Detection of Circulating Gastrointestinal Cancer Cells in Conditionally Reprogrammed Cell Culture.

BACKGROUND/AIM: The aim of this study was to detect circulating tumor cells (CTC) in the peripheral blood of gastrointestinal cancer patients using conditionally reprogrammed cell (CRC) culture. MATERIALS AND METHODS: We confirmed the sensitivity of the CRC culture method. Five ml of blood were obtained from 81 cancer patients (56 colorectal and 25 gastric). The collected mononuclear cells were cultured for 4 weeks in the CRC condition. Finally, cultured cells were characterized by RT-PCR for the expression of hTERT and MAGE A1-6 mRNA. RESULTS: The CRC method had a CTC detection limit of 6 cells for gastric cancer cells. After culture of 81 blood specimens, 38 formed visible cells, including 5 colonies. Among the 38 cells, 13 were hTERT positive and 4 were MAGE A1-6 positive. The final CTC detection rate was 16.0%. CONCLUSION: The CRC culture may potentially be used to evaluate the metastatic cancer cells in the circulation.

Suppression of c-Myc enhances p21WAF1/CIP1 -mediated G1 cell cycle arrest through the modulation of ERK phosphorylation by ascochlorin.

Numerous anti-cancer agents inhibit cell cycle progression via a p53-dependent mechanism; however, other genes such as the proto-oncogene c-Myc are promising targets for anticancer therapy. In the present study, we provide evidence that ascochlorin, an isoprenoid antibiotic, is a non-toxic anti-cancer agent that induces G1 cell cycle arrest and p21WAF1/CIP1 expression by downregulating of c-Myc protein expression. Ascochlorin promoted the G1 arrest, upregulated p53 and p21WAF1/CIP1 , and downregulated c-Myc in HCT116 cells. In p53-deficient cells, ascochlorin enhanced the expression of G1 arrest-related genes except p53. Small interfering RNA (siRNA) mediated c-Myc silencing indicated that the transcriptional repression of c-Myc was related to ascochlorin-mediated modulation of p21WAF1/CIP1 expression. Ascochlorin suppressed the stabilization of the c-Myc protein by inhibiting ERK and P70S6K/4EBP1 phosphorylation, whereas it had no effect on c-Myc degradation mediated by PI3K/Akt/GSK3ß. The ERK inhibitor PD98059 and siRNA-mediated ERK silencing induced G1 arrest and p21WAF1/CIP1 expression by downregulating c-Myc in p53-deficient cells. These results indicated that ascochlorin-induced G1 arrest is associated with the repression of ERK phosphorylation and c-Myc expression. Thus, we reveal a role for ascochlorin in inhibiting tumor growth via G1 arrest, and identify a novel regulatory mechanism for ERK/c-Myc.

Melanoma antigen-encoding gene family member A1-6 and hTERT in the detection of circulating tumor cells following CD45- depletion and RNA extraction.

A total of 76 blood samples from patients without malignant disease and 107 blood samples from patients with malignant disease were investigated for the presence of circulating tumor cells (CTCs). To detect CTCs, hematopoietic cells were removed from the blood samples and different RNA extraction methods were used to amplify the melanoma antigen-encoding gene family member A1-family member A6 (MAGE A1-6) and the human telomerase reverse transcriptase (hTERT) gene as potential CTC markers. Comparison between four methods for extracting RNA from the blood was performed. The samples were enriched by cluster of differentiation 45 (CD45) antibody capturing, and the reverse transcription-quantitative polymerase chain reaction was used to amplify the MAGE A1-6 and hTERT genes. MAGE A1-6 and hTERT gene expression levels were also evaluated in 14 cancer cell lines, and the MAGE A1-6 and hTERT expression levels were 85.7 and 100%, respectively. The RNeasy method demonstrated the most sensitivity in the SNU1 cells mixed with blood, although the differences between methods was non-significant. The positive expression levels of MAGE A1-6 and hTERT was 11.8% in the control group and 58.9% in those with malignant disease. In the 70 patients with colorectal cancer, positive expression levels of MAGE A1-6 or hTERT were significantly higher in stages T3 and T4 compared with in stages T1 and T2. The CTC detection method involving CD45 antibody capture, RNA extraction and MAGE A1-6 and hTERT reverse transcription resulted in good rates of sensitivity and specificity. Thus, the present study concluded that MAGE A1-6 and hTERT genes may be potential and practical markers for CTCs in a clinical setting.

Clinical Practice in the Use of Adjuvant Chemotherapy for Patients with Colon Cancer in South Korea: a Multi-Center, Prospective, Observational Study.

BACKGROUND: Adjuvant chemotherapy is a crucial part of treatment for patients with locally advanced colon cancer. This study was conducted to investigate the actual practice in the use of adjuvant chemotherapy for patients with high-risk stage II or stage III colon cancer in South Korea. METHODS: This was a 24-month open-label, prospective, observational study conducted at 12 centers across South Korea. Patients with high-risk stage II and stage III colon cancer receiving adjuvant chemotherapy after curative surgery were included, and data were collected at baseline, third, and sixth month. RESULTS: A total of 246 patients were included in the analyses. Of five available regimens (FOLFOX, CAPOX, 5-FU/LV, capecitabine, and UFT/LV), FOLFOX was most commonly used (82.5%). Investigators indicated the "efficacy" as the major cause for selecting FOLFOX or CAPOX. For 5-FU/LV, capecitabine, or UFT/LV, the "safety" or "patient's characteristics (age, comorbidity, and stage)" was one of the most important selecting factors. Patients receiving 5-FU/LV, capecitabine, or UFT/LV had older age, worse PS and lower disease stage (stage II) than patients receiving FOLFOX or CAPOX. Hematologic toxicities were the most common cause of dose adjustment and treatment delay. CONCLUSIONS: In South Korea, FOLFOX was the most commonly used regimen for adjuvant chemotherapy and its efficacy was the main cause for selecting this regimen. Patients receiving 5-FU/LV, capecitabine, or UFT/LV had older age, worse PS and lower disease stage (stage II) than patients receiving FOLFOX or CAPOX.

The Clinical Features and Predictive Risk Factors for Reoperation in Patients With Perianal Crohn Diseases; A Multi-Center Study of a Korean Inflammatory Bowel Disease Study Group.

PURPOSE: Perianal lesions are common in Crohn disease, but their clinical course is unpredictable. Nevertheless, predicting the clinical course after surgery for perianal Crohn disease (PCD) is important because repeated operations may decrease patient's quality of life. The aim of this study was to predict the risk of reoperation in patients with PCD. METHODS: From September 1994 to February 2010, 377 patients with PCD were recruited in twelve major tertiary university-affiliated hospitals and two specialized colorectal hospitals in Korea. Data on the patient's demographics, clinical features, and surgical outcomes were analyzed. RESULTS: Among 377 patients, 227 patients were ultimately included in the study. Among the 227 patients, 64 patients underwent at least one reoperation. The median period of reoperation following the first perianal surgery was 94 months. Overall 3-year, 5-year, and 10-year cumulative rates of reoperation-free individuals were 68.8%, 61.2%, and 50.5%, respectively. In multivariate analysis (Cox-regression hazard model), reoperation was significantly correlated with an age of onset less than 20 years (hazard ratio [HR], 1.93; 95% confidence interval [CI], 1.07-3.48; P = 0.03), history of abdominal surgery (HR, 1.99; 95% CI, 1.08-3.64; P = 0.03), and the type of surgery. Among types of surgery, fistulotomy or fistulectomy was associated with a decreased incidence of reoperation in comparison with incision and drainage (HR, 0.19; 95% CI, 0.09-0.42; P < 0.001). CONCLUSION: Young age of onset and a history of abdominal surgery were associated with a high risk of reoperation for PCD, and the risk of reoperation were relatively low in fistulotomy or fistulectomy procedures.

Microenvironmental interactions and expression of molecular markers associated with epithelial-to-mesenchymal transition in colorectal carcinoma.

The tumor microenvironment is known to play a critical role in tumor progression, invasion and metastasis. The epithelial-to-mesenchymal transition (EMT) is understood as a process of tumor invasion and metastasis. Therefore, we investigated the relation between the EMT and the microenvironment of colorectal carcinoma (CRC). The histological features and expression of EMT markers in tumor cells and surrounded stromal cells were obtained from the surgically resected tissues of 39 patients using microscopic review and immunohistochemistry. The loss of expression of E-cadherin was more prominent in the invasive front of tumor than the surface, where α-smooth muscle actin-positive carcinoma-associated fibroblasts (CAFs) are accumulated. The signaling molecules of the Wnt and TGF-ß1-Smad pathway were expressed more frequently in the tumor cells and/or CAFs of the invasive margin than those of the tumor surface. The expressions of related transcription factors, such as SNAIL and ZEB1, were increased in the tumor cells and CAFs. The process of EMT may be activated in the tumor margin of CRC under the control of CAFs. Related signaling molecules and transcription factors might be induced by paracrine effects of the surrounding CAFs.

The promise of mTOR inhibitors in the treatment of colorectal cancer.

INTRODUCTION: Recently, deregulation of protein synthesis has begun to gain attention as a major player in cancer development and progression. Specifically, deregulation of the process of translation initiation appears to play a key role in oncogenesis. The PI3K/Akt/mTOR pathway is vital for cellular metabolism, growth and proliferation and thus an attractive therapeutic target in oncology. Accordingly, several mTOR inhibitors are currently being tested in many cancers including colorectal cancer (CRC). AREAS COVERED: In this review, the key components of the PI3K/Akt/mTOR pathways, their molecular alterations and the inhibitors targeting the mTOR pathway in CRC are described. Complex interactions with other pathways such as the MAPK pathway are analyzed, as are possible drug combinations that target this pathway. In addition, novel strategies for use of mTOR pathway inhibitors in CRC treatment are introduced. EXPERT OPINION: Clinical trials of mTOR inhibitors have been investigated in CRC. mTOR inhibitors may represent an attractive antitumor target in combination with strategies to target other pathways that may overcome resistance. Further research is needed to identify critical molecular effector mechanisms, molecular markers that predict responsiveness and potential toxicities.

The current state of targeted agents in rectal cancer.

Targeted biologic agents have an established role in treating metastatic colorectal cancer (CRC), and the integration of targeted therapies into the treatment of CRC has resulted in significant improvements in outcomes. Rapidly growing insight into the molecular biology of CRC, as well as recent developments in gene sequencing and molecular diagnostics, has led to high expectations for the identification of molecular markers to be used in personalized treatment regimens. The mechanisms of action and toxicities of targeted therapies differ from those of traditional cytotoxic chemotherapy. Targeted therapy has raised new insight about the possibility of tailoring treatment to an individual's disease, the assessment of drug effectiveness and toxicity, and the economics of cancer care. This paper covers the last decade of clinical trials that have explored the toxicity and efficacy of targeted agents in locally advanced and metastatic CRC and how their role may benefit patients with rectal cancer. Future efforts should include prospective studies of these agents in biomarker-defined subpopulations, as well as studies of novel agents that target angiogenesis, tumor-stromal interaction, and the cell signaling pathways implicated in rectal cancer.

Value and interpretation of resection margin after a colonoscopic polypectomy for malignant polyps.

PURPOSE: This study was designed to compare the clinicopathologic findings of an endoscopic polypectomy for malignant polyps with subsequent surgery and to evaluate the appropriateness of the pathologic finding criterion of the resection margin as an indicator for surgery in cases of malignant colorectal polyps. METHODS: We examined the clinicopathologic characteristics, complications and prognoses among the patients who underwent a colonoscopic polypectomy in both our hospitals and at other hospitals from April 2003 and April 2010. These patients were divided into two groups, the group (non-operation group) that only underwent a polypectomy (n = 37) and the group (operation group) that underwent a polypectomy with subsequent surgery (n = 33). RESULTS: There were no differences between two groups in the ratios of the number of men to the number of women, the ages or the comorbidities. In terms of endoscopic findings, we found no differences between the two groups in the locations of the polyps, the sizes of the polyps, or the presence of stalks. However, ulceration of polyps was higher in the non-operation group (51.5% vs. 21.6%; P = 0.009), as was the case with submucosal invasion (75.8% vs. 16.2%; P < 0.005). When an endoscopic polypectomy was performed, incomplete resection margins and specimens with margins involved occurred more frequently in the operation group (93.9% vs. 51.4%; P < 0.005), but no residual tumor was detected in 31 of 33 (93.9%) patients in that group. One pathologist reviewed the specimens of 54 patients (operation group, 19; non-operation group, 36). Six of the 19 polyps (31.6%) in the operation group and fifteen of the 36 polyps (41.7%) in the non-operation group had a margin without cancer cells. CONCLUSION: We may accept the criterion of a safe margin, including a coagulation zone. A multidisciplinary approach has to be developed by surgeons, endoscopists and pathologists based on a discussion of the risk factors for the patient before making a decision on the treatment treatment.

Melanoma-associated antigen (MAGE) expression in the normal mucosa around colorectal cancer after curative resection: presence of undetectable free cancer cells.

PURPOSE: Curative surgical resection is of great importance and some trials have been performed to identify free undetectable cancer cells using molecular markers. The aim of this study is to investigate melanoma-associated antigen (MAGE) expression in normal mucosa around colorectal cancer and its clinical significance.? MATERIAL AND METHOD: From October 2003 to June 2004, we collected 46 colorectal cancer and matched normal mucosal tissues within 20 mm, 20 to 50 mm and more than 50 mm from tumors after the curative operation. Twenty-two mucosal tissues were harvested from patients with benign colorectal diseases as controls. MAGE expression was assayed using nested RT-PCR of MAGE A1-6 mRNA.? RESULTS: The MAGE expression rates in cancer tissue and adjacent normal mucosa were 65.2%, 6.5% (<20 mm), 2.2% (20-50 mm) and 0.0% (>50 mm), respectively, while MAGE was not expressed in the mucosa of benign diseases. The MAGE-positive cases in the normal mucosa around tumors were located in the left colon or rectum, and one patient showed anastomotic mucosal site recurrence.? CONCLUSIONS: MAGE expression in normal-appearing mucosa around colorectal cancer showed some clinical findings suggesting the presence of undetectable free cancer cells after curative resection.

A case of small bowel obstruction due to a paracecal hernia.

Internal hernias are rare causes of small bowel obstruction, and one such internal hernia is the paracecal hernia. We report a case of a small bowel obstruction related to a paracecal hernia in which a preoperative diagnosis was made on computed tomography. A laparotomy was performed for definitive diagnosis and treatment. The surgery achieved a good outcome.

Gene expression profiling: canonical molecular changes and clinicopathological features in sporadic colorectal cancers.

AIM: To investigate alternative or subordinate pathways involved in colorectal tumorigenesis and tumor growth, possibly determining at-risk populations and predicting responses to treatment. METHODS: Using microarray gene-expression analysis, we analyzed patterns of gene expression relative to canonical molecular changes and clinicopathological features in 84 sporadic colorectal cancer patients, standardized by tumor location. Subsets of differentially expressed genes were confirmed by real-time reverse-transcript polymerase chain reaction (RT-PCR). RESULTS: The largest number of genes identified as being differentially expressed was by tumor location, and the next largest number by lymphovascular or neural invasion of tumor cells and by mismatch repair (MMR) defects. Amongst biological processes, the immune response was significantly implicated in entire molecular changes observed during colorectal tumorigenesis (P < 0.001). Amongst 47 differentially expressed genes, seven (PISD, NIBP, BAI2, STOML1, MRPL21, MRPL16, and MKKS) were newly found to correlate with tumorigenesis and tumor growth. Most location-associated molecular changes had distinct effects on gene expression, but the effects of the latter were sometimes contradictory. CONCLUSION: We show that several differentially expressed genes were associated with canonical molecular changes in sporadic colorectal cancers, possibly constituting alternative or subordinate pathways of tumorigenesis. As tumor location was the dominant factor influencing differential gene expression, location-specific analysis may identify location-associated pathways and enhance the accuracy of class prediction.

Risk factors and oncologic impact of anastomotic leakage after rectal cancer surgery.

PURPOSE: The impact of anastomotic leakage on the long-term oncologic outcome is not clear. This retrospective study evaluated risk factors and oncologic impacts of anastomotic leakage after rectal cancer surgery. METHODS: Data were analyzed from 1,391 patients who underwent sphincter preservation for rectal cancer between January 1997 and August 2003. Operations were classified as anterior resection (n = 164), low anterior resection (n = 898), or ultralow anterior resection (n = 329). RESULTS: The anastomotic leakage rate was 2.5 percent. Multivariate analysis identified male (hazard ratio, 3.03), old age (hazard ratio, 2.42), and lower anastomosis level (hazard ratio, 2.68) as risk factors for leakage. The local recurrence rates were 9.6 and 2.2 percent for the leakage and nonleakage groups, respectively but were not significant (P = 0.14). The overall five-year survival rates were 55.1 and 74.1 percent in the leakage and nonleakage groups, respectively (P < 0.05), and the cancer-specific survival rates were 63 and 78.3 percent in the leakage and nonleakage groups, respectively (P = 0.05). However, in subgroup analysis, significant differences were identified only in Stage III patients. CONCLUSIONS: Age, sex, and ultralow anterior resection were found to be risk factors for anastomotic leakage after rectal cancer surgery. In addition, leakage was associated with poor survival.