Relative Gene Expression Values Research Articles

Abstract PO3-24-04: Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer

Abstract Title Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer Background Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several key cellular properties that are closely accompanied with trastuzumab resistance formation, we still have poor knowledge on the genetic and epigenetic variations that promote such transformation. Here we suggested that the epigenetic modification changes on histone H3 together with altered chromatin architecture promote lipid metabolism reprogramming during secondary trastuzumab formation. Materials and methods Induced secondary trastuzumab-resistant SKBR3_HR cell line together with the original trastuzumab-sensitive SKBR3 cell line were applied in this study. Total RNA was collected for transcriptome analysis. Anti-H3K4me3 and K27me3 antibodies were chosen for CUT&Tag sequencing library preparation. Total genome DNA was prepared for Micro-C sequencing library preparation. Activity score of metabolism pathway was calculated as relative gene expression value averaged over all genes in this pathway in certain cell types. Results Upregulation of arachidonic acid metabolism and downregulation of unsaturated fatty acid synthesis, which are mainly characterized by the activation of PTGS1 and PTGES genes and the repression of FASN and SCD genes, respectively, were observed during the formation of secondary trastuzumab resistance from SKBR3 cell to SKBR3_HR cells. Variations of H3k4me3 instead of H3k27me3 regulate the expression of these 4 genes. The accumulation of H3k4me3 was detected at the promoters of PTGS1 and PTGES genes while they were removed from the promoters of FASN and SCD genes in SKBR3_HR cells. More intra-chromosomal interactions were constituted during resistance formation. In detail, 4626 and 4394 topological associated domains, 3125 and 5824 DNA loops were founded in SKBR3 and SKBR3_HR cells, respectively. Furthermore, the lost and gained DNA loops between SCD and PTGS1 genes and distant genome regions may indicate the weaken and strengthen interactions with transcriptional regulators located there. Conclusions During trastuzumab resistance formation, altered histone modifications as well as higher genome structure could regulate the expression of key genes in lipid metabolism pathways, which may further affect cell properties and interactions with cells in tumor microenvironment. Citation Format: Ningjun Duan, Yijia Hua, Yongmei Yin. Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-04.

Abstract 3698: Epigenetic modification induced reprogramming of PUFAs-associated metabolism during trastuzumab resistance formation of HER2-positive breast cancer

Abstract Background: Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several potential reasons that cause trastuzumab resistance, we still have poor knowledge about the changes in cell itself as well as its interaction with tumor microenvironment (TME) components during resistance formation. Here we suggested that altered histone modification reprograms polyunsaturated fatty acids (PUFAs) metabolism, which reduces cellular PUFAs and stimulates prostaglandin E2 (PGE2) production. Excessive PGE2 may further affect both cancer cells as well as immune cells in TME. Materials and Methods: Secondary trastuzumab-resistant cell line SKBR3_HR was generated from sensitive SKBR3 cells by raising trastuzumab concentration gently from 1ug/ml to 20ug/ml in 30 weeks. Cell viabilities were measured by CCK8 test. CUT&Tag with anti-H3K4me3 and anti-H3K27me3 antibodies were applied to make sequencing libraries. Total RNA and protein were collected for transcriptome and proteome analysis. Activity scores of metabolism processes were defined and calculated as the relative gene expression value averaged over all genes in this pathway in certain cell types. Oxidized PUFAs and PGE2 were assessed by flow cytometry with BODIPY-C11 and ELISA, respectively. Results: SKBR3_HR cells showed similar viability as primary resistant JIMT1 cells and were significantly higher than original SKBR3 cells. Pathway analysis indicated reduced PUFAs synthesis and activated arachidonic acid metabolism. As a consequence, oxidized PUFAs decreased while PGE2 accumulated heavily in SKBR3_HR cells, which may promote tumor and vascular endothelial cell proliferation but interrupt immune cell activity in TME. Transcriptional activity of two critical PUFAs synthesis-related genes, FASN and SCD, were reduced in resistant cells, while two key genes in arachidonic acid metabolism, PTGS1 and PTGES, were activated. H3K4me3 and H3K27me3 play opposite but critical rules in transcriptional regulation. During resistance formation, changes happened on 3256 H3K27me3 peaks as well as 316 H3K4me3 peaks. In detail, although little H3K27me3 changes were found at the promoter regions of four genes (FASN, SCD, PTGS1 and PTGES), reduced H3K4me3 levels may inhibit FASN and SCD expression while raised levels could stimulate PTGS1 and PTGES transcription. Conclusions: Combining the transcriptome and histone modification data, we have a deeper understanding of the cellular adaptions to trastuzumab. Altered trimethylation of H3K4 but not H3K27 remodels the expressions of FASN, SCD, PTGS1 and PTGES, drives the reprogramming of PUFAs-associated metabolism, and finally reduces cellular PUFAs while stimulates PGE2 production. High PGE2 level may affect the activities of tumor and TME-related cells. Citation Format: Yongmei Yin, Ningjun Duan, Yijia Hua, Shuang Hu. Epigenetic modification induced reprogramming of PUFAs-associated metabolism during trastuzumab resistance formation of HER2-positive breast cancer. [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 3698.

Abstract P1-13-20: Reprogramming of oncogenic signal pathways and metabolism during trastuzumab resistance formation of HER2 positive breast cancer

Abstract Background The formation of resistance against trastuzumab deeply affects the treatment of HER2 positive breast cancer. Although studies have demonstrated several possible reasons that cause the trastuzumab resistance, the precise changes of cell process as well as the interaction between cancer cells and tumor microenvironment during the formation of resistance are still poorly understood. Here we figured out several crucial changes including oncogenic signal pathways as well as metabolism processes especially amino acids and polyunsaturated fatty acids (PUFAs) during the establishment of trastuzumab resistance. We further suggested that the inducers of ferroptosis may be promising reagents for trastuzumab resistant HER2 positive breast cancer. Methods The trastuzumab resistant cell was generated from a sensitive cell line SKBR3. To simulate the adaptation process of HER2 positive tumor to anti-tumor drug, the concentration of trastuzumab was changed gently to prevent cell death. After 8 weeks treatment, trastuzumab concentration in medium was raised from 1μg/ml to 6μg/ml, and a “persister” cell line SKBR3_HP was obtained. The original cell line SKBR3 was cultured for 8 weeks as well to exclude any changes that caused by culture condition. And each cell line includes 3 biological repeats. The viability of two cell lines is measured by CCK8 cell proliferation test with different concentration of trastuzumab. Total RNA of both SKBR3 and SKBR3_HP cells were prepared for sequencing. Activity scores of oncogenic signal pathways as well as metabolism processes were defined and calculated as the relative gene expression value averaged over all genes in this pathway in certain cell type. Flow cytometry was applied for reactive oxygen species measurement with BODIPY-C11. Results After 8 weeks treatment of trastuzumab, the persister cell line SKBR3_HP showed a significant higher viability than original sensitive cell line SKBR3. Oncogenic signal pathway analysis revealed that RTK-Ras, MYC and HIPPO pathways turn into a more active state in SKBR3_HP cells, as well as the upregulation of several cell cycle genes, which are all the response to the blockage of HER2 signal cascade and together maintains cell proliferation. Beside signal pathway, the reprogramming of metabolism is also the consequence of cell adaptation to trastuzumab. Minor changes in energy metabolism, such as glycolysis, citrate cycle and oxidative phosphorylation were observed in SKBR3_HP cells. However, amino acid metabolism, including the synthesis of phenylalanine, histidine, arginine, proline, cysteine and methionine, was largely enhanced in SKBR3_HP cells. Fatty acid, specifically the metabolism of PUFAs, such as linoleic acid, alpha-linolenic acid and arachidonic acid, was activated during the formation of trastuzumab resistance, which was confirmed by the lipid metabolomics data that most n-3/n-6 PUFAs decreased in SKBR3_HP cells. As cysteine and PUFAs metabolism might closely associate with cellular redox balances, erastin and RSL3 were applied to interrupt the intake of cysteine and potentiate the lipid peroxidation process, respectively. A situation of higher ferroptosis sensitivity accompanies raising peroxidated lipids could be detected in SKBR3_HP cells. Conclusion By analyzing the transcriptome and metabolomics data of trastuzumab sensitive and persister cell lines, we pointed out that the changes of oncogenic signal pathway, together with metabolism variation, particularly amino acids and PUFAs, are all the cellular adaptations to high trastuzumab environment. And the higher ferroptosis sensitivity of persister cells could be a valuable treatment target. Citation Format: Ningjun Duan, Yijia Hua, Shuang Hu, Yongmei Yin. Reprogramming of oncogenic signal pathways and metabolism during trastuzumab resistance formation of HER2 positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-20.

Exploring the multidimensional heterogeneities of glioblastoma multiforme based on sample-specific edge perturbation in gene interaction network.

Glioblastoma multiforme (GBM) is the most malignant brain cancer with great heterogeneities in many aspects, such as prognosis, clinicopathological features, immune landscapes, and immunotherapeutic responses. Considering that gene interaction network is relatively stable in a healthy state but widely perturbed in cancers, we sought to explore the multidimensional heterogeneities of GBM through evaluating the degree of network perturbations. The gene interaction network perturbations of GBM samples (TCGA cohort) and normal samples (GTEx database) were characterized by edge perturbations, which were quantized through evaluating the change in relative gene expression value. An unsupervised consensus clustering analysis was performed to identify edge perturbation-based clusters of GBM samples. Results revealed that the edge perturbation of GBM samples was stronger than that of normal samples. Four edge perturbation-based clusters of GBM samples were identified and showed prominent heterogeneities in prognosis, clinicopathological features, somatic genomic alterations, immune landscapes, and immunotherapeutic responses. In addition, a sample-specific perturbation of gene interaction score (SPGIScore) was constructed based on the differently expressed genes (DEGs) among four clusters, and exhibited a robust ability to predict prognosis. In conclusion, the bioinformatics approach based on sample-specific edge perturbation in gene interaction network provided a new perspective to understanding the multidimensional heterogeneities of GBM.

Sample-Specific Perturbation of Gene Interactions Identifies Pancreatic Cancer Subtypes.

Pancreatic cancer is a highly fatal disease and an increasing common cause of cancer mortality. Mounting evidence now indicates that molecular heterogeneity in pancreatic cancer significantly impacts its clinical features. However, the dynamic nature of gene expression pattern makes it difficult to rely solely on gene expression alterations to estimate disease status. By contrast, biological networks tend to be more stable over time under different situations. In this study, we used a gene interaction network from a new point of view to explore the subtypes of pancreatic cancer based on individual-specific edge perturbations calculated by relative gene expression value. Our study shows that pancreatic cancer patients from the TCGA database could be separated into four subtypes based on gene interaction perturbations at the individual level. The new network-based subtypes of pancreatic cancer exhibited substantial heterogeneity in many aspects, including prognosis, phenotypic traits, genetic mutations, the abundance of infiltrating immune cell, and predictive therapeutic efficacy (chemosensitivity and immunotherapy efficacy). The new network-based subtypes were closely related to previous reported molecular subtypes of pancreatic cancer. This work helps us to better understand the heterogeneity and mechanisms of pancreatic cancer from a network perspective.

Efficiency Correction Is Required for Accurate Quantitative PCR Analysis and Reporting.

Quantitative PCR (qPCR) aims to measure the DNA or RNA concentration in diagnostic and biological samples based on the quantification cycle (Cq) value observed in the amplification curves. Results of qPCR experiments are regularly calculated as if all assays are 100% efficient or reported as just Cq, ΔCq, or ΔΔCq values. When the reaction shows specific amplification, it should be deemed to be positive, regardless of the observed Cq. Because the Cq is highly dependent on amplification efficiency that can vary among targets and samples, accurate calculation of the target quantity and relative gene expression requires that the actual amplification efficiency be taken into account in the analysis and reports. PCR efficiency is frequently derived from standard curves, but this approach is affected by dilution errors and hampered by properties of the standard and the diluent. These factors affect accurate quantification of clinical and biological samples used in diagnostic applications and collected in challenging conditions. PCR efficiencies determined from individual amplification curves avoid these confounders. To obtain unbiased efficiency-corrected results, we recommend absolute quantification with a single undiluted calibrator with a known target concentration and efficiency values derived from the amplification curves of the calibrator and the unknown samples. For meaningful diagnostics or biological interpretation, the reported results of qPCR experiments should be efficiency corrected. To avoid ambiguity, the Minimal Information for Publications on Quantitative Real-Time PCR Experiments (MIQE) guidelines checklist should be extended to require the methods that were used (1) to determine the PCR efficiency and (2) to calculate the reported target quantity and relative gene expression value.

Expression profiles of 11 candidate genes involved in drought tolerance of pedunculate oak (Quercus robur L.). Possibilities for genetic monitoring of the species.

Abstract Pedunculate oak (Quercus robur L.) is one of the most significant broadleaved tree species in Europe. However, various abiotic and biotic agents have influenced pedunculate oak forests, among which drought stress has been frequently described as the main driver of this species forests decline. In this study we assessed relative expression profile of 11 candidate genes involved in many different metabolic pathways and potentially responsible for oak drought tolerance. The obtained results succeed in partially tackling drought tolerance mechanisms of targeted natural pedunculated oak population. This gene pool may represent a base for adaptation and therefore genetic diversity should be conserved. In this paper we described different expression responses of four pedunculate oak ecological groups, characterized by different physiological status (senescent vs vital) and flowering period (early (var. praecox) vs late (var. tardissima)). The most significant differences in relative gene expression levels are shown between the flowering period (tardissima (8 genes upregulated) vs praecox (3 genes upregulated)), more than a physiological status (sene-scent vs vital). Only three genes wrky53, rd22 and sag21 showed upregulated expression pattern in senescent physiological groups, indicating their possible role in the coping mechanisms of oak in stressed environment. Results showed interesting connections of relative gene expression values of identified drought-tolerance related genes with flowering period and provide further recommendations for adequate conservation and monitoring of this important oak gene pool in its southeast refugium.

Sample-specific perturbation of gene interactions identifies breast cancer subtypes.

Breast cancer is a highly heterogeneous disease, and there are many forms of categorization for breast cancer based on gene expression profiles. Gene expression profiles are variables and may show differences if measured at different time points or under different conditions. In contrast, biological networks are relatively stable over time and under different conditions. In this study, we used a gene interaction network from a new point of view to explore the subtypes of breast cancer based on individual-specific edge perturbations measured by relative gene expression value. Our study reveals that there are four breast cancer subtypes based on gene interaction perturbations at the individual level. The new network-based subtypes of breast cancer show strong heterogeneity in prognosis, somatic mutations, phenotypic changes and enriched pathways. The network-based subtypes are closely related to the PAM50 subtypes and immunohistochemistry index. This work helps us to better understand the heterogeneity and mechanisms of breast cancer from a network perspective.

Elevated Lipocalin-2 Can Indicate the Vascular Inflammation in Patients with Ischemic Stroke

Purpose: Elevated level of Lipocalin-2 (LCN2), a new acute phase adipokine, was described after ischemic stroke. A number of researchers feel as though that LCN2 originated from the infiltrating neutrophils and other cells in brain after stroke. Others measured elevated LCN2 expression in arteriosclerotic plaque. Therefore we have investigated LCN2 relative gene expression level of blood neutrophil granulocytes in patients with ischemic stroke to assess if elevated LCN2 is the cause or consequence of ischemic stroke. Methods: Laboratory and anamnestic data were collected, which could have a role in development of thrombo-embolic events in patients with ischemic stroke. RNA based method was used to evaluate the relative gene expression level of LCN2. We calculated Odds Ratio (OR) and Confidence Interval (CI) for the association between LCN2 and ischemic stroke. Results: 34 samples were available for evaluation. The LCN 2 relative gene expression level was decreased in 12 cases. In this group, 91% of patients have Atrial Fibrillation (AF) at the time of hospitalisation. The mean LCN2 relative gene expression value was 64.25% (ranges: 34%-115%) in patients with AF. It was significantly lower than in patients with normal sinus rhythm (409.2%; ranges: 127%-1127%; p=0.0003). The elevated LCN2 relative gene expression level significantly (p=0.012) increases the risk of stroke (OR: 12.6) independently from other factors. Conclusions: High LCN2 expression level seems to have strong positive predictive value on ischemic stroke, and may be useful in thrombotic risk stratification of plaque vulnerability in these patients.

Abstract P1-05-04: Progestin receptor content of breast cancer associated with expression of gene subsets for peptide/protein hormones and cognate receptors in LCM-procured cells that impact clinical outcomes

Abstract Assessing breast cancer prognosis and treatment selection was first enhanced by discovery that estrogen (ER) and progestin receptor (PR) proteins were clinically relevant biomarkers. Later reports suggested expression of certain protein hormones in breast cancer cells appear to be related to clinical behavior. We deciphered clinically relevant relationships between PR content in tissue biopsies and relative expression levels of genes directing synthesis of peptide/protein hormones and their cognate receptors in LCM-procured breast cancer cells. This global approach revealed unique relationships between both PR+ or PR- carcinomas and small subsets of genes for these peptide/protein hormones and cognate receptors as independent predictors of risk of recurrence. Methods: Expression of genes for 61 peptide/protein hormones and 81 cognate receptor proteins were measured by microarray analyses of LCM-procured carcinoma cells from de-identified primary breast carcinoma biopsies. Using an IRB-approved biorepository and database, previously total RNA was extracted from carcinoma cells to determine expression levels of ˜22,000 genes. Univariable and multivariable Cox regressions with interaction of each hormone eceptor gene, individually or paired, and use of LASSO were determined with relative gene expression values of each protein ligand and its cognate receptor. PR content and ligand binding affinity of each carcinoma were quantified by FDA approved assays. Results: Using de-identified clinical outcomes that extended up to 12 years, univariable Cox regression analyses of 142 candidates revealed AVPR1A, AVPR2, CALCR, CRH, LHB, POMC, SCT, SST, SSTR1 and TMSB10 independently predicted PFS or OS. Violin plots identified candidate genes associated with PR content. Multivariable analyses of relative gene expression of 115 hormone-receptor pairs showed IAPP-CALCR, RLN2-RXFP1, GHRH-GHRHR, CGA-TSHR, EDN1-ENDRA and POMC-MC5R exhibited statistically significant interaction for predicting OS among 145 PR+ cancers. Four gene pairs (HCRT-HCRTR2, CRH-CRHR1, HCRT-HCRTR1 and CORT-SSTR4) were associated with OS among 101 PR- lesions. Using LASSO, PR+ lesions expressing either (CGA & SSTR2) or (CGA) predicted OS and PFS, respectively. Similarly, for PR- cancers, gene subsets (GRP, TMSB15, VIP2) or (AGT, GH1, GRP, TMSB15A) predicted OS and PFS, respectively. Three of four signatures were externally validated with SurvExpress. Conclusion: Different gene expression profiles for protein hormones and cognate receptors were identified in PR+ or PR- carcinomas at time of diagnosis that predict PFS and OS regardless of treatment. Combining PR content with gene expression of LCM-procured cells is likely to provide insight into alternative treatments whereby standard of care performed poorly or to identify genes that correlate with cancer progression regardless of treatment modality. Collectively, results suggest that many primary breast cancers exhibit considerable endocrine autonomy for controlling disease progression, supporting investigation of protein products of gene candidates in isolated populations of breast carcinoma cells to develop novel biomarker assays. Citation Format: Daniels MW, Brock GN, James WL. Progestin receptor content of breast cancer associated with expression of gene subsets for peptide/protein hormones and cognate receptors in LCM-procured cells that impact clinical outcomes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-04.

RefEx, a reference gene expression dataset as a web tool for the functional analysis of genes

Gene expression data are exponentially accumulating; thus, the functional annotation of such sequence data from metadata is urgently required. However, life scientists have difficulty utilizing the available data due to its sheer magnitude and complicated access. We have developed a web tool for browsing reference gene expression pattern of mammalian tissues and cell lines measured using different methods, which should facilitate the reuse of the precious data archived in several public databases. The web tool is called Reference Expression dataset (RefEx), and RefEx allows users to search by the gene name, various types of IDs, chromosomal regions in genetic maps, gene family based on InterPro, gene expression patterns, or biological categories based on Gene Ontology. RefEx also provides information about genes with tissue-specific expression, and the relative gene expression values are shown as choropleth maps on 3D human body images from BodyParts3D. Combined with the newly incorporated Functional Annotation of Mammals (FANTOM) dataset, RefEx provides insight regarding the functional interpretation of unfamiliar genes. RefEx is publicly available at http://refex.dbcls.jp/.

Gene Expression Meta-Analysis of Potential Metastatic Breast Cancer Markers.

Background: Breast cancer metastasis is a highly prevalent cause of death for European females. DNA microarray analysis has established that primary tumors, which remain localized, differ in gene expression from those that metastasize. Cross-analysis of these studies allow to revile the differences that may be used as predictive in the disease prognosis and therapy.Objective: The aim of the project was to validate suggested prognostic and therapeutic markers using meta-analysis of data on gene expression in metastatic and primary breast cancer tumors.Method: Data on relative gene expression values from 12 studies on primary breast cancer and breast cancer metastasis were retrieved from Genevestigator (Nebion) database. The results of the data meta-analysis were compared with results of literature mining for suggested metastatic breast cancer markers and vectors and consistency of their reported differential expression.Results: Our analysis suggested that transcriptional expression of the COX2 gene is significantly downregulated in metastatic tissue compared to normal breast tissue, but is not downregulated in primary tumors compared with normal breast tissue and may be used as a differential marker in metastatic breast cancer diagnostics. RRM2 gene expression decreases in metastases when compared to primary breast cancer and could be suggested as a marker to trace breast cancer evolution. Our study also supports MMP1, VCAM1, FZD3, VEGFC, FOXM1 and MUC1 as breast cancer onset markers, as these genes demonstrate significant differential expression in breast neoplasms compared with normal breast tissue.Conclusion: COX2 and RRM2 are suggested to be prominent markers for breast cancer metastasis. The crosstalk between upstream regulators of genes differentially expressed in primary breast tumors and metastasis also suggests pathways involving p53, ER1, ERB-B2, TNF and WNT, as the most promising regulators that may be considered for new complex drug therapeutic interventions in breast cancer metastatic progression.

Abstract 2263: Prognostic significance of REV3like and TYMS gene expression in blood samples from non-small cell lung patients treated with platinum-based chemotherapy plus pemetrexed

Abstract BACKGROUND: Pemetrexed is an effective antineoplastic agent as monotherapy (second line and maintenance setting) or in platinum-based combination regimes (first-line). Pemetrexed inhibits three key enzymes in the folate metabolic pathway: thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT). As a consequence, pemetrexed interferes with the synthesis of both pyrimidine and purine, thereby effectively inhibiting both DNA and RNA synthesis. The aim of this study was to investigate the impact of the PBMC (peripheral blood mononuclear cells) expression levels of AEG1, BRCA1, REV3like, ant TYMS mRNA in the clinical outcome in NSCLC patients treated with Pemetrexed-based therapy. MATERIAL AND METHODS: This is a prospective, observational cohort study, of consecutively selected patients. We included patients with non-squamous NSCLC, stage IV, treated with platinum-based chemotherapy plus Pemetrexed. Blood samples from each patient were collected at the beginning of treatment. Quantitative reverse transcription (qRT)-PCR was performed with the use of the TaqMan gene expression assay (Applied Biosystems) according to the manufacturer's instructions. Amplifications were carried out on the Applied Biosystem 7500 RT-PCR system, and relative gene expression values were calculated by the ΔΔCt method (Sequence Detection System 2.0.5). Results from analysis, done in triplicate, are expressed relative to expression levels of GAPDH. RESULTS: We here present preliminary results of 16 patients included in the study: median age was 63.4 (range 52-82); 12 were males. Histological subtypes were 13 adenocarcinomas, and 3 large cell carcinomas. Pemetrexed was combined with Carboplatin in 9 patients and Cisplatin in 7 patients. Best response obtained was partial response in 7 patients, stable disease in 3 patients, and progression in 5 patients. We found significant differences in the REV3like expression levels between patients with PR&SD and patients with PD (0.69 ± 0.14 vs 0.44 ± 0.07; P = 0.003). In addition there were statistically significant differences in disease free survival (3 ± 0.74 vs 13 ± 1.89, P = 0.035; 12 ± 2.32 vs 3 ± 2.19, P = 0.003, months), and overall survival (10 ± 5.21 vs not reached, P = 0.026; not reached vs 12 ± 2.38, P = 0.007, months) between patients with mRNA levels of REV3like and TYMS under mean value of the cohort and patients with levels over mean value of the our cohort. CONCLUSIONS: This study supports the value of the PBMC expression profiling of the REV3like and TYMS genes in the prognosis of NSCLC treated with platinum-based chemotherapy plus Pemetrexed. Citation Format: Maria Teresa Agullo-Ortuño, C.Vanessa Díaz-García, José A. López-Martín, Elena Prieto-García, Santiago Ponce, Jon Zugazagoitia, Lara Iglesias-Docampo, Ana B. Enguita, Luis Paz-Ares, Juan A. Nuñez-Sobrino. Prognostic significance of REV3like and TYMS gene expression in blood samples from non-small cell lung patients treated with platinum-based chemotherapy plus pemetrexed. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2263.

Abstract 5120: The novel claudin-low molecular subtype of high-grade urothelial bladder cancer is highly immunogenic yet immunosuppressed

Abstract Rationale: High-grade urothelial carcinoma of the bladder is a heterogeneous disease, with molecular subtypes characterized by distinct tumor biologies and prognoses. Our group and others have described the basal and luminal subtypes, and we now report on the discovery of the claudin-low subtype, all of which resemble analogous subtypes in breast cancer. Methods: We analyzed mRNA sequencing and whole exome sequencing data for The Cancer Genome Atlas (TCGA) bladder tumors and tumors collected at UNC. We performed unsupervised hierarchical clustering on relative gene expression values with significance testing using SigClust to identify the claudin-low subtype. Basal, luminal, and claudin-low bladder tumors were compared for enrichment of genetic features (single nucleotide and copy number variation), gene set expression, immune gene signature expression, T cell receptor (TCR) and B cell receptor (BCR) gene segment expression, and number of predicted MHC Class I and Class II neoantigens. We further report on the first use of our VDJician software to reconstruct full-length rearranged BCR sequences from short-read RNA sequencing data in bladder cancer. Results: Claudin-low bladder tumors were defined by low expression of tight junction claudin-proteins, high expression of epithelial-to-mesenchymal transition genes and immune gene signatures, and were associated with reduced overall survival compared to luminal tumors. A minimal gene set classifier to identify claudin-low tumors showed some but not complete overlap with an optimal classifier derived in breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features: increased rates of RB1, EP300, and NCOR1 mutations, increased frequency of EGFR amplification, decreased rates of FGFR3, ELF3, and KDM6A mutations, and decreased frequency of PPARG amplification. Claudin-low tumors showed the highest expression of immune gene signatures (including an immunosuppression signature), however in contrast to basal tumors, increased immune gene signature expression was not associated with prolonged survival. Claudin-low tumors also showed the highest overall expression of rearranged BCR sequences but lowest BCR repertoire diversity. Predicted neoantigen burden did not vary significantly by subtype, however broad cytokine and chemokine expression levels were elevated in claudin-low tumors, potentially related to low PPARG activity driving increased NFKB activity. Conclusions: Claudin-low bladder cancer is a novel molecular subtype with distinct molecular and immunologic features and prognostic significance. Given the presence of dense immune infiltrates, BCR repertoire characteristics consistent with an antigen-driven response, and high expression of immunosuppression genes, claudin-low bladder tumors may be primed to respond to immune checkpoint inhibitor therapy. Citation Format: Benjamin G. Vincent, William Kim, Jordan Kardos, Shengjie Chai, Joel Parker, Lisle Mose, Sara Selitsky, Michael Iglesia, Matthew Milowsky. The novel claudin-low molecular subtype of high-grade urothelial bladder cancer is highly immunogenic yet immunosuppressed. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5120.

TIMP-1 expression in human colorectal cancer is associated with SMAD3 gene expression levels: a pilot study.

The prognosis of colorectal cancer (CRC) varies considerably, and there is a compelling need to identify novel biomarkers with prognostic significance. The aim of the present study was to evaluate the prognostic value of a panel of six genes (CDH1, SMAD3, TGFβ1, ICAM-1, TIMP-1 and MUC12) in CRC patients. METHODS. We evaluated these genes by qRT-PCR in normal and CRC tumor tissue, and correlated the relative gene expression values with clinical, pathological aspects and other biological factors. RESULTS. RNA expression levels of CDH1, SMAD3, TGFβ1, ICAM-1, TIMP-1 and MUC12 were measured by qRT-PCR in a set of 39 tumor samples and non-cancer tissue. Statistically significant increases in expression levels were found for ICAM-1 and TIMP-1 when comparing tumor samples to the non-tumor group. CONCLUSIONS. Among the genes which displayed differential expressions between tumor tissue and adjoining normal tissue, the ones that presented statistically significant correlations were TIMP-1 and SMAD3, possibly with prognostic significance.

Abstract 4666: Profiling mesothelin protein expression by immunohistochemistry and gene expression in adenocarcinoma and squamous cell carcinoma of lung

Abstract Mesothelin is a 40 kDa secreted glycoprotein expressed in normal mesothelial cells and over-expressed in several histological types of tumors. Detection of mesothelin by immunohistochemistry (IHC) may assist in the diagnosis of mesothelioma. Mesotheliomas are positive for mesothelin staining, but carcinomas of the lung may also be positive for this marker. Mesothelin positivity in adenocarcinoma of the lung reportedly ranges from 22% to 71% of cases depending upon the specific subtype. Mesothelin positivity in squamous cell carcinomas (SCC) of lung is reported to be 16% in non-keratinizing and 31% in keratinizing subtypes. We performed mesothelin IHC on a cohort of 50 lung carcinoma samples (16 adenocarcinomas and 34 SCC). Mesothelin expression was observed in 10 out of 16 (62.5%) lung adenocarcinoma samples, of which 8 showed greater than 10% of tumor cells with positive membrane staining of any intensity. Mesothelin expression was observed in 19 out of 34 (55.9%) lung SCC samples, of which only 3 samples showed greater than 10% of tumor cells with positive membrane staining of any intensity. These findings are in concordance with previous reports which show a higher prevalence of mesothelin protein expression in lung adenocarcinoma than in lung SCC. To correlate RNA expression with protein expression, we performed gene expression profiling on a subcohort of these lung cancer specimens. Out of 50 lung carcinoma samples, 28 samples (10 adenocarcinomas and 18 SCCs) provided adequate RNA yield for gene expression profiling for mesothelin. A total mean relative gene expression (mRGE) value of 13.64 with a standard deviation (SD) of ±3.71 was obtained for the adenocarcinoma samples and a mRGE value of 14.78 with a SD of 2.64 was obtained for the SCC samples. We next arbitrarily assigned samples with greater than 10% mesothelin stained cells as “positive” and the remainder as “negative”. Six negative adenocarcinoma samples yielded a mRGE value of 12.98 with a SD of ±3.84, and four positive adenocarcinoma samples yielded a mRGE value of 14.63 with SD of ±3.82. Sixteen negative SCC samples resulted in a mRGE 14.63 with SD of ± 2.75 and two positive SCC samples yielded a mRGE of 15.98 with a SD of ±1.37. There was no apparent correlation between mRGE values and IHC positivity. We also correlated gene expression with p53 mutation status. Sixteen wild type samples, composed of equal number of adenocarcinoma and SSC had a total mRGE of 14.29 with SD of ±3.32. Seven samples with p53 mutations had a total mRGE of 16.09 with SD ±1.93. These data indicate that mesothelin gene expression is not associated with p53 mutation status. Future studies with an increased number of samples may yield significant associations between protein expression, gene expression and mutation status. Citation Format: Jackson Wong, Dana Gaffney, Michael Sharp, Brenda Hertzog, Jayaprakash Karkera, Suso Platero, John Alvarez. Profiling mesothelin protein expression by immunohistochemistry and gene expression in adenocarcinoma and squamous cell carcinoma of lung. [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 4666. doi:10.1158/1538-7445.AM2014-4666

Trends in blood gene expression in non-small cell lung cancer patients treated with bevacizumab.

e22002 Background: Bevacizumab (BV) has shown increased overall response rate and survival in patients (Pt) with non-small cell lung cancer (NSCLC). No biomarkers have been described predictors of response in these Pt. The aim of this study is to investigate the usefulness of gene expression profiles of peripheral blood monocytes from patients with NSCLC before and after chemotherapy plus BV. Methods: A prospective study of blood samples from Stage IV NSCLC Pt treated with BV plus chemotherapy in our institution between 2009 and 2010 was performed. Plasma samples were collected before cycle 1 and after completion of cycle 2 (week 7). Clinical and radiological outcomes were recorded. The TaqMan Human Angiogenesis Array was used for analysis (micro fluidic card). This array contains 96 target involved in human angiogenesis and lymphangiogenesis. Amplifications were carried out on the AB 7900HT RT-PCR system, and relative gene expression values were calculated by the ΔΔCt method. Results: We present prelimin...

The effects of Nigella sativa (Ns), Anthemis hyalina (Ah) and Citrus sinensis (Cs) extracts on the replication of coronavirus and the expression of TRP genes family

Extracts of Anthemis hyalina (Ah), Nigella sativa (Ns) and peels of Citrus sinensis (Cs) have been used as folk medicine to fight antimicrobial diseases. To evaluate the effect of extracts of Ah, Ns and Cs on the replication of coronavirus (CoV) and on the expression of TRP genes during coronavirus infection, HeLa-CEACAM1a (HeLa-epithelial carcinoembryonic antigen-related cell adhesion molecule 1a) cells were inoculated with MHV-A59 (mouse hepatitis virus–A59) at moi of 30. 1/50 dilution of the extracts was found to be the safe active dose. ELISA kits were used to detect the human IL-8 levels. Total RNA was isolated from the infected cells and cDNA was synthesized. Fluidigm Dynamic Array nanofluidic chip 96.96 was used to analyze the mRNA expression of 21 TRP genes and two control genes. Data was analyzed using the BioMark digital array software. Determinations of relative gene expression values were carried out by using the 2−∆∆Ct method (normalized threshold cycle (Ct) value of sample minus normalized Ct value of control). TCID50/ml (tissue culture infectious dose that will produce cytopathic effect in 50 % of the inoculated tissue culture cells) was found for treatments to determine the viral loads. The inflammatory cytokine IL-8 level was found to increase for both 24 and 48 h time points following Ns extract treatment. TRPA1, TRPC4, TRPM6, TRPM7, TRPM8 and TRPV4 were the genes which expression levels changed significantly after Ah, Ns or Cs extract treatments. The virus load decreased when any of the Ah, Ns or Cs extracts was added to the CoV infected cells with Ah extract treatment leading to undetectable virus load for both 6 and 8hpi. Although all the extract treatments had an effect on IL-8 secretion, TRP gene expression and virus load after CoV infection, it was the Ah extract treatment that showed the biggest difference in virus load. Therefore Ah extract is the best candidate in our hands that contains potential treatment molecule(s).Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-014-3019-7) contains supplementary material, which is available to authorized users.

Expression profiles of Toll-like receptors 1-10 in human placenta during viral infection

Event Abstract Back to Event Expression profiles of Toll-like receptors 1-10 in human placenta during viral infection Patrycja Suski1*, Jarosław Kalinka2, Mirosława Studzińska1, Agnieszka Jabłońska1, Zbigniew J. Leśnikowski1 and Edyta Paradowska1 1 Institute of Medical Biology, Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, Poland 2 Medical University of Lodz, Department of Perinatology, First Chair of Gynecology and Obstetrics, Poland Placenta provides an important physical and immunological barrier against infectious agents and a pregnancy-specific component of the innate immune system. Toll-like receptors (TLRs) are essential for the induction of innate immunity responses in different human tissues including the maternal and fetal interface. The objective of this study was to investigate the TLR1-10 gene expressions in the human term placentas during viral infection. The explants of third-trimester chorionic villi and deciduas were exposed to infection with laboratory strains of HCMV, HSV-1 or VSV. Gene expression of TLR1-10 was assessed by quantitative real-time PCR using Power SYBR Green PCR Master Mix. The relative gene expression values were calculated using the comparative Ct method, and data were normalized against YWHAZ levels. This study clearly demonstrated that the human term placenta differentially expressed functional TLR1-10, indicating that these receptors are believed to be important for placental immune responses against viruses. We found that TLR3 mRNA levels were lower than other receptors in all examined placentas. We observed increased expression of TLR1, 9 and 10 in HCMV-infected decidua compared to unstimulated tissue. These findings demonstrated that the decidua was a critical component of the innate immunity response during HCMV infection. In contrast, infection with HSV-1 or VSV did not affect the expression of TLRs. Our results suggest that the TLRs could be important immune regulators in placenta during viral infection. Acknowledgements This work was supported by the European Regional Development Fund under the Operational Programme Innovative Economy, Grant No. POIG.01.01.02-10-107/09. Keywords: TLR expression, human placenta, hcmv, HSV-1, VSV Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Suski P, Kalinka J, Studzińska M, Jabłońska A, Leśnikowski ZJ and Paradowska E (2013). Expression profiles of Toll-like receptors 1-10 in human placenta during viral infection. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00142 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Mrs. Patrycja Suski, Institute of Medical Biology, Polish Academy of Sciences, Laboratory of Molecular Virology and Biological Chemistry, Lodz, Poland, psuski@cbm.pan.pl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Patrycja Suski Jarosław Kalinka Mirosława Studzińska Agnieszka Jabłońska Zbigniew J Leśnikowski Edyta Paradowska Google Patrycja Suski Jarosław Kalinka Mirosława Studzińska Agnieszka Jabłońska Zbigniew J Leśnikowski Edyta Paradowska Google Scholar Patrycja Suski Jarosław Kalinka Mirosława Studzińska Agnieszka Jabłońska Zbigniew J Leśnikowski Edyta Paradowska PubMed Patrycja Suski Jarosław Kalinka Mirosława Studzińska Agnieszka Jabłońska Zbigniew J Leśnikowski Edyta Paradowska Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Transcriptional and Enzymatic Profiling of Pleurotus ostreatus Laccase Genes in Submerged and Solid-State Fermentation Cultures

The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription-quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.