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PURPOSE Pembrolizumab or nivolumab plus chemotherapy was approved as a first-line treatment for high programmed cell death ligand 1 (PD-L1)–expressing esophageal squamous cell carcinoma (ESCC) by the European Medicines Agency, whereas the US Food and Drug Administration approved this regimen regardless of PD-L1 expression. The superiority of programmed death-1 (PD-1) antibody plus chemotherapy over chemotherapy alone in patients with low PD-L1–expressing ESCC remains debatable. METHODS Post hoc analysis of the Chinese JUPITER-06 study focusing on efficacy stratified by PD-L1 tumor proportion score (TPS; using JS311 antibody) was conducted. Electronic databases were searched to identify eligible randomized controlled trials for meta-analysis. Study-level pooled analyses of hazard ratios (HRs) for overall survival and progression-free survival and odds ratios for objective response rate according to PD-L1 expression were performed. RESULTS The post hoc analysis of JUPITER-06 showed more prominent clinical benefit with PD-1 antibody plus chemotherapy than with chemotherapy alone in both the high and low PD-L1–expressing subgroups. Five randomized controlled trials were included in the meta-analysis, and two PD-L1 expression scoring criteria, TPS (≥ 1%/< 1%) and combined positive score (CPS, ≥ 10/< 10), were analyzed. Significant overall survival benefit by adding PD-1 antibody to chemotherapy was observed in both the TPS < 1% (HR, 0.74; 95% CI, 0.56 to 0.97) and CPS < 10 (HR, 0.77; 95% CI, 0.66 to 0.89) subgroups. Similarly, significantly prolonged progression-free survival was observed in both the TPS < 1% (HR, 0.66; 95% CI, 0.50 to 0.86) and CPS < 10 (HR, 0.63; 95% CI, 0.47 to 0.84) subgroups. In addition, the objective response rate of the TPS < 1% subgroup was significantly improved (odds ratio, 1.71; 95% CI, 1.27 to 2.29). In all high PD-L1–expressing subgroups, the pooled benefit of PD-1 antibody plus chemotherapy was significantly better than that of chemotherapy. CONCLUSION This study provided novel evidence supporting the superiority of PD-1 antibody plus chemotherapy to chemotherapy alone in patients with advanced ESCC with low PD-L1 expression. Further studies of predictive biomarkers are warranted.

Growing evidence implies a link between DNA methylation and tumor immunity/immunotherapy. However, the global influence of DNA methylation on the characteristics of the tumor microenvironment and the efficacy of immunotherapy remains to be clarified. In this study, we systematically evaluated the DNA methylation regulator patterns and tumor microenvironment characteristics of 1,619 gastric cancer patients by clustering the gene expression of 20 DNA methylation regulators. Three gastric cancer subtypes that had different DNA methylation modification patterns and distinct tumor microenvironment characteristics were recognized. Then, a DNA methylation score (DMS) was constructed to evaluate DNA methylation modification individually. High DMS was characterized by immune activation status, increased tumor mutation burden, and tumor neoantigens, with a favorable prognosis. Conversely, activation of the stroma and absence of immune cell infiltration were observed in the low DMS group, with relatively poor survival. High DMS was also certified to be correlated with enhanced efficacy of immunotherapy in four immune checkpoint blocking treatment cohorts. In conclusion, the characterization of DNA methylation modification patterns may help to enhance our recognition of the tumor immune microenvironment of gastric cancer and guide more personalized immunotherapy strategies in the future.

Background Numerous reports on microRNAs have illustrated their role in tumor growth and metastasis. Recently, a new prognostic factor, miR‐125b‐2‐3p, has been identified for predicting chemotherapeutic sensitivity in advanced colorectal cancer (CRC). However, the specific mechanisms and biological functions of miR‐125b‐2‐3p in advanced CRC under chemotherapy have yet to be elucidated. Methods MiR‐125b‐2‐3p expression was detected by real‐time PCR (RT‐PCR) in CRC tissues. The effects of miR‐125b‐2‐3p on the growth, metastasis, and drug sensitivity of CRC cells were tested in vitro and in vivo. Based on multiple databases, the upstream competitive endogenous RNAs (ceRNAs) and the downstream genes for miR‐125b‐2‐3p were predicted by bioinformatic analysis, followed by the experiments including luciferase reporter assays, western blot assays, and so on. Results MiR‐125b‐2‐3p was significantly lowly expressed in the tissues and cell lines of CRC. Higher expression of miR‐125b‐2‐3p was associated with relatively lower proliferation rates and fewer metastases. Moreover, overexpressed miR‐125b‐2‐3p remarkably improved chemotherapeutic sensitivity of CRC in vivo and in vitro. Mechanistically, miR‐125b‐2‐3p was absorbed by long noncoding RNA (lncRNA) XIST regulating WEE1 G2 checkpoint kinase (WEE1) expression. The upregulation of miR‐125b‐2‐3p inhibited the proliferation and epithelial‐mesenchymal transition (EMT) of CRC induced by lncRNA XIST. Conclusions Lower miR‐125b‐2‐3p expression resulted in lower sensitivity of CRC to chemotherapy and was correlated with poorer survival of CRC patients. LncRNA XIST promoted CRC metastasis acting as a ceRNA for miR‐125b‐2‐3p to mediate WEE1 expression. LncRNA XIST‐miR‐125b‐2‐3p‐WEE1 axis not only regulated CRC growth and metastasis but also contributed to chemotherapeutic resistance to CRC., Low expression of miR‐125b‐2‐3p in CRC was linked to lower chemotherapeutic sensitivity and poor survival. The lncRNA XIST promoted CRC invasion and migration by functioning as a ceRNA for miR‐125b‐2‐3p to mediate WEE1 expression. Our findings suggest that the lncRNA XIST‐miR‐125b‐2‐3p‐WEE1 axis not only regulated CRC growth and metastasis but also contributed to chemotherapeutic resistance to CRC, which may shed light on their targeted applications.

Gastrointestinal cancer causes countless deaths every year due to therapeutic resistance. However, whether metabolic alterations contribute to chemoresistance is not well understood. In this study, we report that fatty acid (FA) catabolism was activated in gastrointestinal cancer cells treated with oxaliplatin, which exhibited higher expression of the rate-limiting enzymes carnitine palmitoyltransferase 1B (CPT1B) and CPT2. The clinical analysis also showed that high expression of these enzymes was associated with poor oxaliplatin-based chemotherapy outcomes in patients. Furthermore, genetic or pharmacological inhibition of CPT2 with perhexiline disturbed NADPH and redox homeostasis and increased reactive oxygen species (ROS) generation and cell apoptosis in gastrointestinal cancer cells following oxaliplatin treatment. Specifically, the combination of oxaliplatin and perhexiline significantly suppressed the progression of gastrointestinal cancer in cell-based xenograft and patient-derived xenograft (PDX) models. Mechanistically, CPT2 was transcriptionally upregulated by nuclear factor of activated T cells 3 (NFATc3), which translocated to the nucleus in response to oxaliplatin treatment. In summary, our study suggests that the inhibition of CPT-mediated FA catabolism combined with conventional chemotherapy is a promising therapeutic strategy for patients with gastrointestinal cancers.

BackgroundLong noncoding RNAs (lncRNAs) play nonnegligible roles in the epigenetic regulation of cancer cells. This study aimed to identify a specific lncRNA that promotes the colorectal cancer (CRC) progression and could be a potential therapeutic target.MethodsWe screened highly expressed lncRNAs in human CRC samples compared with their matched adjacent normal tissues. The proteins that interact withLINRIS(Long Intergenic Noncoding RNA for IGF2BP2 Stability) were confirmed by RNA pull-down and RNA immunoprecipitation (RIP) assays. The proliferation and metabolic alteration of CRC cells withLINRISinhibited were tested in vitro and in vivo.ResultsLINRISwas upregulated in CRC tissues from patients with poor overall survival (OS), andLINRISinhibition led to the impaired CRC cell line growth. Moreover, knockdown ofLINRISresulted in a decreased level of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), a newly found N6-methyladenosine (m6A) ‘reader’.LINRISblocked K139 ubiquitination of IGF2BP2, maintaining its stability. This process prevented the degradation of IGF2BP2 through the autophagy-lysosome pathway (ALP). Therefore, knockdown ofLINRISattenuated the downstream effects of IGF2BP2, especially MYC-mediated glycolysis in CRC cells. In addition, the transcription ofLINRIScould be inhibited by GATA3 in CRC cells. In vivo experiments showed that the inhibition ofLINRISsuppressed the proliferation of tumors in orthotopic models and in patient-derived xenograft (PDX) models.ConclusionLINRISis an independent prognostic biomarker for CRC. TheLINRIS-IGF2BP2-MYC axis promotes the progression of CRC and is a promising therapeutic target.

4054 Background: Circulating tumor DNA (ctDNA) is a promising biomarker for detecting molecular residual disease (MRD) and relapse after definitive treatment in multiple solid cancers. However, the significance of ctDNA is rarely clarified in locoregional gastric cancer (GC). Here, we conducted a prospective and observational study to evaluate the utility of ctDNA in predicting the recurrence risk of GC. Methods: From October 2016 to June 2019, 100 patients with stage II/III resectable GC were recruited in this study (NCT02887612). Primary tumors and plasma samples were collected perioperatively and after adjuvant chemotherapy (ACT). Somatic variants were captured via a targeted sequencing panel of 425 cancer-related genes. The plasma of patients was defined as ctDNA positive only if one or more variants detected in the plasma presented in at least 2% of the primary tumors. All the patients received curative-intent standard-of-care therapy. Results: Preoperative ctDNA was detectable in 38 (38.0%) patients but showed limited value for predicting recurrence. After surgery (median days, 4), the plasma of 25 (25.0%) patients were still ctDNA positive and they had higher recurrence risk than the non-positive patients (hazard ratio [HR], 2.74 (95% CI: 1.37–5.48); P = 0.003). Forty-one patients had evaluable plasma after ACT and 10 (24.4%) of them who were ctDNA positive had remarkably higher recurrence and death risk compared with ctDNA-negative patients (recurrence-free survival [RFS] HR = 14.99 (95% CI: 3.08–72.96); P < 0.001; overall survival HR, 11.88 (95% CI: 2.38–59.24); P < 0.001). In particular, post-ACT ctDNA achieved better predictive performance (sensitivity, 77.8%; specificity, 90.6%) than both postoperative ctDNA and post-treatment tumor biomarkers (i.e., CEA, CA199, and CA72-4). In all multivariate analyses, ctDNA positivity was an independent factor of RFS. Patients with ERBB4 mutation in their primary tumors had poorer RFS compared to those were ERBB4 wildtype (HR = 3.46 (95% CI: 1.32–9.03); P = 0.007). A comprehensive model incorporating ctDNA status for recurrence risk prediction showed a higher concordance index (0.78; 95%CI, 0.71–0.84) than the model without ctDNA status (0.71; 95%CI, 0.64–0.79; P = 0.009). Conclusions: Postoperative and post-ACT ctDNA was associated with MRD and high risk of relapse in patients with stage II/III GC and can be utilized to guide GC management in post-surgical settings.

The acidic tumor microenvironment provides an energy source driving malignant tumor progression. Adaptation of cells to an acidic environment leads to the emergence of cancer stem cells. The expression of the vitamin D receptor (VDR) is closely related to the initiation and development of colorectal carcinoma (CRC), but its regulatory mechanism in CRC stem cells is still unclear. Our study revealed that acidosis reduced VDR expression by downregulating peroxisome proliferator-activated receptor delta (PPARD) expression. Overexpression of VDR effectively suppressed the stemness and oxaliplatin resistance of cells in acidosis. The nuclear export signal in VDR was sensitive to acidosis, and VDR was exported from the nucleus. Chromatin immunoprecipitation (ChIP) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses showed that VDR transcriptionally repressed SRY-box 2 (SOX2) by binding to the vitamin D response elements in the promoter of SOX2, impairing tumor growth and drug resistance. We demonstrated that a change in the acidic microenvironment combined with overexpression of VDR substantially restricted the occurrence and development of CRC in vivo. These findings reveal a new mechanism by which acidosis could affect the stemness of CRC cells by regulating the expression of SOX2 and show that abnormal VDR expression leads to ineffective activation of vitamin D signaling, resulting in a lack of efficacy of vitamin D in antineoplastic process.

Objectives Although the genomic landscape of small‐cell carcinoma of the oesophagus (SCCE) has been dissected, its transcriptome‐level aberration and immune microenvironment status are unknown. Methods Using ultra‐deep whole transcriptome sequencing, we analysed the expression profile of nine paired SCCE samples and compared the transcriptome with public transcriptomic data set of normal oesophageal mucosa and other cancer types. Based on the transcriptome data, the immune signatures were investigated. The genomic data of 55 SCCE samples were also applied for immune checkpoint blockade therapy (ICBT) biomarker evaluation including microsatellite instability (MSI) status, tumor mutation burden (TMB) and neoantigen burden (TNB). Also, we evaluated the CD8, CD68 and programmed death‐ligand 1 (PD‐L1) in 62 retrospective SCCE samples with IHC assay. Results Differential expression analysis revealed that the cell cycle, p53, and Wnt pathways are significantly deregulated in SCCE. Immune microenvironment analysis showed that high leucocyte infiltration and adaptive immune resistance did occur in certain individuals, while the majority showed a relatively suppressive immune status. Immune checkpoints such as CD276 and LAG‐3 were upregulated, and higher M2 macrophage infiltration in tumor tissues. Furthermore, normal tissues adjacent to the tumors of SCCE presented a more activated inflammatory status than tumor‐free healthy controls. These observations showed that ICBT might benefit SCCE patients. As the critical biomarker of ICBT, TMB of SCCE was 3.64 with the predictive objective response rate 13.2%, while the PD‐L1‐positive rate was 43%. Conclusions Our study systematically characterized the immune microenvironment in small‐cell carcinoma of the esophagus and provided evidence that several patients with SCCE may benefit from immune checkpoint blockade therapy., In this study, transcriptomic aberrance and immune microenvironment of small‐cell carcinoma of the esophagus (SCCE) was comprehensively characterized with transcriptome and immunohistochemistry analysis. Assessment of immune checkpoint blockade (ICB) treatment biomarkers provides a rationale for use of ICB treatment in patients with SCCE.

Background Immune checkpoint inhibitors (ICIs) have achieved impressive success in different cancer types, yet responses vary and predictive biomarkers are urgently needed. Growing evidence points to a link between DNA methylation and anti-tumor immunity, while clinical data on the association of genomic alterations in DNA methylation-related genes and ICI response are lacking. Methods Clinical cohorts with annotated response and survival data and matched mutational data from published studies were collected and consolidated. The predictive function of specific mutated genes was first tested in the discovery cohort and later validated in the validation cohort. The association between specific mutated genes and tumor immunogenicity and anti-tumor immunity was further investigated in the Cancer Genome Altas (TCGA) dataset. Results Among twenty-one key genes involving in the regulation of DNA methylation, TET1-mutant (TET1-MUT) was enriched in patients responding to ICI treatment in the discovery cohort (P 0.05 in both two non-ICI-treated cohorts). In TCGA dataset, TET1-MUT was strongly associated with higher tumor mutational burden and neoantigen load, and inflamed pattern of tumor-infiltrating T lymphocytes, immune signatures and immune-related gene expressions. Conclusions TET1-MUT was strongly associated with higher ORR, better DCB, longer PFS, and improved OS in patients receiving ICI treatment, suggesting that TET1-MUT is a novel predictive biomarker for immune checkpoint blockade across multiple cancer types.

Background Long non-coding RNAs (lncRNAs) are emerging as key molecules in gastric cancer(GC), yet their potential molecular mechanisms are not well understood. The aim of our study is to identify lncRNAs that are up-regulated in gastric cancer tissues and explore their function in gastric cancer metastasis. Methods RNA sequencing of 48 paired gastric tumor and non-tumor tissues in SYSUCC was conducted. The upregulated lncRNAs were selected and overlapped with those in TCGA database. A siRNA library was established using the top 50 lncRNAs highly expressed in GC and used to identify lncRNAs that significantly affected cell migration based on transwell assays. The expression of our focused lncRNA CRCAL-2(ColoRectal Cancer Associated LncRNA-2) was measured using quantitative reverse transcription PCR assays and lncRNA in situ hybridization. In vivo,cell-based and patient-derived xenograft (PDX) models were used to further explore roles of CRCAL-2 in GC metastasis. Then, RNA pull-down, mass spectrometry analyses, western blot and RNA-binding protein immunoprecipitation (RIP) were performed to identify interaction proteins and related mechanisms of CRCAL-2. Results LncRNA CRCAL-2, significantly overexpressed in the tumor tissues, was identified as the key regulator of GC metastasis. Upregulated CRCAL-2 in GC correlated with poor overall survival. Knockdown of CRCAL-2 significantly reduced GC cells migration, invasion, and metastasis of xenograft tumors in nude mice. Mechanistically, CRCAL-2 promoted GC metastasis by directly interacting with and stabilizing the nuclear transport protein RAN, resulting in increasement of nuclear import of beta-catenin and ultimately, activation of downstream targeted EMT(Epithelial-Mesenchymal Transition) genes. Additionally, the transcription factor YY1 could bind to the promoter of CRCAL-2 to upregulate its expression. Conclusions Our results suggest that YY1/CRCAL2/beta-catenin axis play a crucial role in GC metastasis, and the newly identified lncRNA CRCAL-2 might be developed as a biomarker and potential therapeutic target of GC in patients.

Background: Accumulating evidence has demonstrated that microRNAs regulate diverse tumorigenic processes and play important roles in tumor metastasis and growth. Recently, miR-125b-2-3p was identified as a meaningful prognostic factor for predicting chemotherapeutic sensitivity in advanced colorectal cancer (CRC). However, the biological functions and molecular mechanisms of miR-125b-2-3p in the chemotherapy of advanced CRC have yet to be elucidated. Methods: MiR-125b-2-3p expression was detected by real-time PCR in CRC tissues. Gain-of-function experiments were performed to assess the effect of miR-125b-2-3p on CRC growth, metastasis, invasion and drug sensitivity in vitro and in vivo. Based on multiple databases, the competitive endogenous RNAs (ceRNAs) and target genes for miR-125b-2-3p were predicted and have been confirmed by bioinformatic analysis, luciferase reporter assays, rescue experiments and western blot assays. Findings: MiR-125b-2-3p expression was significantly downregulated in CRC tissues and cell lines. MiR-125b-2-3p overexpression was correlated with lower proliferation rates, fewer metastases and better chemotherapeutic sensitivity in vitro and in vivo. Mechanistically, miR-125b-2-3p was regulated by lncRNA XIST and influenced the expression of the WEE1 G2 checkpoint kinase (WEE1). Interpretation: These findings suggested that the lncRNA XIST-miR-125b-2-3p-WEE1 axis not only regulated CRC growth and metastasis but also contributed to chemotherapeutic resistance, thus identifying a potential therapeutic target for CRC. Funding: National Natural Science Foundation of China (81572392), Guangdong special support program (2016TQ03R614), National Key RD Natural Science Foundation of Guangdong Province (2014A030312015); Science and Technology Program of Guangdong (2019B020227002); Science and Technology Program of Guangzhou (201904020046, 201803040019, 201704020228). Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: This study was approved by the institutional ethics review board of SYSUCC (Guangzhou, China). Our animal study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

Background/Aims: Small cell carcinoma of the esophagus (SCCE) is a rare and highly deadly malignancy with rapid disease progress and extensive metastasis, whereas present therapy for the cancer is limited, making the identification of a new treatment strategy an area of intense research. We previous reported the genomic characteristics and alterations of the SCCE, but the transcriptome abnormality of the cancer has not been investigated yet. Method: we performed an RNA Sequencing on nine paired samples from patients with SCCE and conducted a series of comprehensive analysis on the data. DESeq2 and GSEA were applied on transcriptome of SCCE tumor tissue and their para-normal to identify differential expressive genes and associated pathways. Besides, with MCP-counter and a list of immunity related genes, we also compared immune signal of the expression data in pair. By applying a rigorous batch removing strategy, including preprocessing raw data in identical method and RUVSeq, transcriptome data of healthy esophageal mucosa from GTEx was involved as control samples in comparison. In addition, we applied CIBERSORT to calculate the fraction of different tumor infiltrated leukocytes (TILs) of SCCE and compare the number with those of other cancers that have been proven to be potentially responsive to immunotherapy. Results: We identified 1486 and 1782 genes were significantly up-regulated and down-regulated in SCCE tumor tissue against their para-normal, respectively. Pathway analysis revealed the E2F Target and G2M checkpoint are significantly enriched in tumor tissue. Immune signal analysis across the samples in pair showed that in 3 out of 9 paired sample, immunity reaction upregulates in tumor tissue. T-SNE cluster across transcriptome data of SCCE tumor tissue, their para-normal and healthy esophageal mucosa showed the para-normal tissue, namely the normal tissue adjacent to tumor, is more similar to tumor tissue than the healthy. CIBERSORT analysis showed the difference in TILs fraction between SCCE and the compared cancers. For example, the relative fraction of plasma cell of SCCE is higher than those of ESCC(P=0.040), CIN type of STAD(P=0.040), while lower than those of SCLC(P=0.020). M2 macrophages is higher in SCCE than ESCC(P=0.002), EAC(P Conclusion: In summary, our analysis provides transcriptome evidence that immunotherapy might be an efficient treatment strategy for small cell esophagus cancer. Citation Format: Qi Zhao, Yan-xing Chen, Ying-nan Wang, Qi-nian Wu, Hui Sheng, JIa-jia Hu, Yun-xin Lu, Ze-xian Liu, Zhi-xiang Zuo, Feng Wang, Rui-hua Xu. Systematic transcriptome analysis of small cell carcinoma of esophagus suggests a possibility for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4242.