The Cancer Genome Atlas RNA Sequencing Data Research Articles

CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

Hypoxia-inducible factor 2α protein and mRNA expression correlate with histomorphological features in clear cell renal cell carcinoma

Hypoxia-inducible factor 2α (HIF2α) has been identified as a potential biomarker and novel target for systemic therapy in clear cell renal cell carcinoma (ccRCC). The present study aims to evaluate the association of HIF2α protein and HIF2A mRNA expression with clinicopathological factors and histomorphological features related to vasculature and inflammation of ccRCC using a localized ccRCC cohort (n = 428) and The Cancer Genome Atlas (TCGA)-KIRC cohort (n = 433). HIF2α protein expression was immunohistochemically assessed using tissue microarrays and HIF2A mRNA expression was assessed using the TCGA RNA-sequencing data. Positive HIF2α protein and high HIF2A mRNA expression were observed in 145 (33.9 %) and 142 (32.8 %) patients, respectively. Positive nuclear HIF2α protein expression was significantly associated with the clear histological phenotype and architectural patterns related to rich vascular networks (p < 0.001), and no tumor-associated immune cells status (p < 0.05) in addition to favorable prognostic factors such as lower TNM stage, lower WHO/ISUP grade, or the absence of necrosis (p < 0.001). The HIF2A mRNA expression profile by the TCGA cohort showed similar trends as the HIF2α protein profile. In addition, positive HIF2α protein and high HIF2A mRNA expression were associated with higher recurrence-free survival and overall survival, respectively (both p < 0.001). In conclusion, we comprehensively demonstrated the association of HIF2α profiles with clinicopathological factors and histomorphological features related to vasculature and inflammation at both protein and mRNA levels. Histomorphological features expressing HIF2α may provide information on HIF2α targeted therapeutic response as well as prognosis in ccRCC patients.

Integrative Analysis Reveals the Potential Role and Prognostic Value of GOLM1 in Hepatocellular Carcinoma.

Overexpression of Golgi membrane protein 1 (GOLM1) is closely associated with hepatocellular carcinoma (HCC) vascular invasion. How GOLM1 may be involved in angiogenesis in HCC remains unclear. We explored how GOLM1 promotes angiogenesis in HCC and potential prognostic value. Expression levels of GOLM1 in HCC patients and healthy controls were obtained from The Cancer Genome Atlas (TCGA). Differentially expressed genes (DEGs) between HCC patients and controls were compared. GOLM1 was knocked out in the HCC cell line, and RNA sequencing and DEG expression analysis were performed compared with control cells. Based on TCGA data and cell line RNA sequencing data, DEGs affected by a high expression of GOLM1 were identified. Subsequently, enrichment analysis was performed to explore the functions and pathways of the DEGs affected by a high expression of GOLM1. A relevant network analysis was built. Cox regression, genomic variance analysis scores, minimum absolute shrinkage and selection operator regression, and random forest regression models were applied to determine the best prognostic model and validated using the GSE54236 dataset from the Gene Expression Omnibus (GEO). We determined the effect of GOLM1 expression on immune cell infiltration in liver cancer. GOLM1 was overexpressed in HCC tissues compared with controls, and its level correlated with tumor purity and prognosis. 400 DEGs affected by highly expressed GOLM1 were identified in TCGA and cell line RNA sequencing data. Enrichment analysis revealed that these DEGs may be related to biological processes of oxidative stress and angiogenesis and involved in the VEGF signaling pathway and protein processing in endoplasmic reticulum. We predicted a comprehensive regulatory network in which GOLM1 activated VEGF signaling to promote HCC angiogenesis. GOLM1 may interact with E2F1 and IGF2BP3 to promote angiogenesis. GOLM1 overexpression was associated with greater immune cell infiltration. A random forest regression model was the best prognostic model. Our study reveals a potential molecular mechanism of GOLM1 in promoting HCC. We developed two prognostic models based on DEG associated with GOLM1 overexpression to help stratify HCC prognosis and improve individualized treatment.

Glutathione peroxidase 2 is a metabolic driver of the tumor immune microenvironment and immune checkpoint inhibitor response

BackgroundThe existence of immunologically ‘cold tumors’ frequently found across a wide spectrum of tumor types represents a significant challenge for cancer immunotherapy. Cold tumors have poor baseline pan-leukocyte infiltration, including...

Abstract 6172: Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes reveals immune-excluded phenotype is correlated with TGF-beta pathway related genomic aberrations

Abstract Aberrant transforming growth factor-beta(TGF-B) pathway in the tumor microenvironment has been highlighted as one of the core resistance pathways of immunotherapy, by excluding tumor-infiltrating lymphocytes (TIL) out of the tumor area. However, no studies have coupled immune phenotypes classified by spatial analysis of TIL in whole slide images (WSI) with TGF-B pathway analysis on a large-scale database. Here, we hypothesized that the immune-excluded phenotype classified by a deep-learning spatial analysis model, Lunit SCOPE IO, would be correlated with the aberrant TGF-B pathway in The Cancer Genome Atlas (TCGA) cohorts. Aberrant TGF-B pathway was measured by Trimmed Mean of M-values (TMM) normalized and transformed to log2 of counts-per-million of previously published gene sets of fibroblast-specific TGF-beta responsive gene signature, using edgeR packages from TCGA RNA-sequencing data (n=6,709) across the 23 cancer types. Lunit SCOPE IO was developed to identify immune phenotypes trained and validated from 3,166 multi-cancer H&amp;E WSI with sections of 2.8e+9 mm2 tumor tissue containing 5.9e+6 TIL annotated by 52 board-certified pathologists. Lunit SCOPE IO classified immune phenotypes as immune-inflamed and -excluded according to the proportion of TIL density either highly conserved in cancer epithelium (CE) or cancer stroma (CS), respectively, and otherwise, classified as immune-desert with low TIL density in CE and CS. Aberrant TGF-B expression was highly enriched in multiple cancer types including pancreatic cancer, head and neck cancer, kidney clear cell carcinoma, lung squamous cell carcinoma, and breast cancer, in ascending order. TGF-B expression was increased in microsatellite-stable tumor samples (p = 7.4e-15) or samples with low tumor mutational burden (TMB, &amp;lt; 10/megabase, p = 4.9e-8), compared to those with microsatellite instability-high or high TMB, respectively. Interestingly, TGF-B expression was highly correlated with the proportion of cancer stroma in WSI (R = 0.315, p &amp;lt; 2.2e-16) and the proportion of immune-excluded phenotype (R = 0.115, p &amp;lt; 2.2e-16) across multiple cancer types. Tumor samples with SMAD4 mutations (n = 161, 2.4%) had significantly higher TGF-B expression (p = 0.0190), and a higher proportion of immune-excluded phenotype (p &amp;lt; 2.2e-16) in WSI, compared to wild-type SMAD4. Aberrant TGF-B pathway is clearly associated with increased proportion of cancer stroma, and excluded TIL, or immune-excluded phenotype in a large-scale pan-carcinoma analysis. Citation Format: Gahee Park, Sanghoon Song, Hyung-Gyo Cho, Soo Ick Cho, Wonkyung Jung, Lunit AI team, Sergio Pereira, Donggeun Yoo, Kyunghyun Paeng, Chan-Young Ock. Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes reveals immune-excluded phenotype is correlated with TGF-beta pathway related genomic aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6172.

Expression of colorectal neoplasia differentially expressed in anaplastic thyroid carcinoma and its effect on cancer cell proliferation.

BackgroundThe incidence of anaplastic thyroid cancer (ATC) is high among human cancers. Colorectal neoplasia differentially expressed (CRNDE) is highly expressed in common tumors, and is therefore a potential molecular target for anti-tumor therapy. However, the function of CRNDE in ATC remains elusive.MethodsThe Gene Expression Omnibus (GEO) database was used to screen the differential expression of long-noncoding RNA (lncRNA) in ATC tissues. The Cancer Genome Atlas (TCGA) database was used to analyze the expression of CRNDE in thyroid cancer (THCA) tissues and its impact on patient prognosis. Quantitative real-time PCR (qRT-PCR) was used to determine the expression level of CRNDE in tumor and control tissues. The biological function of CRNDE in THCA was explored using TCGA RNA sequencing (RNA-seq) data analysis. ATC cell lines with low and high CRNDE expression were selected for CRNDE siRNA transfection, and the proliferation of cells was detected in each group.ResultsThe GEO and TCGA databases analysis results showed that CRNDE was highly expressed in ATC tissues, which is related to the poor prognosis of THCA patients. Also, the expression of CRNDE in the ATC cell line, ARO (human thyroid cancer cell line), was relatively high, while the expression in sw579 is relatively low. Therefore, ARO and sw579 were chosen for CRNDE small interfering RNA (siRNA) transfection. Compared with negative control (si-NC), the expression of CRNDE in si-CRNDE-1, si-CRNDE-2, and si-CRNDE-3 was reduced, indicating that the inhibitory effect was significantly enhanced and the cell proliferation ability was reduced, and the cell cycle is arrested in the G0/G1 phase. Finally, it was found that the wnt3a, β-catenin, and cyclinD1 protein expressions of si-CRNDE-1 and si-CRNDE-2 were significantly reduced.ConclusionsThe high expression of CRNDE in ATC tissues may promote the proliferation of ATC cells by regulating the Wnt/β-catenin signaling pathway. CRNDE may be a potential molecular target for the treatment of ATC.

Histologic-Based Tumor-Associated Immune Cells Status in Clear Cell Renal Cell Carcinoma Correlates with Gene Signatures Related to Cancer Immunity and Clinical Outcomes.

The three-tier immunophenotype (desert, excluded, and inflamed) and the four-tier immunophenotype (cold, immunosuppressed, excluded, and hot) have been linked to prognosis and immunotherapy response. This study aims to evaluate whether immunophenotypes of clear cell renal cell carcinoma, identified on hematoxylin and eosin-stained slides, correlate with gene expression signatures related to cancer immunity, and clinical outcomes. We evaluated tumor-associated immune cells (TAICs) status using three methodologies: three-tier immunophenotype based on the location of TAICs, four-tier immunophenotype considering both the location and degree of TAICs and inflammation score focusing only on the degree of TAICs, using a localized clear cell renal cell carcinoma cohort (n = 436) and The Cancer Genome Atlas (TCGA)-KIRC cohort (n = 162). We evaluated the association of the TAICs status assessed by three methodologies with CD8 and PD-L1 immunohistochemistry and immune gene expression signatures by TCGA RNA-sequencing data. All three methodologies correlated with immunohistochemical and immune gene expression signatures. The inflammation score and the four-tier immunophenotype showed similarly higher accuracy in predicting recurrence-free survival and overall survival compared to the three-tier immunophenotype. In conclusion, a simple histologic assessment of TIACs may predict clinical outcomes and immunotherapy responses.

FAM83A as a Potential Biological Marker Is Regulated by miR-206 to Promote Cervical Cancer Progression Through PI3K/AKT/mTOR Pathway

Background and Objective: Chemotherapy and radiotherapy are effective treatment options for cervical cancer (CC), but their efficacy is limited by short survival rate of about 5 years particularly for advance stage CC. Bioinformatics analysis combined with experimental in vivo and in vitro data can identify potential markers of tumorigenesis and cancer progression to improve CC prognosis and survival rate of the patients. This study aims to investigate the prognostic value of family with sequence similarity 83, member A (FAM83A) gene and miR-206 in promoting CC progression and the involved genetic signaling pathways.Method: This was a bioinformatic analysis study based on RNA sequencing data of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and verification by in vivo and in vitro experimental data. It was designed to evaluate whether the aberrantly expressed gene signatures could serve as new potential biomarker to improve prognosis prediction in CC. The TCGA RNA sequencing data [306 cervical squamous cell carcinoma (SCC) and endocervical adenocarcinoma samples and 13 adjacent samples] and GEO data (GSE9750 and GSE52903 datasets) were integrated and performed a bioinformatics analysis.Results: The results showed that CC-associated FAM83A gene serves as a key regulator of CC development and progression. Functionally, we observed that FAM83A is significantly overexpressed in CC, which is linked to poor overall survival as well as disease-free survival in CC patients. The in-vitro and in-vivo assessments performed after silencing FAM83A revealed that cell proliferation was significantly inhibited and the S-phase cell cycle arrest was induced. Mechanistically, FAM83A plays a role in PI3K/AKT signaling, and its downstream molecules could promote CC cell proliferation. Furthermore, functionality assessments by in-vitro luciferase reporter system and immunoblot analysis showed that miR-206 was the upstream of FAM83A and negatively correlated with FAM83A.Conclusion: The miR-206/FAM83A/PI3K/AKT signaling pathway possibly serves as a critical effector in CC progression indicating the potential prognostic value of FAM83A gene as a novel biomarker for CC progression.

Infiltrative tumour growth pattern correlates with poor outcome in oesophageal cancer

ObjectiveOesophageal cancer (OEC) is an aggressive disease with a poor survival rate. Prognostic markers are thus urgently needed. Due to the demonstrated prognostic value of histopathological growth pattern (HGP) in...

Frequency of MicroRNA Response Elements Identifies Pathologically Relevant Signaling Pathways in Triple-Negative Breast Cancer.

SummaryComplex interactions between mRNAs and microRNAs influence cellular functions. The mRNA-microRNA interactions also determine the post-transcriptional availability of mRNAs and unbound microRNAs. MicroRNAs binds to one or more microRNA response elements (MREs) located on the 3′UTR of mRNAs. In this study, we leveraged MREs and their frequencies in cancer and matched normal tissues to obtain insights into disease-specific interactions between mRNAs and microRNAs. We developed a bioinformatics method “ReMIx” that utilizes RNA sequencing (RNA-Seq) data to quantify MRE frequencies across the transcriptome. We applied ReMIx to triple-negative (TN) breast cancer tumor-normal adjacent pairs and identified MREs specific to TN tumors. ReMIx identified candidate mRNAs and microRNAs in the MAPK signaling cascade. Further analysis of MAPK gene regulatory networks revealed microRNA partners that influence and modulate MAPK signaling. In conclusion, we demonstrate a novel method of using MREs in the identification of functionally relevant mRNA-microRNA interactions in TN breast cancer.

Mining featured biomarkers associated with vascular invasion in HCC by bioinformatics analysis with TCGA RNA sequencing data

This study aims to identify the feature genes associated with vascular invasion in hepatocellular carcinoma (HCC). Here, the RNA sequencing data related to vascular invasion in The Cancer Genome Atlas (TCGA) database, including 292 HCC patients with complete clinical data were included in our study as the training dataset for construction and E-TABM-36, including 41 HCC patients with complete clinical data was used as the validation dataset. Following data normalization, differentially expressed mRNA and copy number (CN) were selected between with and without vascular invasion samples. A support vector machine (SVM) classifier was constructed and validated in GSE9828 and GSE20017 datasets. Total 59 feature genes were found by the SVM classifier. Using Cox regression analysis, three clinical features, including Patholigic T, Stage and vascular invasion and 6 optimal prognostic genes, including ANO1, EPHX2, GFRA1, OLFM2, SERPINA10 and TKT were significantly correlated with prognosis. A risk score formula was developed to assess the prognostic value of 6 optimal prognostic genes, which were identified to possess the most remarkable correlation with overall survival in HCC patients. By performing in vitro experiments, we observed TKT was significantly increased, but OLFM2 was decreased in high metastatic potential HCC cell lines (SK-HEP-1 and MHCC-97 H) compared with low metastatic potential cell line Huh7 and normal human liver cell line LO2 using western blotting analysis. Knockdown of TKT in MHCC-97H or overexpression of OLFM2 in SK-HEP-1 significantly suppressed cell migration and invasion using transwell assays. Our results demonstrated that TKT and OLFM2 might be novel independent biomarkers for predicting survival based on the presence of vascular invasion in patients with HCC.

Identification of key genes and pathways by bioinformatics analysis with TCGA RNA sequencing data in hepatocellular carcinoma.

Improved insight into the molecular characteristics of hepatocellular carcinoma (HCC) is required to predict prognosis and to develop a new rationale for targeted therapeutic strategy. Bioinformatics methods, including functional enrichment and network analysis combined with survival analysis, are required to process a large volume of data to obtain further information on differentially expressed genes (DEGs). The RNA sequencing data related to HCC in The Cancer Genome Atlas (TCGA) database were analyzed to screen DEGs, which were separately submitted to perform gene enrichment analysis to identify gene sets and signaling pathways, and to construct a protein-protein interaction (PPI) network. Subsequently, hub genes were selected by the core level in the network, and the top hub genes were focused on gene expression analysis and survival analysis. A total of 610 DEGs were identified, including 444 upregulated and 166 downregulated genes. The upregulated DEGs were significantly enriched in the Gene Ontology analysis (GO): Cell division and in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway: Cell cycle, whereas the downregulated DEGs were enriched in GO: Negative regulation of growth and in the KEGG pathway: Retinol metabolism, with significant differences. Cyclin-dependent kinase (CDK)1 was selected as the top hub gene by the PPI network, which exhibited a similar expression trend with the data from the Gene Expression Omnibus (GEO) database. Survival analysis revealed a significantly negative correlation between CDK1 expression level and overall survival in the TCGA group (P<0.01) and the GEO group (P<0.01). Therefore, high-throughput TCGA data analysis appears to be an effective method for screening tumor molecular markers, and high expression of CDK1 is a prognostic factor for HCC.

Analysis of key genes and signaling pathways involved in Helicobacter pylori-associated gastric cancer based on The Cancer Genome Atlas database and RNA sequencing data.

Helicobacter pylori (H. pylori) infection is associated with the development of gastric cancer, although the mechanism is unclear. Herein, this study aimed to clarify the key genes and signaling pathways involved in H. pylori pathogenesis based on The Cancer Genome Atlas (TCGA) database and RNA sequencing analysis. Forty-nine gastric cancer samples (16 with H. pylori and 33 without H. pylori) and 35 cancer-adjacent normal samples from TCGA database were analyzed by bioinformatics. The differentially expressed genes between H. pylori-positive and H. pylori-negative patients were verified in 18 gastric cancer (GC) samples (9 with H. pylori and 9 without H. pylori), which were analyzed using RNA sequencing. Survival analysis was carried out to explore associations between the differentially expressed genes and prognosis. Bioinformatics analysis was performed to determine the signaling pathways associated with H. pylori. The baseline level of clinical features from TCGA database and RNA sequencing showed no differences between the H. pylori-positive and H. pylori-negative GC groups (P>0.05). TP53 was shown to be upregulated in the H. pylori-positive group in both TCGA database and RNA sequencing data, which also showed higher expression in the GC tissues than in adjacent normal tissues (P<0.05). CCDC151, CHRNB2, GMPR2, HDGFRP2, and VSTM2L were shown to be downregulated in the H. pylori-positive group by both TCGA database and RNA sequencing, which also showed lower expression in the GC tissues than in adjacent normal tissues (P<0.05). GC patients with low expression levels of HDGFRP2 had a poor prognosis (P<0.05). Thirty-three signaling pathways and 10 biological processes were found to be positively associated with H. pylori infection (P<0.05, FDR<0.05). These results indicate that some genes (TP53, CCDC151, CHRNB2, GMPR2, HDGFRP2, VSTM2L) and previously unidentified signaling pathways (eg, the Hippo signaling pathway) might play an important role in H. pylori-associated GC.

ZNF154 is a promising diagnosis biomarker and predicts biochemical recurrence in prostate cancer

ObjectiveTo screen the methylated genes for early diagnosis and biochemical recurrence (BCR) prediction in prostate cancer (PCa) patients. MethodsDifferentially methylated CpG sites (DMCs) of PCa were screened out from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database. Combined with TCGA RNA sequencing data and clinical information, the DMCs associated genes with different expression and related to BCR were selected as candidate genes. Then, the expression level of the best candidate gene ZNF154 was validated by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, the prognosis potential of the hypermethylation gene ZNF154 was assessed by Kaplan-Meier, univariate and multivariate cox regression analysis. ResultsA total of 87 candidate genes were screened out. Compared to benign prostate (BP) tissues, ZNF154 has three hypermethylation sites (cg03234186, cg12506930, cg26465391) in the promoter region in PCa tissues. qRT-PCR results showed that ZNF154 expression level was reduced in PCa tissues than in BP tissues (P = 0.004). Besides, the ZNF154 methylation level was negatively correlated with mRNA expression (r = −0.766, P < 0.001), and was highly cancer-specific in PCa (area under the curves (AUCs) = 90.030%). In addition, Kaplan-Meier analysis showed ZNF154 methylation level was associated with BCR (P = 0.005), and ZNF154 could be an independent factor for BCR prediction in PCa by using univariate and multivariate cox regression analysis (P = 0.035, HR = 8.218). Conclusions87 PCa specific genes were obtained. Further analysis gave the evidence that ZNF154 can be used as a specific maker for PCa diagnosis. Hypermethylation level of ZNF154 lead to gene expression inhibition and function loss, which contribute to the development and poor outcomes in PCa. In addition, the mean methylation level of ZNF154 can be used as an independent risk factor to predict BCR.

Abstract LB-235: DNA methylation of individual repetitive elements in HCV infection-induced HCC

Abstract Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. Hepatitis C virus (HCV) infection is a major risk factor for HCC in the US. HCV infection may cause cancer by suppressing host defense that controls the propagation of repetitive element (RE) sequences, including LINE-1 and Alu, the most representative REs in the human genome. DNA methylation of RE helps stabilize the genome, while reduced RE methylation, also referred as global hypomethylation, leads to genomic instability and is known as a hallmark of cancer. However, current available high-throughput technology for DNA methylation profiling only enables genomewide RE methylation study on a global scale. The role of individual RE methylation in cancer is largely understudied. In this study, using our recently developed and published machine learning algorithm to enhance the RE coverage of the Illumina methylation array platform, we examined the role of individual RE methylation in HCV-induced HCC across the entire genome. We conducted an epigenome-wide association study on 22 HCC, 20 cirrhotic, and 44 normal liver tissues. We included equal numbers of HCV-induced and alcohol-induced HCC/cirrhosis cases. The Illumina 450k array profiled 1939 REs and we computationally predicted 33184 REs; correlations between the overlapping 1541 profiled and predicted RE methylation were &amp;gt; 0.91. Sex, tumor differentiation, and tumor stage were similarly distributed across the HCV and alcohol groups. We adjusted for batch effects and used Benjamini-Hochberg adjusted false discovery rate (FDR) to account for multiple testing. Relative to normal liver tissue, we observed over twice as many differentially methylated (FDR &amp;lt; 0.05) REs in HCV-HCC compared to alcohol-HCC tissue (2911 vs 1304, 749 in common). We validated our results in The Cancer Genome Atlas (TCGA) (325 vs 129, 74 in common) with high correlation (r = 0.90). We also observed that 169 REs were significantly altered in HCV-cirrhosis tissue (compared to none in alcohol-cirrhosis tissue), of which, 17 REs in 13 genes, including PIK3CD, LY9, GCK, NLRC3, and PRKCZ, were consistently hypomethylated in both HCC and cirrhotic liver tissue induced by HCV (r = 0.87). Using TCGA RNA sequencing data, we found that these REs were located in gene body and their hypomethylation was associated with lower expression of their host genes (r &amp;gt; 0.3, p &amp;lt; 0.05). Gene Ontology Enrichment analysis showed that these genes were enriched in T-cell activation &amp; differentiation and cytokine production (FDR &amp;lt; 0.05). Our findings indicate that HCV infection has a more extensive impact on the DNA methylation landscape in RE than alcohol consumption, and that HCV infection may also induce similar hypomethylation in cirrhotic liver. Hypomethylated RE may alter local chromatin environment and disrupt expression of genes involved in immune and inflammatory responses, both of which may promote HCC development. RE hypomethylation in specific genomic locus may serve as early detection biomarker of HCC in patients with HCV-induced cirrhosis. Citation Format: Yinan Zheng, Jimmy Ren, Ryan A. Hlady, Keith D. Robertson, Robert L. Murphy, Lewis R. Roberts, Lifang Hou. DNA methylation of individual repetitive elements in HCV infection-induced HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-235.

Lyc-55716: A novel small-molecule RORγ agonist immuno-oncology agent: Rationale for tumor selection and clinical evaluation of gastric and esophageal carcinoma in phase 2a expansion.

67 Background: Type 17 effector T cell differentiation and function are regulated by the master transcription factor retinoic acid receptor–related orphan receptor γt (RORγ). Synthetic RORγ agonists, which modulate immune cell gene expression to enhance effector functions and decrease Treg and checkpoint pathways, have shown promise as mono- and combination therapy in syngeneic tumor models. Translational and bioinformatics assessments were performed during Phase 1 testing of the investigational RORγ agonist LYC-55716 to support inclusion of patients with gastric carcinoma (GC) and esophageal carcinoma (EC) in a Phase 2a expansion of clinical trial LYC-55716-1001 (NCT02929862). Methods: Transcriptional profiling of murine and human T cells treated ± RORγ agonists identified a gene signature. Using The Cancer Genome Atlas (TCGA), and other public datasets, data on GC and EC patients were evaluated to determine: (a) RORγ and RORγ-inducing cytokine expression; (b) signature genes associated with RORγ biology, surrogate biomarkers for endogenous RORγ ligands, and correlations with prognosis; (c) tumor microenvironment (TME) immune profiles. Results: Expression: In TCGA RNA sequencing data analysis, 50%-70% of GC and EC samples expressed moderate to high levels of RORγ, indicating infiltration of Type 17 T cells into the tumors. Furthermore, genes that support expression of RORγ were highly expressed in ~75% of GC and EC tumors. Biology: TCGA data showed that GC and EC tumors express low levels of sterol efflux genes, suggesting low levels of endogenous agonists in the TME. In analysis of public datasets, expression of the RORγ target gene IL17A correlated with a favorable prognosis for EC. In addition, some of the other RORγ signature genes also correlated with good prognosis in GC and EC. Immune profile: TCGA showed infiltration of T cells and a high mutational burden in GC and EC, which are positively associated with efficacy of immunotherapy. Conclusions: Bioinformatics assessments of RORγ expression, biology, and tumor immune profiles support the inclusion of GC and EC patients in an ongoing Phase 2 clinical trial of LYC-55716.

Lowly methylated region analysis identifies EBF1 as a potential epigenetic modifier in breast cancer

ABSTRACTBreast cancer (BC) encompasses heterogeneous pathologies with different subtypes exhibiting distinct molecular changes, including those related to DNA methylation. However, the role of these changes in mediating BC heterogeneity is poorly understood. Lowly methylated regions (LMRs), non-CpG island loci that usually contain transcription factor (TF) binding sites, have been suggested to act as regulatory elements that define cellular identity. In this study, we aimed to identify the key subtype-specific TFs that may lead to LMR generation and shape the BC methylome and transcription program. We initially used whole-genome bisulfite sequencing (WGBS) data available at The Cancer Genome Atlas (TCGA) portal to identify subtype-specific LMRs. Differentially methylated regions (DMRs) within the BC PAM50 subtype-specific LMRs were selected by comparing tumors and normal tissues in a larger TCGA cohort assessed by HumanMethylation450 BeadChip (450K) arrays and TF enrichment analyses were performed. To assess the impact of LMRs on gene expression, TCGA RNA sequencing data were downloaded and Pearson correlations between methylation levels of loci presenting subtype-specific TF motifs and expression of the nearest genes were calculated. WGBS methylome data revealed a large number of LMRs for each of the BC subtypes. Analysis of these LMRs in the 450K datasets available for a larger sample set identified 7,765, 5,657, and 19 differentially methylated positions (DMPs) between normal adjacent tissues and tumor tissues from basal, luminal, and HER2-enriched subtypes, respectively. Unsupervised clustering showed that the discriminatory power of the top DMPs was remarkably strong for basal BC. Interestingly, in this particular subtype, we found 4,409 differentially hypomethylated positions grouped into 1,185 DMRs with a strong enrichment for the early B-cell factor 1 (EBF1) motifs. The methylation levels of the DMRs containing EBF1 motifs showed a strong negative correlation with the expression of 719 nearby genes, including BTS2 and CD74, two oncogenes known to be specific for basal BC subtype and for poor outcome. This study identifies LMRs specific to the three main BC subtypes and reveals EBF1 as a potentially important regulator of BC subtype-specific methylation and gene expression program.

Predictive role of galectin-1 and integrin α5β1 in cisplatin-based neoadjuvant chemotherapy of bulky squamous cervical cancer.

Although galectin-1 and integrin α5β1 confer chemoresistance to certain types of cancer, whether their expression predicts the response to cisplatin-based neoadjuvant chemotherapy (NACT) in squamous cervical cancer remains unclear. Paired tumor samples (pre- and post-chemotherapy) were obtained from 35 bulky squamous cervical cancer patients treated with cisplatin-based NACT and radical hysterectomy at our hospital between January 2007 and August 2014. The expression of galectin-1 and integrin α5β1 in tumor cells and stromal cells was analyzed by immunohistochemistry. The correlation between galectin-1/integrin α5β1 and apoptosis-associated markers was investigated by using the The Cancer Genome Atlas (TCGA) RNA-sequencing data. Seventeen patients were identified as chemotherapy responders and 18 as non-responders. Galectin-1 and integrin α5β1-positive immunostaining was more frequently observed in stromal cells than its in tumor cells. The expression of galectin-1 and integrin α5β1 in stromal and tumor cells was significantly down-regulated in postchemotherapy cervical cancer tissues. High levels of galectin-1 and integrin α5β1 in stromal were associated with a negative chemotherapy response in squamous cervical cancer patients treated with cisplatin-based NACT. Additionally, the expression of galectin-1 and integrin α5 correlated negatively with caspase 3/caspase 8 by using the TCGA RNA-sequencing data. Galectin-1 and integrin α5β1 expression in stromal may serve as a prediction of the responses to cisplatin-based NACT for patients with bulky squamous cervical cancer. Galectin-1 and integrin α5β1 may be implicated in the development of chemoresistance in cervical cancer via suppressing apoptosis.

Abstract P6-04-02: Identification of ESR1 splice variants associated with prognosis in estrogen receptor positive breast cancer

Abstract Background: Splice variants play a major role in carcinogenesis and disease progression. It is well known that androgen receptor splice variants are associated with resistance to prostate cancer treatment. Estrogen receptor (ER)-positive breast cancers constitute about 70% of all breast cancers and have better prognosis compared to ER-negative cancers. However, there are ER-positive breast cancers that acquire resistance to anti-estrogen therapy, and 12-55% of those tumors were shown to possess ESR1 mutations. The aim of this study was to identify common splice variants in the ESR1 gene and investigate their association with disease outcome. Methods: Whole transcriptome sequencing was performed on breast cancer specimens from 120 invasive breast cancer patients who underwent operation at Seoul National University Hospital (SNUH) and data from SNUH, GEO, and The Cancer Genome Atlas (TCGA) was used for normal breast tissue sequencing. Exon-exon junctions were identified on aligned RNA sequencing data and was used to construct exon graphs. Splice variant candidates were selected from exon graphs and were merged according to variant subtypes of samples. Subtypes were accessed differentially in relation to how frequent the junctions appear in tumor samples and common exon skipping types with frequent junctions were identified. TCGA RNA sequencing data was then used to search for the common exon skipping subtypes detected from SNUH RNA sequencing data. Results: Of the 120 tumor samples, 50 were clinically ER-positive by immunohistochemistry. Among exon paths logically possible, 125 paths were not observed in normal breast tissues. Exon 4-5 junction was the most commonly observed junction in the tumor samples. In a search for exon skipping type that results in missing ligand-binding domain of ER, three exon skipping types were identified. Exon skipping with exon 5-10 junction (type 1), exon 9-12 junction (type 2), and exon 10-12 (type 3) was seen in 4 (8%), 4 (8%), and 10 (20%) ER-positive samples, respectively. Retrospective medical chart review of the 18 patients showed recurrence in 4 (100%), 2 (50%), and 4 (40%) patients with type 1, 2, and 3 exon skipping, respectively. Evaluation of TCGA RNA sequencing data of 872 ER-positive samples suggested exon 4-5 junction as the most common junction. A search for exon skipping types in TCGA revealed 1 (0.1%), 9 (1.0%), and 454 (52.1%) samples with type 1, 2, and 3 exon skipping, respectively. However, none of the patients with type 1 or 2 had metastasis or had expired. Of the 454 patients with type 3 exon skipping, 54 patients had died, constituting 61.4% of 88 mortalities in the whole ER-positive population. Conclusion: Certain splice variants of ESR1 gene yields exon skipping subtypes commonly observed in the ER-positive breast cancer. Estrogen receptor-positive breast cancer with these exon skipping types resulting in a missing ligand-binding domain of ER may be associated with poorer disease outcome. Further investigation is warranted to validate the role of ESR1 exon skipping subtypes in the disease progression of breast cancer. Citation Format: Lee H-B, Han W, Ko S, Kim M-S, Lim S, Lee K-M, Kang YJ, Han JH, Kim Y, Yoo T-K, Moon H-G, Noh D-Y, Kim S, Han W. Identification of ESR1 splice variants associated with prognosis in estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-02.

Abstract 1544: The clinical behavior of endometrioid and serous endometrial carcinomas is governed by distinct biological processes

Abstract Background: Endometrioid and serous carcinomas of the uterus, the two most common histological types of endometrial carcinoma, have been shown to have distinct clinicopathological characteristics and to be underpinned by distinct constellations of molecular aberrations. We sought to define the biological processes that govern the clinical behavior of endometrial cancers based on their transcriptomic characteristics as defined by RNA-sequencing. Material and Methods: RNA-sequencing-based gene expression and clinicopathological data from 323 endometrial cancers were retrieved from The Cancer Genome Atlas (TCGA). Sixteen prototype genes representative of different biological processes that would likely play a role in endometrial and other hormone-driven cancers were defined. TCGA RNA-sequencing data were used to determine the transcription module of each prototype gene. The expression of prototype genes and modules and their association with outcome was assessed in univariate and multivariate survival models in the whole cohort, and in specific subtypes, including endometrioid vs serous, grade 1, 2 and 3 cancers, and the genomic subtypes as described in the TCGA study. Results: The clinical behavior of endometrial cancers was found to be associated with hormone receptor signaling, PI3K pathway signaling and DNA mismatch repair processes. Furthermore, when adjusted for the known prognostic clinicopathological parameters, we found that MSH6 mRNA expression was independently associated with the outcome of patients with endometrial cancer. At variance with other hormone-driven tumors, such as breast cancer, the level of expression of proliferation-related genes was not a predictor of outcome in patients with endometrial cancers. The biological processes governing the outcome of endometrioid and serous endometrial cancers were shown to be distinct; whilst in endometrioid carcinomas hormone receptor, PI3K and DNA mismatch repair modules were associated significantly with outcome in univariate analysis, the clinical behavior of serous cancers was significantly associated with the expression of the apoptosis-related gene CASP3 and Wnt signaling (CTNNB1). When stratified according to grade, the PLAU prototype gene and its invasion module were found to be significantly associated with the survival of patients with grade 3 endometrial cancer, but not of those with grade 1 or 2 cancers. Conclusion: Our data suggest that stratification of endometrial cancers according to histological type (i.e. endometrioid vs serous) and potentially grade may be of importance for the design of clinical trials testing targeted therapeutics as these parameters define subgroups of endometrial cancers that not only have distinct genetic aberrations but also distinct molecular pathways that define the outcome of patients with this disease. Citation Format: Britta Weigelt, Christophe Lemetre, Charlotte K.Y. Ng, Salvatore Piscuoglio, Jorge S. Reis-Filho. The clinical behavior of endometrioid and serous endometrial carcinomas is governed by distinct biological processes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1544. doi:10.1158/1538-7445.AM2014-1544