Masazumi Okajima

Frequent alterations in the Wnt signaling pathway in colorectal cancer with microsatellite instability

It is generally accepted that both dysfunction of the Wnt signaling pathway, including mutations in the adenomatous polyposis coli (APC) and β‐catenin genes, and genetic instability play important roles in colorectal carcinogenesis. However, alteration of the components in the Wnt signaling pathway in colorectal cancer (CRC) with microsatellite instability (MSI) has not been elucidated. In order to assess the status of the Wnt signaling components in CRC with MSI, mutational analyses of the β‐catenin, APC, Axin 1, and T cell factor 4 (TCF4) genes were performed. Three of 33 samples had mutations in exon 3 of the β‐catenin gene and two in the APC gene. Eight mutations in seven samples were detected by single‐strand conformation polymorphism and subsequent direct sequence analysis of the entire coding region of the Axin 1 gene. Furthermore, TCF4, which is one of the transcriptional factors in the Wnt signaling pathway and has a mononucleotide repeat sequence (a nine‐ adenine repeat, (A)9) in its C‐terminal region, was mutated in 13 of the 33 samples. Thus, alteration in the Wnt signaling pathway is frequently observed in CRC with MSI, including hereditary nonpolyposis colorectal cancer, as well as in familial adenomatous polyposis and sporadic CRC without MSI.

High miR-21 expression from FFPE tissues is associated with poor survival and response to adjuvant chemotherapy in colon cancer.

Colon cancer (CC) is a leading cause of cancer mortality. Novel biomarkers are needed to identify CC patients at high risk of recurrence and those who may benefit from therapeutic intervention. The aim of this study is to investigate if miR‐21 expression from RNA isolated from formalin‐fixed paraffin‐embedded (FFPE) tissue sections is associated with prognosis and therapeutic outcome for patients with CC. The expression of miR‐21 was measured by quantitative reverse transcriptase‐polymerase chain reaction in a Japanese cohort (stage I‐IV, n = 156) and a German cohort (stage II, n = 145). High miR‐21 expression in tumors was associated with poor survival in both the stage II/III Japanese (p = 0.0008) and stage II German (p = 0.047) cohorts. These associations were independent of other clinical covariates in multivariable models. Receipt of adjuvant chemotherapy was not beneficial in patients with high miR‐21 in either cohort. In the Japanese cohort, high miR‐21 expression was significantly associated with poor therapeutic outcome (p = 0.0001) and adjuvant therapy was associated with improved survival in patients with low miR‐21 (p = 0.001). These results suggest that miR‐21 is a promising biomarker to identify patients with poor prognosis and can be accurately measured in FFPE tissues. The expression of miR‐21 may also identify patients who will benefit from adjuvant chemotherapy.

Combined immunohistochemistry of β-catenin, cytokeratin 7, and cytokeratin 20 is useful in discriminating primary lung adenocarcinomas from metastatic colorectal cancer

BackgroundIt is important to discriminate between primary and secondary lung cancer. However, often, the discriminating diagnosis of primary lung acinar adenocarcinoma and lung metastasis of colorectal cancer based on morphological and pathological findings is difficult. The purpose of this study was to evaluate the clinical usefulness of immunohistochemistry of β-catenin, cytokeratin (CK) 7, and CK20 for the discriminating diagnosis of lung cancer.MethodsWe performed immunohistochemistry of β-catenin, CK7, and CK20 in 19 lung metastasis of colorectal cancer samples, 10 corresponding primary colorectal cancer samples and 11 primary lung acinar adenocarcinoma samples and compared the levels of accuracy of the discriminating diagnosis by using antibodies against these antigens.ResultsPositive staining of β-catenin was observed in all the lung metastasis of colorectal cancer samples as well as in the primary colorectal cancer samples but in none of the primary lung acinar adenocarcinoma samples. Positive staining of CK7 was observed in 90.9% of the primary lung acinar adenocarcinoma samples and in 5.3% of the lung metastasis of colorectal cancer samples, but in none of the primary colorectal cancer samples. Positive staining of CK20 was observed in all the primary colorectal cancer samples and in 84.2% of the lung metastasis of colorectal cancer samples, but in none of the primary lung acinar adenocarcinoma samples.ConclusionCombined immunohistochemistry of β-catenin, CK7, and CK20 is useful for making a discriminating diagnosis between lung metastasis of colorectal cancer and primary lung acinar adenocarcinoma. This method will enable accurate diagnosis of a lung tumor and will be useful for selecting appropriate therapeutic strategies, including chemotherapeutic agents and operation methods.

Objective assessment of endoscopic surgical skills by analyzing direction-dependent dexterity using the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD)

PurposeWe evaluated our system of objectively assessing endoscopic surgical skills.MethodsWe developed the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD), which records the movement of the tip of an endoscopic instrument precisely. The orbits of experienced surgeons (expert group) and those of medical students (novice group) were evaluated by measuring the deviation from the ideal course on horizontal and vertical planes. These data were integrated with the time taken to move the tip of an endoscopic instrument between a distal side pole (A) and a proximal side pole (C) (Task 1), and between a left side pole (D) and a right side pole (B) (Task 2).ResultsThe integrated deviation of the expert group was significantly lower than that of the novice group on both the horizontal and vertical planes in Task 1 (P = 0.0004, P = 0.009) and Task 2 (P < 0.0001, P = 0.0002). Thus, the spatial perception of experts was significantly better than that of novices. We also found that the direction of the scope and the movement of the endoscopic instrument were related to difficulties in spatial perception for both experts and novices. HUESAD detected and resolved these differences based on the directions of the scope and movement of the endoscopic instruments.ConclusionsThe HUESAD is a reliable system for assessing a surgeon’s dexterity, based on direction and movement. It helps us to attain a higher degree of accuracy and to create an ideal setting for optimal endoscopic surgery.

Rho-associated kinase inhibitor reduces tumor recurrence after liver transplantation in a rat hepatoma model.

Tumor recurrence after liver transplantation still remains a significant problem in patients with hepatocellular carcinoma. The small GTPase Rho/Rho‐associated kinase (ROCK) pathway is involved in the motility and invasiveness of cancer cells. We investigated whether tacrolimus activated the Rho/ROCK signal pathway to promote the invasiveness of rat hepatocellular carcinoma cells. We also investigated whether the ROCK inhibitor Y‐27632 suppressed tumor recurrence after experimental liver transplantation in a rat hepatocellular carcinoma model. Orthotopic liver transplantation was performed in hepatocellular carcinoma cell line McA‐RH7777‐bearing rats. Tacrolimus was administered to liver transplant rats and these rats were divided into two groups: the Y‐27632‐treated (10 mg/kg, for 28 days) group and the Y‐27632‐untreated group. Tacrolimus enhanced the cancer cell migration and stimulated phosphorylation of the myosin light chain (MLC), a downstream effector of Rho/ROCK signaling. Y‐27632 suppressed the cancer cell migration and tacrolimus‐induced MLC phosphorylation. Suppression of tumor recurrence after liver transplantation and significant prolongation of survival were observed in the Y‐27632‐treated rats in comparison with theY‐27632‐untreated rats. Tacrolimus stimulates the Rho/ROCK signal pathway to enhance the invasiveness of hepatocellular carcinoma, and the ROCK inhibitor Y‐27632 can be used as a new antimetastatic agent for the prevention of tumor recurrence after liver transplantation.

Search for transmembrane protein in gastric cancer by the Escherichia coli ampicillin secretion trap: expression of DSC2 in gastric cancer with intestinal phenotype†

Gastric cancer (GC) is one of the most common malignancies worldwide. Genes expressed only in cancer tissue, and especially on the cell membrane, will be useful molecular markers for diagnosis and may also be good therapeutic targets. To identify genes that encode transmembrane proteins present in GC, we generated Escherichia coli ampicillin secretion trap (CAST) libraries from two GC cell lines and normal stomach. By sequencing 4320 colonies from CAST libraries, we identified 30 candidate genes that encode transmembrane proteins present in GC. Quantitative reverse transcription‐polymerase chain reaction analysis of these candidates revealed that ZDHHC14, BST2, DRAM2, and DSC2 were expressed much more highly in GC than in 14 kinds of normal tissues. Among these, DSC2 encodes desmocollin 2, which is one of three known desmocollins. Immunohistochemical analysis demonstrated that 22 (28%) of 80 GC cases were positive for desmocollin 2, and desmocollin 2 expression was observed frequently in GC with the intestinal mucin phenotype. Furthermore, desmocollin 2 expression was correlated with CDX2 expression. These results suggest that expression of desmocollin 2, induced by CDX2, may be a key regulator for GC with the intestinal mucin phenotype. Our results provide a list of genes that have high potential as a diagnostic and therapeutic target for GC. Copyright

Regulation of T helper type-1 immunity in hapten-induced colitis by host pretreatment with granulocyte colony-stimulating factor☆ ☆

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is an immunoregulatory drug whose effects include modulation of antigen-presentation. We investigated the potential ameliorative effect of pretreatment with rhG-CSF in a hapten-induced colitis animal model. Sprague-Dawley rats were given rhG-CSF (125 microg/kg subcutaneously twice a day for 5 days) before a colonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Consequent colonic damage was evaluated pathologically, and cytokine mRNA expression levels in macroscopically inflamed sites were measured by real-time quantitative reverse transcription-polymerase chain reaction. Pretreatment with rhG-CSF remarkably attenuated both the loss of body weight and colonic wall thickening due to progressive transmural inflammation. In the control, treatment with TNBS led to a statistically significant (p < 0.05) upregulation of IFN-gamma mRNA expression in the inflammatory sites measured at post-treatment day 7. In the experimental group, pretreatment with rhG-CSF abrogated transcription of IFN-gamma (p < 0.05), but was not, however, associated with an upregulation of IL-4 or the regulatory cytokines TGF-beta and IL-10. Furthermore, transcription of IL-12p35 (a rate-limiting factor for the production of IL-12) was significantly (p < 0.05) downregulated by rhG-CSF at 24h post-TNBS instillation, whereas IL-12p40 was not affected. Pretreatment with rhG-CSF drastically attenuated the degree of TNBS-induced colitis through selective downregulation of Th1-associated cytokines.

CDX2 Regulates Multidrug Resistance 1 Gene Expression in Malignant Intestinal Epithelium

The caudal-related homeobox transcription factor CDX2 has a key role in intestinal development and differentiation. CDX2 heterozygous mutant mice develop colonic polyps, and loss of CDX2 expression is seen in a subset of colon carcinomas in humans. Ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression is frequently detected in intestinal metaplasia in the stomach and esophagus. We sought to define CDX2-regulated genes to enhance knowledge of CDX2 function. HT-29 colorectal cancer cells have minimal endogenous CDX2 expression, and HT-29 cells with ectopic CDX2 expression were generated. Microarray-based gene expression studies revealed that the Multidrug Resistance 1 (MDR1/P-glycoprotein/ABCB1) gene was activated by CDX2. Evidence that the MDR1 gene was a direct transcriptional target of CDX2 was obtained, including analyses with MDR1 reporter gene constructs and chromatin immunoprecipitation assays. RNA interference-mediated inhibition of CDX2 decreased endogenous MDR1 expression. In various colorectal cancer cell lines and human tissues, endogenous MDR1 expression was well correlated to CDX2 expression. Overexpression of CDX2 in HT-29 cells revealed increased resistance to the known substrate of MDR1, vincristine and paclitaxel, which was reversed by an MDR1 inhibitor, verapamil. These data indicate that CDX2 directly regulates MDR1 gene expression through binding to elements in the promoter region. Thus, CDX2 is probably important for basal expression of MDR1, regulating drug excretion and absorption in the lower gastrointestinal tract, as well as for multidrug resistance to chemotherapy reagent in CDX2-positive gastrointestinal cancers.

Active Strobe Imager for Visualizing Dynamic Behavior of Tumors

This paper newly proposes the active strobe imager (ASI) that can visualize the dynamic behaviors of internal organ to medical doctors within Human dynamic eye sight, so that they can get a hint for detecting the location of tumors in real time during operation. The ASI is composed of a nozzle for supplying a pulsated air jet to an internal organ, a strobe system for visualizing the dynamic behaviors, a camera for capturing the image at the moment of strobe flashing, and a monitor for displaying the dynamic motion of internal organ, respectively. Without the assistance of strobe, medical doctors can not see what is really happening in real time, since the frequency of air jet is even more than the range of human dynamic eye sight. After explaining the basic principle, we show a couple of experiments for both artificial and real human lungs. Through these experiments, we newly found that the ASI is really helpful for discovering tumors or other diseases hidden in internal organs. Movies are available in Website.

Scientific assessment of endoscopic surgical skills.

Abstract Recently, significant attention has been focused on training and education for safe endoscopic surgery. A new assessment method, the Hiroshima University Endoscopic Surgical Assessment Device (HUESAD), has been designed at Hiroshima University to evaluate the smoothness of the movement of endoscopic instruments from velocity. Experts (with experience in performing more than 100 laparoscopic surgeries) and novices (with no experience in performing laparoscopic surgery) were recruited for this study. The aim of task 1 was to move the tip of the endoscopic instrument on the tops of poles from A to C, and task 2 was to move it from the right pole B to the left pole D. The peak velocity (Vp) and the time when peak velocity appears (Tp) were analyzed. Both the peak velocity (Vp) and the time when peak velocity appears (Tp) to perform task 1 and task 2 were significantly faster in the expert group than in the novice group. The peak velocity (Vp) and the time when peak velocity appears (Tp) in HUESAD, which indicate the smoothnes of the endoscopic procedure, are among the most important factors for assessing endoscopic surgical skills.