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Supplementary Table 6. HLA reference set

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.

Supplementary Table 8. CD28_co-stimulatory signal

Supplementary Table 7. All differentially expressed genes

Supplementary Table 1-5 and 10-12. Supplementary Figure 1-10.

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.

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.

3609 Background: Total neoadjuvant therapy (TNT) with induction chemotherapy and chemoradiation is the standard of care for locally advanced rectal cancers. Incorporation of PD-1 blockade in the neoadjuvant setting is promising for mismatch repair deficient (dMMR) patients, yet the efficacy of TNT combined with PD-1 blockage for pMMR/MSS patients is little unknown. The purpose of this study is to evaluate the clinical benefit of neoadjuvant PD-1 blockade combined with TNT in pMMR/MSS locally advanced rectal cancer. Methods: We designed a prospective, single-arm, phase II study on locally advanced dMMR/MSS rectal cancer (PKUCH 04 trial). Patients will receiving neoadjuvant regimen (PD-1 inhibitor, camrelizumab, also called SHR01210, 200mg d1, oxaliplatin 130mg/m2 d1, capecitabine 1250mg/m2 bid1-14, for three cycles, then long course chemoradiation (IMRT, 1.8Gy/f*25f), then another two cycles of CapeOx (oxaliplatin 130mg/m2 d1, capecitabine 1250mg/m2 bid1-14, q3wks) if no disease progression occurs. The primary endpoint was pathologic complete response (pCR) rate, and the secondary outcome include adverse event and surgical complication. Tumor assessment was carried out after induction, chemoradiation, and before the decision of either surgery or watch and wait. Results: A total of 27 patients were screened and finally 25 patients were eligible for further analysis, with median age 58 years (range 31-70). The median distance from the anal verge was 5.3 cm (1.2-10). 76% were male, 76% had N2 disease, 56% positive MRF, 80% positive EMVI. All patients have completed the induction and chemoradiation phases. 21 patients underwent TME surgery, of which 7 had pathological complete response (33.3%). Four achieved clinical complete or near complete response and chose watch and wait. 7 had major pathological remission (over 90% remission). Another 7 had partial pathological remission. No progressive disease was found. 14 had LAR, 7 APR, and. The surgical complication rate was 14.3%, and the mortality rate was 0. The common adverse events included nausea (80%), lymphopenia (80%), paresthesia (76%), reactive cutaneous capillary endothelial proliferation (72%), neutropenia (68%). Grade 3 adverse events include lymphopenia (24%), diarrhea (8%), and thrombocytopenia (4%) and no grade 4 or 5 events were observed. Till January 2022, no recurrence or regrowth was found. Conclusions: Total neoadjuvant chemoradiation combined with neoadjuvant PD-1 blockade is safe and effective which can achieve good regression and sphincter preservation for pMMR/MSS rectal cancer. This suggests a potential new paradigm for treatment of pMMR/MSS locally advanced rectal cancer and necessitates further investigation. Clinical trial information: NCT04340401.

3611 Background: Total neoadjuvant therapy (TNT) with chemotherapy and chemoradiotherapy (CRT) is the standard treatment for locally advanced rectal cancer patients, especially for those with high risk factors. And the effects of immune checkpoint inhibitors (ICIs) on rectal cancer with defect mismatch repair (dMMR) have been demonstrated. However, the efficacy of TNT combined with PD-1 blockage for rectal cancer patients with proficient mismatch repair (pMMR) is unknown. The purpose of this study is to evaluate the clinical safety and efficacy of neoadjuvant PD-1 blockade combined with TNT in locally advanced rectal cancer with pMMR. Methods: We designed a prospective, single-arm, phase II study on stage II and III dMMR rectal cancer with high risk factors. Patients received neoadjuvant POC regimen (PD-1 inhibitor, camrelizumab, also called SHR01210, 200mg d1, Oxaliplatin 130mg/m2 d1 and Capecitabine 1250mg/m2 bid1-14, q3wks) for three cycles, then with long course chemoradiation (IMRT, 1.8Gy/f*25f). If there were no disease progression occurs, patients would be treated with another two cycles of CapeOx. The primary endpoint was pathological complete response rate (pCR rate), and the secondary outcome include adverse events and surgical complication. Biopsies and plasma pre-neoadjuvant therapy were collected and performed with whole-exome sequencing (WES), and ctDNA sequencing, respectively. Results: 27 patients were screened, of whom 25 patients were enrolled. All patients have completed the TNT. After neoadjuvant treatment, 21 patients underwent TME surgery. 7 (33.3%) patients were pCR, 3 (14.3%) achieved clinical complete or near complete response and 7 (33.3%) had a major pathological response. No progressive disease was found. WES and ctDNA sequencing analysis showed that tumor mutation burden (TMB) and level of ctDNA did not differ significantly between responders and non-responders. However, 3 responders with TMB high were found to be defective in other DNA damage repair pathways. Conclusions: Total neoadjuvant chemoradiation combined with neoadjuvant PD-1 blockade can achieve high pathological response rate, demonstrating its antitumor efficacy in pMMR locally advanced rectal cancer patients with high risk factors.

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.

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-1β, IL-2 and MIP-1β 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.

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.

Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, with high heterogeneity. The significant difference in prognosis and therapeutic response of left and right colon cancer is a hot spot in the field of colorectal cancer (CRC) in recent years. The prognosis and drug response of right-sided colon cancer were significantly worse than that of the left-sided colon cancer. Clinical trials have proved that MSI-H colon cancer is mainly seen in the right-sided colon cancer, and the prognosis is better than that of MSS. Because MSS and MSI-H patients have different molecular characteristics and prognosis, we searched for key genes of RCC and LCC in MSS and MSI-H groups respectively. The difference between the prognosis and the curative effect of the left and right-sided colon cancer is essentially the difference between the molecular pathology and the immune environment. Therefore, the aim of this study is to explore the molecular mechanism related to the prognosis of left and right-sided colon cancer in order to provide a molecular target for accurate treatment of left and right-sided colon cancer. First of all, bioinformatics methods were used to screen the right and left-sided CRC differentially expressed genes in MSS and MSI-H. The expression levels of these key genes in clinical samples and cell lines were detected by RT-qPCR and IHC, and their correlation with survival prognosis was analyzed. Homeobox C6(HOXC6)were selected. First, HOXC6 stable overexpressed and knockout colorectal cancer cell lines were established and its biological function was preliminarily studied, including transwell, wound-healing assay and CCK-8 tests. Finally, the ChIP-seq data were used to analyze the carcinogenic mechanism of HOXC6. Bioinformatics methods were used to screen 30 differentially expressed genes in MSS right and left CRC, of which HOXC6 in the right colon cancer was significantly higher than that in the left-sided colon cancer, which was positively correlated with TNM staging and lymph node metastasis. Kaplan-Meier survival analysis showed that the expression of HOXC6 was negatively correlated with the total survival of CRC patients (p=0.002). RT-qPCR and IHC showed that the expression of HOXC6 in the right-sided colon cancer was higher than that in the left, P Citation Format: Xin li, Jinhong Xia, Dengbo Ji, Jin Gu. HOXC6 promotes tumorigenesis of right-sided colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2430.

Additional file 4: Table S2. The concentration of CEA of CRC patients; Figure S1. The relationship between CEA concentration and CRC stages.

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

AIM: To investigate the correlation of miR-193a-5p with lymph node metastasis and postoperative survival of colorectal cancer (CRC) patients. METHODS: A total of 304 formalin-fixed, paraffin-embedded specimens (69 paired cancer and normal tissues, 55 primary tumors of stage III CRC and matched lymph nodes, and 56 primary tumors of stage II CRC) were included in this study. The relative expression levels of miR-193a-5p in the normal mucosa, primary cancer, and metastatic lymph node lesions were measured by quantitative real-time reverse transcriptase polymerase chain reaction. We evaluated the association of its expression with colorectal cancer lymph node metastasis, clinicopathological factors, and patient survival. RESULTS: The relative expression level of miR-193a-5p was significantly lower in CRC tissues than in the normal mucosa (P = 0.0060). The expression levels of miR-193a-5p were lower in primary CRC tissues with lymph node metastases than in those without metastases (P = 0.0006), and decreased expression of miR-193a-5p correlated with advanced lymph node metastatic stage (P = 0.0007). Kaplan-Meier analysis showed that patients with low miR-193a-5p expression had decreased disease-free survival (DFS) (P = 0.0026) and poor overall survival (OS) (P = 0.0003). Interestingly, for the group of patients with lymph node metastases, miR-193a-5p expression was also related to survival. Patients with low miR-193a-5p expression had decreased DFS (P = 0.0262) and poor OS (P = 0.0230). Moreover, multivariate analysis indicated that downregulation of miR-193a-5p was an independent predictor of poor OS. CONCLUSION: Downregulation of miR-193a-5p correlates with lymph node metastasis and poor survival of CRC. miR-193a-5p may be a useful biomarker for CRC diagnosis, metastasis and prognosis prediction.

Background Given the emerging role of microRNA in tumor disease progression, we investigated the association between microRNA expression, liver metastasis and prognosis of colorectal cancer. Methods Colorectal cancer tissues from patients with or without liver metastases were profiled to identify differentially expressed microRNA. Expression profile was further assessed using quantitative reverse transcription PCR and in situ hybridization. Correlation between miR-181a expression, the most differentially expressed microRNA, between patients with and without liver metastasis, and its downstream target genes were investigated using qRT-PCR. Luciferase reporter assay was conducted to establish functional association between miR-181a and its target genes. Manipulation of miR-181a expression and its consequences in tumor growth and metastasis were demonstrated in various in vitro and in vivo models. Results miR-181a was revealed being the most elevated in CRC with liver metastases. miR-181a expression correlated with advanced stage, distant metastasis, and served as an independent prognostic factor of poor overall survival. Stable transfection of CRC cell lines with miR-181a promoted cell motility and invasion, as well as tumor growth and liver metastasis,while silencing its expression resulted in reduced migration and invasion. Additionally, we identified WIF-1 as direct and functional targets of miR-181a. Ectopic expression of miR-181a suppressed the epithelial markers E-cadherin and β-catenin, while enhanced the mesenchymal markers vimentin. Conclusion Our data demonstrate that miR-181a expression is associated with CRC liver metastasis and survival. miR-181a has strong tumor-promoting effects through inhibiting the expression of WIF-1, and its potential role in promoting epithelial-mesenchymal transition.

This study was designed to develop novel and better reliable serum prognostic biomarkers for colorectal cancer (CRC). A 50 sample set including CRC, adenoma and healthy control sera was used to identify the serum proteins involved in CRC carcinogenesis using serum proteomic approach. Alpha-2-glycoprotein 1, zinc-binding (AZGP1), pigment epithelium derived factor (PEDF) and peroxiredoxin 2 (PRDX2) were selected as good candidates. Two independent cohorts of 868 individuals were enrolled. The expression of selected proteins in serum from cohort 1 (n = 534) was quantified with enzyme-linked immunosorbent assays. CRC sera of this cohort (n = 405) were assigned to training and test sets, which were used to identify and verify the prognostic markers. The prognostic values of identified proteins were further validated in cohort 2 (n = 334) using quantitative reverse transcription PCR and immunohistochemical staining. Our data showed that the elevated AZGP1 and decreased PEDF and PRDX2 expressions in CRC serum and tissues were correlated with liver metastases. In the training set, higher AZGP1 and lower PEDF levels in sera were significantly associated with a poorer overall survival (OS), higher AZGP1 was also associated with a poorer disease-free survival (DFS). This association was verified in the testing set and further validated in patients in cohort 2. Patients with lower PEDF or PRDX2 levels in their CRC tissues had a significantly poorer DFS or OS than patients with high levels of these proteins in cohort 2. Univariate and multivariate analyses indicated that the prognostic performance of serum AZGP1 and PEDF was independent of other clinicopathological factors. We propose that they may serve as prognostic markers and potential therapeutic targets in CRC.