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Yu Liu,1,2,* Wenju Chang,1– 3,* Wentao Tang,1– 3,* Ye Wei,1– 3 Tianyu Liu,1,2 Yijiao Chen,1,2 Meiling Ji,1– 3 Fei Liang,4 Li Ren,1– 3 Jianmin Xu1– 3 1Colorectal Cancer Center, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China; 2Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China; 3Shanghai Engineering Research Center of Colorectal Cancer Minimally Invasive, Shanghai, People’s Republic of China; 4Department of Biostatistics, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jianmin XuDepartment of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of ChinaTel +86 13501984869Email xujmin@aliyun.comBackground: Rectal gastrointestinal stromal tumors (GISTs) are rare digestive tumors. The treatment methods of rectal GISTs are diverse, while no standardized treatment was recommended. The aim of this study was to report the treatment mode of rectal GISTs in our center.Methods: Patients with pathologically confirmed rectal GISTs who received neoadjuvant therapy and surgical resection were retrospectively collected. Perioperative complications and long-term prognosis were evaluated.Results: From January 2010 to December 2019, 36 patients were pathologically diagnosed with primary rectal GISTs. After excluding patients who were treated with surgery or imatinib alone, 21 patients received neoadjuvant therapy and surgery was enrolled. During neoadjuvant treatment, tumors shrank significantly (6.53cm to 4.68cm, p< 0.001) without toxicities over grade 2. The total postoperative complication rate was 42.9% (all grade). R0 resection was achieved in 76.2% patients. Transanal resection had advantages on anus preservation and postoperative recovery. No patients died during the follow-up period, 4 patients relapsed and the relapse-free survival was 81.0%.Conclusion: The combination of neoadjuvant therapy and surgical resection was a safe and effective treatment for rectal gastrointestinal stromal tumors.Keywords: rectal gastrointestinal stromal tumors, GISTs, neoadjuvant therapy, surgical resection

Background Anastomotic leakage (AL) is a severe complication of low anterior resection (LAR) for rectal cancer, and effective prevention is urgently needed. In the robotic era, this study aimed to explore the role of innovative techniques in preventing AL in rectal cancer patients undergoing robotic LAR. Methods From May 2012 to May 2017, a total of 601 patients underwent robotic LAR, with 191 patients participated as control subjects (non-PST group) and 410 patients are subjected to a trinity technique (PST group). The AL rate, short-term and long-term outcomes are analyzed and compared. Results The overall rate of AL was 6.8% out of 601 patients, with Grade B at 5.7% and Grade C at 1.1%, using the ISREC grading system. The PST group presented lower incidence of both overall AL (5.1% vs 10.5%, P = 0.015) and major AL (0.2% vs 3.2%, P = 0.005), when compared with the non-PST group, respectively. Furthermore, the PST group had similar surgical complications (17.3% vs 20.9%, P = 0.286), while with lower re-hospitalization rate (2.7% vs 6.3%, P = 0.038) and reoperation rate (0.2% vs 4.2%, P = 0.001), compared with the non-PST group, respectively. Short-term recovery and long-term oncological outcomes were not significant in the two groups. By multivariate logistic regression models, the risk factors of AL of robotic LAR are confirmed as non-PST technique, estimated blood loss ≥100 mL, anastomosis from anal verge Conclusions The innovative PST technique may shed light on an effective method for preventing occurrence of AL in robotic LAR.

Researches focusing on the effects of alternative splicing (AS) on relapse of rectal cancer is little and signature based on the AS is blank. In this study, bioinformatic analysis was performed to identify and analyze the relapse-associated ASs, a signature was also constructed. In conclusion, 829 relapse-associated ASs of 676 mRNA were identified. 603 proteins with 2119 interactions were involved in the PPI (protein-protein interactions) network. 43 relapse-associated ASs and 64 SFs (splicing factors) with 160 interactions were indicated. Finally, we built a robust signature to predict the relapse of I–III rectal cancer with a high AUC (0.98) of ROC at 1 year. Based on the ASs involved in the signature, 4 molecular subgroups that could distinguish the relapse rate in diverse groups were identified. Our research provided an overview of relapse-associated ASs in I–III rectal cancer.

Introduction Major histocompatibility complex (MHC) plays an important role in colorectal cancer (CRC) immunity. However, the function of MHC class I chain-related B (MICB) molecule is not very clear. In this study, we explored the prognostic effect of MICB in colorectal cancer. Material and methods From 2008-05 to 2012-11, consecutive CRC patients of Zhongshan Hospital, Fudan University were retrospectively enrolled as primary cohort. The inclusion criteria were as follows: receiving primary radical resection, pathologically confirmed colorectal adenocarcinoma, no treatment before surgery, clinicopathological data available. Another cohort of CRC patients were collected from a public dataset GSE39582 of GEO database from 1987 to 2007 in the same criteria for validation. MICB was detected using immunochemistry and evaluated as prognostic biomarker. The cut-off value of MICB expression was calculated using X-tile software. Results Finally, 863 patients were enrolled in the primary cohort, and 556 patients were enrolled in the validation cohort. MICB expression was significantly associated with tumor size and primary histological type in primary cohort, and with primary tumor location and distant metastases in validation cohort. The survival analysis showed that patients with high MICB expression had significantly better overall survival in both primary (P = 0.002) and validation (P = 0.001) cohorts. The multivariate analysis also confirmed that high MICB expression was a significantly independent protective factor for overall survival in both primary (hazard ratio HR = 0.741, 95% CI 0.594–0.924) and validation (HR = 0.699, 95% CI 0.508–0.961) cohorts. Conclusion For stage I–IV CRC patients, MICB was confirmed a novel independent prognostic factor. It could help better stratification of CRC prognosis.

Background: The tumour microenvironment (TME) plays a crucial role in tumour progression and metastasis. However, the infiltration pattern of TME cells in CRC samples and the immune escape mechanism underneath have not been studied sufficiently. Methods: Transcriptomic data from public datasets were retrieved online. In total, 1802 samples from the microarray dataset and 619 samples from the TCGA dataset were enrolled. The ssGSEA algorithm and unsupervised clustering were used for TME cells infiltration speculation and infiltration pattern recognition. Findings: CRC samples can be classified into three distinct TME cell subtypes. Subtype 1, the immune-active subtype, was characterised by high infiltration of activated adaptive immune cells. Subtype 2, the immune-desert subtype, featured high tumour purity and low infiltration of immune and stromal cells. And subtype 3, stroma-rich subtype, had high infiltration of stromal cells. The stroma-rich subtype conferred a significantly worse prognosis. Three subtypes had different immune escape mechanisms. The immune-active subtype has the highest immune checkpoint expression level. In comparison, the immune-desert subtype had the lowest immunogenicity and the defective antigen presentation. And the stroma-rich subtype lacked activated immune cells. Interpretation: Distinct TME cell subtypes and immune escape mechanisms may provide inspiration and direction for further researches on CRC immunotherapy. Funding Information: This work was supported by the National Natural Science Foundation of China (No. 82002517, 82072678), the Shanghai Sailing Program (20YF1407100), and Shanghai Science and Technology Committee Project (18140903200). Declaration of Interests: The authors report no conflict of interest.

Backgrounds Colorectal cancer (CRC) is one of the cancers with the highest mortality rate and understanding the mechanism behind its progression is the key to improve patients’ prognosis. The role of miR-500a-3p has been demonstrated to be involved in the progression of several human cancers but its role in CRC is unclear. The aim of this study was to disclose the function of miR-500a-3p in CRC progression. Methods The expression of miR-500a-3p and Cyclin-dependent kinases 6 (CDK6) in 134 CRC tissues were tested respectively. The effects on cell proliferation by miR-500a-3p were explored in vitro and in vivo. The glycolysis of CRC cells was determined by Mass Spectrometry and Seahorse XF 96 Extracellular Flux Analyzer. Dual-luciferase reporter assay were performed to validate the relationship between miR-500a-3p and CDK6. Results miR-500a-3p was abnormally down-regulated in CRC tissues and cell lines and was negatively associated with malignancies and worse prognosis. miR-500a-3p mimics impeded CRC cells proliferation in vitro and in vivo. miR-500a-3p inhibited glucose consumption, lactate and ATP production, down-regulated the expression of hexokinase2 (HK2). Bioinformatics database prediction combined with western blot and luciferase assay confirmed that CDK6 is the direct target of miR-500a-3p. CDK6 overexpression was able to rescue the inhibitory function of miR-500a-3p on the proliferation and glycolysis in CRC cells. Conclusions The results of this study reveal a potential tumor suppressor role of miR-500a-3p by inhibiting proliferation and aerobic glycolysis through targeting CDK6 in CRC, which may provide new insights into novel prognostic biomarker and therapeutic targets for CRC.

The molecular classification of patients with colon cancer is inconclusive. The gene set enrichment analysis (GSEA) of dysregulated genes among normal and tumor tissues indicated that the cell cycle played a crucial role in colon cancer. We performed univariate Cox regression analysis to find out the prognostic-related genes, and these genes were then intersected with cell cycle-associated genes and were further recognized as prognostic and cell cycle-associated genes. Unsupervised non-negative matrix factorization (NMF) clustering was performed based on cell cycle-associated genes. Two subgroups were identified with different overall survival, clinical features, cell cycle enrichment profile, and mutation profile. Through nearest template prediction (NTP), the molecular classification could be effectively repeated in the original data set and validated in several independent data sets indicating that the classification is highly repeatable. Furthermore, we constructed two prognostic signatures in two subgroups, respectively. Our molecular classification based on cell cycle may provide novel insight into the treatment and the prognosis of colon cancer.

Colorectal cancer is a major cause of death worldwide, and the identification of new diagnostic and prognostic biomarkers is crucial to develop new strategies to avoid colorectal cancer-related deaths. Small nuclear ribonucleoprotein polypeptide N (SNRPN) is an imprinted gene that plays an important role in various neurodevelopmental disabilities. In this study, SNRPN was highly expressed in colorectal cancer tissues and involved in the progression of this disease. Immunohistochemistry analysis of 1,310 colorectal cancer tissue samples showed that SNRPN highly expressed in cancer tissues than in adjacent tissues and was mainly localized in the nucleus. Clinical pathological factor analysis demonstrated that higher expression of SNRPN was significantly associated with larger tumor size, location of the tumor on the left-sided colon, neural invasion, and distant metastasis. Univariate and multivariate analyses showed that SNRPN expression was an independent risk factor for survival, with high expression levels indicating worse overall survival. Both in vitro and in vivo experiments confirmed that high expression of SNRPN was associated with tumor proliferation, cell cycle, and metastasis. Knocking down SNRPN blocked the cell cycle at the G2/M phase transition and promoted tumor cell apoptosis, inhibiting the progression of colorectal cancer. To explore the up-steam of SNRPN, we found by luciferase reporter assay and chromosomal immunoprecipitation assay that E2F8 was a transcriptional regulator up-steam of SNRPN in colorectal cancer. Systematic studies of SNRPN will help us discover new regulatory molecules and provide a theoretical basis for finding new molecular targets for this disease.

Background: Existing studies for ferroptosis and prognosis in colorectal cancer (CRC) were limited. In this study, we aim to investigate the prognostic role of ferroptosis markers in patients with CRC and exploration of its micro-environmental distributions. Methods: A total of 911 patients from 2008 to 2013 with CRC were enrolled. Immunohistochemical staining was performed for CRC patients’ tissue microarray. Selection and prognostic validation of markers were based on mRNA data from the cancer genome atlas (TCGA) database. Gene Set Enrichment Analysis (GSEA) was performed to indicate relative immune landmarks and hallmarks. Ferroptosis and immune contexture were examined by CIBERSORT. Survival outcomes were analyzed by Kaplan-meier analysis and cox analysis. Results: A panel of 42 genes was selected. Through mRNA expression difference and prognosis analysis, GPX4, NOX1 and ACSL4 were selected as candidate markers. By IHC, increased GPX4, decreased NOX1 and decreased FACL4 indicate poor prognosis and worse clinical characteristics. Ferroptosis score based on GPX4, NOX1 and ACSL4 was constructed and validated with high C-index. Low ferroptosis score can also demonstrate the better progression free survival and better adjuvant chemotherapy (ACT) responsiveness. Moreover, tumor with low ferroptosis score tend to be infiltrated with more CD4+ T cells, CD8+ T cells and less M1 macrophage. Finally, we found that IFN-γ was potentially the central molecule at the crossroad between ferroptosis and onco-immune response. Conclusion: Ferroptosis plays important role on CRC tumor progression, ACT response and prognosis. Ferroptosis contributes to immune-supportive responses and IFN-γ was the central molecule for this process. Funding Statement: This work was supported by National Natural Science Foundation of China (Grant No. 81602040, 81903067 and 81402341), Clinical science and technology innovation project of Shanghai (SHDC12016104) and Shanghai Science and Technology Committee Project (17411951300). Declaration of Interests: The authors declare no conflicts of interest for the publication of this manuscript. Ethics Approval Statement: Written informed consent was obtained by all the patients. The study protocol followed the ethical guidelines of the Declaration of Helsinki and was approved by the Ethical Committee of Zhongshan Hospital of Fudan University. The ethics approval ID was B2017-166R.

Backgrounds Cancer-related mortality in patients with colorectal cancer (CRC) is predominantly caused by development of colorectal liver metastases (CLMs). How to screen the sensitive chemotherapy and targeted therapy is the key element to improve the prognosis of CLMs patients. The study aims to develop patient-derived organoids-based xenografted liver metastases (PDOX-LM) model of CRC, to recapitulate the clinical drug response. Methods We transplanted human CRC primary tumor derived organoids in murine spleen to obtain xenografted liver metastases in murine liver. Immunohistochemistry (IHC) staining, whole-exome and RNA sequencing, and drug response testing were utilized to identify the homogeneity in biological and genetic characteristics, and drug response between the PDOX-LM models and donor liver metastases. Results We successfully established PDOX-LM models from patients with CLMs. IHC staining showed that positive expression of CEA, Ki67, VEGF, FGFR2 in donor liver metastases were also well preserved in matched xenografted liver metastases. Whole-exon sequencing and transcriptome analysis showed that both xenografted and donor liver metastases were highly concordant in somatic variants (≥ 0.90 frequency of concordance) and co-expression of driver genes (Pearson’s correlation coefficient reach up to 0.99, P = 0.001). Furthermore, drug response testing showed that the PDOX-LM models can closely recapitulated the clinical response to mFOLFOX6 regiments. Conclusions This PDOX-LM model provides a more convenient and informative platform for preclinical testing of individual tumors by retaining the histologic and genetic features of donor liver metastases. This technology holds great promise to predict treatment sensitivity for patients with CLMs undergoing chemotherapy.

Liver metastasis is the main reason for the poor prognosis of colorectal cancer, and identifying molecules involved in liver metastases of colorectal cancer may provide effective therapeutic targets. Zinc-α2-glycoprotein 1(AZGP1) is a candidate biomarker for diagnosis and prognosis in cancer. However, its function and molecular mechanism in metastatic colorectal cancer remains largely unknown. We previously found that up-regulated AZGP1 promotes proliferation, migration and invasion in colorectal cancer cell line, here we elucidated the mechanism of AZGP1 in regulating metastasis. In this article, we found that AZGP1 was also highly expressed in colorectal cancer tissues with liver metastasis relative to those without metastasis, and abundant expression of AZGP1 was associated with poor prognosis, also, AZGP1 down regulation prevented cell metastasis in vivo and in vitro. We further demonstrated that AZGP1 promotes metastasis by regulating the epithelial-mesenchymal transition (EMT) and associating with molecules involved in the focal adhesion pathway, including the adhesion molecule FLNA, which acts as an important protein interactor. More importantly, AZGP1 down regulation inhibited the phosphorylation of FLNA mediated by the restrain of PAK2 kinase, thereby inducing its proteolysis and subsequently affecting its subcellular localization, where it regulates the EMT and promotes metastasis. Collectively, these results highlight AZGP1 as a new and promising therapeutic molecule for liver metastatic colorectal cancer.

The role of mast cells (MCs) in colorectal cancer (CRC) progression was controversial. Thus, our study was designed to evaluate the prognostic value of MCs as well as their correlation with immune microenvironment. A retrospective cohort of CRC patients of stages I-IV was enrolled in our study. Consecutive patients (854) were divided into training set (427 patients) and validation set (427 patients) randomly. The findings were further validated in a GEO cohort, GSE39582 (556 patients). The mast cell density (MCD) was measured by immunohistochemical staining of tryptase or by CIBERSORT algorithm. Low MCD predicted prolonged overall survival (OS) in training and validation set. Moreover, MCD was identified as an independent prognostic indicator in both sets. Better stratification for CRC prognosis can be achieved by building a MCD based nomogram. The prognostic role of MCD was further validated in GSE39582. In addition, MCD predicted improved survival in stages II and III CRC patients receiving adjuvant chemotherapy (ACT). Multiple immune pathways were enriched in low MCD group while cytokines/chemokines promoting anti-tumor immunity were highly expressed in such group. Furthermore, MCD was negatively correlated with CD8+ T cells infiltration. In conclusion, MCD was identified as an independent prognostic factor, as well as a potential biomarker for ACT benefit in stages II and III CRC. Better stratification of CRC prognosis could be achieved by building a MCD based nomogram. Moreover, immunoactivation in low MCD tumors may contributed to improved prognosis.

Peng Zheng,* Li Ren,* Qingyang Feng,* Dexiang Zhu,* Wenju Chang, Guodong He, Meiling Ji, Mi Jian, Qi Lin, Tuo Yi, Ye Wei, Jianmin Xu Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China *These authors contributed equally to this work Purpose: To investigate differences in clinical characteristics and mutational patterns between synchronous and metachronous colorectal liver metastases (CLMs). Patients and methods: From June 2008 to December 2014, patients with RAS wild-type CLMs treated at Zhongshan Hospital, Fudan University were included. DNA extracted from formalin-fixed paraffin-embedded tissue of primary tumors was sequenced with next-generation sequencing for single-nucleotide polymorphism of 96 genes according to custom panel. Mutations were compared between synchronous and metachronous liver metastases and correlated with clinical characteristics. Results: A total of 161 patients were included: 93 patients with synchronous CLM and 68 patients with metachronous CLM. Patients with metachronous CLM were obviously elder. For pathology of primary tumors, synchronous CLMs were larger in size, poorly differentiated, and more frequently local advanced and lymph node positive. For evaluation of liver metastases, synchronous CLM had more and larger metastatic lesions. The median number of mutations in synchronous CLMs was significantly higher than in metachronous group (22 vs. 18, p

Decidual protein induced by progesterone (DEPP/DEPP1/C10ORF10) is induced by denying access to food and reduced by refeeding in insulin-sensitive organs in vivo. The negative regulation of DEPP by insulin is also proven in several cell lines. However, the functions of DEPP in insulin-sensitive organs remain unknown. In the present study, we investigated the impact of DEPP on hepatic energy metabolism and addressed the underlying mechanisms. The metabolic effects of DEPP were investigated in mice with adenovirus-mediated hepatic overexpression. Liver triglyceride (TG), glycogen, and serum metabolites were detected by biochemical assays. Energy homeostasis was measured by indirect calorimetry. Quantitative PCR was used to examine expression of genes involved in fatty acid oxidation, ketogenesis, lipogenesis, and gluconeogenesis. To evaluate the role of fibroblast growth factor 21 (FGF21) mediating the metabolic effects of DEPP, FGF21 antibody was administrated intraperitoneally to mice at 24 h after the delivery of adenovirus, and the metabolic alterations were examined. Reactive oxygen species (ROS) levels were measured by catalase activity assay, live cell fluorescence, or quantitative PCR. Effects of DEPP on the phenotype of db/db mice were also assessed. Acute hepatic overexpression of DEPP significantly reduced serum glucose and TG levels, dramatically elevated β-hydroxybutyrate levels, and improved glucose clearance. Compared with controls, DEPP overexpression reduced food intake, the energy expenditure rate, and the respiratory quotient. DEPP overexpression significantly increased fatty acid oxidation and ketogenesis but suppressed lipid synthesis and gluconeogenesis. Investigations of the underlying mechanisms revealed that DEPP regulates energy metabolism by inducing oxidative stress. With the impairment of the ROS scavenging system and promotion of ROS formation, DEPP overexpression leads to ROS accumulation. FGF21 is upregulated in response to oxidative stress and mediates the effects of DEPP on fatty acid oxidation, ketogenesis, and lipid synthesis but not gluconeogenesis, as evidenced by the fact that the FGF21 antibody dramatically suppressed a DEPP-induced increase of fatty acid oxidation and ketogenesis, reversed the reduction of lipid synthesis, but did not change the suppression of gluconeogenesis. Moreover, overexpression of DEPP in db/ db mice led to a marked reduction in body weight and serum glucose levels and significantly improved insulin sensitivity. Hepatic overexpression of DEPP in mice promotes fatty acid oxidation and ketogenesis and suppresses lipogenesis and gluconeogenesis, which is partly mediated by FGF21 induced by elevated cellular ROS levels.-Li, W., Ji, M., Lin, Y., Miao, Y., Chen, S., Li, H. DEPP/DEPP1/C10ORF10 regulates hepatic glucose and fat metabolism partly via ROS-induced FGF21.

An increasing number of studies have demonstrated that circular RNAs (circRNAs) can regulate gene expression through interacting with microRNAs. In this study, we analyzed the expression of antisense to CDR1as in colorectal cancer (CRC). CDR1as had a higher expression in CRC tissues compared to adjacent, normal mucosa and was positively associated with tumor size, T stage, lymph node metastasis, and poor overall survival (OS). Downregulation of CDR1as suppressed CRC cell proliferation and invasion and increased microRNA-7 (miR-7) expression. Intriguingly, ectopic expression of miR-7 in CRC cells consistently inhibited proliferation and invasion, and the miR-7 inhibitor was able to rescue the function of CDR1as knockdown. Mechanistic studies demonstrated that CDR1as silencing suppressed EGFR and IGF-1R expression, which could be partially blocked by the miR-7 inhibitor. Finally, positive correlations between CDR1as expression and EGFR and IGF-1R expression were observed in CRC samples. Thus, given the importance of CDR1as in blocking miR-7 and positively regulating EGFR and IGF-1R, dysregulated CDR1as expression may play an important role in CRC progression.

Chunzhi Qin,1,* Li Ren,1,* Meiling Ji,1,* Shixu Lv,2,* Ye Wei,1 Dexiang Zhu,1 Qi Lin,1 Pingping Xu,1 Wenju Chang,1 Jianmin Xu1 1Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, 2Department of Surgical Oncology, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China *These authors contributed equally to this work Background: CDKL1 is a member of the cell division cycle 2 (CDC2)-related serine threonine protein kinase family and is overexpressed in malignant tumors such as melanoma, breast cancer, and gastric cancer.Objective: This study aimed to evaluate whether CDKL1 can serve as a potential molecular target for colorectal cancer therapy.Materials and methods: Expression of CDKL1 in colorectal cancer tissues and cell lines was measured by immunohistochemistry and Western blot, respectively. To investigate the role of CDKL1 in colorectal cancer, CDKL1-small hairpin RNA-expressing lentivirus was constructed and infected into HCT116 and Caco2 cells. The effects of RNA interference (RNAi)-mediated CDKL1 downregulation on cell proliferation and invasion were assessed by CCK-8, colony formation, transwell, and tumorigenicity assays in nude mice. The effects of CDKL1 downregulation on cell cycle and apoptosis were analyzed by flow cytometry. Furthermore, microarray method and data analysis elucidated the molecular mechanisms underlying the phenomenon.Results: CDKL1 protein was overexpressed in colorectal cancer tissues compared with paired normal tissues. Knockdown of CDKL1 in HCT116 and Caco2 significantly inhibited cell growth, colony formation ability, tumor invasion, and G1–S phase transition of the cell cycle. The knockdown of CDKL1 stimulated the upregulation of p15 and retinoblastoma protein.Conclusion: CDKL1 plays a vital role in tumor proliferation and invasion in colorectal cancer in vitro and in vivo and, thus, may be considered as a valuable target for therapeutic intervention. Keywords: CDKL1, cell cycle, protein kinases, colorectal cancer

Many genes and mutations have been reported for colorectal cancer (CRC); however, very few have been associated with colorectal cancer liver metastasis (CRLM). We performed gene expression profiling experiments to identify genetic markers for CRLM and elucidate the molecular mechanisms. Microarray experiments were performed on CRC primary tumor samples with or without liver metastasis (LM) using the Affymetrix U133 plus 2.0 GeneChip Array. A new identified gene-scinderin (SCIN) was overexpressed with synchronous LM at both the RNA level evaluated with quantitative real-time PCR and protein level evaluated with immunohistochemistry and also with short overall survival analyzed with Kaplan-Meier method. With multivariate analysis indicated that SCIN served as an independent poor prognostic predictor for CRC patients. Disease-free survival was also significantly lower in SCIN overexpressing CRC patients with metachronous LM. In addition, SCIN knockdown significantly reduced cell proliferation, induced cell cycle arrest, and promoted the expression of some cell cycle apoptosis-related protein. Moreover, the DIAPH1, STAT3, CDK2, CDK4, and EGFR levels were downregulated, whereas CDKN2B and COL4A1 were upregulated in DLD-1-shSCIN cells by microarray analysis compared with DLD-1 shCon cells. These findings revealed that SCIN may serve as an important predictor of CRLM and poor outcome for CRC patients. SCIN may be a potential therapeutic target in human CRC. However, translation of its roles into clinical practice will require further investigation and additional experimental validation.

Background: In this paper, we aim to explore clinical value of skeletal muscle index (SMI) and prognostic nutrition index (PNI) on resected colorectal cancer liver metastasis (CRLM). Results: Among the 539 patients, 355 were males. Baseline lower SMI was associated with smaller BMI, smaller PNI, smaller pre-albumin and longer hospitalization days (P

Colorectal cancer (CRC) is the third leading cause of death from cancer worldwide. Immature colon carcinoma transcript-1 (ICT1) has been reported to be correlated with lung cancer; however, whether ICT1 is a functional gene in CRC, as well as the molecular mechanism underlying ICT1 mediation of colorectal-tumor formation, remains unknown.The expression of ICT1 was firstly determined by using immunohistochemistry in 861 CRC specimens. The correlation of ICT1 expression with clinicopathological parameters and the survival rate was analyzed. Furthermore, we investigated the effect of ICT1 silencing on CRC cell proliferation and migration by MTT, colony formation, flow cytometry, and transwell in vitro.ICT1 is highly expressed in a cohort of human CRCs, and that higher ICT1 expression may lead to reduced overall survival rate of CRC. Likewise, ICT1 silencing lowered the cell viability through cell-cycle arrest, inhibited cell migration, and induced apoptosis in CRC. We further revealed a novel mechanism in which ICT1 promoted CRC growth via the intracellular AMPK, SAPK/JNK, and PARP signaling pathways.Our data showed that ICT1 could be an important target for CRC diagnosis and treatment.

Yihao Mao,1,* Qingyang Feng,1,2,* Peng Zheng,1,* Liangliang Yang,1,* Tianyu Liu,1 Yuqiu Xu,1 Dexiang Zhu,1,2 Wenju Chang,1,2 Meiling Ji,1,2 Li Ren,1,2 Ye Wei,1,2 Guodong He,1,2 Jianmin Xu1,2 1Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200030, China; 2Shanghai Engineering Research Center of Colorectal Cancer Minimally Invasive, Shanghai 200030, China *These authors contributed equally to this work Purpose: Tumor purity is defined as the proportion of cancer cells in the tumor tissue. The impact of tumor purity on colon cancer (CC) prognosis, genetic profile, and microenvironment has not been thoroughly accessed.Materials and methods: Clinical and transcriptomic data from three public datasets, GSE17536/17537, GSE39582, and TCGA, were retrospectively collected (n=1,248). Tumor purity of each sample was inferred by a computational method based on transcriptomic data. Survival-related analyses were performed on microarray dataset containing GSE17536/17537 and GSE39582 (n=794), whereas TCGA dataset was utilized for subsequent genomic analysis (n=454).Results: Right-sided CC patients showed a significantly lower tumor purity. Low purity CC conferred worse survival, and tumor purity was identified as an independent prognostic factor. Moreover, high tumor purity CC patients benefited more from adjuvant chemotherapy. Subsequent genomic analysis found that the mutation burden was negatively associated with tumor purity, with only APC and KRAS significantly more mutated in high purity CC. However, no somatic copy number alteration event was correlated with tumor purity. Furthermore, immune-related pathways and immunotherapy-associated markers (programmed cell death protein 1 [PD-1], programmed death-ligand 1 [PD-L1], cytotoxic T-lymphocyte-associated protein 4 [CTLA-4], Lymphocyte-activation gene 3 [LAG-3] and T-cell immunoglobulin and mucin-domain containing-3 [TIM-3]) were highly enriched in low purity samples. Notably, the relative proportion of M2 macrophages and neutrophils, which indicated worse survival in CC, was negatively associated with tumor purity.Conclusion: Tumor purity exhibited potential value for CC prognostic stratification as well as adjuvant chemotherapy benefit prediction. The relative worse survival in low purity CC may attribute to higher mutation frequency in key pathways and purity-related microenvironmental changing. Keywords: adjuvant chemotherapy, colon cancer, prognosis, tumor microenvironment, tumor purity

Purpose: For stage II colon cancer, the efficacy of postoperative adjuvant chemotherapy remains controversial. It is well known that tumor-associated macrophages (TAMs) are important in tumor progression. In this study, TAMs were investigated as prognostic and predictive biomarkers for the efficacy of adjuvant chemotherapy for stage II colon cancer after radical resection. Experimental Design: This study enrolled two independent cohorts of consecutive patients from one medical center with pathologic stage II colon cancer after radical resections. Macrophages were detected using IHC staining of CD68 and CD206. Infiltration densities of CD68+ TAMs, CD206+ TAMs, and ratio of CD206+ TAMs/CD68+ TAMs (CD206/CD68 ratio) were calculated as prognostic and predictive biomarkers. Results: The primary and validation cohorts consisted of 521 and 314 patients, respectively. In both cohorts, high CD206/CD68 ratio was significantly associated with poor disease-free survival (DFS) and overall survival (OS). As an independent risk factor, CD206/CD68 ratio also had significantly better prognostic efficacy than CD68+ TAM density, CD206+ TAM density, and traditional clinicopathologic high-risk factors. Moreover, adjuvant chemotherapy significantly improved DFS and OS for patients with high CD206/CD68 ratio but not for those with low CD206/CD68 ratio. The interaction analyses were also significant for DFS. In subgroup analysis, CD206/CD68 ratio was still a significant predictor for adjuvant chemotherapy for patients in traditional high-risk group of recurrence (significant interaction for DFS). Conclusions: For stage II colon cancer, CD206/CD68 ratio is a better prognostic and predictive biomarker for postoperative adjuvant chemotherapy. Together with clinicopathologic high-risk factors, it will aid in precision treatment.

Circulating cell-free DNA (cfDNA) has become a potential diagnostic and prognostic biomarker for colorectal cancer (CRC). In non-cancerous diseases, it has been confirmed that cfDNA can be recognized by Toll-like receptor 9 (TLR9), leading to a significant biological change. Nevertheless, the biological significance of cfDNA and its relationship with TLR9 in tumor malignancy is still unclear. Therefore, the purpose of this study is to explore the biological role of cfDNA in colorectal cancer (CRC). The expression of TLR9 was measured in different CRC cell lines and cancerous samples by RT-PCR or immunohistochemistry, which showed that high expression of TLR9 was significantly correlated with the tumor metastasis, advanced TNM stage and poor prognosis of patients. Then, cfDNA was obtained from fluorouracil (5FU)-induced apoptotic cancer cells in vitro and transfection techniques were used to transfect siRNA and cDNA plasmid for TLR9. Cancer cells were stimulated using isolated cfDNA fragments, and results showed that cfDNA could promote colorectal cancer cell proliferation via TLR9. Meanwhile, we demonstrated that the cfDNA binding to TLR9 could facilitate cell migration and invasion. Finally, we demonstrated that cfDNA initiated downstream TLR9-MyD88 signaling and induced robust release of chemokine interleukin 8 (IL-8), which helped to elucidate the mechanisms underlying these phenomena. Our data suggest that cancer cell-derived cfDNA contributes to cancer progression through activation of TLR9-MyD88 signaling and IL-8 secretion in CRC. These findings provide a novel perspective for understanding of tumor progression and provoke a potential therapeutic target for CRC treatment.

Purpose. We aimed to identify new predictive biomarkers for cetuximab in first-line treatment for patients with RAS wild-type metastatic colorectal cancer (mCRC). Methods. The study included patients with KRAS wild-type unresectable liver-limited mCRC treated with chemotherapy with or without cetuximab. Next-generation sequencing was done for single nucleotide polymorphism according to custom panel. Potential predictive biomarkers were identified and integrated into a predictive model within a training cohort. The model was validated in a validation cohort. Results. Thirty-one of 247(12.6%) patients harbored RAS mutations. In training cohort (N=93), six potential predictive genes, namely, ATP6V1B1, CUL9, ERBB2, LY6G6D, PTCH1, and RBMXL3, were identified. According to predictive model, patients were divided into responsive group (n=66) or refractory group (n=27). In responsive group, efficacy outcomes were significantly improved by addition of cetuximab to chemotherapy. In refractory group, no benefit was observed. Interaction test was significant across all endpoints. In validation cohort (N=123), similar results were also observed. Conclusions. In the first-line treatment of mCRC, the predictive model integrating six new predictive mutations divided patients well, indicating a promising approach to further refine patient selection for cetuximab on the basis of RAS mutations.

There are currently no accurate predictive markers of metachronous liver metastasis from colorectal cancer. Recent studies demonstrated that the expression patterns of sphingosine-1-phosphate receptor 1 (S1PR1) are altered in several tumors, but in colorectal cancer, the patterns remain unknown. Our study was designed to evaluate the expression and prognostic significance of S1PR1 protein in patients with colorectal cancer. The expression of S1PR1 was detected using the tissue microarray technique and immunohistochemical method and compared with clinicopathological parameters in 153 colorectal cancer patients. The prognostic value of S1PR1 expression was evaluated by Kaplan-Meier and Cox regression analysis. A molecular prognostic stratification scheme incorporating S1PR1 expression was determined by using receiver operating characteristic (ROC) analysis. S1PR1 was significantly highly expressed in 70.6 % (108/153) of the colorectal cancer lesions compared to their high expressions in only 5.9 % (9/153) of the adjacent non-cancerous tissues. Upregulated expression of S1PR1 was significantly associated with depth invasion and metachronous liver metastasis. Increased S1PR1 expression in colorectal cancer was positively correlated with poor overall survival. Multivariate survival analysis suggested that S1PR1 expression was an independent prognostic indicator for the disease. Applying the prognostic value of S1PR1 density to TNM stage system showed a better prognostic value in patients with colorectal cancer. Aberrant S1PR1 expression in colorectal cancer was associated with metachronous liver metastasis and worse survival outcome, and also, it was an independent prognostic factor. According to our analysis, combined TNM stage and intratumoral expression of S1PR1 demonstrated a better prognostic value than any of these two parameters alone. Conclusively, we suggest that detection and analysis of S1PR1 expression in colorectal cancer tissue might be used for predicting prognosis of colorectal cancer.

The effects of paracrine action from early activated hepatic stellate cells (HSCs) on resident liver epithelium cells are not clear. Here, we investigated whether a systemic infusion of early activated HSC-derived paracrine factors (HSC-CM) would evoke an enhanced liver protective response in acetaminophen (APAP)-induced acute liver injury (ALI) in mice and explored the possible underlying mechanisms. The survival rate, liver injury, and liver regeneration were analyzed in mice with or without HSC-CM treatment in vivo. A systemic infusion of HSC-CM provided a significant survival benefit in APAP-induced ALI. HSC-CM therapy resulted in a reduction of hepatocellular death and increased numbers of both proliferating hepatocytes and adult hepatic progenitor cells (AHPCs) with up-regulation of liver regeneration relevant genes. The HSC-CM treatment reduced leukocyte infiltration and down-regulated systemic inflammation with decreases in IFN-γ, IL-1ra, IL-1β, TNF-α, and increases in IL-10. The direct anti-death and pro-regeneration effects of HSC-CM on AHPCs were demonstrated using in vitro assays. Treatment with HSC-CM promoted AHPCs proliferation and resulted in increased pAkt expression in vitro, and this effect was abolished by the PI3K/Akt inhibitor LY294002. These data provide evidence that early activated HSC-CM therapy offered trophic support to the acutely injured liver by inhibiting liver cell death and stimulating regeneration, potentially creating a new method for the treatment of ALI.

e15582Background: For stage II colon cancer, the effect of postoperative adjuvant chemotherapy is still controversial. M2 subtype of tumor-associated macrophages (TAMs) play an important role in pr...

e15646Background: Tumor purity is the proportion of cancer cells in the tumor tissue. The impact of tumor purity on colon cancer (CC) prognosis, genetic profile and microenvironment has not been th...

Objective Currently, no published studies have compared the clinical outcomes of the medial-to-lateral approach (MA) and lateral-to-medial approach (LA) for open right hemicolectomy. Thus, the present study aimed to assess whether one of these approaches has any potential benefits over the other. Methods A retrospective study was performed of all patients who underwent open right hemicolectomy with pathologically confirmed disease who met the eligibility criteria between June 2008 and June 2012. The population was divided into an MA group and an LA group by propensity scoring. We compared patient demographic and clinical characteristic variables between the two groups and assessed short-term and long-term outcomes. Results A total of 450 patients (MA, n = 150; LA, n = 300) were evaluated. The operation time (MA,138.4 minutesvs.LA,166.2 minutes; P < .05) and blood loss (MA,52.0mL vs. LA,62.6mL; P < .05)were significantly lower in the MA group. No differences in the number of harvested lymph nodes and oncologic outcomes were observed between the two groups. Further subgroup analysis for stage III colon cancer revealed that the MA group had significantly more retrieved lymph nodes (MA,18.8vs. LA,16.0; P = .028). There were no differences in other variables between the two groups. Conclusions The MA reduced operative time and blood loss compared with the LA. We thus concluded that the MA provided short-term benefits compared with the LA in open right hemicolectomy for right-sided colon cancer.

Homeobox C6 (HOXC6), a member of the homeobox family that encodes highly conserved transcription factors, plays a vital role in various carcinomas. In this study, we used a tissue microarray (TMA) consisting of 462 CRC samples to demonstrate that HOXC6 is more abundantly expressed in colorectal cancer (CRC) tissues than adjacent normal mucosa. Clinicopathological data indicated that higher HOXC6 expression correlated with poor overall survival and was associated with primary tumor location in the right colon, primary tumor (pT) stage 3/4 and primary node (pN) stage 1/2. Multivariate analysis showed that high HOXC6 expression was an independent risk factor for poor CRC patient prognosis. HOXC6 downregulation via lentivirus-mediated expression of HOXC6-targeting shRNA reduced HCT116 cell viability and colony formation in vitro, and reduced growth of subcutaneous xenografts in nude mouse. HOXC6 thus appears to promote CRC cell proliferation and tumorigenesis through autophagy inhibition and mTOR pathway activation.

620 Background: For stage II colon cancer, the effect of postoperative adjuvant chemotherapy is still controversial. It is well known that tumor-associated macrophages (TAMs) play an important role in tumor progression. The aim of this study is to determine the effect of TAMs as predictor for adjuvant chemotherapy for stage II colon cancer. Methods: From July 2009 to June 2012, 521 patients with pathological stage II colon cancer were included. TAMs were detected using tissue microarray and immunohistochemistry (all TAMs detected by CD68; M2 subtype detected by CD206). The density of CD68+ TAMs, CD206+ TAMs and the ratio of CD206+ TAMs / CD68+ TAMs (CD206 / CD68 ratio) were calculated. The cut-off values were defined using X-Tile software. Results: High CD206+ TAMs density and high CD206 / CD68 ratio were significantly associated with reduced disease-free survival (DFS, P < 0.001 and P < 0.001, respectively) and overall survival (OS, P < 0.001 and P < 0.001, respectively). And CD206 / CD68 ratio had a better prognostic power. Furthermore, for patients with low CD206 / CD68 ratio, adjuvant chemotherapy made no benefit. But for high CD206 / CD68 ratio, adjuvant chemotherapy significantly improved DFS and OS (as shown in Table 1). In subgroup analysis, for T3 with high-risk factors or T4 tumors, CD206 / CD68 ratio was also a significant predictor for adjuvant chemotherapy (interaction P = 0.024 in DFS). Conclusions: For stage II colon cancer, CD206 / CD68 ratio was a good prognostic and predictive biomarker for adjuvant chemotherapy. Together with clinicopathological high-risk factors, it might facilitate patient counselling and individualise management. [Table: see text]

Qi Lin,# Ye Wei,# Li Ren,# Yunshi Zhong,# Chunzhi Qin, Peng Zheng, Pingping Xu, Dexiang Zhu, Meiling Ji, Jianmin XuDepartment of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, People's Republic of China#These authors contributed equally to this workBackground: Tumor deposits are one of the important influencing factors among the different editions of Tumor, Node, Metastasis classification. Incidence and prognosis of tumor deposits in stage I, II, and III colorectal cancer patients has been explored. The aim of this study was to determine the prognostic value of tumor deposits in stage IV colorectal cancer patients who underwent simultaneous resection for synchronous colorectal liver metastases (SCRLM).Methods: Clinicopathological and outcome data of 146 consecutive SCRLM patients who underwent simultaneous R0 resection between July 2003 and July 2013 were collected from our prospectively established SCRLM database. The prognostic value of tumor deposits was evaluated by Kaplan–Meier and Cox regression analysis.Results: Tumor deposits were detected in 41.8% (61/146) of these SCRLM patients. Tumor deposits were significantly correlated with lymph node metastasis and nerve invasion of the primary tumors (P=0.002, P=0.041; respectively). The Kaplan–Meier survival analysis revealed that the overall survival (OS) and disease-free survival (DFS) of SCRLM patients with tumor deposits were significantly poorer than those with no tumor deposits (P=0.039, P=0.001; respectively). And with multivariate analysis, we found that positive tumor deposits were significantly associated with shorter DFS independent of lymph node status (P=0.002). Subgroup analysis found that of the 57 SCRLM patients with negative lymph node status, the OS and DFS of patients with positive tumor deposits were significantly shorter than those with negative tumor deposits (P=0.002 and P=0.031, respectively). Of the 89 patients with positive lymph node status, the OS of patients with tumor deposits was not significantly different than those without tumor deposits (P=0.965); however, the DFS of patients with tumor deposits was significantly shorter than those with no tumor deposits (P=0.034).Conclusion: Tumor deposits may be an independent adverse prognostic factor in SCRLM patients who underwent simultaneous R0 resection.Keywords: tumor deposits, synchronous colorectal liver metastases, prognostic factors&nbsp

e15079Background: To identify predictive biomarkers for the efficacy of cetuximab. Materials and Methods: The DNA samples from 106 patients were sequenced for single nucleotide polymorphisms (SNPs)...