Daisuke Shimizu

Vascular Endothelial Growth Factor Messenger RNA Expression Level Is Preserved in Liver Metastases Compared with Corresponding Primary Colorectal Cancer

Purpose: Increased vascular endothelial growth factor (VEGF) expression is associated with colorectal cancer liver metastases. It is reasonable to expect that measurement of VEGF in liver metastases would provide the best prediction of therapy benefit for VEGF-targeted drugs, such as bevacizumab (Avastin). In this study, we evaluated how VEGF mRNA level in primary colorectal cancer was related to that in corresponding liver metastases. Thirty-one pairs of primary colorectal cancer and corresponding liver metastases were analyzed. Experimental Design: Formalin-fixed, paraffin-embedded tumor specimens were dissected by using laser-captured microdissection. RNA was extracted and cDNA was prepared by reverse transcription. Quantitation of VEGF and internal reference gene (β-actin) was done using real-time PCR (Taqman PCR). Results: There was no difference between median VEGF mRNA levels of primary colorectal cancer and liver metastases (median value 3.79 versus 3.97: P = 0.989). On an individual basis, there was a significant correlation in VEGF mRNA expression between primary colorectal cancer and corresponding liver metastases (rs = 0.6627, P < 0.0001). In addition, the VEGF mRNA levels of the patients who had two or more liver metastatic tumors were significantly higher than those of the patient who had solitary liver metastatic tumor in both primary cancer (5.02 versus 3.34: P = 0.0483) and liver metastases (4.38 versus 3.25: P = 0.0358). Conclusion: Good prediction of VEGF mRNA levels in liver metastases can be obtained by measuring those of primary colorectal cancer. The risk of multiple liver metastatic tumors might be predictable by measuring VEGF mRNA expression in primary colorectal cancer. Further study is required to confirm these preliminary results.

Predicting 5-fluorouracil chemosensitivity of liver metastases from colorectal cancer using primary tumor specimens: Three-gene expression model predicts clinical response

We identified genes related to 5‐fluorouracil (5‐FU) sensitivity in colorectal cancer and utilized these genes for predicting the 5‐FU sensitivity of liver metastases. Eighty‐one candidate genes involved in 5‐FU resistance in gastric and colon cancer cell lines were previously identified using a cDNA microarray. In this study, the mRNA expression levels of these 81 selected genes and the genes of 5‐FU‐related enzymes, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT), were measured using real‐time quantitative RT‐PCR assays of surgically resected materials from primary colorectal tumors in 22 patients. Clinical responses were estimated by evaluating the effects of 5‐FU‐based hepatic artery injection (HAI) chemotherapy for synchronous liver metastases. Four genes (TNFRSF1B, SLC35F5, NAG‐1 and OPRT) had significantly different expression profiles in 5‐FU‐nonresponding and responding tumors (p < 0.05). A “Response Index” system using three genes (TNFRSF1B, SLC35F5 and OPRT) was then developed using a discriminate analysis; the results were well correlated with the individual chemosensitivities. Among the 11 cases with positive scores in our response index, 9 achieved a reduction in their liver metastases after 5‐FU‐based chemotherapy, whereas only 1 of the 11 cases with negative scores responded well to chemotherapy. Our “Response Index” system, consisting of TNFRSF1B, SLC35F5 and OPRT, has great potential for predicting the efficacy of 5‐FU‐based chemotherapy against liver metastases from colorectal cancer.

Quantitative, tissue-specific analysis of cyclooxygenase gene expression in the pathogenesis of Barrett's adenocarcinoma.

Cyclooxygenase (Cox-2) is implicated in the pathogenesis of many cancers including esophageal adenocarcinoma (EAC), whereas the role of the isoform Cox-1 in carcinogenesis is not well understood. To further elucidate the role of these factors in the development of EAC, we measured the gene expressions (mRNA levels) of Cox-2 and Cox-1 by real-time quantitative polymerase chain reaction (QRTPCR) in tissues from normal esophagus with and without erosive gastroesophageal reflux disease (GERD), Barrett’s esophagus (BE), dysplasia, adenocarcinoma, and in healthy gastric antrum. All tissues were purified by laser capture microdissection from endoscopic or surgical resection specimens. Median Cox-2 gene expression did not differ significantly among the esophageal control groups but was elevated 5-fold in BE, 8-fold in dysplasia and 16-fold in EAC compared to normal esophageal controls with no erosive GERD. Erosive GERD tissue had slightly higher median Cox-2 expression but Cox-2 expression in normal antrum was much higher than that in a normal esophagus, close to that of dysplasia. In contrast to that of Cox-2, Cox-1 expression was significantly decreased in all neoplastic tissues compared to normal controls. Cox-1 and Cox-2 expression varied over a wide range in the neoplastic tissues but over a relatively narrow range in the esophageal normal tissues. The occurrence of substantial alterations in Cox-1 and Cox-2 expression at the BE stage indicates that these are early events in the development of EAC. These results confirm the important role of Cox-2 amplification in the pathogenesis of esophageal adenocarcinoma, but the unexpected down-regulation of Cox-1 raises questions about its role in carcinogenesis.

5-Fluorouracil-related gene expression levels in primary colorectal cancer and corresponding liver metastasis

Gene expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) have been shown to be associated with response to 5‐fluorouracil‐based therapies. Analyzing these gene expression levels in liver metastases is important to obtain the best prediction of therapy. Our aim was to determine how TS, DPD, TP and OPRT gene expression levels in primary colorectal cancer (CRC) were related to those in liver metastases. Formalin‐fixed, paraffin‐embedded tumor specimens from 31 pairs of primary CRC and corresponding liver metastases were dissected by using laser‐captured microdissection. RNA was extracted and cDNA was prepared by reverse‐transcription. Quantitation of target gene and internal reference gene was performed using real‐time PCR. No significant difference was seen between median mRNA expression levels of TS, DPD, TP and OPRT in primary cancer and those in corresponding liver metastases (median value: TS 1.48 vs. 1.43; p = 0.92, DPD 0.19 vs.0.12; p = 0.10, TP 1.20 vs. 0.98; p = 0.39, OPRT 1.17 vs. 0.95; p = 0.10). When matched tissue sets were compared on an individual basis, there was a significant correlation for TS mRNA expression between primary cancer and corresponding liver metastases (rs = 0.52, p = 0.0026). However, no correlation was seen between matched sets for DPD, TP or OPRT. Significant correlation was seen between DPD and TP expression levels in both primary CRC (rs = 0.38, p = 0.03) and liver metastases (rs = 0.72, p < 0.0001). A good prediction of TS mRNA levels in liver metastases can be obtained by measuring those of primary CRC, although no correlation was seen for DPD, TP and OPRT.

Antireflux Surgery Normalizes Cyclooxygenase-2 Expression in Squamous Epithelium of the Distal Esophagus

BACKGROUND:In some patients GERD presents with heartburn and regurgitation symptoms but a relative paucity of endoscopic and clinical findings, while in others symptoms may be minor or absent yet there is significant mucosal damage on endoscopy including the presence of Barretts esophagus. The initial injury of gastroesophageal reflux is to the squamous esophageal mucosa, but while substantial research has been devoted to determining which genes are involved in the progression of Barretts to dysplasia and cancer, little is known about the gene expression alterations in the squamous mucosa of patients with reflux. We hypothesized that the expression of cyclooxygenase-2 (Cox-2) might be increased in the squamous esophageal mucosal of patients with reflux, and might be a molecular indicator of reflux injury. Further, we hypothesized that Cox-2 expression in the squamous mucosa would be reduced following the elimination of reflux with an antireflux operation.METHODS:Biopsies of the distal esophageal squamous mucosa were taken 3 cm above the squamocolumnar junction (SCJ) in 28 GERD patients before and after Nissen fundoplication. Following microdissection and RNA isolation, quantitative real-time PCR was used to measure Cox-2 gene expression in paraffin-embedded (N = 16) and fresh frozen (N = 12) tissue. Biopsies from patients (paraffin N = 15, frozen N = 14) with normal acid exposure and no evidence of mucosal injury were analyzed as controls.RESULTS:Median Cox-2 expression in the squamous epithelium from paraffin embedded biopsies in patients with reflux disease was significantly increased compared to controls (p = 0.04). The presence of esophagitis or Barretts esophagus did not significantly alter the expression of Cox-2 compared to patients with nonerosive reflux disease (NERD). After antireflux surgery median Cox-2 expression values were significantly reduced (p = 0.0003) and were normalized to levels similar to controls without reflux (p = 0.74). Similar results were observed in the prospectively obtained fresh frozen tissue.CONCLUSIONS:Cox-2 gene expression is increased in the distal esophageal squamous mucosa of most patients with GERD, and the elevation was similar whether there was mucosal injury in the form of esophagitis or Barretts or no visible mucosal injury. This suggests that increased Cox-2 expression may serve as a molecular marker of reflux disease. The increased Cox-2 expression in patients with reflux was usually normalized following antireflux surgery. These findings demonstrate for the first time that gene expression can be altered by surgical correction of reflux. Thus, in addition to symptom control and improvement in the quality of life, perhaps future studies assessing the efficacy of antireflux therapy should also focus on the impact of the therapy on gene expression in the esophageal squamous mucosa.

Unusual carbon-sulfur bond cleavage in the reaction of a new type of bulky hexathioether with a zerovalent palladium complex

The reaction of a bulky hexathioether, TbtS(o-Phen)S(o-Phen)SS(o-Phen)S(o-Phen)STbt (o-Phen = o-phenylene, Tbt = 2,4,6-tris[bis(trimethylsilyl)methyl]phenyl) (1), with 3 molar amounts of Pd(PPh3)4 afforded trinuclear palladium complex bridged by two benzenedithiolato ligands via a three-step palladium insertion reaction into one sulfur-sulfur and two carbon-sulfur bonds of 1.

Preliminary analysis of Nacholapithecus scapula and clavicle from Nachola, Kenya

The Miocene ape Nacholapithecus is known from rather complete skeletons; some of them preserve the shoulder joint, identified by three scapulae and one clavicle. Comparisons made with other Miocene and living apes (Proconsul, Equatorius, Ugandapithecus) suggest that the mobility of the scapulohumeral joint was important, and scapular features such as the morphology and position of the spine and the morphology of the acromion and axillary border resemble those of climbing arboreal primates except for chimpanzees, gorillas, or orang-utans. From the size of the scapula (male Nasalis size), it is clear that the animal is smaller than an adult chimpanzee, but the clavicle is almost as relatively long as those of chimpanzees. Some features closer to colobine morphology reinforce the hypothesis that Nacholapithecus was probably a good climber and was definitely adapted for an arboreal life.

Increasing cyclooxygenase-2 (cox-2) gene expression in the progression of Barrett's esophagus to adenocarcinoma correlates with that of Bcl-2.

Previous studies from our laboratory and others have suggested that increased expression of cox‐2 is important in the genesis of esophageal adenocarcinoma. In vitro studies suggest that cox‐2 regulates expression of the anti‐apoptotic protein bcl‐2, thus possibly accounting for reduced apoptosis in carcinogenesis. The aim of this study was to investigate the relationship of these 2 genes in the development of Barretts‐associated adenocarcinoma. Histologic sections from endoscopic biopsies or esophagectomy specimens were classified as non‐dysplastic Barretts (n = 30), intraepithelial neoplasia (n = 12) and adenocarcinoma (n = 48). The desired tissue was isolated by laser capture microdissection and expression levels of cox‐2 and bcl‐2 were measured by quantitative real‐time PCR (Taqman®). Gene expression levels were compared to samples of the distal esophageal squamous epithelium (n = 55) and reflux‐esophagitis (n = 25), without Barretts or cancer. Expression of both bcl‐2 and cox‐2 were increased in non‐dysplastic Barretts (p = 0.0077, p = 0.0037), intraepithelial neoplasia (p = 0.0053, p = 0.0220) and adenocarcinoma (p < 0.0001, p < 0.0001) compared to squamous epithelium or reflux‐esophagitis. Furthermore, there is a significant correlation between these two genes, especially in carcinoma (p < 0.0001).

Current progress in the prediction of chemosensitivity for breast cancer.

Advances in chemotherapy have improved the prognosis of patients with breast cancer significantly. Individualization is important for optimization of chemotherapy. The prediction of tumor sensitivity to anticancer agents has been intensively investigated for that purpose. There have been 2 approaches to predict the efficacy of drugs against individual tumors, drug-sensitivity tests and molecular marker genes. Although some of these tests are already available clinically, the prediction of chemosensitivity remains a goal to be achieved.Several studies with microarrays revealed that comprehensive analyses of genes may provide useful information for determining the chemosensitivity of cancer. We have started to use a cDNA microarray to study the chemosensitivity of breast cancer. Taken together with recent data, studies for drug sensitivity should provide insights into the mechanisms of drug sensitivity and the optimal design of more effective treatment strategies in breast cancer.

Breast cancer manifested by hematologic disorders

Breast cancer is the most common type of cancer in women. However, it is very rarely manifested as hematologic disorders. A 35-year-old woman was admitted because of disseminated intravascular coagulation. Examinations revealed the presence of breast cancer in her left breast; therefore, paclitaxel was administered weekly. Although disseminated intravascular coagulation was controlled, pulmonary dysfunction due to lymphangitis carcinomatosa suddenly occurred 10 weeks after treatment. Pulmonary dysfunction was effectively treated with epirubicin and cyclophosphamide. Twenty-three weeks after treatment, the patient developed liver dysfunction accompanied with jaundice due to progressive metastatic lesions in the liver; liver dysfunction improved after the administration of vinorelbine. Subsequently, because of the recurrence of pulmonary dysfunction, rechallenge with epirubicin and cyclophosphamide was performed and was effective; however, this therapy was discontinued because of its adverse effects. She expired of liver failure 33 weeks after the occurrence of disseminated intravascular coagulation. Metastatic tumors in the bone marrow, lung, and liver showed different sensitivities to different anti-cancer agents. We report a case of breast cancer manifested by hematologic disorders which was treated by a sequential chemotherapy.