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Supplementary Figure 1, Figure 2, Figure 3. Representative photomicrographs detailing immunohistochemical analysis of ANGPTL2 expression, Figure S2: ANGPTL2 expression in 5 colorectal cancer cell lines, Figure S3: Receiver operating characteristics curve analysis using serum ANGPTL2 for distinguishing patients with CRC with normal CEA levels from normal controls (NC).

Purpose: Angiopoietin-like protein 2 (ANGPTL2) is a mediator of chronic inflammation and inflammatory carcinogenesis. The biologic and clinical significance of ANGPTL2 remains unknown in human cancer. Therefore, we investigated the function of ANGPTL2 and evaluated its clinical significance in both primary tumors and matched sera in patients with colorectal cancer.Experimental Design: A colorectal cancer cell line was transfected with siRNA against ANGPTL2 for the assessment of its function. We examined ANGPTL2 expression in colorectal cancer tissues (n = 195) by immunohistochemistry. Finally, we screened serum ANGPTL2 levels from 32 colorectal cancers and 23 normal controls (NC), and validated these results in serum samples obtained from 195 colorectal cancers and 45 NCs by ELISA.Results: Knockdown of ANGPTL2 in vitro significantly inhibited cell proliferation, migration, and invasion, whereas it enhanced anoikis. ANGPTL2 was overexpressed in colorectal cancer tissues, and was significantly associated with advanced T stage, lymph node, and liver metastasis. Likewise, serum ANGPTL2 levels in colorectal cancers were significantly higher than NCs (P < 0.01), and allowed distinguishing of colorectal cancers from NCs with high accuracy (AUC = 0.837). The subsequent validation step confirmed that serum ANGPTL2 levels in colorectal cancers were significantly higher than in NCs (P < 0.0001), and had a high AUC value (0.885) for distinguishing colorectal cancers from NCs. High serum ANGPTL2 was significantly associated with advanced T stage, lymph node and liver metastasis, early relapse, and poor prognosis in colorectal cancers.Conclusion: Serum ANGPTL2 is a novel diagnostic and recurrence-predictive biomarker in patients with colorectal cancer. Clin Cancer Res; 20(23); 6175–86. ©2014 AACR.

Supplementary Tables S1-3. Table S1:Patient characteristics in the validation set, Table S2:Primer Sequences for KRAS and BRAF mutation analysis, Table S3:Comparison between ROC from screening step and validation step.

Supplementary material and methods. Detailed patient characteristics in the validation step, Cell culture condition, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, yellow tetrazole (MTT) assay, Anoikis assay, Invasion assay, Wound healing assay,Immunohistochemistry, Enzyme-Linked Immunosorbent Assay (ELISA).

MINI: In the present study, we systemically and comprehensively evaluated the prognostic significance of a combination of inflammatory factors using preoperative blood examination, and focused on the potential feasibility of our newly developed lymphocyte-CRP ratio (LCR) as a prognostic biomarker in CRC patients. We have firstly identified that a combination of lymphocytic counts along with CRP levels, which we defined as LCR, is a more reliable indicator of poor prognosis compared with other combinations of inflammatory markers, in CRC patients. Furthermore, preoperative LCR could also identify CRC patients who are at higher risk for postoperative infectious complications. Background Systemic inflammation via host-tumor interactions is currently recognized as a hallmark of cancer. The aim of this study was to evaluate the prognostic value of various combinations of inflammatory factors using preoperative blood, and to assess the clinical significance of our newly developed inflammatory score in colorectal cancer (CRC) patients. Method In total 477 CRC patients from the discovery and validation cohorts were enrolled in this study. We assessed the predictive impact for recurrence using a combination of nine inflammatory markers in the discovery set, and focused on lymphocyte-C-reactive protein ratio (LCR) to elucidate its prognostic and predictive value for peri-operative risk in both cohorts. Results A combination of lymphocytic count along with C-reactive protein levels demonstrated the highest correlation with recurrence compared with other parameters in CRC patients. Lower levels of preoperative LCR significantly correlated with undifferentiated histology, advanced T stage, presence of lymph node metastasis, distant metastasis, and advanced stage classification. Decreased preoperative LCR (using an optimal cut-off threshold of 6000) was an independent prognostic factor for both disease-free survival and overall survival, and emerged as an independent risk factor for postoperative complications and surgical-site infections in CRC patients. Finally, we assessed the clinical feasibility of LCR in an independent validation cohort, and confirmed that decreased preoperative LCR was an independent prognostic factor for both disease-free survival and overall survival, and was an independent predictor for postoperative complications and surgical-site infections in CRC patients. Conclusion Preoperative LCR is a useful marker for perioperative and postoperative management of CRC patients.

Background Malnutrition can adversely affect treatment responses and oncological outcomes in cancer patients. However, among patients with rectal cancer who undergo chemoradiotherapy (CRT), the significance of peri-treatment nutrition assessment as a predictor of treatment response and outcome remains unclear. Objective The aim of this study was to determine whether the Prognostic Nutrition Index (PNI) based on peri-treatment serum can be used as a predictor of treatment response and outcome in patients with rectal cancer who undergo CRT. Design, setting, and patients We analyzed 114 patients with rectal cancer who received preoperative CRT followed by total mesorectal excision at our institution. Results Post-CRT PNI was significantly lower than pre-CRT PNI in rectal cancer patients. Although post-CRT PNI did not significantly correlate with either overall survival or disease-free survival, low pre-CRT PNI was significantly associated with shorter overall survival and disease-free survival in this population and was also an independent risk factor for ineffectiveness of long-course preoperative CRT. Finally, low pre-CRT PNIs were a stronger indicator of poor prognosis and early recurrence in patients with pathological lymph node metastasis (who generally need to receive postoperative chemotherapy), than in those with no pathological lymph node metastasis. Conclusion Pretreatment PNI could be useful in evaluating and managing patients with rectal cancer who undergo CRT followed by curative resection.

Aim To identify predictors of poor prognosis of patients with colon cancer (CC) who underwent surgery with curative intent, we investigated the association between the albumin to globulin ratio (AGR) with clinicopathological findings such as overall (OS) and disease-free (DFS) survival. Patients and methods We conducted a retrospective study of clinicopathological findings, including preoperative laboratory data, for 248 patients with stage I-III CC. Results Patients with low AGR had shorter DFS and OS compared to those with high AGR. Multivariate analyses identified low AGR as an independent variable independently associated with recurrence and poor prognosis of patients with CC who underwent surgery with curative intent regardless of lymphnode metastasis. Conclusion The preoperative AGR was an independent predictor of recurrence and poor prognosis of patients with CC who underwent surgery with curative intent. The AGR indicates that these patients may benefit from intensive adjuvant therapy.

Although patients with metastatic colorectal cancer (CRC) are often unable to undergo treatment after resection of primary tumors, identifying such patients before surgery is not easy. In this study, we evaluated the association among clinicopathological findings, survival outcomes, and ability to undergo multimodal therapy after primary tumor resection in patients with Stage IV CRC. We collected clinicopathological findings and preoperative laboratory data, including carcinoembryonic antigen (CEA) and systemic inflammatory response markers for 92 patients who were treated for Stage IV CRC between 2005 and 2014. We used multivariate analysis on factors that affect prognosis and ability to undergo postoperative treatment. Postoperative multimodal therapy improved overall survival (OS) significantly. Among serum markers, elevated CEA, neutrophil-to-lymphocyte ratio, and modified Glasgow prognosis score (mGPS) were significant indicators of shorter OS. In multivariate analysis, low performance status (P = 0.003), undifferentiated histology type (P = 0.019), and elevated mGPS (P = 0.042) were independent predictors of worse prognosis; and older age (P = 0.016), right-sided colon cancer (P = 0.043), and elevated mGPS (P = 0.031) were independent risk factors for difficulty of introducing postoperative multimodal therapy. Preoperative mGPS is a useful objective indicator for CRC patients with multiple metastases who are able to undergo primary site resection followed by postoperative multimodal therapy.

Surgical site infection (SSI) is a potentially morbid and costly complication of surgery. The purpose of this study was to determine preoperative and operative predictors of SSIs after gastric resection with lymphadenectomy in patients with gastric cancer (GC). Data on clinicopathologic factors, including operative and preoperative laboratory factors, for 384 patients with GC who had undergone curative surgery were analyzed in this retrospective study to assess their associations with SSIs. Superficial/deep incisional SSIs (iSSIs) and organ/space SSIs (o/sSSIs) occurred in 18 (4.6%), and 27 (7.0%), respectively. The o/sSSIs were significantly associated with surgery-related factors such as duration of operation, blood loss, and extent of tumor. Additionally, high levels of preoperative indicators of systemic inflammation, including neutrophil counts, neutrophil/lymphocyte ratio, and C-reactive protein concentrations, were significantly associated with o/sSSIs. Multivariate analyses demonstrated that preoperative neutrophil counts and duration of surgery were independent predictors for o/sSSIs, whereas only preoperative serum albumin concentration was predicted for iSSIs. In patients with GC undergoing curative surgery, preoperative neutrophil count and operation time are potentially valuable predictors of o/sSSIs, whereas only preoperative serum albumin predicts iSSIs.

The discovery of biomarkers to predict the potential for lymph node (LN) metastasis in patients with colorectal cancer (CRC) is essential for developing improved strategies for treating CRC. In the present study, they used isobaric tags for relative and absolute quantitation to conduct a proteomic analysis designed to identify novel biomarkers for predicting LN metastasis in patients with CRC. They identified 60 differentially expressed proteins specifically associated with LN metastasis in CRC patients and classified the molecular and functional characteristics of these proteins by bioinformatic approaches. A literature search led them to select heat shock protein 47 (HSP47) as the most suitable candidate biomarker for predicting LN metastasis. Validation analysis by immunohistochemistry showed that HSP47 expression in patients with CRC and the number of HSP47-positive spindle cells in the tumor stroma were significantly higher compared with those in adjacent normal colonic mucosa, and the number of the latter cells increased with tumor progression. Further, the number of HSP47-positive spindle cells in stroma was a more informative marker for identifying LN metastasis than HSP47expression. Multivariate analysis identified spindle cells that expressed elevated levels of HSP47 as an independent predictive biomarker for CRC with LN metastasis. Moreover, these cells served as an independent marker of disease-free and overall survival of patients with CRC. Their data indicate that the number of HSP47-positive spindle cells in the stroma of CRC may serve as a novel predictive biomarker of LN metastasis, early recurrence and poor prognosis.

BACKGROUND Circulating microRNAs (miRNAs) are attracting major interest as potential non-invasive biomarkers for colorectal cancer (CRC). This study aimed to identify a novel serum miRNA biomarker for the early detection and/or evaluating prognosis of CRC patients. PATIENTS AND METHODS Comprehensive miRNA array analysis was carried out using serum samples from patients with colorectal neoplasia and healthy controls. Next, to verify whether the candidate miRNA possessed a secretory potential, we screened miRNA expression levels in culture medium from 2 CRC cell lines, followed by serum analysis from 12 stage IV CRC, 12 adenoma, and 12 control subjects. Thereafter, we validated expression of candidate miRNAs in 179 primary CRC tissues, as well as serum samples from an independent cohort of 211 CRCs, 56 adenomas, and 57 control subjects. RESULTS Through microarray analysis, we identified significantly higher levels of miRNA-1290 (miR-1290) in serum from patients with colorectal adenomas and cancers. We verified miR-1290 overexpression in serum of CRC patients in a training cohort. In the validation cohort, serum miR-1290 levels were significantly up-regulated in patients with colorectal adenomas (P < 0.0001) and cancers (P < 0.0001). Serum miR-1290 levels could robustly distinguish adenoma [area under the curve (AUC) = 0.718] and CRC patients (AUC = 0.830) from normal subjects. High miR-1290 expression in serum and tissue was significantly associated with tumor aggressiveness and poor prognosis. Moreover, serum miR-1290 levels were an independent prognostic factor [hazard ratio (HR) = 4.51; 95% confidence interval (CI) = 1.23-23.69; P = 0.0096] and an independent predictor for tumor recurrence (hazard ratio = 3.92; 95% confidence interval = 1.11-25.14; P = 0.032) in CRC. CONCLUSIONS Serum miR-1290 is a novel biomarker for early detection, recurrence, and prognosis in CRC.

Objectives: MicroRNA (miR)-503 is downregulated in several cancers and plays a tumor-suppressive role in carcinogenesis. However, the miR-503 expression pattern, its clinical significance and its molecular mechanism in colorectal cancer (CRC) have not been investigated. Methods: We analyzed miR-503 expression in normal mucosa (n = 20), adenoma (n = 27) and CRC (n = 20). We quantified miR-503 expression in an independent cohort (n = 191) and investigated the clinical significance of miR-503 in CRC. CRC cell lines were transfected with anti-miR-503 to assess its function and target gene. Results: miR-503 expression increased according to the adenoma-carcinoma sequence. High miR-503 expression was significantly associated with large tumor size, serosal invasion, lymphatic and venous invasion as well as lymph node metastasis. CRC patients with high miR-503 expression had significantly earlier relapse and poorer prognosis than those with low expression. miR-503 was an independent recurrence marker in stage I/II CRC. In vitro, attenuated miR-503 expression resulted in inhibition of proliferation, invasion and migration and acquisition of anoikis of CRC cells. The putative target gene (calcium-sensing receptor) was significantly upregulated after miR-503 attenuation. Conclusions: miR-503 acts as an ‘onco-miR' in CRC. High miR-503 expression is associated with early recurrence and poor prognosis in CRC.

Background Despite advances in local control of rectal cancer, recurrence in distant organs is still one of the main causes of mortality. Prognostic biomarkers would be valuable for the treatment of patients who have rectal cancer. Objective The aim of our study was to investigate the prognostic impact of lymphocyte-to-monocyte ratio in patients with rectal cancer receiving preoperative chemoradiotherapy, and to clarify the clinical significance of lymphocyte-to-monocyte ratio. Design Prospectively maintained data of patients with rectal cancer were retrospectively evaluated to clarify the clinical relevance of the lymphocyte-to-monocyte ratio. Setting This study was conducted at a single expert center. Patients A total of 119 consecutive patients with rectal cancer through chemoradiotherapy followed by total mesorectal excision at our institute were enrolled in this study. Eight patients were excluded because of a lack of laboratory data, and finally 111 patients were assessed in this study. Main outcome measures The primary outcome measured was the clinical relevance of the lymphocyte-to-monocyte ratio in patients with rectal cancer receiving chemoradiotherapy. Results Patients with a low pretreatment lymphocyte-to-monocyte ratio showed poor prognosis significantly both in overall survival and disease-free survival of those with rectal cancer receiving chemoradiotherapy. Multivariate analyses showed that low pretreatment lymphocyte-to-monocyte ratio level, presence of pathological lymph node metastasis (ypN(+)), and high pretreatment serum C-reactive protein level were independent prognostic factors of overall survival and disease-free survival. In addition, time-to-event analysis divided into 2 groups by ypN status showed that low pretreatment lymphocyte-to-monocyte ratio was correlated with poor overall survival and disease-free survival not only in group ypN(-) but also in group ypN(+). Limitations The present study had several limitations, including that it was a retrospective observational and single institutional study with Japanese patients. Conclusions The combination of lymphocyte-to-monocyte ratio and ypN status can be a predictive marker of poor prognosis and recurrence among patients with rectal cancer undergoing preoperative chemoradiotherapy. See Video Abstract at http://links.lww.com/DCR/A780.

Diagnostic markers facilitate more selective screening and treatment strategies for ulcerative colitis (UC)-associated cancer (UCAC). The expression of trefoil factor-3 (TFF3), which is involved in mucosal protection and repair in the gastrointestinal tract, was analyzed and its significance for UCAC was evaluated. A total of 145 patients with UC who underwent proctocolectomies were enrolled, including 15 patients (10.8%) with UCAC. TFF3 expression in the rectal mucosa and in cancer cells was assessed using immunohistochemistry, and the expression in UCAC and sporadic colorectal cancer was compared. Analyzing the mucinous granules of goblet cells located in crypts revealed that the non-cancerous rectal mucosa of patients with UCAC had significantly lower mean TFF3 staining scores compared with patients with UC without UCAC or patients with sporadic cancer. TFF3 staining score was revealed to be an independent predictor of UCAC development. These results indicated that low TFF3 expression in the rectal mucosa was associated with the development of UCAC. Thus, TFF3 expression in the rectal mucosa may be a useful biomarker for monitoring patients with UC.

Objective: This study aimed to investigate clinicopathological responses and oncological outcome in patients receiving short- or long-course chemoradiotherapy (CRT) and to assess the predictive factor for recurrence in each treatment. Methods: A total of 118 rectal cancer patients receiving preoperative CRT were enrolled. Clinicopathological responses and oncological outcome in patients receiving short- or long-course CRT were investigated. Results: Despite there being no significant differences in the prognosis of disease-free survival (DFS) based on TNM stage classification in patients receiving long-course CRT, patients with advanced stage demonstrated poor DFS after short-course CRT. The presence of lymph node metastasis was a predictor of poor DFS in short-course CRT, whereas poor pathological response was a predictor of recurrence in long-course CRT. Conclusions: Distinct predictors of recurrence depending on the CRT course might be needed to discriminate candidates from rectal cancer patients receiving preoperative CRT who might benefit from more intensive adjuvant therapy after surgery.

Colony‑stimulating‑factor‑1 (CSF‑1) is a hematopoietic growth factor that exerts its effects through the c‑fms/CSF‑1 receptor (CSF‑1R). The CSF‑1/CSF‑1R axis is thought to be involved in the development of several types of cancer. This study aimed to clarify the clinical and biological significance of the CSF‑1/CSF‑1R axis in gastric cancer (GC). For this purpose, we evaluated CSF‑1 and CSF‑1R expression in GC tissues from 148 patients by RT‑qPCR and immunohistochemistry. The biological roles of the CSF‑1/CSF‑1R axis were investigated by measuring the cell proliferation and migration, and anoikis resistance in a human GC cell line following treatment with recombinant human CSF‑1 and/or CSF‑1R inhibitor. The results revealed that an elevated expression of CSF‑1 or CSF‑1R significantly correlated with disease progression and with a poor overall survival (OS, P=0.037 and 0.016, respectively) and disease‑free survival (DFS, P

Background/aim This study aimed to clarify the potential of modified Glasgow Prognostic Score (mGPS) as a prognostic biomarker and reveal the significance of fish oil (FO)-enriched nutrition in colorectal cancer (CRC). Patients and methods A total of 738 CRC patients from three different patient cohorts, including 670 patients in the biomarker study and 68 patients in the nutrition-intervention study, were analyzed. Results High preoperative mGPS was significantly correlated with well-recognized disease progression factors and advanced UICC stage classification. In addition, high mGPS was an independent prognostic factor in both cohorts, especially in stage III and IV patients. These statuses were maintained in postoperative course and correlated with sarcopenia. Furthermore, FO-enriched nutrition suppressed systemic inflammatory reaction and improved skeletal muscle mass and prognosis, especially in CRC patients with mGPS 1 or 2. Conclusion Assessment of mGPS could identify patients with high-risk CRC, who might be candidates for FO-enriched nutrition.

CD26 is a transmembrane glycoprotein whose role in various types of malignancies, along with the potential therapeutic and diagnostic targets, has been evaluated. Preoperative chemoradiotherapy (CRT) is an effective tool for local control of rectal cancer, but the rate of disease recurrence remains high. The aim of this study was to clarify the association between CD26 expression and rectal cancer after preoperative CRT. A total of 85 patients with rectal cancer who had undergone preoperative CRT were enrolled in this study. We investigated CD26 expression in residual tumors and the surrounding stromal tissue using immunohistochemistry. Additionally, stromal CD26 gene expression was assessed by real-time quantitative polymerase chain reaction. Patients with high CD26 expression in cancer tissue more frequently had serosal invasion, vascular invasion, and a poor pathological response. High expression of CD26 in the tumor stroma was significantly correlated with histology and tumor recurrence. High CD26 expression in the stroma, but not the tumor itself, was significantly correlated with a poor prognosis. Patients expressing CD26 in the tumor stroma, based on transcriptional analysis, also had a significantly poorer prognosis than those without the expression. In multivariate analysis, lymph node metastasis and high stromal CD26 expression were identified as independent prognostic factors in patients with rectal cancer after neoadjuvant CRT. Stromal CD26 expression after preoperative CRT was significantly associated with tumor recurrence and prognosis in rectal cancer patients. Our data suggest that stromal CD26 plays an important role and is a potential therapeutic target in tumor relapse.

Metastasis is a major cause of death in patients with gastric cancer (GC). MicroRNAs (miRNAs) relating to the epithelial–mesenchymal transition (EMT) control GC progression and metastasis. The aim of this study was to evaluate serum EMT-associated miRNAs for metastatic and prognostic noninvasive biomarkers in GC. In the first step of this study (preliminary experiments), we selected candidate miRNAs associated with metastasis by analyzing the expression of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) and miR-203 in serum samples from stage I (n = 12) and stage IV (n = 12) GC patients. The second phase involved the independent validation of candidate miRNAs in serum specimens from 130 patients with GC and 22 controls. Based on the preliminary experiments, miR-203 was selected as the candidate serum miRNA that was most closely associated with metastasis. Validation analysis revealed that serum miR-203 levels were significantly lower in stage IV than stage I–III GC patients. Serum miR-203 expression was significantly lower in GC patients with a higher T stage, vessel invasion, and lymph node, peritoneal, and distant metastases. Low expression of serum miR-203 was significantly associated with poor disease-free and overall survival. Multivariate analysis revealed that low serum miR-203 expression was an independent predictive marker for lymph node, peritoneal, and distant metastases and a poor prognosis in patients with GC. Serum miR-203 has the potential to serve as a noninvasive biomarker for prognosis and to predict metastasis in patients with GC.

Preoperative serum systemic inflammatory response (SIR) in patients with colorectal cancer (CRC) has been reported to be a predictive biomarker of early recurrence. The molecular status of CRC, including microsatellite instability (MSI), BRAF and KRAS mutations, and tumor-infiltrating lymphocytes (TILs), has also been associated with recurrence in CRC patients treated with curative surgery. We investigated the impacts of SIR status, TILs, and MSI on recurrence in curative CRC patients. In this retrospective study, we enrolled 157 patients with stage I–III CRC undergoing curative surgery, for whom preoperative neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and C-reactive protein (CRP) data were available as indicators of SIR status. Molecular status was evaluated by counting TILs as the numbers of intratumoral Foxp3- and CD8-positive T cells by immunohistochemistry. MSI status was determined using five mononucleotide repeat microsatellite markers. Kaplan–Meier analysis of SIR indicators revealed that higher CRP, NLR, and PLR were associated with significantly poorer disease-free survival (DFS). Low levels of infiltrating CD8-positive T cells in CRC tissue was a significant predictor of poor DFS. Multivariate analysis showed that few infiltrating CD8-positive T cells and high serum CRP levels were independent predictive factors for recurrence. Furthermore, the combination of high CRP and few infiltrating CD8-positive T cells increased the predictive accuracy in these patients. The results of this study suggest that both CRP levels in preoperative serum and CD8 T cells in CRC tissue are useful biomarkers for predicting early relapse in CRC patients treated with curative surgery.

Colorectal cancer (CRC) is predominantly a disease of the elderly. Elderly patients may also exhibit poorer outcomes due to the increased burden of comorbidities, functional dependency and limited life expectancy. The aim of this study was to evaluate the outcome of laparoscopic surgery in elderly patients with CRC. A total of 148 patients who underwent laparoscopic surgery at our institution between January, 2000 and December, 2011 were enrolled. We compared the differences between elderly patients (aged >75 years, n=48) and non-elderly patients (aged

Rac GTPase-activating protein (RacGAP) 1 plays a key role in controlling various cellular phenomena including cytokinesis, transformation, invasive migration and metastasis. This study investigated the function and clinical significance of RacGAP1 expression in colorectal cancer (CRC). The intrinsic functions of RacGAP1 in CRC cells were analyzed using small interfering RNA (siRNA). We analyzed RacGAP1 mRNA expression in surgical specimens from 193 CRC patients (Cohort 1) by real-time PCR. Finally, we validated RacGAP1 protein expression using formalin-fixed paraffin-embedded samples from 298 CRC patients (Cohort 2) by immunohistochemistry. Reduced RacGAP1 expression by siRNA in CRC cell lines showed significantly decreased cellular proliferation, migration and invasion. In Cohort 1, RacGAP1 expression in CRC was significantly higher than in adjacent normal mucosa and increased according to tumor node metastasis stage progression. High RacGAP1 expression in tumors was significantly associated with progression and prognosis. In Cohort 2, RacGAP1 protein was overexpressed mainly in the nuclei of CRC cells; however, its expression was scarcely observed in normal colorectal mucosa. RacGAP1 protein expression was significantly higher in CRC patients with higher T stage, vessel invasion and lymph node and distant metastasis. Increased expression of RacGAP1 protein was significantly associated with poor disease-free and overall survival. Multivariate analyses revealed that high RacGAP1 expression was an independent predictive marker for lymph node metastasis, recurrence and poor prognosis in CRC. Our data provide novel evidence for the biological and clinical significance of RacGAP1 as a potential biomarker for identifying patients with lymph node metastasis and poor prognosis in CRC.

A 41-year-old man experienced a swollen scrotum three days after a motorcycle accident and presented to our hospital. He had had a primary suture repair for anoperineal trauma in an outside hospital at the time of the injury. He presented to us with general fatigue, low grade fevers, and perineal pain. Abdominal computed tomography showed subcutaneous emphysema from the scrotum to the left chest. The sutured wound had foul-smelling discharge and white exudate. We made the diagnosis of necrotizing fasciitis and immediately opened the sutured wound and performed initial debridement and lavage with copious irrigation. We continued antibiotics and lavage of the wound until the infection was controlled. Fortunately, the necrotizing fasciitis did not worsen and he was discharged after 15 days. Our experience indicates that anoperineal injuries should not be closed without careful and intensive follow-up due to the potential of developing necrotizing fasciitis.

A 41-year-old man presented to our hospital with lower abdominal pain and a high-grade fever. Physical examination revealed rebound tenderness and guarding in the lower abdomen. Abdominal X-ray examination showed a radiopaque object in the right lower quadrant of the abdomen. Abdominal computed tomography (CT) demonstrated that the object had a strong artifact with over 10,000 Hounsfield units, as well as ascites around the terminal ileum. We diagnosed acute peritonitis with a suspicion of the perforation due to unknown foreign body and performed an emergency laparotomy. Operative findings showed a contained perforation of a phlegmonous appendicitis, and appendectomy was performed. The resected specimen demonstrated that the appendix contained a fecalith, and histopathological examination showed the crystal structure of barium sulfate in the lumen of the appendix. Unfortunately, we did not obtain the history of screening for gastric cancer using a barium examination one month prior to our appendectomy. Our experience demonstrates the importance of establishing a history of barium examinations of the gastrointestinal tract in a patient with a radiopaque object in the right lower quadrant of the abdomen for early diagnosis of barium appendicitis. Additionally, early diagnosis of barium appendicitis may affect the selection of surgical procedures.

// Koichiro Mori 1 , Yuji Toiyama 1 , Kohei Otake 1 , Shozo Ide 1 , Hiroki Imaoka 1 , Masato Okigami 1 , Yoshinaga Okugawa 1 , Hiroyuki Fujikawa 1 , Susumu Saigusa 1 , Junichiro Hiro 1 , Minako Kobayashi 1 , Masaki Ohi 1 , Koji Tanaka 1 , Yasuhiro Inoue 1 , Yuhko Kobayashi 2 , Yasuhiko Mohri 1 , Issei Kobayashi 2 , Ajay Goel 3 and Masato Kusunoki 1 1 Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie, Japan 2 Center for Molecular Biology and Genetics, Mie University, Mie, Japan 3 Center for Gastrointestinal Research & Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX USA Correspondence to: Yuji Toiyama, email: ytoi0725@clin.medic.mie-u.ac.jp Ajay Goel, email: Ajay.Goel@BSWHealth.org Keywords: iTRAQ-based quantitative proteomic analysis; ezrin; lymph node metastasis; colorectal cancer; biomarker Received: February 02, 2017 Accepted: June 29, 2017 Published: October 30, 2017 ABSTRACT Colorectal cancer (CRC)-associated mortality is primarily caused by lymph node (LN) and distant metastasis, highlighting the need for biomarkers that predict LN metastasis and facilitate better therapeutic strategies. We used an I sobaric T ags for R elative and A bsolute Q uantification (iTRAQ)-based comparative proteomics approach to identify novel biomarkers for predicting LN metastasis in CRC patients. We analyzed five paired samples of CRC with or without LN metastasis, adjacent normal mucosa, and normal colon mucosa, and differentially expressed proteins were identified and subsequently validated at the protein and/or mRNA levels by immunohistochemistry and qRT-PCR, respectively. We identified 55 proteins specifically associated with LN metastasis, from which we selected ezrin for further analysis and functional assessment. Expression of ezrin at both the protein and mRNA levels was significantly higher in CRC tissues than in adjacent normal colonic mucosa. In univariate analysis, high ezrin expression was significantly associated with tumor progression and poor prognosis, which was consistent with our in vitro findings that ezrin promotes the metastatic capacity of CRC cells by enabling cell invasion and migration. In multivariate analysis, high levels of ezrin protein and mRNA in CRC samples were independent predictors of LN metastasis. Our data thus identify ezrin as a novel protein and mRNA biomarker for predicting LN metastasis in CRC patients.

Elderly patients are regarded as being at increased risk during major abdominal surgery because of a lack of functional reserve and an increased number of comorbidities. The aim of this study was to compare short- and long-term outcomes of laparoscopic gastrectomy between elderly and young gastric cancer patients. Two-hundred ten patients who underwent laparoscopic gastrectomy for gastric cancer at our institution between January 2001 and December 2011 were included in this retrospective study. Patients were divided into two age groups (younger than 70 years and older than 70 years) and were evaluated with respect to postoperative morbidity, quality of life (QOL), and survival. Postoperative morbidity was similar in elderly and young groups (18.3 vs. 21.6 %; P = 0.718). Overall survival of the elderly group was significantly worse than that of the young group (P

Introduction The aim of this study was to clarify the factors associated with postoperative complications and prognoses following laparoscopy-assisted gastrectomy for gastric cancer. Methods A total of 214 patients who underwent laparoscopy-assisted gastrectomy for gastric cancer were enrolled in this retrospective study. Factors that were potentially associated with postoperative complications and prognosis were assessed using the following clinical and perioperative parameters: the Estimation of Physiologic Ability and Surgical Stress scoring system, the Prognostic Nutritional Index, and the Clavien–Dindo classification. Results Postoperative complications developed in 18.7% of patients. Blood loss ≥ 1% of body weight was significantly correlated with anastomotic leakage (P = 0.0108). Severe complications, based on the Clavien–Dindo classification, were more frequent in patients with proximal or total gastrectomies (P = 0.0324). A preoperative C-reactive protein level > 0.5 mg/dL, blood loss > 1% of body weight, age ≥ 70 years at the time of operation, and an Estimation of Physiologic Ability and Surgical Stress score ≥ 0.2 were significantly correlated with poor prognosis. Multivariate analysis revealed that two of these factors, preoperative C-reactive protein level ≥ 0.5 mg/dL and operative blood loss ≥ 1% of body weight, were independent prognostic factors (P = 0.0376 and P = 0.0112, respectively). The Prognostic Nutritional Index had no significant correlation with prognosis or the occurrence of postoperative complications. Conclusion Operative blood loss ≥ 1% of body weight and type of resection were associated with an increased frequency of postoperative complications, while preoperative C-reactive protein levels and operative blood loss may be prognostic predictors for gastric cancer patients following laparoscopy-assisted gastrectomy.

Interleukin-13 (IL-13) is an immunosuppressive cytokine produced by several immune cells and cancer cells. The aim of this retrospective study was to determine if serum IL-13 levels have an association with clinical outcome in patients with colorectal cancer. A total of 241 patients with colorectal cancer were enrolled in the present study. Preoperative serum IL-13 concentrations were measured by enzyme-linked immunosorbent assay. We analyzed the association of serum IL-13 levels with clinicopathological variables. Patients with lymph node metastasis, lymphatic invasion, vascular invasion, distant metastases or advanced stage of disease had significantly lower serum IL-13 levels. Low serum IL-13 was significantly associated with both poor recurrence-free and overall survival. Multivariate analysis showed that low IL-13 levels were an independent predictive marker for poor prognosis. In conclusion, our data suggest that low serum IL-13 levels may be a useful predictive marker for poor prognosis in colorectal cancer.

Rac GTPase activating protein 1 (RacGAP1) plays a regulatory role in cell growth, transformation and metastasis. The aim of this study was to clarify the association between RacGAP1 expression and clinical outcome in patients with gastric cancer. A total of 232 gastric cancer patients in our institute who underwent surgery without preoperative treatments were enrolled in this study. We investigated RacGAP1 expression using immunohistochemistry (IHC) and evaluated IHC scores calculated by the percentage of positive cells and intensity and its expression at the invasive front. RACGAP1 expression was also assessed. RacGAP1 expression was observed in the nuclei of gastric cancer cells. Evaluation by IHC score showed no significant correlations with clinicopathological variables except for histological differentiation. In transcriptional analyses, RACGAP1 expression was elevated in diffuse type gastric cancer than intestinal type without a significant difference. We observed significant correlations of RacGAP1 protein expression at the invasive front with older age, tumor size, lymph node metastasis, lymphatic invasion, vascular invasion and advanced stage. Patients with RacGAP1 protein expression at the invasive front had significantly poorer prognosis than those without it (P

Neoadjuvant therapy followed by surgery can improve long-term survival and reduce local recurrence in patients with esophageal squamous cell carcinoma (ESCC). Extracapsular lymph node involvement (ECLNI) reflects tumor progression in gastrointestinal malignancies. The aim of this study was to clarify the correlation between ECLNI and clinical outcome in ESCC following neoadjuvant therapy. A total of 36 patients with ESCC who underwent neoadjuvant therapy followed by surgery were enrolled in this study (CT: n = 16; CRT: n = 20). The correlation between ECLNI and clinicopathological variables was investigated. In addition, we also evaluated whether differences in pathological response existed between primary tumors and metastatic lymph nodes (LNs), and whether chemotherapy (CT) and chemoradiotherapy (CRT) had different effects on LNs. Of 36 patients, 22.2 % had detectable ECLNI. ECLNI was significantly correlated with tumor size (>40 mm), LN density (≧20 %), advanced stage, lymphatic invasion, non-R0 resection, and poor pathological response. Patients with ECLNI had a significantly poorer prognosis than those without ECLNI (P = 0.0040). No differences in pathological response were observed between primary tumors and metastatic LNs for each type of therapy. The median number of dissected LNs was 21, 45, and 14 in the surgery alone (n = 22), CT, and CRT groups, respectively (P

We previously visualized in vivo responses to chemotherapy in a colorectal liver metastatic xenograft model using in vivo real-time and time-series intravital two-photon laser scanning microscopy (TPLSM). In this study, we established the method for evaluating the response of peritoneal xenografts to chemotherapy of metastatic gastric cancer using intravital TPLSM.Red fluorescent protein-expressing gastric cancer cells (NUGC4) were inoculated into the peritoneal cavity of green fluorescent protein nude mice.Laparotomy revealed that 2 weeks after inoculation, macroscopic peritoneal metastatic nodules were formed. The first intravital TPLSM session revealed that they were composed of red tumor cell clusters and green surrounding stroma. Paclitaxel was administered intraperitoneally after the first TPLSM three times a week for 7 days in the treatment group. At the second laparotomy, there were significantly fewer and smaller nodules in the treated mice than in the controls. The second intravital TPLSM session showed tumor cell fragmentation, swelling, and nuclear condensation in the metastatic nodules--a response to chemotherapy. There were multinuclear tumor cells in the paclitaxel-treated living mice.Our method may become a powerful tool for evaluating the efficacy of novel anti-gastric cancer drugs in a preclinical murine model with minimum interindividual variation.

We report a case involving a minimally invasive single-incision laparoscopic and endoscopic cooperative local excision of a duodenal gastrointestinal stromal tumor. A 59-year-old man presented with a 35-mm lesion located in the second portion of the duodenum. A local resection was performed via single-incision laparoscopic and endoscopic cooperative surgery. Intraluminal endoscopic dissection of the duodenal mucosa and submucosa was performed circumferentially around the tumor. The resection was then completed by laparoscopic dissection of the seromuscular layer around the tumor. The tumor was retrieved laparoscopically. After confirming that the resection achieved clear surgical margins, we closed the duodenal wall with a laparoscopic stapling device. There were no postoperative complications, including stenosis. Single-incision laparoscopic and endoscopic cooperative surgery can be safely and effectively performed for a duodenal submucosal tumor.

To clarify whether the expression of nitrative and oxidative DNA damage markers in the rectal mucosa of patients with ulcerative colitis (UC) could be used to predict UC-associated neoplasia.A longer duration of UC can increase the risk of developing UC-associated cancer (UCAC). Effective diagnostic markers are being sought to provide more selective screening and treatment strategies for patients with long-standing UC.A total of 141 patients with UC who underwent a proctocolectomy were enrolled in this study. The expression of 8-nitroguanine (8-NG), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and inducible nitric oxide synthase (iNOS) in the rectal mucosa were evaluated using immunohistochemistry (IHC) and assessed relative to the pathogenesis of UC-associated neoplasia.Eighteen patients (12.8%) had UC-associated neoplasia including low-grade or high-grade dysplasia and UCAC. IHC scores of 8-NG in UC-associated neoplasia group was significantly higher than in non-neoplasia group (P0.0001). In contrast, IHC score of 8-oxodG in non-neoplasia group was significantly decreased compared with UC-associated neoplasia group (P=0.0028). In logistic regression analysis, duration of disease8 years, high IHC scores of 8-NG, and low 8-oxodG in the rectal mucosa were significantly associated with the development of UC-associated neoplasia (P0.01). The expression of 8-NG was more frequently observed in patients with UCAC than in patients with sporadic colorectal cancer (P0.01).These results suggest that evaluating the expression levels of 8-NG in the rectal mucosa may be a useful biomarker for detecting patients with UC-associated neoplasia.

Preoperative chemoradiotherapy (CRT) is an effective tool for local control that functions by inducing cancer cell apoptosis and inhibiting cell growth. The aim of this study was to evaluate the expression of caspase-cleaved keratin 18 cytoskeletal protein, M30, which is known as an apoptotic marker in residual rectal cancer following preoperative CRT. A total of 72 patients with rectal cancer who had undergone preoperative CRT were enrolled in this study. Immunostaining with M30 cytodeath antibody was performed and the correlation between M30 staining and clinicopathological variables was analyzed. Furthermore, we examined the correlation of M30 staining with the expression of Bax, Bcl-2, Ki67 and PCNA using transcriptional and immunohistochemical analyses. The results showed that 34 (47%) patients were positive for M30 staining. Lack of M30 expression was significantly correlated with advanced T stage, postoperative stage and tumor recurrence (P

Slug and Vimentin genes play a critical role in regulating epithelial-mesenchymal transition (EMT) via downregulation of epithelial markers and upregulation of mesenchymal markers. The present study evaluated the clinical significance of Slug and Vimentin expression as potential disease biomarkers in colorectal cancer (CRC). At first, the biological role of Slug in CRC was assessed by RNA interference in CRC cell lines to assess tumor progression, invasion and migration. Next, we analyzed Slug and Vimentin expression in surgical tissue specimens from 181 CRC patients (Cohort 1) by quantitative real-time reverse transcription-PCR and 208 patients (Cohort 2) by immunohistochemistry. Knockdown of Slug using small interfering RNA in CRC cell lines resulted in inhibition of EMT, reduced cell proliferation, invasion and migration in CRC cells. Interestingly, Slug and Vimentin expression in cancer tissues was significantly higher in patients with higher T stage, lymph node involvement, liver metastasis and advanced tumor node metastasis stages. A significant correlation was observed between Slug and Vimentin expression in CRC (messenger RNA: ρ = 0.546, protein: ρ = 0.405), and increased expression of Slug and Vimentin was significantly associated with poor prognosis. Furthermore, increased expression of Slug emerged as an independent prognostic factor and a predictive marker of lymph node metastasis in CRC patients. Our data provide novel evidence for the biological and clinical significance of Slug and Vimentin expression as potential predictive biomarkers for identifying patients with lymph node metastasis or poor prognosis in CRC.

Chronic inflammation increases the risk of developing several gastrointestinal malignancies. Chemokines that are produced by colonic epithelial cells play significant roles in the maintenance and repair of the epithelial barrier. The present study aimed to clarify whether the expression of CCL20 and its receptor, CCR6, was correlated with the development of ulcerative colitis (UC)-associated neoplasia. A total of 93 patients with UC who underwent proctocolectomies were enrolled in the present study. Immunohistochemical analysis for CCL20 and CCR6 expression in the rectal mucosa was performed and the correlation between expression and the pathogenesis of UC-associated neoplasia was investigated. A total of 16 (17.2%) patients presented with UC-associated neoplasia. The immunohistochemistry (IHC) score for CCL20 was significantly increased in the patients with a mild form of the disease (P=0.0363). The IHC score for CCL20 expression in the patients with UC-associated neoplasia was higher compared with the patients without neoplasia (P=0.0294). In contrast, there was no significant correlation between CCR6 expression and the clinicopathological variables. The logistic regression analysis revealed that a high IHC score for CCL20 expression in the rectal mucosa and a disease duration of more than eight years were significantly correlated with the development of UC-associated neoplasia (P