RNA Tumor Research Articles

Identification of Survival Risk and Immune-Related Characteristics of Kidney Renal Clear Cell Carcinoma

Background Immunity exerts momentous functions in the progression and treatment of kidney renal clear cell carcinoma (KIRC). A better understanding of the relationship between KIRC and immunity may make a great contribution to evaluating the prognosis and immune-related therapeutic response of KIRC. Methods A series of information such as RNA sequence, clinical data, and tumor mutation burden (TMB) of KIRC patients were downloaded through The Cancer Genome Atlas (TCGA). Next, combining the survival information and gene expression data of TCGA and Gene Expression Omnibus (GEO), we established an immune gene-related prognosis model (IGRPM) and analyzed it. Then we constructed a nomogram which was convenient for clinicians to judge the prognosis of KIRC. Last but not the least, the expressions of some genes used to construct IGRPM in early KIRC, and adjacent normal tissues were verified through real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Perl (strawberry-perl-5.30.0.1-64bit), R software (4.0.3), and GraphPad Prism 7 were used to process the relevant data. Results The single-sample gene set enrichment analysis (ssGSEA) showed that there were significant differences in StromalScore, ImmuneScore, ESTIMATEScore, TumorPurity, 22 kinds of human immune cells infiltration, and HLA genes expression between high immunity group (Immunity_H) and low immunity group (Immunity_L). The Immunity_H expressed more immune-related genes and enriched more immune-related functions than the Immunity_L. In addition, compared with the low-risk group, the high-risk group had worse survival outcome and higher TMB. Combining IGRPM-based risk characteristic and TMB, we found that low-TMB + low-risk was the most beneficial to the survival outcome of KIRC patients. The risk characteristic based on IGRPM could be used as an independent prognostic factor for KIRC, and the nomogram constructed for evaluating the prognosis of KIRC showed excellent predictive potential. The RT-qPCR results suggested that not all the genes used to construct IGRPM showed differential expression in early KIRC compared with adjacent normal tissues, but all these genes had significant influence on the prognosis of KIRC. Conclusion These comprehensive immune assessments and survival predictions, integrating multiple aspects of data and clinical information, can provide additional value to the current Tumor Node Metastasis staging system for risk stratification of KIRC and may facilitate the development of KIRC immunotherapy.

Abstract 4231: Hypoxia reduction in tandem with anti-angiogenic therapy remodels the pancreatic tumor microenvironment

Abstract Pancreatic cancer patients remain largely unresponsive to single agent immune checkpoint blockade (ICB) therapy. This refractory state originates from PDAC’s unique immunosuppressive microenvironment that is densely populated by suppressive myeloid cells, exhibits poor vascularity, and is highly hypoxic. We previously showed that the hypoxia-activated prodrug TH-302 (Evofosfamide) reduces intratumoral hypoxia through a tissue remodeling process, initiates tumor vasculature reorganization, and sensitizes aggressive, spontaneous murine models of prostate cancer to ICB. In a clinical trial testing the combination of Evofosfamide with cytotoxic T-lymphocyte-associated protein (CTLA-4) blockade (NCT03098160) a subset of metastatic, immune checkpoint blockade refractory patients showed prolonged progression free survival. While these studies highlight hypoxia reduction as therapeutically tractable, we lack complete understanding of the contribution of the tumor vasculature to hypoxia reduction therapy, as well as the downstream consequences of hypoxia reduction on the cellular composition of the tumor microenvironment and the responsiveness to immunotherapy. We used a transplantable, orthotopic pancreatic tumor model derived from Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre mice and a syngeneic prostate tumor model to study tumor responses to hypoxia-reduction, anti-angiogenic therapy, and combination immunotherapy. We find that Evofosfamide with anti-VEGFR-2 (DC101) significantly extends mouse survival. Combination therapy reduces intratumoral hypoxia, improves vessel integrity, and increases intratumoral DNA damage. In response to the improved metabolic microenvironment, CD8 T cells gain enhanced effector function and lose expression of exhaustion-associated features. Like other anti-angiogenic regimens, combination Evofosfamide and DC101 leads to an increased frequency of PD-L1 expressing cells within the tumor, however blockade of PD-1 failed to prolong survival. Bulk-tumor RNA sequencing and tumor infiltrating lymphocyte analysis implicates immature myeloid cells as mediators of resistance. Re-education of the infiltrating myeloid stroma with CD40 agonist could improve responses to hypoxia-reducing, anti-angiogenic therapy. Put together, these data indicate that targeted hypoxic reduction with anti-angiogenic therapy remodels the pancreatic tumor microenvironment. In this setting, CD40 agonist over PD-1 blockade provides an additive benefit in prolonging mouse survival. Citation Format: Arthur J. Liu, Michael A. Curran. Hypoxia reduction in tandem with anti-angiogenic therapy remodels the pancreatic tumor microenvironment [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 4231.

The Molecular Twin platform: a novel machine learning tool for democratization of precision cancer medicine.

e13546 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers. Contemporary analyses focused on a handful of molecular and clinical variables combined with machine learning algorithms (MLA) are unable to accurately predict therapy outcomes. Here, we use the Molecular Twin multi-omic analytical platform that evaluates tumor and host features extracted from 10 multi-omic analytes and provides an array of MLA, including a Parsimonious Biomarker Model that can predict survival and recurrence with limited analytic burden, while maintaining a high degree of fidelity. Methods: Retrospectively collected serum and tissue samples from 74 patients with Stage I/II resectable PDAC were subjected to targeted NGS DNA sequencing, whole transcriptome RNA sequencing, paired tissue proteomics, unpaired serum proteomics, lipidomics and computational pathology. Analytes including plasma proteins, RNA fusions, tissue proteins, plasma lipids, RNA gene expressions, CNVs, INDELS, SNVs and tumor nuclei characteristics, were processed to obtain a panel of 6363 features. 1024 single-omic and multi-omics feature combinations generated from this panel served as input for 7 different types of MLA to predict binary survival (SR) and disease recurrence (DR) outcomes. The resultant 70 single and 7098 multi-omic biomarker models were evaluated for positive predictive value (PPV) and accuracy (ACC) in predicting DR and SR, and feature proportions learned by each ML model using leave-one-patient-out cross-validation strategy. By recursively eliminating features with low importance, we developed progressively parsimonious biomarker models for predicting SR and DR. Results: Our top model was multi-omic and predicted the SR with ACC = 0.85, PPV = 0.87 and the DR with ACC = 0.90, and PPV = 0.91. It outperformed all models based only on one single analyte type including plasma protein, RNA fusion, tissue protein, plasma lipid, clinical, RNA gene expression, tumor nuclei characteristics, CNV, INDEL and SNV, in predicting the SR. This model contained predominantly plasma protein features. Interestingly, less accurate models contained a greater proportion of other features in addition to plasma proteins. Parsimonious feature reduction of the top model stabilized at 589 features yielding an ACC = 0.85, and PPV = 0.85, comparable to the intact model. Conclusions: This proof-of-concept of the Molecular Twin precision medicine platform applied in PDAC reveals the potential of our unique MLA to provide a novel parsimonious biomarker panel with similar fidelity as much larger biomarker panels. If these results are reproduced on larger datasets, across tumor types, the Molecular Twin platform would have significant potential to democratize precision cancer medicine by discovering smaller biomarker panels with the predictive performance of much larger ones thus reducing cost and simplifying assays.

Abstract P3-09-15: Value-added clinical tumor/normal whole exome and whole transcriptome sequencing versus a DNA and RNA tumor only gene panel for managing breast cancer

Abstract As sequencing technologies advance, there is increasing interest in the value of more comprehensive molecular profiling techniques. Our laboratory has clinically validated two next generation sequencing (NGS) assays for detecting somatic variants in solid tumors. The Sema4 Signal Solid Tumor Panel, utilizes the Thermo Fisher 161 gene Oncomine Comprehensive v3 assay and the Sema4 Signal Whole Exome/Transcriptome Sequencing (WES/WTS) assay consists of whole transcriptome RNAseq for the tumor and whole exome sequencing for the tumor and normal DNA on the Illumina NovaSeq. We compiled a cohort of 224 breast tumors from 211 patients encompassing 10 histological subtypes (BRCA (102), IDC (74), ILC (21), BRCANOS (11), BRCNOS (4), IBC (1), ACBC (1), IMMC (1), MBC (2), MDLC (2), Other (5)). A total of 209 specimens were analyzed by our Solid Tumor panel, and 15 were analyzed by WES/WTS. ER/PR/HER2 status was available for 212 tumors with 36 triple-negative, 123 ER(+)/PR(+)/HER2(-), and 23 ER(+)/PR(+)/HER2(+). Treatment history was available for 151 patients. Of the 118 patients who self-reported racial identity, 8 identified as Asian, 33 identified as Black or African American, 77 identified as white. Sequencing with either the 161 gene panel or WES/WTS detected clinically significant variants in 95% (213/224) of the tumors sequenced. Targetable PIK3CA variants were reported in 36% (80/224) of tumors, as well as CCND1 amplification (16%, 26/224) and ESR1 resistance variants (10%, 24/224). Tier 1 drugs were reported in 35% (79/224) of tumors, as well as tier 2 drugs (4%, 10/224). At least 2 patients had tumor specimens obtained at time points spanning 18 or more months and acquired ESR1 p.Y537S and p.Y537C resistance mutations in the later specimens. A patient with triple-negative specimens, obtained in 2018 and 2020, had no change in clinically significant variants but had loss of variants of unknown significance in the later sample. Panel sequencing and WES/WTS are comparable in terms of known variant detection, but panel-based testing has considerable limitations for assessing disease progression and genomic complexity. For the 15 WES/WTS specimens, we were able to determine additional molecular features, including complex copy number state, which has relevance to prognostic information, tumor mutation burden, homologous recombination deficiency, and differential gene expression. Moreover, WES of the tumor and normal specimens resolved multiple germline variants that had been reported as somatic variants in a tumor-only report from a patient with triple-negative breast cancer. Our findings support the adoption of tumor/normal WES/WTS as a standard diagnostic tool for breast cancer patients. Citation Format: Kimberly S. Cole, Zhiqiang Li, Amy Tiersten, Wanying Zhang, Hussam Al-Kateb, Feras M. Hantash, Michael R. Rossi, Scott Newman, Xiang Zhou, Eric E. Schadt, Rong Chen, William K. Oh, Marc Y. Fink. Value-added clinical tumor/normal whole exome and whole transcriptome sequencing versus a DNA and RNA tumor only gene panel for managing breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-15.

RNA N6-Methyladenosine Regulators Contribute to Tumor Immune Microenvironment and Have Clinical Prognostic Impact in Breast Cancer.

Purpose: This study aims to reveal the relationship between RNA N6-methyladenosine (m6A) regulators and tumor immune microenvironment (TME) in breast cancer, and to establish a risk model for predicting the occurrence and development of tumors. Patients and methods: In the present study, we respectively downloaded the transcriptome dataset of breast cancer from Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database to analyze the mutation characteristics of m6A regulators and their expression profile in different clinicopathological groups. Then we used the weighted correlation network analysis (WGCNA), the least absolute shrinkage and selection operator (LASSO), and cox regression to construct a risk prediction model based on m6A-associated hub genes. In addition, Immune infiltration analysis and gene set enrichment analysis (GSEA) was used to evaluate the immune cell context and the enriched gene sets among the subgroups. Results: Compared with adjacent normal tissue, differentially expressed 24 m6A regulators were identified in breast cancer. According to the expression features of m6A regulators above, we established two subgroups of breast cancer, which were also surprisingly distinguished by the feature of the immune microenvironment. The Model based on modification patterns of m6A regulators could predict the patient’s T stage and evaluate their prognosis. Besides, the low m6aRiskscore group presents an immune-activated phenotype as well as a lower tumor mutation load, and its 5-years survival rate was 90.5%, while that of the high m6ariskscore group was only 74.1%. Finally, the cohort confirmed that age (p < 0.001) and m6aRiskscore (p < 0.001) are both risk factors for breast cancer in the multivariate regression. Conclusion: The m6A regulators play an important role in the regulation of breast tumor immune microenvironment and is helpful to provide guidance for clinical immunotherapy.

Use of comprehensive next-generation sequencing to identify pathogenic germline variants with therapeutic relevance in metastatic breast cancer.

10527 Background: Among patients with early-stage breast cancer, approximately 6-10% have a pathogenic germline variant (PGV) conferring inherited cancer predisposition. In contrast, few studies have explored the frequency and types of PGVs identified in patients with metastatic breast cancer (MBC); therefore, additional data is needed. Methods: From 2011-2020, 278 patients with MBC underwent fresh tumor biopsy and blood sample collection for paired tumor/normal DNA (targeted exome capture with analysis of 1700 genes) and RNA (tumor transcriptome) sequencing through the Michigan Oncology Sequencing (Mi-ONCOSEQ) program. Somatic and germline alterations were annotated and classified according to degree of clinical actionability with results returned to treating oncologists. Retrospective chart review was performed to determine if: 1) a PGV was identified prior to Mi-ONCOSEQ testing, 2) patients met National Comprehensive Cancer Network (NCCN) guideline criteria for genetic testing on the basis of personal or family cancer history and 3) patients received subsequent therapy informed by a PGV. Results: Forty-eight of the 278 patients (17.3%) had at least one PGV identified, with a total of 50 PGVs identified in this cohort. Only twelve of these PGVs (24%) had been identified prior to Mi-ONCOSEQ testing. The most frequent PGVs identified were in CHEK 2 (n = 9, 18%), MUTYH (n = 6, 12%), BRCA 1 (n = 5, 10%), BRCA2 (n = 5, 10%), ATM (n = 4, 8%) and PALB2 (n = 4, 8%). Somatic loss of heterozygosity events (LOH) occurred in 30 of the 50 cases with PGVs identified (60%). LOH events were observed in 83.3% of BRCA1, BRCA2, ATM and PALB2 PGVs, but were less frequently observed with CHEK2 (33.3%) and MUTYH (66.7%). Two hundred sixteen out of 278 patients (77.7%) in this cohort met NCCN criteria for genetic testing, although six patients with a PGV identified (CHEK2: n = 5; MUTYH: n = 1) did not meet NCCN criteria. Twenty-nine PGVs identified (58%) had potential therapeutic relevance and 11 patients (22.9%) received targeted therapy based on the PGV. Conclusions: The frequency of PGVs identified in this cohort is nearly double the frequency reported for patients with early-stage disease, suggesting that certain PGVs may confer worse prognosis. CHEK2, the most frequently identified PGV, was less likely to have an identifiable LOH event. The direct role of CHEK2 PGVs in tumor pathogenesis is uncertain, but other mechanisms of silencing the wild type allele must be considered. Despite the majority of patients meeting NCCN criteria for genetic testing, those with PGVs in CHEK2 were less reliably identified by this mechanism. The majority of PGVs identified were of potential therapeutic relevance, supporting the recommendation for genetic testing in all patients with MBC.

Multi gene mutation signatures in colorectal cancer patients: predict for the diagnosis, pathological classification, staging and prognosis

BackgroundIdentifying gene mutation signatures will enable a better understanding for the occurrence and development of colorectal cancer (CRC), and provide some potential biomarkers for clinical practice. Currently, however, there is still few effective biomarkers for early diagnosis and prognostic judgment in CRC patients. The purpose was to identify novel mutation signatures for the diagnosis and prognosis of CRC.MethodsClinical information of 531 CRC patients and their sequencing data were downloaded from TCGA database (training group), and 53 clinical patients were collected and sequenced with targeted next generation sequencing (NGS) technology (validation group). The relationship between the mutation genes and the diagnosis, pathological type, stage and prognosis of CRC were compared to construct signatures for CRC, and then analyzed their relationship with RNA expression, immunocyte infiltration and tumor microenvironment (TME).ResultsMutations of TP53, APC, KRAS, BRAF and ATM covered 97.55% of TCGA population and 83.02% validation patients. Moreover, 57.14% validation samples and 22.06% TCGA samples indicated that patients with mucinous adenocarcinoma tended to have BRAF mutation, but no TP53 mutation. Mutations of TP53, PIK3CA, FAT4, FMN2 and TRRAP had a remarkable difference between I-II and III-IV stage patients (P < 0.0001). Besides, the combination of PIK3CA, LRP1B, FAT4 and ROS1 formed signatures for the prognosis and survival of CRC patients. The mutations of TP53, APC, KRAS, BRAF, ATM, PIK3CA, FAT4, FMN2, TRRAP, LRP1B, and ROS1 formed the signatures for predicting diagnosis and prognosis of CRC. Among them, mutation of TP53, APC, KRAS, BRAF, ATM, PIK3CA, FAT4 and TRRAP significantly reduced their RNA expression level. Stromal score, immune score and ESTIMATE score were lower in patients with TP53, APC, KRAS, PIK3CA mutation compared non-mutation patients. All the 11 gene mutations affected the distributions of immune cells.ConclusionThis study constructed gene mutation signatures for the diagnosis, treatment and prognosis in CRC, and proved that their mutations affected RNA expression levels, TME and immunocyte infiltration. Our results put forward further insights into the genotype of CRC.

Abstract PO048: Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma

Abstract Cutaneous melanoma (CM) has one of the highest response rates across cancers to immune checkpoint blockade (ICB) therapies, but mucosal melanoma (MM) responds poorly to ICB. MM patients have very few effective treatments options, making it critical to understand and improve anti-tumor immunity in these patients. MM is a rare subtype of melanoma, accounting for approximately 1% of all malignant melanoma diagnoses. MM occurs on mucosal surfaces such as nasal, vaginal, and anorectal, which provide a highly unique tumor microenvironment compared to CM arising in the skin. We compared the tumor immune microenvironment between CM and MM by multiplex immunofluorescence and discovered MM has decreased frequency of infiltrating immune cells. Using RNA sequencing, we investigated intra-tumoral differences in global gene expression between CM and MM that might explain the lack of immune cell infiltration and ICB response in MM. Compared to CM, MM had decreased expression of numerous innate immune genes in the RIG-I pathogen-sensing pathway. The RIG-I pathway produces an inflammatory response to destroy foreign pathogens, but is also important for ICB sensitivity and anti-tumor immunity in CM. Studies in CM demonstrate the RIG-I pathway enhances antigen presentation and promotes a favorable immune cell profile in the tumor microenvironment. Interestingly, the tumor microbiome, which is vastly different between CM and MM, has been shown to regulate the RIG-I pathway. We measured total fungal and bacterial load in MM and CM primary tumors and found increased levels of both bacteria and fungi in MM compared to CM. This raises the possibility that microbiome-mediated suppression of the RIG-I pathway underlies poor anti-tumor immunity and ICB response in MM, and that re-activation of this pathway may be a novel therapeutic strategy for overcoming ICB resistance in MM patients. Direct RIG-I agonists are still in clinical development, but FDA-approved hypomethylating agents, particularly 5’ aza-deoxycytidine (decitabine), can activate the RIG-I pathway and improve anti-tumor immunity by increasing the expression of RIG-I pathway activating genes, endogenous RNA retroviruses, tumor antigens, and natural killer (NK) cell ligands. We treated MM cell lines with decitabine and observed strong induction of RIG-I pathway genes, NK ligands, and tumor antigens. In conclusion, we have identified the loss of intra-tumoral RIG-I signaling as a potential microbiome-mediated mechanism underlying the poor tumor immune microenvironment and ICB response in MM. Combining decitabine, which is FDA-approved and now available orally, with ICB represents a novel treatment strategy for these difficult to treat MM patients that can be rapidly translated into the clinic. Citation Format: Morgan MacBeth, Richard Tobin, Robert Van Gulick, Martin D. McCarter, William A. Robinson, Kasey L. Couts. Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO048.

Analysis of RNA m6A methylation regulators and tumour immune cell infiltration characterization in prostate cancer

Potential roles of RNA N6-methyladenosine (m6A) modification in tumour microenvironment (TME) cell infiltration has been demonstrated in recent studies. Nonetheless, the mechanism of its regulation remains unknown and immunotherapy has been marginal in prostate cancer. We demonstrated the expression of different m6A regulators within prostate cancer related to genetic variation, alternative splicing (AS), tumour mutational burden (TMB) and TME. Unsupervised clustering and risk prediction model constructed by 24 m6A regulators could predict scores of TME and prostate cancer patients prognosis. T cells CD8 was the intersection of immune cells which are related to multiple biological processes, and the fraction of T cells CD8 strongly correlates with immune associated gene sets. m6A methylation modification and immune cells infiltration played a nonnegligible role in prostate cancer. Our study represents a step towards personalized immunotherapy for prostate cancer patients.

Abstract A22: Simultaneous multiparametric profiling of cell-free RNA (cfRNA) and cell-free tumor DNA (ctDNA) in advanced-stage colorectal cancer

Abstract Background: It has been recognized that intratumoral heterogeneity together with therapeutic intervention-associated multiple genomic alterations added complexity for precision therapy in late-stage colorectal cancer (CRC). The clinical utility/validity of ctDNA assays have been systematically analyzed and proved under clinical settings in select cancer types with demonstrated specificity and sensitivity. Notably, cfRNA sequencing confirmed and enhanced detection sensitivity for variants with low allele frequency. In this study, plasma-based ctDNA mutation and cfRNA gene expression in patients with advanced CRC were simultaneously tested using NGS liquid biopsy panels. Methods: ctDNA and cfRNA were extracted from plasma of 25 treatment-naive patients with advanced CRC (stage III/IV), then were tested using the 600-gene PredicineATLAS ctDNA NGS assay and 180-gene PredicinePLUS cfRNA NGS assay, respectively. Various genomic alterations including single-nucleotide variation (SNV), Indel, copy number variation (CNV) and genome rearrangement are detected, along with tumor mutation burdens (TMB) evaluation/measurement. RNA-based variants and gene expression are also analyzed. Results: At ctDNA level, 96% (24/25) ctDNA samples were detected with at least 1 genomic alteration and total 265 SNV/Indels and 27 CNVs were identified across samples, including 180 genes with reported SNVs/indels and 23 genes with CNV. 20% (5/25) of the patients with RAS somatic mutations, 32% (8/25) with TP53 mutations, 8% (2/25) with APC mutations, and 8% (2/25) with PIK3CA mutations. TPM3-NTRK1 fusion was also detected in one patient. A subset of these variants detected at ctDNA level were also confirmed by cfRNA sequencing. Conclusions: Our results demonstrated the clinical utility/feasibility of simultaneous multiparametric profiling of ctDNA and cfRNA for comprehensive molecular insight/characterization in advanced-stage cancer. cfRNA-derived variant detection confirms the variants originally detected in ctDNA test and further validates the high-specificity results for report interpretation. Further investigation of exploring concordance between temporally matched tissue and plasma samples is ongoing. The ctDNA and cfRNA-based combined liquid biopsy solution provides holistic molecular overview for detecting driver mutations, monitoring efficacy/disease burden, and identifying subclonal alterations that may related to drug resistance and tumor heterogeneity. Citation Format: Zheng Feng, Danyi Wang, Dennis Merkle, Jianjun Yu, Shidong Jia, Juergen Scheuenpflug. Simultaneous multiparametric profiling of cell-free RNA (cfRNA) and cell-free tumor DNA (ctDNA) in advanced-stage colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A22.

Updates on human equilibrative nucleoside transporter 1 in neoplastic diseases

The human equilibrative nucleoside transporter 1(hENT1)is a multi-integrated membrane protein that mediates the transport of nucleosides, nucleobases and nucleoside analogs.It is mainly distributed on the cell membrane in humans.hENT1 maintains the homeostasis of cells and the intracellular concentration of nucleoside drugs through facilitating diffusion, thereby affecting DNA and RNA synthesis, cell signaling, metabolic regulation and tumor chemotherapy.This article reviews the progress in the structure and function of hENT1, the distribution in tumor tissues, the nucleoside analogs response and the prognosis of neoplastic diseases. Key words: Human equilibrative nucleoside transporter 1; Nucleoside analogs; Neoplasm

Abstract LB-036: Citrullinome analysis identifies the role of PADI4 in regulating RNA processing and tumor immunogenicity

Abstract Recent proteome analyses have provided a comprehensive overview of various posttranscriptional modifications (PTM); however, PTMs involving protein citrullination remain unclear. Thus, we performed a proteomic analysis of citrullinated proteins and identified more than 100 PADI4 substrates, the majority of which are related to RNA processing and protein translation. Citrullination of RBMX (RNA binding motif protein, X-linked) inhibited the interaction with other hnRNP family members. RNA sequence analyses revealed altered splicing patterns in more than 300 genes in bone marrow cells in Padi4-/- mice. Approximately one-fifth of the PADI4 substrates contained an RG/RGG motif, and citrullination by PADI4 competitively inhibited the methylation of the RGG motif in FET proteins (EWS and TAF15). In addition, PADI4-mediated citrullination inhibited the aggregation of TAF15 proteins, which is a frequently-observed feature in neurodegenerative diseases, such as ALS. An antibody against citrullinated RGG motifs was observed in 11.5% (27 among 234) of serum samples from patients with HCV-related hepatocellular carcinoma, and female patients with high serum antibody exhibited poor prognosis. Our findings imply that PADI4-mediated citrullination plays a key role in RNA processing, protein aggregation and tumor immunogenicity, and suggest that PADI4 activation has potential therapeutic implications for neurodegenerative disease and cancer immunotherapy. Citation Format: Chizu Tanikawa, Koichi Matsuda. Citrullinome analysis identifies the role of PADI4 in regulating RNA processing and tumor immunogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-036. doi:10.1158/1538-7445.AM2017-LB-036

RNA turbulence by tumor type: Overexpression of actionable mRNA signaling pathways by histology.

e23213 Background: Overexpression of mRNA provides an oncogenic mechanism, downstream of DNA level changes detected by sequencing, that can be treated with targeted therapies. It is still unknown why a subset of patients experience substantial responses to targeted therapies, while others experience minimal benefit. We hypothesize that the occurrence of multiple mRNA drivers may segment by cancer type in a similar manner to mutation burden. Methods: We examined results from 2088 patients with the 23 most common histologies that had received molecular testing at Paradigm Diagnostics by a panel of 56 mRNA targets, including 21 from the MAPK pathway, 14 from the P53 pathway, and 18 from the PI3K pathway. We calculated RNA turbulence, defined as the number of mRNA overexpressed in each case, as well as the pathway-specific RNA turbulence for the MAPK, PI3K and TP53 pathways. Results: Significant differences in overall and pathway-specific RNA turbulence across cancer types was observed. Colon and rectal cancers had high turbulence in the MAPK and TP53 pathways, with 87% having multiple putative drivers in the MAPK pathway and 29% having multiple drivers in the PI3K pathway. Small cell lung cancer and kidney cancers had high turbulence in the PI3K pathway, with 33% and 39% having multiple RNA drivers respectively. Overall, pancreatic, kidney and colon cancers had the highest turbulence and GIST, melanoma and cholangiocarcinoma had the lowest. There was a slight inverse relationship in tumor type when ranked by mutation burden vs. RNA turbulence Conclusions: RNA turbulence represents a unique mechanism by which to analyze tumors and correlates with disease type. Multiplex diagnostics assaying DNA, RNA, and protein level tumor changes will likely be needed to guide cancer therapeutics.

Abstract 3547: Systemically delivered human telomerase reverse transcriptase (hTERT)-targeting p53-laden adenovirus shows strong antitumor effects in intrahepatic Hep3B xenograft mouse model

Abstract Human telomerase reverse transcriptase (hTERT) RNA and p53 tumour suppressor were previously suggested as useful targets for cancer gene therapy. In this study, we investigated the effects of systemically delivered adenoviruses harboring trans-splicing ribozyme that could target hTERT and induce p53 transcript in targeted cancer cells in vivo using mice with Hep3B human hepatocellular carcinoma cells that metastasized to the liver. We observed that intravenous injection of this virus (0.5 X1011 Virus) produced efficient regression of Hep 3B tumor metastasized nodules. Next we performed in vitro study to know whether it has a synergistic effects with CDDP, we observed that CDDP (2.5 ug/ml) increased cytotoxic effects in vitro given with the virus (10 MOI), but in vivo study revealed that addition of CDDP or arsenic hexoxide (As4O6) did not improve the therapeutic efficacy of the virus treatment. Next we performed toxicity test with 4 doses (0.5 X1011, 1 X1011, 2 X1011, and 5 X1011, Virus), which revealed that there were no toxic death, but 2-4 fold increased in AST and ALT with no changes in bilirubin, BUN, Creatinine levels. In conclusion, this study demonstrates that the adenoviruses harboring trans-splicing ribozyme targeting hTERT and inducing p53 transcript represents a safe and promising cancer treatment for hepatocellular carcinoma. [This study was supported by a grant from the National R&amp;D Program for Cancer Control, Ministry for Health, Welfare &amp; Family Affairs, Republic of Korea. (0820050), and National Research Foundation of Korea (NRF) grant funded by the Korea government(MEST) (20120002631).] Keywords: Adenovirus, trans-splicing ribozyme, p53 tumour suppressor, Human Telomerase Reverse Transcriptase, cancer Citation Format: Won Sup Lee, Hye Lim Kang, Arulkumar Nagappan, Jeong Won Yun, GonSup Kim, Soon Chan Hong, Sang-Jin Lee, In-Hoo Kim. Systemically delivered human telomerase reverse transcriptase (hTERT)-targeting p53-laden adenovirus shows strong antitumor effects in intrahepatic Hep3B xenograft mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3547. doi:10.1158/1538-7445.AM2015-3547

Abstract LB-311: Whole-exome sequencing of polycythemia vera revealed novel driver genes and somatic mutation shared by T cells and granulocytes

Abstract Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by augmented myelopoiesis leading to increased red cell mass with frequent excessive proliferation of other myeloid lineages. Over 95% of PV patients harbor JAK2V617F mutation, which confers proliferative advantage to affected myeloid progenitors. JAK2V617F, which is thought to be necessary but not sufficient for the disease, is often associated with acquired uniparental disomy on chromosome 9p (9pUPD). To better understand the underlying molecular basis of PV, we performed whole-exome sequencing and DNA copy-number analysis of 31 JAK2V617F-positive patients and further investigated the evolution of somatic mutations using longitudinal samples. In addition to JAK2 V617F and 9pUPD, we identified recurrent somatic mutation in an additional 5 genes including ASXL1, TET2, DNMT3A, and SF3B1 and recurrent DNA copy-number alterations at 1q and 18p. Forty-two percent of patients had at least one somatic mutation in an epigenetic modifier gene, and 23% of patients harbored mutation in RTK/RAS, RNA processing pathways, tumor suppressors and protein kinases. In 4 of 31 cases, variant allele abundance suggested somatic mutations of ASXL1, DNMT3A and SF3B1 may have preceded JAK2V617F. Interestingly, in 7 PV patients mutations also present in COSMIC were shared between the patients’ T-cell controls and granulocytes: 4 NF1, 2 ASXL1, and one each in TET2, IDH2, and DNMT3A, suggesting these genes mutated early in hematopoietic development. Absence of JAK2V617F in these T-cells ruled out cross-contamination of cell lineages. In an additional 4 cases, a minor JAK2V617F subclone acquired new mutations in DNMT3A, SF3B1, SMARCA2, and TET2 during PV progression. In summary, seventy percent of patients had mutation of at least one gene important to development of myeloid neoplasms, other than JAK2V617F. Our data reveal that these mutations, in particular NF1, may have arisen in some cases early in hematopoiesis but also in some patients during progression after diagnosis. These results suggest that the order of mutation is not as important as the total mutational burden in the development of PV. Citation Format: David A. Wheeler, Linghua Wang, Sabina Swierczek, Kimberly Hickman, Jennifer A. Drummond, Donna Muzny, Richard A. Gibbs, Joseph Prchal. Whole-exome sequencing of polycythemia vera revealed novel driver genes and somatic mutation shared by T cells and granulocytes. [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 LB-311. doi:10.1158/1538-7445.AM2014-LB-311

A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

BackgroundDeproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression.ResultsWe report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression.ConclusionThe data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

Erratum: The Classic: Integration of Deoxyribonucleic Acid Specific for Rous Sarcoma Virus after Infection of Permissive and Nonpermissive Hosts

Erratum to: Clin Orthop Relat Res DOI 10.1007/s11999–008–0355–8 In the online version of this article, the affiliations of Harold E. Varmus, Peter K. Vogt, and J. Michael Bishop are not current. They are current as displayed here. Note that Dr. Varmus's correct email address is [email protected]. In the original publication of Varmus HE, Vogt PK, Bishop JM. Integration of deoxyribonucleic acid specific for Rous sarcoma virus after infection of permissive and nonpermissive hosts (RNA tumor viruses/reassociation kinetics/duck cells). Proc Nat Acad Sci USA. 1973;70:3067–3071, the authors’ affiliations were listed as follows: Harold E. Varmus, J. Michael Bishop Department of Microbiology, University of California, San Francisco, Calif. 94122 Peter K. Vogt Department of Microbiology, University of Southern California Medical School, 2025 Zonal Ave., Los Angeles, Calif. 90033

Reductions in tumor RNA integrity associated with clinical response to epirubicin/docetaxel chemotherapy in breast cancer patients.

Abstract Abstract #6068 Background: Optimal doses and dosing intervals for neoadjuvant anthracycline/taxane chemotherapy are poorly studied. Moreover, biomarkers for measuring response to such combination regimens are unknown. This study investigated these issues in sequential phase I/II cohorts of women with locally advanced or inflammatory breast cancer treated with epirubicin and docetaxel with pegfilgrastim support at 3- or 2-weekly intervals in association with the NCIC-CTG MA.22 clinical trial.&amp;#x2028; Methods: Accrual has been completed for the 3-weekly regimen (maximum tolerated dose: epirubicin 105 mg/m2, taxotere 75 mg/m2), and continues for the phase II portion of the 2-weekly regimen. Six core biopsies were obtained from 50 patients pre-, mid-, and post-treatment. Immunohistochemical staining was performed to determine baseline levels of ER, PR, HER2 and Topo II expressed as % positive stain. Tumour RNA integrity (RIN) and tumor extent were measured pre-, mid- and post-treatment by capillary electrophoresis and light microscopy after haematoxylin/eosin staining, respectively. Associations between maximum and average RIN at the three time points and tumour extent, clinical response, pathologic complete response, or baseline levels of ER, PR, HER2 and Topo II were assessed using Spearman correlation coefficients after data transformation to improve symmetry and stabilize variances. The association between both RIN and tumour extent, and baseline drug dose was assessed using a 1-way ANOVA.&amp;#x2028; Results: Low mid-treatment maximum RIN was associated with high drug dose level (p=0.05) and eventual pathologic complete response (p=0.01). Post-treatment, low maximum and average RIN were found to be associated with low tumor extent (p=0.004 and p=0.01, respectively). As well, low average RIN was significantly associated with clinical complete response post-treatment (p=0.01). As expected, post-treatment low tumor extent was significantly associated with pathologic complete response (p=0.01). High pre-treatment Topo II levels were also significantly associated with high RIN (p = 0.03). No association was observed between RIN and HER2, ER or PR.&amp;#x2028; Discussion: The association of RIN with tumour extent, pathologic complete response, clinical response, a known risk factor (Topo II), and drug dose suggests that the RIN may represent an important new biomarker for measuring response to anthracycline/taxane combinations (and possible other chemotherapy regimens) in breast cancer patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6068.

RNA interference and tumor gene therapy

RNA interference(RNAi)refers to homologous complementary mRNA degradation in- duced specifically by double stranded RNA,which actually interdicts the expression of corresponding genes.The continual development of RNAi study shows its tremendous advantage as a novel gene therapy technique on tumor.

Expression of telomerase RNA component correlates with the MIB-1 proliferation index in ependymomas.

Although there is general agreement that certain morphologic subtypes of ependymoma are benign, the biologic behavior of other ependymal neoplasms is poorly understood and not clearly related to conventional histopathologic criteria. The absence of universally accepted standards has prompted the search for more objective biologic markers. Telomerase is an RNA-containing enzyme associated with immortality in proliferating stem cells and many tumors. We investigated the proliferative activity of 26 ependymomas as determined by MIB-1 immunolabeling and compared the results with the in situ expression of human telomerase RNA (hTR) and WHO tumor grade. The study included 9 WHO grade I ependymomas (6 subependymomas and 3 myxopapillary ependymomas), 13 WHO grade II ependymomas, and 4 anaplastic (WHO grade III) ependymomas. The proliferation index (PI) and telomerase RNA expression were significantly increased in grade III ependymomas (p < 0.0001 for PI and p = 0.0015 for hTR). In these tumors, the PI and hTR expression were highly correlated (p = 0.0001). Of note, a single case designated grade II showed both increased proliferative activity and the highest hTR expression detected in this series of ependymal neoplasms. Our results suggest that the PI and hTR expression may be important biologic markers, independent of other histopathologic criteria of tumor grade. Future studies examining the correlation of MIB-1 cell kinetics and hTR expression with clinical parameters in selected ependymoma subtypes are needed to determine the prognostic relevance of these markers.