Chikao Miki

Correlation of CD133, OCT4, and SOX2 in Rectal Cancer and Their Association with Distant Recurrence After Chemoradiotherapy

BackgroundCancer stem cells are associated with metastatic potential, treatment resistance, and poor patient prognosis. Distant recurrence remains the major cause of mortality in rectal cancer patients with preoperative chemoradiotherapy (CRT). We investigated the role of three stem cell markers (CD133, OCT4, and SOX2) in rectal cancer and evaluated the association between these gene levels and clinical outcome in rectal cancer patients with preoperative CRT.MethodsThirty-three patients with rectal cancer underwent preoperative CRT. Total RNAs of rectal cancer cells before and after CRT were isolated. Residual cancer cells after CRT were obtained from formalin-fixed paraffin-embedded (FFPE) specimens using microdissection. The expression levels of three stem cell genes were measured using real-time reverse-transcription polymerase chain reaction (RT-PCR). The association between these gene levels and radiation was evaluated using colon cancer cell lines. Immunohistochemical staining of these markers after CRT was also investigated.ResultsThere were significant positive correlations among the three genes after CRT. Patients who developed distant recurrence had higher levels of the three genes compared with those without recurrence in residual cancer after CRT. These elevated gene levels were significantly associated with poor disease-free survival. The radiation caused upregulation of these gene levels in LoVo and SW480 in vitro. Immunohistochemically, CD133 staining was observed in not only luminal surface but also cytoplasm.ConclusionsExpression of CD133, OCT4, and SOX2 may predict distant recurrence and poor prognosis of rectal cancer patients treated with preoperative CRT. Correlations among these genes may be associated with tumor regrowth and metastatic relapse after CRT.

Serum interleukin-6 level reflects the tumor proliferative activity in patients with colorectal carcinoma

Interleukin (IL)‐6 plays a central role as a differentiation and growth factor of tumor cells. It may be hypothesized that increased serum IL‐6 derives from the tumor tissue, and that serum IL‐6 levels consequently reflect the biologic characteristics of the tumor. The authors investigated the association between the serum levels of IL‐6 in colorectal carcinoma patients and the biologic characteristics of the tumor as well as clinicopathologic status of the patients.

Bioresorbable Hyaluronate-Carboxymethylcellulose Membrane (Seprafilm) in Surgery for Rectal Carcinoma: A Prospective Randomized Clinical Trial

PurposeTo evaluate the effectiveness of Seprafilm in preventing abdominal adhesions after radical resection of rectal carcinoma, and to observe whether Seprafilm had any adverse effects in patients treated with radiotherapy and chemotherapy.MethodsA total of 62 patients participated in this prospective randomized clinical study, which was conducted to compare the outcomes of patients operated on with Seprafilm (SEPRA+) with those operated on without Seprafilm (SEPRA−). All patients received preoperative radiotherapy, followed by a two-stage operation, and 5-fluorouracil (5-FU)-based systemic chemotherapy. The primary endpoint of severity and extent of adhesions were evaluated at the time of ileostomy closure. The secondary endpoint included the recurrence of tumors, late complications, and outcome.ResultsSeprafilm significantly reduced the adhesions in both the midline incision area and peristomal area. This in turn reduced the operation time, blood loss, and extent of the incision at ileostomy closure. Seprafilm was not associated with any postoperative complications or chemoradiation-related toxicity, nor did it affect recurrence or survival rates.ConclusionSeprafilm effectively reduced abdominal adhesions in chemoradiated patients, and had no adverse effects on the oncologic results of fully introduced adjuvant therapy. Thus, Seprafilm is a safe and effective tool for use in rectal carcinoma surgery.

C-Reactive Protein as a Prognostic Variable That Reflects Uncontrolled Up-Regulation of the IL-1-IL-6 Network System in Colorectal Carcinoma

Up-regulation of the IL-1-IL-6 network stimulates systemic expression of C-reactive protein (CRP).This cytokine network system plays a pivotal role in inducing angiogenic growth factors in in-testinalmucosa. Serum CRP level and tissue concentrations of cytokines in colorectal cancerpatients were determined and an in vitro model was employed to determine the time course ofinduction of IL-6 in Caco-2 cells. Increased serum CRP was associated with recurrent diseaseand shorter survival time. Intense surgical stress and the presence of an acute phase reactantwere independently associated with overexpression of IL-6 in the tumor. Enhanced IL-6 pro-teinexpression in Caco-2 cells induced by the initial treatment with IL-1β or lipopolysaccha-ridecould be abrogated by additional presupplementation of IL-1ra. The presence of an acutephase reactant reflects uncontrolled up-regulation of the local IL-1-IL-6 network system in thetumor, which may enhance the survival and proliferation of remnant cancer cells after tumorresection.

Preoperative C‐reactive protein as a prognostic and therapeutic marker for colorectal cancer

This study aimed to evaluate the significance of preoperative C‐reactive protein (CRP) as a prognostic marker for carcinoembryonic antigen (CEA)‐independent stage I or II colorectal cancer (CRC) patients.

Serum hepatocyte growth factor as an index of disease status of patients with colorectal carcinoma.

To evaluate the clinical significance of serum levels of hepatocyte growth factor (HGF) in colorectal cancer patients, we measured the venous and portal concentrations of HGF in 60 patients. The tissue concentrations in the tumour and adjacent normal mucosa were also determined. The serum HGF concentration for the peripheral venous blood of the patients was significantly higher than that in normal controls. The content of HGF in cancer tissue was also significantly higher than that in normal mucosa, and it was correlated with the serum HGF concentration for the peripheral venous blood. The serum concentration of HGF reflected pathological features, including tumour size and lymph node or liver metastasis, and it showed an association with various preoperative nutritional parameters and the preoperative haemoglobin level. The serum HGF concentration was also correlated with the serum concentrations of immunosuppressive acidic protein and interleukin-6, indices of the hosts immunological condition. Serum HGF seems to be a useful index of the disease status of patients with colorectal carcinoma.

Mitotic Checkpoint Protein hsMAD2 as a Marker Predicting Liver Metastasis of Human Gastric Cancers

hsMAD2, the human homologue of mitotic arrest deficient 2 (MAD2), is a key component of the mitotic checkpoint system. Recently, mutations and decreased expression of mitotic checkpoint genes including hsMAD2 have been reported in cancer cell lines with defective mitotic checkpoint. However, the genetic alterations in the genomic hsMAD2 gene have not been determined in gastric cancers. Moreover, the biological implications of the overexpressed hsMAD2 in primary cancers are unknown. In this study, we analyzed 32 primary gastric cancers with polymerase chain reaction (PCR) amplification of all exons, including flanking intronic sequences, of the genomic hsMAD2 gene followed by direct DNA sequencing. We also measured the hsMAD2 protein levels in cancer and normal tissues by semi‐quantitative immunoblotting. No mutations were found in the coding sequences, although three single nucleotide polymorphisms (SNPs) were identified in the noncoding sequences in 13 of 32 patients. These SNPs were not associated with either hsMAD2 expression or disease progression. The semi‐quantitative western blot analysis showed hsMAD2 was significantly overexpressed in gastric cancer tissues compared with corresponding normal tissues (P<0.001). The calculated ratio of the hsMAD2 protein in cancer tissue (C) to that in corresponding normal tissue (N) (C/N ratio) was significantly higher in patients with well differentiated adenocarcinoma (P=0.0274) or with synchronous liver metastasis (P=0.0025). A C/N ratio greater than 3 was observed more frequently in patients with synchronous liver metastasis. Therefore, C/N ratio >3 may be clinically important as a predictive indicator for metachronous liver metastasis of gastric cancers.

Radiation induces epithelial-mesenchymal transition in colorectal cancer cells

Radiotherapy remains a major approach to adjuvant therapy for patients with advanced rectal cancer. Nevertheless, the effects of radiation on malignant processes have yet to be clarified. The aim of this study was to assess the biological effects of radiation on colorectal cancer (CRC) cells with special reference to epithelial-mesenchymal transition (EMT), a key developmental program often activated during cancer invasion and metastasis. We investigated the effect of radiation on two colorectal cancer cell lines, CaR1 and DLD1, assessing cell morphology, motility, migration and invasive ability. Expression of molecules associated with EMT was determined using RT-PCR, Western blotting, and immunofluorescence staining in control and irradiated cells. We also used real-time RT-PCR to examine the expression of molecules associated with EMT before and after chemoradiotherapy. Thus, we studied 26 rectal cancer patients who received preoperative chemoradiotherapy followed by radical surgery. In addition, we examined the relationship between disease recurrence and the expression of a number of proteins. Irradiation caused CRC cells to undergo phenotypic changes characteristic of EMT: spindle-cell shape, loss of polarity, intercellular separation and pseudopodia formation. Irradiation enhanced cell migration and invasiveness. In irradiated CRC cells, molecular changes consistent with EMT were observed. In clinical samples, we observed molecular changes consistent with EMT, and those changes were significantly enhanced in patients with recurring disease. These results indicate that irradiation induces an alteration to a malignant phenotype consistent with EMT in colorectal cancer cells.

Serum hepatocyte growth factor as a prognostic marker for stage II or III colorectal cancer patients

We previously reported that a combination of serum hepatocyte growth factor (HGF) and carcinoembryonic antigen (CEA) was useful for selecting early‐stage colorectal cancer patients with aggressive disease. The aim of the present study was to determine whether serum HGF could provide CEA‐independent prognostic information on patients undergoing surgery with curative intent. Serum samples were collected from 184 patients with colorectal cancer and 30 controls. Reverse‐transcription polymerase chain reaction was used to detect HGF expression in colorectal cancer cell lines. Serum and tissue levels of HGF were measured by enzyme‐linked immunosorbent assay. The serum HGF levels in colorectal cancer patients were compared with those in healthy controls, and we retrospectively assessed the association between serum HGF levels and clinicopathological findings and survival. Expression of HGF was significantly higher in colorectal cancer tissues compared with non‐tumor tissues. The serum HGF levels were closely correlated with the HGF levels in cancer tissue. The mean serum HGF level in patients was significantly higher than that in controls, and significantly higher in patients with large tumor, lymph‐node involvement and distant metastasis. According to the receiver operating characteristic (ROC) analysis, elevated serum HGF levels can predict patients with larger tumor, lymph‐node and distant metastasis. Elevated serum HGF level demonstrated a significant association with poor survival, and was only an independent risk factor for poor survival in Stage II or/and III. Elevated serum HGF level is significantly associated with colorectal cancer development, lymph or distant invasive phenotypes and survival, especially in Stage II or III patients.

The expression patterns of Toll-like receptors in the ileal pouch mucosa of postoperative ulcerative colitis patients.

The aim of this study was to evaluate the expression pattern of Toll-like receptors (TLRs) in the pouch mucosa of ulcerative colitis patients in comparison with that in the ileum mucosa of noninflammatory bowel disease patients. Pouch mucosal biopsy specimens were collected from postoperative patients who had undergone surgery for ulcerative colitis. Normal ileum specimens were collected from colon cancer patients. The specimens were assessed by immunofluorescence histochemistry using TLR2, TLR3, TLR4, and TLR5 polyclonal antibodies. The normal ileal mucosa constitutively expressed TLR3 and TLR5, whereas TLR2 and TLR4 were barely detectable. In the mucosa of active pouchitis, TLR2 and TLR4 was strongly upregulated, and TLR4 was upregulated even in a noninflamed pouch. No TLR3 or TLR5 expression was detectable. These data suggest that pouchitis may be associated with distinctive changes in selective TLR expression in the pouch mucosa, and that TLR4 alterations in the innate response system may contribute to the pathogenesis of these disorders in particular.