impact of Smad4 protein expression on liver metastasis in colorectal cancer. We also evaluated the association between chromosome18q deletion and liver metastasis. We selected 20 colorectal cancers with liver metastasis for the experimental group, and 20 cases without liver metastasis for the control. In order to exclude the influence of lymph node metastasis, all cases were lymph node negative. In addition, the two groups were matched for tumour depth, tumour differentiation and tumour location. We compared the expression level of Smad4 protein immunohistochemically in these 20 matched pairs. We also compared the loss of heterozygosity status at chromosome 18q in these 20 matched pairs. Immunohistochemical staining revealed a significant difference (P=0.024) in the level of Smad4 protein between the two groups. We also observed a significantly different (P=0.0054) ratio of allelic deletion at chromosome 18q21. Smad4 protein expression level and allelic loss at 18q21 are associated with the process of liver metastasis in colorectal cancers evaluated when excluding clinical and pathological features except for liver metastasis.

Prediction of liver metastasis after colorectal cancer using reverse transcription-polymerase chain reaction analysis of 10 genes

PURPOSE Liver metastasis is one of the major types of recurrence after surgery for colorectal cancer. Traditional methods of predicting liver metastasis are limited in their accuracy, suggesting the need to develop new predictors. We developed a 10-gene signature that is closely associated with the development of liver metastasis after colorectal cancer. PATIENTS AND METHODS We examined a total of 189 frozen specimens of primary colorectal cancers using both microarray and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Initially, we studied gene expression in colorectal cancer tissue from 160 randomly selected patients who had undergone surgical resection of colorectal cancer and evaluated the association between the level of gene expression and the occurrence of liver metastasis. We developed a gene-expression model for the prediction of liver metastasis based on the RT-PCR findings. We then used specimens from 29 other patients for validation. RESULTS The expression of 14 genes was correlated with liver metastasis according to both microarray and RT-PCR analysis. We constructed an accurate predictive model based on the results for 10 of these genes, which included epiregulin (EREG), amphiregulin (AREG), cyclooxygenase 2 (COX-2) and lymphocyte-specific protein tyrosine kinase (LCK). The 10-gene signature was an independent predictor of liver metastasis. The model was validated in the independent set of 29 patients. The predictive accuracy of the model in a test set of patients was 86.2%. CONCLUSION The 10-gene signature identified in this study is closely associated with the occurrence of liver metastasis in colorectal cancer patients.

Gene expression of mesenchyme forkhead 1 (FOXC2) significantly correlates with the degree of lymph node metastasis in colorectal cancer.

In stage III colorectal cancer, patients with N1 stage tumors show poorer outcome than patients with N2 stage tumors. Our objective was to identify genes that are predictive for the presence of lymph node metastasis, and to characterize the aggressiveness of lymph node metastases. Gene expression profiles of colorectal cancer were determined by microarray in training (n = 116) and test (n = 25) sets of patients. We identified 40 discriminating probes in patients with and without lymph node metastases. Using these probes, we could predict the presence of lymph node metastasis with an accuracy of 87.1% (training set) and 76.0% (test set). Among discriminating probes, FOXC2 expression was significantly correlated with the degree of lymph node metastasis. FOXC2 was expressed significantly and disparately in patients with N1 and N2 stage tumors as analyzed by real-time reverse transcriptase-polymerase chain reaction. FOXC2 appears to be involved in determining the aggressiveness of lymph node metastasis in colorectal cancer.

Microsatellite instability in poorly differentiated adenocarcinomas of the colon and rectum: relationship to clinicopathological features

Background: Poorly differentiated adenocarcinomas of the colon and rectum (Por) feature the worst prognosis among the various types of colorectal carcinomas. Por is highly associated with microsatellite instability (MSI), although MSI is associated with an improved prognosis in colorectal cancers. Aim: To investigate the influence of MSI on clinicopathological features and survival of patients affected by Por. Methods: 53 patients affected by Por were investigated. DNA extracted from tumour sections and the corresponding normal tissue was analysed by PCR at five microsatellite loci: BAT25, BAT26, D2S123, D5S346 and D17S250. Tumours with alterations at two or more loci were classified as MSI-Por. The others were classified as microsatellite stability (MSS)-Por. The clinicopathological features and survival of patients with MSI-Por and MSS-Por were investigated. Results: Of the 53 patients who were examined, 12 (22.6%) were MSI-Por, whereas 41 (77.4%) were MSS-Por. Significant differences were found between MSI-Por and MSS-Por regarding the following clinicopathological features: age, gender, lymph-node metastasis (MSI-Por: 4/12; MSS-Por: 33/41), TNM stage (MSI-Por: T1/T2/T3/T4 = 2/6/2/2; MSS-Por: 3/3/19/16) and lymphatic invasion (MSI-Por: 4/10; MSS-Por: 27/35). Kaplan–Meier survival curves and log-rank analysis showed that MSI-Por was associated with better prognosis than MSS-Por, although no significant difference was found. Conclusions: Compared with MSS-Por, MSI-Por is significantly associated with a low incidence of lymph-node metastases and a low stage. This indicates that MSI-Por is a less aggressive subtype.