Yoshihiro Kazama

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.

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.

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.

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.

Chromosome 18q deletion as a novel molecular predictor for colorectal cancer with simultaneous hepatic metastasis.

Currently, surgical treatment for colorectal hepatic metastasis is performed with low mortality and morbidity rates. However, there is no definitive marker that predicts patient outcome. The aim of this study is to identify the molecular predictor of survival along with its clinical properties. Fifty-six patients were surgically treated for colorectal cancer and synchronous hepatic metastasis from January 1994 to December 2004. Clinicopathologic and molecular factors were reviewed in association with overall survival (OS) and disease-free survival (DFS). Chromosome 18q deletion in the primary tumor was a molecular predictor that affected OS (P=0.021). Decreased expression of the Smad4 protein tended to affect the outcome; however, no statistical significance was observed (P=0.29:OS, P=0.45:DFS). Preoperative carcinoembryonic antigen (P=0.013) and carbohydrate antigen 19-9 (CA19-9) (P<0.0001) levels were poor clinical predictors of OS. The number of primary lymph nodes was the only pathologic factor that affected DFS (P=0.0052). The number and diameter of hepatic metastasis had no influence on survival. In conclusion, we demonstrated that chromosome 18q deletion, in conjunction with high carcinoembryonic antigen and CA19-9 levels, is an unfavorable prognostic factor. This novel molecular predictor is helpful in identifying patients who would benefit from surgical resection.

Crohn's disease with life-threatening hemorrhage from terminal ileum : successful control by superselective arterial embolization

A case of life-threatening lower gastrointestinal hemorrhage from Crohns disease is reported. Several promising studies have recently been published that describe superselective embolization for the treatment of massive lower gastrointestinal hemorrhage in patients with bleeding colonic diverticular disease and angiodysplasia, and success rates of 74%–93% have been reported. But in patients with Crohns disease, successful superselective embolization has rarely been reported. This is a report of successful superselective embolization in a patient with Crohns disease; this should be the initial treatment of choice in Crohns disease in an attempt to avoid surgical resection, because repeated resections predispose patients to the development of short-bowel syndrome.

Mucinous colorectal cancers with chromosomal instability: a biologically distinct and aggressive subtype.

Colorectal cancers can progress through 2 pathways of genomic instability: microsatellite instability (MSI) and chromosomal instability (CSI). We investigated the influence of CSI and MSI on clinicopathological features and survival of 35 patients affected by mucinous colorectal cancers (MCRC). MSI status was determined by PCR amplification using 5 standard markers. Evidence for CSI was gathered by identifying loss of heterozygosity (LOH) of 4 loci (2p, 5q, 17p, 18q). We defined “MSI-MCRC” as those that showed MSI-H, and “CSI-MCRC” as those that showed LOH at 1 or more of these sites but did not show MSI-H. Among 35 cases, 18 cases (51.4%) were CSI-MCRC, whereas 11 cases (31.4%) were MSI-MCRC. Significant differences were found between CSI-MCRC and MSI-MCRC regarding the following clinicopathological features: tumor location (P=0.00026), lymph node metastasis (P=0.026), and TNM stage (P=0.026). Kaplan-Meier survival curves and log-rank analysis demonstrated that MSI-MCRC was associated with better prognosis than CSI-MCRC, although no significant difference was found (P=0.10). CSI-MCRC correlates more strongly with lymph node metastasis and advanced stage than MSI-MCRC. This indicates that CSI-MCRC is an aggressive subtype.

SMAD4 levels and allelic imbalance in 18q21 in colorectal cancer.

To the Editor: We read with interest Alhopuro et al.s article in a recent issue of Clinical Cancer Research ([1][1]). They showed the significance of SMAD4 levels as an important predictor of poor prognosis in colon cancer, whereas allelic imbalance in chromosome 18q21 was of no prognostic

Adjuvant Chemotherapy in Colorectal Cancer Patients with Microsatellite Instability

To the Editor: In a recent issue of Clinical Cancer Research ([1][1]), we read Benatti et al.s article with interest, therein they showed significantly better survival in microsatellite instability high (MSI-H) patients than microsatellite stable patients, and concluded that the type of genomic