Whole-transcriptome Sequencing Research Articles

Abstract 6073: Distinct subtype of multiple myeloma revealed by whole genome and transcriptome sequencing

Abstract Background: Advanced sequencing technologies have contributed to identifying genomic and transcriptomic features of various types of tumors. Despite several sequencing data from multiple myeloma (MM) being generated, understanding various properties of MM remained insufficient. Methods: The patients confirmed with MM by bone marrow examination were analyzed in the study. Myeloma cells were enriched from the bone marrow aspirates of the patients using CD138. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) were performed for isolated myeloma cells. Mutational signatures were analyzed using somatic mutations detected by WGS. Results: A total of 37 MM patients (median age 67) were enrolled in this study. WGS revealed somatic mutations of an average of 8872 single nucleotide variants (SNVs) and 934 insertions and deletions (Indels) from the patients, respectively. By the analysis of structural variants, 7 chromothripsis and 4 chromoplexy patients were identified. Mutational signature analysis showed that single base substitution (SBS) 9 signature largely varied between patients. SBS9 signature high patients were mostly diagnosed with non-IgG/non-IgA heavy chain and lambda light chain type myeloma. Notably, IgD myeloma patients were solely detected in the SBS9 signature high group compared to the low group (p = 0.001). Gene expression patterns obtained from WTS were well-divided into SBS9 signature high and low groups, and analysis of differentially expressed genes (DEG) between SBS9 signature high and low groups implied IgD myeloma features including high expression of IGHD. As a validation test, we tried mutational signature analysis on whole exome sequencing (WES) data of 784 MM patients from the MMRF database. Of the 38 patients with more than 500 exonic mutations, only the SBS9 signature high group had IgD myeloma candidates, whereas the SBS9 signature low group had no IgD myeloma candidate (p = 0.014). Conclusions: DNA and RNA sequencing of myeloma patients could classify specific subtypes of disease categories. Further study for SBS 9 signature high and IgD myeloma is needed to discover the underlying mechanism of distinct features and to find out their clinical implication. Citation Format: Sheehyun Kim, Hyundong Yoon, Youngil Koh, Sung-Soo Yoon. Distinct subtype of multiple myeloma revealed by whole genome and transcriptome sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6073.

Abstract 5895: Analysis of microbiome in gastric MALT lymphoma

Abstract Gastric MALT (mucosa-associated lymphoid tissue) lymphoma is found at a rate of 50% or more among the types of MALT lymphoma. Majority of patients with gastric MALT lymphoma are infected with Helicobacter pylori and also have clinical improvement with eradication therapy. However, it is unclear how H. pylori cause gastric MALT lymphoma. To explore it, we prospectively collected 13 patients who are newly diagnosed with gastric MALT lymphoma with H. pylori. We gathered samples from normal regions and lesions during endoscopy for diagnosis. Samples of lesion were harvested again after eradication of H. pylori. we sequenced 10 of WES (whole exome sequencing) and 13 of WTS (whole transcriptome sequencing) with these samples. With sequencing data, we aligned the sequenced reads to human reference genome (hg19) using BWA and called the variants with Mutect2. We aligned the reads and calculated TPM (Transcripts Per Million) from WTS with STAR and RSEM. We performed DEG (Differentially Expressed Genes) and GSEA (Gene Set Enrichment Analysis) for three comparison groups which are divided into normal, lesion, and after eradication. Gene fusions were detected using STAR-Fusion and Arriba with WTS. To detect potential microbiome from the samples, we used Kraken2, a taxonomic classification tool. We found that MUC6 was mutated from all samples and some samples have mutated MUC3A, KMT2C, ZNF595, CDC27, etc., and two have BIRC3-MALT1 fusion. From DEG and GSEA analysis, G protein-coupled receptor activity was activated in the group of lesion samples and suppressed in normal samples and eradication samples. Lymphocyte activation and immune response were also activated in the cancerous lesion. Kraken2 classified the reads which were not aligned to the human reference genome as reads from microbiota including H. pylori. The reads of H. pylori were more abundant in the lesion group than the other groups. According to the result of differentially expressed species analysis between the groups of lesion and eradication, the following species were also more abundant in the lesion group; Veillonella dispar, Helicobacter hepaticus, Helicobacter winghamensis, etc. We analyzed genetic alterations and differentially expressed genes and also classified non-human reads as microbiome with sequencing data from gastric MALT lymphoma. Our results suggest how H.pylori infection is related to carcinogenesis of gastric MALT lymphoma. Citation Format: SeungJoo Yoo, Dong-Yeop Shin, Ja Min Byun, Junshik Hong, Hongseok Yun, Youngil Koh, Sung-Soo Yoon. Analysis of microbiome in gastric MALT lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5895.

Abstract 6165: Identification of biomarkers of early onset colorectal cancer from transciptome and whole genome data

Abstract Colorectal cancer (CRC) is not only the third most commonly occurred cancer worldwide, but it is the leading cause of cancer deaths in 2018. However, the overall incidence of CRC and death from CRC has declined since 1998, the case of early-onset CRC (EOCRC) defined as CRC diagnosed under age 50 has been increasing. Including the etiology of EOCRC, the reasons underlying this upward trend are not well understood. Moreover, some suggested that EOCRC patients have difficulty in early diagnosis compared to late-onset colorectal cancer (LOCRC). For this reason, EOCRC patients tend to be diagnosed at advanced stages. In addition to the clinical feature, EOCRC tumors have different features in pathology and molecularity compared with LOCRC tumors. With these distinct characteristics of EOCRC, there is necessary to characterize EOCRC compared to LOCRC patients. In this study, patients under 50 years (from hereon referred to as ‘‘early-onset’’) and over 70 (‘‘late-onset’’) were considered for comparison. Whole transcriptome sequencing (WTS) for profiling gene expression and whole genome sequencing for mutations were carried out with tumor tissues of early-onset (n=49) and late-onset (n=50) CRC patients. Subsequently, we performed bioinformatic analysis to identify genomic and transcriptomic features. For the precise selection of differentially expressed genes, we selected constantly differentially expressed genes (DEG) in TCGA COAD and READ data. Genes up-regulated in both data set were CPS1, CCL19, CHGB, GLDC, WASF3, RPL39L and MAP9. The down-regulated were PRSS33, ANPEP, DKK4 and NTS. To identify whether CMS classification had different feature in each type if age onset was different, we classified CMS subtypes using transcriptome data. The ratio of each CMS subtypes both early and late-onset group had no significant difference. In terms of somatic mutations reported for driving colorectal cancer, three genes (ATM, BCL9L, NF1) had significantly mutated in early-onset group. Moreover, Non-synonymous mutation frequency had no significant difference while total mutation frequency had significantly difference between early and late-onset group. Lastly, Mutational signature had difference in both groups, assuming that the early-onset group had been through different event attributing to mutational changes. Although these findings should be further validated by detailed functional experiments, our genomic and transcriptomic analysis provide additional insights into understanding of young CRC patients. Citation Format: Yejin Ha, Yun-Jae Shin, Ka Hee Tak, Jong-Lyul Park, Jeong-Hwan Kim, Jong Lyul Lee, Seon-Young Kim, Chan Wook Kim. Identification of biomarkers of early onset colorectal cancer from transciptome and whole genome data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6165.

Abstract 2054: Whole-genome sequencing based homologous recombination deficiency testing for precision oncology of breast cancers

Abstract Breast cancers (BCs) with defects in homologous recombination repair (HRR) pathway depend on the Poly (ADP-ribose) polymerase enzymes (PARP) for their survival and thus are sensitive to PARP inhibitors (PARPi). To screen out BCs with homologous recombination deficiency (HRD), germline mutations in BRCA genes have been tested, which constitute approximately 5% of unselected BC patients. However, a substantial proportion of BCs show HRD phenotypes despite the absence of BRCA inactivating mutations in germline. Herein, we apply whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS) in 332 samples from 325 BC patients for characterizing their HRD phenotypes for PARPi treatments. Of 325 BC patients, 75 cases (23.1%) showed remarkable HRD phenotypes, based on mutational signatures of HRR abrogation. Intriguingly, 51 cases do not harbor any germline mutations in HR-related genes including BRCA1 and BRCA2. The whole-genome portrait of these samples was similar to that of HRD-positive BCs with germline mutation in HRR pathway, but showed markedly distinctive features from HRD-negative BCs. With a notable exception of BRCA1, the second most common somatic mutation in HRD-positive BCs without germline mutation, the mutational landscape of the whole study samples was primarily determined by hormone receptor and HER2 positivity, not by HRD status. Likewise, WTS-based clusters determined in an unsupervised manner were highly dependent on the histological subtype of BC, not on HRD status or germline mutations in the HRR pathway. Notably, differentially expressed gene and gene set enrichment analysis revealed that HRD-positive BCs had an increased inflammatory activity compared to BCs with proficient HR. Overall, our analysis demonstrate that WGS enables better classification of the HR status in BC than conventional BRCA germline testing, suggesting WGS as a rapid and sensitive tool for screening breast cancers for PARPi treatment. Citation Format: Ryul Kim, Seongyeol Park, Joonoh Lim, Boram Yi, Jaemo Koo, Sangmoon Lee, Jeong Seok Lee, Yeon Hee Park, Young Seok Ju. Whole-genome sequencing based homologous recombination deficiency testing for precision oncology of breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2054.

Abstract 239: Whole-genome sequencing based homologous recombination deficiency testing for precision oncology of breast cancers

Abstract Breast cancers (BCs) with defects in homologous recombination repair (HRR) pathway depend on the Poly (ADP-ribose) polymerase enzymes (PARP) for their survival and thus are sensitive to PARP inhibitors (PARPi). To screen out BCs with homologous recombination deficiency (HRD), germline mutations in BRCA genes have been tested, which constitute approximately 5% of unselected BC patients. However, a substantial proportion of BCs show HRD phenotypes despite the absence of BRCA inactivating mutations in germline. Herein, we apply whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS) in 332 samples from 325 BC patients for characterizing their HRD phenotypes for PARPi treatments. Of 325 BC patients, 75 cases (23.1%) showed remarkable HRD phenotypes, based on mutational signatures of HRR abrogation. Intriguingly, 51 cases do not harbor any germline mutations in HR-related genes including BRCA1 and BRCA2. The whole-genome portrait of these samples was similar to that of HRD-positive BCs with germline mutation in HRR pathway, but showed markedly distinctive features from HRD-negative BCs. With a notable exception of BRCA1, the second most common somatic mutation in HRD-positive BCs without germline mutation, the mutational landscape of the whole study samples was primarily determined by hormone receptor and HER2 positivity, not by HRD status. Likewise, WTS-based clusters determined in an unsupervised manner were highly dependent on the histological subtype of BC, not on HRD status or germline mutations in the HRR pathway. Notably, differentially expressed gene and gene set enrichment analysis revealed that HRD-positive BCs had an increased inflammatory activity compared to BCs with proficient HR. Overall, our analysis demonstrate that WGS enables better classification of the HR status in BC than conventional BRCA germline testing, suggesting WGS as a rapid and sensitive tool for screening breast cancers for PARPi treatment. Citation Format: Ryul Kim, Seongyeol Park, Joonoh Lim, Boram Yi, Jaemo Koo, Sangmoon Lee, Jeong Seok Lee, Yeon Hee Park, Young Seok Ju. Whole-genome sequencing based homologous recombination deficiency testing for precision oncology of breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 239.

Abstract 4865: Mechanisms of EGFR inhibitor sensitivity and resistance in chordoma

Abstract Target discovery studies have identified EGFR inhibition as a promising therapeutic strategy in chordoma, motivating ongoing clinical trials. Yet EGFR is not mutated in chordoma, leaving the mechanisms associated with EGFR inhibitor (EGFRi) sensitivity and resistance unclear. In this study, we profiled a panel of over 10 chordoma cell lines to categorize their sensitivity to afatinib, a second-generation EGFRi with potency against the wild-type receptor. Several cell lines, including U-CHCF365, UM-Chor1, MUG-Chor1, and U-CH1, were highly sensitive to afatinib in cell viability assays, with absolute IC50 values below 50 nanomolar. In contrast, other chordoma cell lines, including UM-Chor5C and U-CHCF359B, exhibited limited response to EGFRi at concentrations exceeding 1 micromolar. Among sensitive cell lines, we found that afatinib promoted cell death in some cases (e.g., U-CH1) whereas in others it induced a profound cell cycle arrest. Moreover, in U-CH1 cells, acute afatinib treatment enriched a population of drug-tolerant persister cells with a fusiform morphology, potentially indicative of an epithelial-mesenchymal transition. Ongoing studies are focused on understanding the mechanisms of afatinib-induced cell death, drug tolerance, and acquired resistance, with a view to designing rational combination strategies capable of enhancing the magnitude and duration of therapeutic response. In cell lines with matched xenograft models, in vitro responses to afatinib were consistent in vivo. In a panel of 12 chordoma patient-derived xenograft (PDX) models treated with afatinib or cetuximab, differential sensitivity to EGFRi was recapitulated - with EGFRi promoting complete or negligent tumor growth inhibition in sensitive or resistant models, respectively. Finally, we combined drug sensitivity profiling of chordoma cell lines and PDXs with whole-exome and whole-transcriptome sequencing to identify genomic and transcriptomic features associated with sensitivity and resistance to EGFRi in chordoma. Collectively, our data identify a striking degree of differential sensitivity to EGFRi in chordoma, and begin to shed light on factors associated with primary and acquired resistance. These results provide a framework for guiding patient selection and identifying potential combination therapy regimens to improve EGFRi efficacy and address resistance. Citation Format: Nindo Punturi, Lee Dolat, Joan Levy, Josh Sommer, Daniel M. Freed. Mechanisms of EGFR inhibitor sensitivity and resistance in chordoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4865.

Abstract 613: T cell antitumor function requires potassium transport

Abstract Background: The past 20 years of translational oncology provide a framework in which the ability to eradicate cancer has increased in direct relation to our understanding of T cell biology. The cancer-killing functions of T cells are often compromised within tumors, allowing disease progression. Tumor-derived factors and the hostile microenvironment contribute to constraining of T cell effector functions and driving terminal differentiation. We previously identified that elevated extracellular K+ characteristics of the tumor microenvironment limits both TCR dependent activation and effector functions, while preserving coincidently preserving T cell stemness (Eil R, Nature 2016 & Science 2019). Despite these findings and the centrality of ion gradients to cellular physiology broadly, our understanding of T cell K+ transport remains rudimentary and cannot account for the pleiotropic role K+ abundance plays in antitumor immunity. In fact, little is known about what K+ transporters T cell express nor their functional regulation. Findings: Here, we report the canonical Na+-K+-ATPase to be intertwined with T cell metabolic homeostasis, memory formation, and antitumor activity and therefore an appealing avenue for T cell reprogramming. We first performed whole-transcriptomic sequencing of CD8+ memory subsets and found that components of the Na+-K+-ATPase were expressed at robust levels that were notably enriched in naïve and memory cells. Despite partially impairing Ca2+ influx, T cells genetically deficient for the Na+-K+-ATPase exhibited constitutive activity in both TCR and Akt-mTOR associated signaling pathways along with rapid acquisition of co-inhibitory surface receptors, mitotic arrest, and functional exhaustion. Mechanistically, we identified pathologic ROS accumulation as the driver of unregulated signaling and rapid exhaustion. In vivo, T cells lacking deficient in Na+-K+-ATPase activity failed to achieve proliferative burst following pathogen challenge and demonstrated severely compromised antitumor function. Our results support a model in which the Na+- K+- ATPase primarily functions in T cells to maintain ROS metabolism, thereby preventing its pathologic accumulation and preserving T cell stemness and antitumor potential. Conclusion: These results uncover a fundamental yet underappreciated aspect of T cell biology with translational implications for the ongoing development of cancer immunotherapies. Immediate applications include pharmacologic and genetic targeting of ion transporters in the setting of immune checkpoint blockade or T cell transfer therapies (i.e. CAR, TCR, TIL). Citation Format: Camille Collier, Matthew McWhorter, Kelly Wucherer, Chelsea Jenkins, Alexandra Bartlett, Robert Eil. T cell antitumor function requires potassium transport [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 613.

MP69-01 ESTABLISHMENT OF A NOVEL ANIMAL MODEL IN CARCINOGENESIS OF UPPER TRACT UROTHELIAL CARCINOMA AND ITS GENE PROFILE

You have accessJournal of UrologyCME1 Apr 2023MP69-01 ESTABLISHMENT OF A NOVEL ANIMAL MODEL IN CARCINOGENESIS OF UPPER TRACT UROTHELIAL CARCINOMA AND ITS GENE PROFILE Akinaru Yamamoto, Atsunari Kawashima, Kentaro Jingushi, Yuichi Motoyama, Satoshi Nojima, Sassi Nesrine, Yohei Okuda, Toshiki Oka, Toshihiro Uemura, Gaku Yamamichi, Eisuke Tomiyama, Kosuke Nakano, Makoto Matsushita, Yu Ishizuya, Yoshiyuki Yamamoto, Taigo Kato, Koji Hatano, Tsujikawa Kazutake, and Norio Nonomura Akinaru YamamotoAkinaru Yamamoto More articles by this author , Atsunari KawashimaAtsunari Kawashima More articles by this author , Kentaro JingushiKentaro Jingushi More articles by this author , Yuichi MotoyamaYuichi Motoyama More articles by this author , Satoshi NojimaSatoshi Nojima More articles by this author , Sassi NesrineSassi Nesrine More articles by this author , Yohei OkudaYohei Okuda More articles by this author , Toshiki OkaToshiki Oka More articles by this author , Toshihiro UemuraToshihiro Uemura More articles by this author , Gaku YamamichiGaku Yamamichi More articles by this author , Eisuke TomiyamaEisuke Tomiyama More articles by this author , Kosuke NakanoKosuke Nakano More articles by this author , Makoto MatsushitaMakoto Matsushita More articles by this author , Yu IshizuyaYu Ishizuya More articles by this author , Yoshiyuki YamamotoYoshiyuki Yamamoto More articles by this author , Taigo KatoTaigo Kato More articles by this author , Koji HatanoKoji Hatano More articles by this author , Tsujikawa KazutakeTsujikawa Kazutake More articles by this author , and Norio NonomuraNorio Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003332.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper urinary tract (UT) and bladder are known to differ from the embryology stage. There have been numerous reports of mouse models of bladder carcinogenesis, but there are few reports of mouse models of upper tract urothelial carcinogenesis. The aim of this study is to establish a novel mouse model of upper tract urothelial carcinogenesis. METHODS: We used two strains including C57/BL6 of male and female mice. We fed 0.05% N-butyl-N-(4-hydro-oxybutyl) nitorosamine (BBN) from 6 weeks old to the harvest day. The harvested organs were diagnosed by two different pathologists with histopathological evaluation using Hematoxylin and Eosin staining. Both sides of UT from mouse drinking BBN were subjected to whole transcriptome sequencing (WTS). In human samples, 74 upper tract urothelial carcinoma (UTUC) tissues and 73 adjacent normal (AN) tissues also subjected to WTS from 74 UTUC patients. RESULTS: Female mice of certain strain showed early weight loss compared to the others. They showed high incidence of UTUC with less incidence of bladder cancer (Figure 1), while the others rarely had UTUC. The total incidence of UTUC in this mouse strain was 83.3%, with the earliest UTUC observed at 8 weeks after the start of BBN drinking. The incidence of bilateral UTUC was 22.2%, and these individuals showed weight loss relatively early after the start of BBN drinking. The cancerous side of UT expressed a basal type of mRNA expression profile, with increased expression of Cd44, Cdh3, and Krt14, similar to previous reports of bladder carcinogenesis mouse model (Figure 2). We detected 593 differential expressed genes between cancerous side and non-cancerous side of UT in this mouse model, which could also classify human UTUC and AN samples. In pathway enrichment analysis of these genes, several pathways associated with DNA replication were commonly upregulated in cancer tissues between mouse and human samples. CONCLUSIONS: We have established the novel animal model developing UTUC with high probability. This model could reflect the human UTUC. Source of Funding: This work was supported by a KAKENHI grant (21K20968, 22K09523) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e963 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akinaru Yamamoto More articles by this author Atsunari Kawashima More articles by this author Kentaro Jingushi More articles by this author Yuichi Motoyama More articles by this author Satoshi Nojima More articles by this author Sassi Nesrine More articles by this author Yohei Okuda More articles by this author Toshiki Oka More articles by this author Toshihiro Uemura More articles by this author Gaku Yamamichi More articles by this author Eisuke Tomiyama More articles by this author Kosuke Nakano More articles by this author Makoto Matsushita More articles by this author Yu Ishizuya More articles by this author Yoshiyuki Yamamoto More articles by this author Taigo Kato More articles by this author Koji Hatano More articles by this author Tsujikawa Kazutake More articles by this author Norio Nonomura More articles by this author Expand All Advertisement PDF downloadLoading ...

PD17-11 MOLECULAR PROFILING REVEALS IMMUNOSUPPRESSIVE TUMOR IMMUNE MICROENVIRONMENT IN PRIMARY VERSUS PAIRED ASYNCHRONOUS METASTATIC CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023PD17-11 MOLECULAR PROFILING REVEALS IMMUNOSUPPRESSIVE TUMOR IMMUNE MICROENVIRONMENT IN PRIMARY VERSUS PAIRED ASYNCHRONOUS METASTATIC CLEAR CELL RENAL CELL CARCINOMA Brittney Cotta, Srinivas Nallandhighal, Daniel Triner, Nathan Graham, Rohit Mehra, Marcin Cieslik, Amy Kasputis, Todd Morgan, and Simpa Salami Brittney CottaBrittney Cotta More articles by this author , Srinivas NallandhighalSrinivas Nallandhighal More articles by this author , Daniel TrinerDaniel Triner More articles by this author , Nathan GrahamNathan Graham More articles by this author , Rohit MehraRohit Mehra More articles by this author , Marcin CieslikMarcin Cieslik More articles by this author , Amy KasputisAmy Kasputis More articles by this author , Todd MorganTodd Morgan More articles by this author , and Simpa SalamiSimpa Salami More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Clear cell renal cell carcinoma (ccRCC) is a highly immune infiltrated cancer, but how the tumor immune microenvironment (TIME) evolves during disease progression is unknown. Insights into heterogeneity within primary ccRCC raise questions about potential heterogeneity between primary and paired metastatic tumors. This information may improve understanding of immune evasion leading to metastatic disease and response to immunotherapy. In this study, we characterize and compare the TIME of primary ccRCC with paired asynchronous metastases. METHODS: Analysis of ccRCC patients who developed recurrence post radical nephrectomy and had both primary and metastatic tissue available. Whole-transcriptome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) specimens using the illumina platform. Differential expression gene (DEG) analysis was performed using R package edgeR. Gene set enrichment analyses (GSEA) to identify hallmark pathway enrichment was performed using R package fgsea. TIME deconvolution was quantified using CIBEROSRT, an in silico flow cytometry tool. RESULTS: 42 tumor samples from 19 patients (19 primary tumors with 23 matched metastases) were analyzed. Metastasis sites included lung (n=6), bone (n=6), adrenal (n=4), liver (n=2), lymph node (n= 2), and soft tissue (n=3). Angiogenesis and epithelial to mesenchymal transition (EMT) were the most significantly enriched pathways in metastases compared to primary tumors (FDR < 5%). The primary tumors displayed a more immunosuppressive TIME than their matched metastases when comparing immune cell types. (Figure 1). The higher proportion of T regulatory cells (Tregs) in primary tumors was consistent compared against all metastasis tissue types. Metastasis samples displayed higher proportion of M2 macrophages (p=0.003), with the highest proportion in bone and soft tissue. Bone metastases also appeared to be more immunosuppressive in their TIME when compared to lung metastases, with greater Tregs and a lower immune content score. CONCLUSIONS: We demonstrate higher infiltration of immunosuppressive cells in the TIME of primary ccRCC compared with metastatic sites. Further studies are necessary to determine the prognostic and therapeutic significance of these findings. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e500 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Brittney Cotta More articles by this author Srinivas Nallandhighal More articles by this author Daniel Triner More articles by this author Nathan Graham More articles by this author Rohit Mehra More articles by this author Marcin Cieslik More articles by this author Amy Kasputis More articles by this author Todd Morgan More articles by this author Simpa Salami More articles by this author Expand All Advertisement PDF downloadLoading ...

Whole-transcriptome sequencing identifies key differentially expressed circRNAs/lncRNAs/miRNAs/mRNAs and linked ceRNA networks in adult degenerative scoliosis.

Adult degenerative scoliosis (ADS) is forecast to be a prevalent disabling condition in an aging society. Universally, its pathogenesis is perceived as intervertebral disc degeneration (IDD), however, a thought-provoking issue is why precisely a subset of patients with disc degeneration develop ADS. Exploring the diversities between common IDD and ADS would contribute to unraveling the etiological mechanisms of ADS. Therefore, we aimed to integrate the circRNA, lncRNA, miRNA, and mRNA expression profiles from normal adults (Normal), patients with lumbar disc herniation (LDH), and ADS by whole transcriptome sequencing, which identifies critical functional ncRNA and ceRNA networks and crosstalk between the various transcripts. The fresh whole blood samples (n = 3/group) were collected from ADS patients, LDH patients, and healthy volunteers (Normal group), which were examined for mRNA, miRNA, lncRNA, and circRNA expression and screened for differentially expressed (DE) ncRNAs. Then, Gene Ontology (GO) and KEGG analyses were performed for gene annotation and enrichment pathways on the DE RNAs, which were constructed as a lncRNA-miRNA-mRNA network. Eventually, DE RNAs were validated by qRT-PCR targeting disc nucleus pulposus (NP) tissue in ADS and LDH group (n = 10/group). Compared to the LDH group, we identified 3322 DE mRNAs, 221 DE lncRNAs, 20 DE miRNAs, and 15 DE circRNAs in the ADS. In contrast to Normal, 21 miRNAs and 19 circRNAs were differentially expressed in the ADS. The expression of multiple differentially expressed ncRNAs was confirmed by qRT-PCR analysis to be consistent with the sequencing results. In addition, GO, and KEGG analysis demonstrated that most DE mRNAs and ncRNAs target genes are involved in various biological processes, including Endocytosis, Apoptosis, Rap1 signaling pathway, Notch signaling pathway, and others. The constructed lncRNA-miRNA-mRNA co-expression network was primarily related to angiogenesis and regulation. By focusing on comparing asymmetric and symmetric disc degeneration, whole-transcriptome sequencing and bioinformatics analysis systematically screened for key ncRNAs in the development of ADS, which provided an abundance of valuable candidates for the elucidation of regulatory mechanisms. The DE ncRNAs and the lncRNA-miRNA-mRNA network are intrinsically involved in the regulation of mediator and angiogenesis, which may contribute to the insight into the pathogenesis of ADS.

Construction and analysis of circular RNA-associated competing endogenous RNA network in the hippocampus of aged mice for the occurrence of postoperative cognitive dysfunction.

Circular RNAs are highly stable single-stranded circular RNAs and enriched in the brain. Previous studies showed that circRNAs, as part of competing endogenous RNAs (ceRNAs) network, play an important role in neurodegenerative and psychiatric diseases. However, the mechanism of circRNA-related ceRNA networks in postoperative cognitive dysfunction (POCD) has not been elucidated yet. POCD usually occurs in elderly patients and is characterized by hippocampal dysfunction. Here, aged C57BL/6 mice were subjected to exploratory laparotomy under sevoflurane anesthesia, and this POCD model was verified by Morris water maze test. Whole-transcriptome sequencing was performed on the hippocampus of control group (Con) and surgery group. One hundred and seventy-seven DEcircRNAs, 221 DEmiRNAs and 2,052 DEmRNAs were identified between two groups. A ceRNA network was established with 92 DEcircRNAs having binding sites with 76 DEmiRNAs and 549 target DEmRNAs. In functional enrichment analysis, a pathological pattern of POCD was highlighted in the ceRNA network: Abnormal metabolic process in neural cells, including oxygen metabolism, could promote apoptosis and then affect the synaptic function, which may undermine the neural plasticity and eventually lead to changes in cognitive function and other behavioral patterns. In conclusion, this specific ceRNA network of circRNAs-miRNAs-mRNAs has provided novel insights into the regulatory mechanisms of POCD and revealed potential therapeutic gene targets.

Extensive search of genetic sex markers in Siberian (Acipenser baerii) and Atlantic (A. oxyrinchus) sturgeons

Sturgeons belong to an extraordinarily old and highly endangered group of fish. Although the sex determining region in this taxon was recently discovered, there is still a great need for the development of reliable gene expression markers for sturgeon sexing in their different life stages which could be easily applied in field conditions. In this study an extensive screening of the Atlantic sturgeon genome was performed using: AFLP (202 PC) and MS-AFLP (256 PC) markers likewise the Siberian sturgeon transcripts deriving from the Whole Transcriptome Sequencing (WTS) and cDNA-AFLP (128 PC). Genetic material extracted from female and male fins was used. AFLP reactions were minimized and automatized and a novel method for SCAR identification was proposed (SCAR-NGS). The 204 primary selected markers were subjected to gDNA or cDNA PCR amplifications, semi- and RT-qPCRs in order to confirm the presence of unique sex-marker sequences in the genomes or sex-linked expression differences. We identified eleven loci in the Atlantic sturgeon genome which exhibited sex-linked polymorphisms of the restriction sites (SNP based). In case of the Siberian sturgeon, 41 transcripts were confirmed to be expressed in a sex specific manner; three of which were also significantly differentially amplified in the female and male genomes. Six of those markers were highly overexpressed in males, which was quantified using RT-qPCRs. The approach enabled the discovery of DNA and RNA candidate sequences suitable for application in sturgeon sexing, being important both for commercial aquaculture optimization and genetic variability maintenance of sturgeons.

The long noncoding RNA THBS1-AS1 promotes cardiac fibroblast activation in cardiac fibrosis by regulating TGFBR1.

Cardiac fibrosis is associated with an adverse prognosis in cardiovascular disease that results in a decreased cardiac compliance and, ultimately, heart failure. Recent studies have identified the role of long noncoding RNA (lncRNA) in cardiac fibrosis. However, the functions of many lncRNAs in cardiac fibrosis remain to be characterized. Through a whole-transcriptome sequencing and bioinformatics analysis on a mouse model of pressure overload-induced cardiac fibrosis, we screened a key lncRNA termed thrombospondin 1 antisense 1 (THBS1-AS1), which was positively associated with cardiac fibrosis. In vitro functional studies demonstrated that the silencing of THBS1-AS1 ameliorated TGF-β1 effects on cardiac fibroblast (CF) activation, and the overexpression of THBS1-AS1 displayed the opposite effect. A mechanistic study revealed that THBS1-AS1 could sponge miR-221/222 to regulate the expression of TGFBR1. Moreover, under TGF-β1 stimulation, the forced expression of miR-221/222 or the knockdown TGFBR1 significantly reversed the THBS1-AS1 overexpression induced by further CF activation. In vivo, specific knockdown of THBS1-AS1 in activated CFs significantly alleviated transverse aorta constriction-induced (TAC-induced) cardiac fibrosis in mice. Finally, we demonstrated that the human THBS1-AS1 can also affect the activation of CFs by regulating TGFBR1. In conclusion, this study reveals that lncRNA THBS1-AS1 is a potentially novel regulator of cardiac fibrosis and may serve as a target for the treatment of cardiac fibrosis.

Identification of Key Differentially Expressed mRNAs, miRNAs, lncRNAs, and circRNAs for Xp11 Translocation Renal Cell Carcinoma (RCC) Based on Whole-Transcriptome Sequencing

We carried out whole transcriptome sequencing (WTS) on the tumor and the matching adjacent normal tissues from five patients having Xp11 translocation renal cell carcinoma (RCC). This was performed in terms of obtaining more understanding of the genomic panorama and molecular basis of this cancer. To examine gene-regulatory networks in XP11 translocation RCC, variance expression analysis was carried out, followed by functional enrichment analysis. Gene Expression Omnibus (GEO) of Xp11 translocation RCC data was used to validate the results. As per inclusion criteria, a total of 1886 differentially expressed mRNAs (DEmRNAs), 56 differentially expressed miRNAs (DEmiRNAs), 223 differentially expressed lncRNAs (DElncRNAs), and 1764 differentially expressed circRNAs (DEcircRNAs) were found. KEGG enrichment study of DEmiRNA, DElncRNA, and DEcircRNA target genes identified the function of protein processing in the endoplasmic reticulum, lysosome, and neutrophil-mediated immunity. Three subnetwork modules integrated from the PPI network also revealed the genes involved in protein processing in the endoplasmic reticulum, lysosome, and protein degradation processes, which may regulate the Xp11 translocation RCC process. The ceRNA complex network was created by Cytoscape, which included three upregulated circRNAs, five upregulated lncRNAs, 24 upregulated mRNAs, and two downregulated miRNAs (hsa-let-7d-5p and hsa-miR-433-3p). The genes as a prominent component of the complex ceRNA network may be key factors in the pathogenesis of Xp11 translocation RCC. Our findings clarified the genomic and transcriptional complexity of Xp11 translocation RCC while also pointing to possible new targets for Xp11 translocation RCC characterization.

Cancer-associated fibroblast-derived secreted phosphoprotein 1 contributes to resistance of hepatocellular carcinoma to sorafenib and lenvatinib.

Cancer-associated fibroblasts (CAFs) play an important role in the induction of chemo-resistance. This study aimed to clarify the mechanism underlying CAF-mediated resistance to two tyrosine kinase inhibitors (TKIs), sorafenib and lenvatinib, and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma (HCC). We performed a systematic integrative analysis of publicly available gene expression datasets and whole-transcriptome sequencing data from 9 pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively, to identify key molecules that might induce resistance to TKIs. We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms. The associations of plasma secreted phosphoprotein 1 (SPP1) expression levels before sorafenib/lenvatinib treatment with progression-free survival (PFS) and overall survival (OS) of 54 patients with advanced HCC were evaluated using Kaplan-Meier and Cox regression analysis. Bioinformatic analysis identified CAF-derived SPP1 as a candidate molecule driving TKI resistance. SPP1 inhibitors reversed CAF-induced TKI resistance in vitro and in vivo. CAF-derived SPP1 activated rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) through the integrin-protein kinase C-alpha (PKCα) signaling pathway and promoted epithelial-to-mesenchymal transition (EMT). A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS (P = 0.026) and OS (P = 0.047) in patients with advanced HCC after TKI treatment. CAF-derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion. Its inhibition represents a promising therapeutic strategy against TKI resistance in HCC. Moreover, plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.

Tumor-associated macrophages respond to chemotherapy by detrimental transcriptional reprogramming and suppressing stabilin-1 mediated clearance of EGF.

Tumor resistance to chemotherapy and metastatic relapse account for more than 90% of cancer specific mortality. Tumor-associated macrophages (TAMs) can process chemotherapeutic agents and impair their action. Little is known about the direct effects of chemotherapy on TAMs. The effect of chemotherapeutic platinum agent cisplatin was assessed in the model system of human ex vivo TAMs. Whole-transcriptome sequencing for paired TAMs stimulated and not stimulated by cisplatin was analysed by NGS. Endocytic uptake of EGF was quantified by flow cytometry. Confocal microscopy was used to visualize stabilin-1-mediated internalization and endocytic trafficking of EGF in CHO cells expressing ectopically recombinant stabilin-1 and in stabilin-1+ TAMs. In cohort of patients with breast cancer, the effect of platinum therapy on the transcriptome of TAMs was validated, and differential expression of regulators of endocytosis was identified. Here we show that chemotherapeutic agent cisplatin can initiate detrimental transcriptional and functional programs in TAMs, without significant impairment of their viability. We focused on the clearance function of TAMs that controls composition of tumor microenvironment. For the first time we demonstrated that TAMs' scavenger receptor stabilin-1 is responsible for the clearance of epidermal growth factor (EGF), a potent stimulator of tumor growth. Cisplatin suppressed both overall and EGF-specific endocytosis in TAMs by bidirectional mode: suppression of positive regulators and stimulation of negative regulators of endocytosis, with strongest effect on synaptotagmin-11 (SYT11), confirmed in patients with breast cancer. Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance.

Genome-wide analyses of lung cancer after single high-dose radiation at five time points (2, 6, 12, 24, and 48h).

Background: An increasing number of clinicians are experimenting with high-dose radiation. This study focuses on the genomic effects of high-dose single-shot radiotherapy and aims to provide a dynamic map for non-small cell lung cancer (NSCLC). Methods: We used whole-transcriptome sequencing to understand the evolution at molecular levels in A549 and H1299 exposed to 10 Gy X-rays at different times (2, 6, 12, 24, and 48h) in comparison with the no radiation group. Ingenuity pathway analysis, ceRNA analysis, enrichment analysis, and cell cycle experiments are performed for molecular analyses and function analyses. Results: Whole-transcriptome sequencing of NSCLC showed a significant dynamic change after radiotherapy within 48h. MiR-219-1-3p and miR-221-3p, miR-503-5p, hsa-miR-455-5p, hsa-miR-29-3p, and hsa-miR-339-5p were in the core of the ceRNA related to time change. GO and KEGG analyses of the top 30 mRNA included DNA repair, autophagy, apoptosis, and ferroptosis pathways. Regulation of the cell cycle-related transcription factor E2F1 might have a key role in the early stage of radiotherapy (2.6h) and in the later stage of autophagy (24 and 48h). Functions involving different genes/proteins over multiple periods implied a dose of 10Gy was related to the kidney and liver pathway. Radiation-induced cell cycle arrest at the G2/M phase was evident at 24h. We also observed the increased expression of CCNB1 at 24h in PCR and WB experiments. Conclusion: Our transcriptomic and experimental analyses showed a dynamic change after radiation therapy in 48h and highlighted the key molecules and pathways in NSCLC after high-dose single-shot radiotherapy.

Systematic analysis of circRNA-related ceRNA networks of black rockfish (Sebastes schlegelii) in response to Aeromonas salmonicides infection

Aeromonas salmonicides is a type of Gram-negative bacteria and has become the main fish pathogen in aquaculture because of its characteristics of worldwide distribution, broad host range and potentially devastating impacts. In the past years, studies have been focused to explore the regulatory roles of circRNA-miRNA-mRNA network in fish diseases. However, there are only few systematic studies linked to the anti-bacterial roles of circRNA-related ceRNA networks in the spleen immune system of black rockfish (Sebastes schlegelii). In this study, the whole-transcriptome sequencing (RNA-seq) was conducted in the black rockfish spleen with A. salmonicida challenging. The differentially expressed (DE) circRNAs were identified comprehensively for the following enrichment analysis. Interactions of miRNA-circRNA pairs and miRNA-mRNA pairs were predicted for the construction of circRNA-related ceRNA regulatory networks. Then, protein-protein interaction (PPI) analysis of mRNAs from these ceRNA networks were conducted. Finally, a total number of 39 circRNAs exhibited significantly differential expressions during A. salmonicida infection in the black rockfish spleen in 4338 identified circRNAs from 12 samples in 4 libraries. Functional enrichment analysis suggested that they were significantly enriched in several immune-related pathways, including Endocytosis, FoxO signaling pathway, Jak-STST signaling pathway, Herpes simplex infection, etc. Subsequently, 290 circRNA-miRNA-mRNA pathways (91 at 2 h, 142 at 12 h and 65 at 24 h) were constructed including 31 circRNAs, 50 miRNAs, and 156 mRNAs. In conclusion, the circRNA-related ceRNA networks were established, which will provide some novel insights in molecular mechanistic investigations of anti-bacterial immune response in teleost. Also, these findings will propose significant predictive values for the development of methods of treatment and prevention in black rockfish after bacterial infection in the future.

CCR4-NOT subunit CCF-1/CNOT7 promotes transcriptional activation to multiple stress responses in Caenorhabditis elegans.

CCR4-NOT is a versatile eukaryotic protein complex that controls multiple steps in gene expression regulation from synthesis to decay. In yeast, CCR4-NOT has been implicated in stress response regulation, though this function in other organisms remains unclear. In a genome-wide RNAi screen, we identified a subunit of the CCR4-NOT complex, ccf-1, as a requirement for the C. elegans transcriptional response to cadmium and acrylamide stress. Using whole-transcriptome RNA sequencing, we show that the knockdown of ccf-1 attenuates the activation of a broad range of stress-protective genes in response to cadmium and acrylamide, including those encoding heat shock proteins and xenobiotic detoxification. Consistently, survival assays show that the knockdown of ccf-1 decreases C. elegans stress resistance and normal lifespan. A yeast2-hybrid screen using a CCF-1 bait identified the homeobox transcription factor PAL-1 as a physical interactor. Knockdown of pal-1 inhibits the activation of ccf-1dependent stress genes and reduces C. elegans stress resistance. Gene expression analysis reveals that knockdown of ccf-1 and pal-1 attenuates the activation of elt-2 and elt-3 under stress that encode master transcriptional co-regulators of stress response in the C. elegans, and that overexpression of ELT-2 can suppress ccf-1's requirement for gene transcription in a stress-dependent manner. Our findings reveal a new role for CCR4-NOT in the environmental stress response and define its role in stress resistance and longevity in C. elegans.

Electroacupuncture regulates microglia polarization via lncRNA-mediated hippo pathway after ischemic stroke

ABSTRACT Microglia polarization and microglia-mediated inflammation play a crucial role in the development of ischaemic brain injury. Electroacupuncture (EA) has the function of anti-inflammatory, which has been thoroughly validated and utilized to treat ischemic brain damage. The fundamental mechanism by which EA alleviates ischemic brain damage by decreasing microglia polarization and microglia-mediated inflammation, however, remains unknown. In the current study, the activation of microglia and inflammatory cytokines was analyzed to confirm the anti-inflammatory function of EA in middle cerebral artery occlusion (MCAO) rats. Whole-transcriptome sequencing was used to examine the differentially expressed lncRNAs in the control, MCAO, and MCAO +EA groups. Our findings demonstrated that EA treatment reduced microglia activation and inflammatory cytokine production. In addition, there are 44 lncRNAs were found significantly different in three groups, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of the predicted targets of these lncRNAs suggested that the Hippo pathway may contribute to the development of ischaemic brain injury and to the anti-inflammatory function of EA. Moreover, our data showed that lncRNA TCONS_00022826 (Lnc826) was upregulated in MCAO group, whereas blocked by EA treatment. Furthermore, in vitro OGD cell model data showed that Lnc826 promoted M1 polarization of microglia by regulating the Hippo pathway. Our data suggested that regulating microglia polarization via Lnc826-mediated hippo pathway is a possible mechanism of the EA treatment on ischemic brain injury.