Total Neoadjuvant Therapy With PD-1 Blockade for High-Risk Proficient Mismatch Repair Rectal Cancer.

Importance: Total neoadjuvant therapy (TNT) is the standard treatment for locally advanced rectal cancer, especially for patients with high-risk factors. However, the efficacy of TNT combined with immunotherapy for patients with proficient mismatch repair (pMMR) rectal cancer is unknown. Objectives: To evaluate the safety and efficacy of TNT with induction chemoimmunotherapy followed by long-course chemoradiation in patients with high-risk, pMMR rectal cancer and to identify potential molecular biomarkers associated with treatment efficacy. Design, Setting, and Participants: This cohort study was a single-arm phase 2 trial conducted at Gastrointestinal Cancer Center, Peking University Cancer Hospital & Institute, from June 2020 to October 2021. Biopsies and plasma were collected before treatment for whole-exome sequencing and cell-free DNA sequencing, respectively. Data were analyzed from May 2022 to September 2022. Interventions: Participants received 3 cycles of induction oxaliplatin and capecitabine combined with camrelizumab and radiotherapy (50.6 Gy in 22 fractions) with concurrent capecitabine. Patients without disease progression received 2 cycles of consolidation oxaliplatin/capecitabine. Main Outcomes and Measures: The primary end point was pathologic complete response rate. Results: Of 25 patients enrolled (19 men [76%]; 6 women [24%]; median [IQR] age, 58 [48-64] years), 22 patients (88%) completed the TNT schedule. The pathologic complete response rate was 33.3% (7/21). Twelve patients (48%) achieved clinical complete response, and 4 patients (16%) chose to watch and wait. R0 resection was achieved in 21 of 21 patients, and the major pathologic response rate was 38.1% (8/21). The most common adverse event was nausea (80%, 20/25); grade 3 toxic effects occurred in 9 of 25 patients (36%). Patients with tumor shrinkage of 50% or greater after induction oxaliplatin/capecitabine and camrelizumab or clinical complete response had higher percentages of LRP1B mutation. Mutation of LRP1B was associated with high tumor mutation burden and tumor neoantigen burden. Patients with high tumor mutation burden all benefited from therapy. Conclusions and Relevance: This study found that TNT with induction chemoimmunotherapy followed by long-course chemoradiation was safe and effective for patients with high-risk rectal cancer with pMMR status. Longer follow-up and larger clinical studies are needed to validate this innovative regimen. There is also an urgent need to further validate the predictive value of LRP1B and discover other novel biomarkers with potential predictive value for rectal cancer.

Design and synthesis of doublecortin-like kinase 1 inhibitors and their bioactivity evaluation.

Doublecortin-like kinase 1 (DCLK) is a microtubule-associated serine/threonine kinase that is upregulated in a wide range of cancers and is believed to be related to tumour growth and development. Upregulated DCLK1 has been used to identify patients at high risk of cancer progression and tumours with chemotherapy-resistance. Moreover, DCLK1 has been identified as a cancer stem cell (CSC) biomarker in various cancers, which has received considerable attention recently. Herein, a series of DCLK1 inhibitors were prepared based on the previously reported XMD8-92 structure. Among all the synthesised compounds, D1, D2, D6, D7, D8, D12, D14, and D15 showed higher DCLK1 inhibitory activities (IC50 40-74 nM) than XMD8-92 (IC50 161 nM). Compounds D1 and D2 were selective DCLK1 inhibitors as they showed a rather weak inhibitory effect on LRRK2. The antiproliferative activities of these compounds were also preliminarily evaluated. The structure-activity relationship revealed by our compounds provides useful guidance for the further development of DCLK1 inhibitors.

Design, Synthesis, and Biological Evaluation of Proteolysis-Targeting Chimeras as Highly Selective and Efficient Degraders of Extracellular Signal-Regulated Kinase 5.

Extracellular signal-regulated kinase 5 (ERK5) is recognized as a key member of the mitogen-activated protein kinase family and is involved in tumor growth, migration, and angiogenesis. However, the results of ERK5 inhibition in multiple studies are controversial, and a highly specific ERK5-targeting agent is required to confirm physiological functions. Using proteolysis-targeting chimera technology, we designed the selective ERK5 degrader PPM-3 and examined its biological effect on cancer cells. Interestingly, the selective degradation of ERK5 with PPM-3 did not influence tumor cell growth directly. Based on proteomics analysis, the ERK5 deletion may be associated with tumor immunity. PPM-3 influences tumor development by affecting the differentiation of macrophages. Therefore, PPM-3 is an effective small-molecule tool for studying ERK5 and a promising immunotherapy drug candidate.

UVRAG Promotes Tumor Progression through Regulating SP1 in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common type of cancer. The ultraviolet radiation resistance-associated gene (UVRAG) plays a role in autophagy and has been implicated in tumor progression and prognosis. However, the role of UVRAG expression in CRC has remained elusive. In this study, the prognosis was analyzed via immunohistochemistry, and the genetic changes were compared between the high UVRAG expression group and the low UVRAG expression group using RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq) data, and genetic changes were then identified by in vitro experiments. It was found that UVRAG could enhance tumor migration, drug resistance, and CC motif chemokine ligand 2 (CCL2) expression to recruit macrophages by upregulating SP1 expression, resulting in poor prognosis of CRC patients. In addition, UVRAG could upregulate the expression of programmed death-ligand 1 (PD-L1). In summary, the relationship between UVRAG expression and the prognosis of CRC patients as well as the potential mechanisms in CRC were explored, providing evidence for the treatment of CRC.

FAP promotes metastasis and chemoresistance via regulating YAP1 and macrophages in mucinous colorectal adenocarcinoma.

Mucinous colorectal adenocarcinoma (MC) is less likely to respond to chemotherapy and is associated with poorer prognosis compared with non-MC (NMC). Fibroblast activation protein (FAP) was found and validated to be upregulated in MC patients and was negatively correlated with prognosis and therapeutic outcomes in colorectal cancer (CRC) patients who were treated with adjuvant chemotherapy. Overexpression of FAP promoted CRC cell growth, invasion and metastasis, and enhanced chemoresistance. Myosin phosphatase Rho-interacting protein (MPRIP) was identified as a direct interacting protein of FAP. FAP may influence the efficiency of chemotherapy and prognosis by promoting the crucial functions of CRC and inducing tumor-associated macrophages (TAMs) recruitment and M2 polarization through regulating theRas Homolog Family Member/Hippo/Yes-associated protein (Rho/Hippo/YAP) signaling pathway. Knockdown of FAP could reverse tumorigenicity and chemoresistance in CRC cells. Thus, FAP may serve as a marker for prognosis and therapeutic outcome, as well as a potential therapeutic target to overcome chemoresistance in MC patients.

Single-cell profiling reveals differences between human classical adenocarcinoma and mucinous adenocarcinoma.

Colorectal cancer is a highly heterogeneous disease. Most colorectal cancers are classical adenocarcinoma, and mucinous adenocarcinoma is a unique histological subtype that is known to respond poorly to chemoradiotherapy. The difference in prognosis between mucinous adenocarcinoma and classical adenocarcinoma is controversial. Here, to gain insight into the differences between classical adenocarcinoma and mucinous adenocarcinoma, we analyse 7 surgical tumour samples from 4 classical adenocarcinoma and 3 mucinous adenocarcinoma patients by single-cell RNA sequencing. Our results indicate that mucinous adenocarcinoma cancer cells have goblet cell-like properties, and express high levels of goblet cell markers (REG4, SPINK4, FCGBP and MUC2) compared to classical adenocarcinoma cancer cells. TFF3 is essential for the transcriptional regulation of these molecules, and may cooperate with RPS4X to eventually lead to the mucinous adenocarcinoma mucus phenotype. The observed molecular characteristics may be critical in the specific biological behavior of mucinous adenocarcinoma.

The implications from the interplay of neoadjuvant chemoradiotherapy and the immune microenvironment in rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) is recommended for the treatment of locally advanced rectal cancer. Even though the combination of nCRT and immune checkpoint inhibitors (ICIs) has received much attention, the specific combination modes and dose fractions in radiotherapy (RT) are still indistinct. This review focuses on the immunological mechanism involved in nCRT, the clinical efficacy, the immunological effect of different combined strategies, concurrent or sequential nCRT plus ICIs, long-course RT and short-course RT. This review discusses the impact of nCRT on tumor immunity and summarizes the availability of different dose fractions in RT and distinct combined strategies, aiming at providing clues for optimal neoadjuvant therapy options that maximize efficacy and minimize side effects.

Radiotherapy (RT) and/or chemotherapy before surgery is the most common therapeutic strategy for patients with rectal cancer. This review summarizes the changes in tumor immunity, including immune cells and immune components, after receiving RT and chemotherapy. The authors looked for clues to the proper strategy of combined RT, chemotherapy and other novel agents to magnify antitumor immunity based on these changes. In addition, it emphasizes the RT regimens implicated in distinct immune alterations, which might help improve the efficacy of RT in clinical applications.

Predictive value of proteomic markers for advanced rectal cancer with neoadjuvant chemoradiotherapy.

BACKGROUND: Preoperative neoadjuvant chemoradiation (nCRT) has been the standard treatment for locally advanced rectal cancer. Serum biomarkers to stratify patients with respect to prognosis and response to nCRT are needed due to the diverse response to the therapy. METHODS: Thirteen paired pre- and post-nCRT sera from rectal cancer patients were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) method. Twenty-five proteins were selected for validation by parallel reaction monitoring (PRM) in ninety-one patients. RESULTS: Totally, 310 proteins were identified and quantified in sera samples. Reactome pathway analysis showed that the immune activation-related pathways were enriched in response to nCRT. Twenty-five proteins were selected for further validation. PRM result showed that the level of PZP was higher in pathological complete response (pCR) patients than non-pCR patients. The Random Forest algorithm identified a prediction model composed of 10 protein markers, which allowed discrimination between pCR patients and non-pCR patients (area under the curve (AUC) = 0.886 on testing set). Higher HEP2 and GELS or lower S10A8 in baseline sera were associated with better prognosis. Higher APOA1 in post nCRT sera was associated with better disease-free survival (DFS). CONCLUSIONS: We identified and confirmed a 10-protein panel for nCRT response prediction and four potential biomarkers HEP2, GELS, S10A8 and APOA1 for prognosis of rectal cancer based on iTRAQ-based comparative proteomics screening and PRM-based targeted proteomic validation.

Surgery may not benefit patients with locally advanced rectal cancer who achieved clinical complete response following neoadjuvant chemoradiotherapy.

PURPOSE: We compared the long-term outcome of the watch and wait (WW) strategy and surgery in patients with locally advanced rectal cancer. PATIENTS AND METHODS: This prospective cohort study included 84 patients who achieved clinical complete response (cCR) after neoadjuvant chemoradiotherapy (NCRT). They were divided into the WW group (n = 58) and surgery group (SG, n = 26). Patients in the SG underwent total mesorectal excision. The study site was the Peking University Cancer Hospital. RESULTS: Eighty-four patients were included (58 and 26 in the WW group and SG, respectively). A total of 76·9% of the patients in the SG achieved pathological complete response (pCR) and 23·1% of the patients had a residual tumor. The total recurrence and metastasis rate was 15·4% (4/26) in the SG and 18·9% (11/58) in the WW group. There was no significant difference in the recurrence and metastasis rate between the two groups. In the WW group, 9 cases developed tumor regrowth during follow-up and underwent salvage surgery. The overall survival rate of the WW group (96·6% vs 92·3%) was not significantly different from that of the SG (P > 0·05). The WW patients also retained their anal sphincter function and avoided surgery-related complications. CONCLUSION: The WW strategy is a feasible treatment option in patients with cCR after NCRT. Surgery may not bring benefits to these cCR patients.

Tumor mutation burden in blood predicts benefit from neoadjuvant chemo/radiotherapy in locally advanced rectal cancer.

Distant metastasis has been the major concern of prognosis in patients with locally advanced rectal cancer (LARC). The purpose of this study was to investigate the prognostic value of TMB in blood (bTMB) in LARC patients after receiving neoadjuvant chemoradiotherapy (nCRT) and surgery. Using targeted ctDNA sequencing, we revealed that bTMB level at baseline was positively correlated with recurrence-free survival (RFS). Following nCRT, the patients with decreasing TMB tends to have a longer median RFS. bTMB level after surgery was negatively correlated with RFS. The serum cytokines including IFNγ, IFNα2, IL-1ß, IL-2 and MIP-1ß were significantly higher in pre-nCRT serum with higher bTMB group than that of lower bTMB group. Clonal evolution analysis showed that the pre- and post-nCRT ctDNAs of most cases had shared mutations. In conclusion, we presume that bTMB could potentially improve pre- and post-treatment risk assessment and facilitate individualized therapy for patients with LARC.

Combination of radiotherapy and suppression of Tregs enhances abscopal antitumor effect and inhibits metastasis in rectal cancer.

BACKGROUND: Distant metastasis is the major cause of mortality in patients with locally advanced rectal cancer (LARC) following neoadjuvant chemoradiotherapy. Local radiotherapy can trigger an abscopal response to metastatic tumor cells. However, the abscopal effect is a rare event. CD4+ regulatory T (Treg) cell is a highly immune-suppressive subset which impedes immune surveillance against cancer, prevents the development of effective antitumor immunity and promotes tumor progression. We assume that the exploitation of the proimmunogenic effects of radiotherapy with anti-CD25 or anti-Cytotoxic T-Lymphocyte Associated Protein 4 (anti-CTLA4) monoclonal antibodies (mAbs) may enhance the local and abscopal effects in rectal cancer and improve the therapeutic outcome. METHODS: mRNA expression profiling of 81 pretreatment biopsy samples from LARC patients who received neoadjuvant radiotherapy (nRT) was performed to analyze the correlation between gene expression and prognosis. A retrospective analysis of patients with rectal cancer with distant metastasis or synchronous extracolonic cancers was performed to evaluate the abscopal effect of radiotherapy on rectal cancer. Two different dual-tumor mouse models were established to investigate the efficacy of single dose and dose-fractionated radiotherapy combined with anti-CD25 or anti-CTLA4 and anti-Programmed cell death 1 ligand 1 (anti-PD1) mAbs on the local tumor growth and liver metastasis. The univariate Cox regression analysis, one-way analysis of variance, Dunnett's test, a mixed-effect linear model and Kaplan-Meier survival analysis were used to calculate p values. RESULTS: The proportion of Tregs in pre-nRT biopsies was negatively correlated with prognosis (p=0.007). The retrospective analysis showed that regressing liver metastases were infiltrated by CD8+ T cells. In contrast, stable/progressing metastases and synchronous extracolonic cancers were characterized by PD1+ T cells and Tregs infiltration. Animal experiment results demonstrated that the combination of radiotherapy and anti-CD25/CTLA4 mAb resulted in a significant increase in CD8+ T cells and CD8+/CD4+ ratio in primary and secondary tumors compared with the irradiation alone group (all p<0.05 or p<0.01). The combined treatment was able to decrease Tregs, PD1+CD8+ and PD1+CD4+ T cells (p<0.05), suppress locally irradiated and distal unirradiated tumor growth, and improve overall survival rate. Radiotherapy in conjunction with anti-CTLA4 reduced liver metastasis (p<0.05). CONCLUSIONS: These data indicated that radiotherapy plus depletion of Tregs was able to improve the antitumor response and generate an abscopal effect.

Organoid in colorectal cancer: progress and challenges.

Patient-derived tumor organoids (PDOs) currently represent important modeling tools in pre-clinical investigation of malignancies. Organoid cultures conserve the genetic and phenotypic characteristics of the original tumor and maintain its heterogeneity, allowing their application in many research fields. PDOs derived from colorectal cancer (CRC) have been used for genetic modeling to investigate the function of driver genes. Some researchers have been exploring the value of CRC PDOs in chemotherapy, targeted therapy, and radiotherapy response prediction. The successful generation of PDOs derived from CRC could deepen our understanding of CRC biology and provide novel tools for cancer modeling, for realizing precision medicine by assessing specimens from individual patients ex vivo. The present review discusses recently reported advances in CRC PDOs and the challenges they face as pre-clinical models in CRC research.

Hemolysin BL from novel Bacillus toyonensis BV-17 induces antitumor activity both in vitro and in vivo.

The gut microbiota plays an important role in cancer development and immunotherapy. Bacterial toxins have enormous antitumor potential due to their cytotoxicity and ability to activate the immune system. Using 16S rRNA gene sequencing, we compared the gut microbiota composition of fecal samples from healthy individuals and patients with colorectal cancer (CRC) and observed that the genus Bacillus was common in the healthy donors but was absent in the CRC patients. Further, we isolated a novel Bacillus toyonensis BV-17 from the fecal samples of the healthy individuals. Our results showed that the supernatant of the Bacillus toyonensis BV-17 cultures could quickly kill various tumor cell lines within minutes in vitro, by causing cell membrane disruption, blebbing, and leakage of cytoplasmic content. Fast protein liquid chromatography (FPLC) and mass spectrometry analysis identified hemolysin BL (HBL) as the effector molecule, which exhibits a different cytotoxicity mechanism compared to previous studies. Intra-tumor injection of low dose HBL inhibited the growth of both treated and untreated tumors in mice. The outcomes of this pioneer study suggest that HBL exhibits antitumor activity and is a potential chemotherapeutic agent that could be engineered to target only tumor cells in future.

Enhancement of Sensitivity to Chemo/Radiation Therapy by Using miR-15b against DCLK1 in Colorectal Cancer.

Chemo-/radiotherapy resistance is the main cause accounting for most treatment failure in colorectal cancer (CRC). Tumor-initiating cells (TICs) are the culprit leading to CRC chemo-/radiotherapy resistance. The underlying regulation mechanism of TICs in CRC remains unclear. Here we discovered that miR-15b expression positively correlated with therapeutic outcome in CRC. Expression of miR-15b in pretreatment biopsy tissue samples predicted tumor regression grade (TRG) in rectal cancer patients after receiving neoadjuvant radiotherapy (nRT). Expression of miR-15b in post-nRT tissue samples was associated with therapeutic outcome. DCLK1 was identified as the direct target gene for miR-15b and its suppression was associated with self-renewal and tumorigenic properties of DCLK1+ TICs. We identified B lymphoma Mo-MLV insertion region l homolog (BMI1) as a downstream target regulated by miR-15b/DCLK1 signaling. Thus, miR-15b may serve as a valuable marker for prognosis and therapeutic outcome prediction. DCLK1 could be a potential therapeutic target to overcome chemo-/radioresistance in CRC.

Somatic Mutations and Immune Alternation in Rectal Cancer Following Neoadjuvant Chemoradiotherapy.

Checkpoint blockade therapy triggers tumor-specific immune responses in a variety of cancer types. We presumed that rectal cancer patients could have become sensitive to immunotherapy after receiving neoadjuvant chemoradiotherapy (nCRT). In this study, we report immune alternation in post-nCRT patients compared with pretreatment conditions from gene-expression omnibus (GEO) data. Whole-exome sequencing of 14 locally advanced rectal cancer (LARC) patient samples showed that nCRT induced new mutations compared with the paired pretreatment biopsies, evidenced by appearance of a neoantigen landscape. An association was identified between mutation burden and enrichment of immune activation-related pathways. Animal experiment results further demonstrated that radiotherapy enhanced the efficacy of anti-PD-1. Mutation burden and the neoantigens of LARC patients were associated with response to nCRT. The mRNA expression profiling of 66 pretreatment biopsy samples from LARC patients showed that immune activation-related pathways were enriched in response to nCRT. PD-L1 expression was negatively correlated with disease-free survival in the CD8-low expression patient group who received nCRT in a cohort of 296 samples. Thus, nCRT was able to alter immune function in LARC patients, which may be associated with the appearance of neoantigens. Neoantigens could make rectal cancer patients potential candidates to receive checkpoint blockade immunotherapy, and mutation burden could be a useful biomarker to stratify patients into responding and nonresponding groups for immunotherapy. Cancer Immunol Res; 6(11); 1401-16. ©2018 AACR.

Serum-based microRNA signature predicts relapse and therapeutic outcome of adjuvant chemotherapy in colorectal cancer patients.

BACKGROUND: Approximately 60% of patients with colorectal cancer (CRC) undergo either local recurrence or distant metastases after surgery. Current prognostic biomarkers are insufficient to predict recurrence of CRC and provide little forecast information about what patients are likely to receive benefit from the adjuvant chemotherapy. As microRNAs (miRNAs) constantly exist in human serum and being used to predict the prognosis of a various cancers, this study was designed to identify miRNA-based circulating biomarkers to predict clinical outcomes of CRC. METHODS: A serum-focused miRNA expression was used to investigate if miRNA expression profiles could predict the clinical outcomes of patients with CRC. We created miRNA signature profiles associated in the training set (n = 40), and further validated its prediction in two independent testing cohorts. RESULTS: Using Cox regression and risk-score analysis, we identified a four-miRNA signature (miR-652-3p, miR-342-3p, miR-501-3p and miR-328-3p) for the prediction of tumor relapse and the overall survival(OS) of patients with CRC in the training set (n = 40). This miRNA signature was further validated in a testing set (n = 226) and another independent cohort (n = 56). A high-risk signature score was significantly associated with CRC tumor recurrence and poor treatment outcome. Multivariable Cox regression models indicated that the risk score, based on the four-miRNA signature, was an independent prognostic classifier for patients with CRC. CONCLUSIONS: The serum miRNA signature may serve as a minimally invasive predictor for tumor relapse and treatment outcome in patients with CRC and provide a useful reference for treatment selection.

Glycan analysis of colorectal cancer samples reveals stage-dependent changes in CEA glycosylation patterns.

BACKGROUND: Carcinoembryonic antigen (CEA) is a glycoprotein associated with colorectal cancer (CRC). While the functions of its gene and protein have been fully characterized, its post-translational modifications in the context of CRC development remain undefined. METHODS: To show the correlation between the different stages of CRC development and changes in the glycosylation patterns of CEA, we analyzed CEA in tumor tissues (CEA-T) and paired tumor-adjacent normal tissues (CEA-A) from 53 colorectal cancer patients using a high-density lectin microarray containing 56 plant lectins. RESULTS: We detected higher expression levels of fucose, mannose and Thomsen-Friedenreich antigen, and lower expression levels of N-acetylgalactosamine, N-acetylglucosamine, galactose, branched and bisecting N-glycans on CEA in the tumor tissues relative to the tumor-adjacent normal tissues. Furthermore, a combinatorial assessment of 9 lectins is sufficient to distinguish CRC tumor tissues from tumor-adjacent normal tissues with 83% sensitivity and ~ 90% specificity. Moreover, the levels of N-acetylgalactosamine, mannose, galactose, N-acetylglucosamine on CEA showed a downward trend after first experiencing an increase at Stage II with the stages of CRC. CONCLUSIONS: Our insights into the changing CEA glycosylation patterns and their role in the development of CRC highlight the importance of glycan variants on CEA for early clinical detection and staging of CRC.

Overexpression of LAMC2 predicts poor prognosis in colorectal cancer patients and promotes cancer cell proliferation, migration, and invasion.

Laminin γ2 (LAMC2) has been reported to be involved in the development and progression of a variety of tumors. However, its function in human colorectal cancer is unclear. Our study aimed to investigate the role of laminin γ2 in colorectal cancer. We first performed the multiple Kaplan-Meier survival analysis of laminin γ2 in a cohort of Gene Expression Omnibus datasets and evaluated its relationship with clinical outcomes of colorectal cancer patients. Then, we established stable colorectal cancer cell lines with laminin γ2 overexpression and examined the functional assays in vitro. Finally the expression pattern of laminin γ2 in colorectal cancer clinical samples was analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction. We found that laminin γ2 was significantly correlated with poor clinical outcomes such as disease-specific, recurrence-free, disease-free, and overall survival in colorectal cancer. Moreover, stably overexpressing laminin γ2 promoted proliferation, migration, and invasion of colorectal cancer cells. In addition, overexpressed laminin γ2 was identified in tumor tissues compared with paired adjacent normal tissues and was related to tumor-node-metastasis stage (p = 0.001) and lymph node metastasis (p < 0.001). In summary, our results strongly suggest that laminin γ2 may be a potential prognostic biomarker and therapeutic target in colorectal cancer.

Prognostic value of pigment epithelium-derived factor for neoadjuvant radiation therapy in patients with locally advanced rectal carcinoma.

The aim of the study was to investigate the prognostic value of pigment epithelium-derived factor (PEDF) in locally advanced rectal carcinoma (LARC) treated with neoadjuvant radiation therapy (nRT). The level of PEDF expressing was examined in LARC tissues treated with nRT by immunohistochemistry and the prognostic significance of PEDF was analysed by univariate and multivariate survival analyses. We forced expression of PEDF in highly metastatic LoVo cells. The clonogenic survival assay was used to test the cellular sensitivity to radiation. Wound healing and Boyden chamber assays were used to detect cell migration and invasion. To assess the contribution of PEDF in vivo, we established tumor xenografts. The mechanisms of PEDF on cancer cells was analysed by bioinformatics. Our immunohistochemical staining of tissue samples revealed that prolonged DFS (77.1 vs 49.0%) and OS (87.1 vs 56.3%) was observed in PEDF-positive cases (P<0.001) following nRT. PEDF could be an independent factor for DFS [P=0.001; HR, 0.422 (95% CI, 0.249-0.717)] and OS [P=0.003; HR, 0.418 (95% CI, 0.234-0.749)]. Positive-expression of PEDF was negatively correlated with tumor differentiation (P<0.016), ypT stage (P<0.037), ypTNM stage (P<0.033), and ypN stage (P=0.006). Overexpression of PEDF in high metastatic cells enhanced radiosensitivity and, suppressed migration and invasion in vitro. In tumor xenografts, PEDF significantly suppressed tumor growth. Furthermore, by bioinformatics analysis, we found PEDF performs functions via activating P53 to regulate double-strand break repair pathway and activate the G protein activation pathway. Our findings indicate that PEDF was identified as a predictive candidate for nRT responsiveness. These findings may be used to stratify LARC patients and make alternative strategies for adjuvant treatment.

Glycomic profiling of carcinoembryonic antigen isolated from human tumor tissue.

BACKGROUND: Carcinoembryonic antigen (CEA) is a protein commonly found in human serum, with elevated CEA levels being linked to the progression of a wide range of tumors. It is currently used as a biomarker for malign tumors such as lung cancer and colorectal cancer [Urol Oncol: Semin Orig Invest 352: 644-648, 2013 and Lung Cancer 80: 45-49, 2013]. However, due to its low specificity in clinical applications, CEA can be used for monitoring only, rather than tumor diagnosis. The function of many glycoproteins is critically dependent on their glycosylation pattern, which in turn has the potential to serve in tumor detection. However, little is known about the detailed glycan patterns of CEA. METHODS: To determine these patterns, we isolated and purified CEA proteins from human tumor tissues using immunoaffinity chromatography. The glycan patterns of CEA were then analyzed using a Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry(3) (MALDI-TOF-MS(3)) approach. RESULTS: We identified 61 glycoforms in tumor tissue, where CEA is upregulated. These glycosylation entities were identified as bi-antennary, tri-antennary and tetra-antennary structures carrying sialic acid and fucose residues, and include a multitude of glycans previously not reported for CEA. CONCLUSION: Our findings should facilitate a more precise tumor prediction than currently possible, ultimately resulting in improved tumor diagnosis and treatment.