Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells

BACKGROUND: The retinoic acid receptor beta 2 (RARβ2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARβ2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype. METHODS: RNA from MDA-MB-435 human breast cancer cells transduced with RARβ2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors. RESULTS: RARβ2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 × 10(-8)), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARβ2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007). CONCLUSION: Antimetastatic RARβ2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARβ2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells-2

<p><b>Copyright information:</b></p><p>Taken from "Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells"</p><p>BMC Cancer 2005;5():140-140.</p><p>Published online 28 Oct 2005</p><p>PMCID:PMC1283145.</p><p>Copyright © 2005 Wallden et al; licensee BioMed Central Ltd.</p>on of cancer antigens, tumor suppressors, and genes involved in interferon signalling. Other gene activities are suppressed through RARβ2 action: AP1, cell adhesion, and nutrient processes. All gene activities in this diagram have been confirmed by cell culture qRT-PCR. Gene names in italics (red), have also been confirmed using randomly selected xenograft primary tumors, comparing two pairs of vector control- and RARβ2-resected tumors

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells-0

<p><b>Copyright information:</b></p><p>Taken from "Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells"</p><p>BMC Cancer 2005;5():140-140.</p><p>Published online 28 Oct 2005</p><p>PMCID:PMC1283145.</p><p>Copyright © 2005 Wallden et al; licensee BioMed Central Ltd.</p>Using total RNA used in the arrays, Northern blots were probed with P-dCTP labelled RARβ2 or CTAG1 cDNA. The RARβ2 probe consists of ~1.2 kb KpnI-BamH1 digest fragment of pSG RARβ2 ([75], from Pierre Chambon). The CTAG1 probe was generated from a 463 bp reverse transcriptase PCR product that encompasses the Agilent 60-mer. Blots were sequentially probed with a cDNA for RPLP0 (36B4) as a loading and transfer control. Quantitation of relative transcript levels was normalized to RPLP0 by phosphorimaging using exposure levels within the linear range of detection, avoiding saturation. The RARβ2 mRNA includes elements transcribed from the retroviral vector [8]

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells-1

<p><b>Copyright information:</b></p><p>Taken from "Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells"</p><p>BMC Cancer 2005;5():140-140.</p><p>Published online 28 Oct 2005</p><p>PMCID:PMC1283145.</p><p>Copyright © 2005 Wallden et al; licensee BioMed Central Ltd.</p> were probed with a 1384 bp fragment of SPP1 (see methods) or 800 bp, fragment of RPLP0 (36B4). Phosphorimaging was used for detection and quantitation. B. Western immunoblot analysis. Two independent preparations of each clonal cell line were used. Three mL of serum free-media from 3 × 10cell equivalents was immunoblotted, using a goat anti-SPP1/osteopontin polyclonal antibody (antibody and recombinant protein were a gift from Dr. CM Giachelli) and a secondary rabbit anti-goat antibody (Pierce)

Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens

Background: NanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories. Methods: Analytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test. Results: The measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area). Conclusions: The analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories.Non UBCPathology and Laboratory Medicine, Department ofMedicine, Faculty ofReviewedFacult

Pan-cancer adaptive immune resistance as defined by the Tumor Inflammation Signature (TIS): results from The Cancer Genome Atlas (TCGA)

Abstract The Tumor Inflammation Signature (TIS) is an investigational use only (IUO) 18-gene signature that measures a pre-existing but suppressed adaptive immune response within tumors. The TIS has been shown to enrich for patients who respond to the anti-PD1 agent pembrolizumab. To explore this immune phenotype within and across tumor types, we applied the TIS algorithm to over 9000 tumor gene expression profiles downloaded from The Cancer Genome Atlas (TCGA). As expected based on prior evidence, tumors with known clinical sensitivity to anti-programmed cell death protein 1 (PD-1) blockade had higher average TIS scores. Furthermore, TIS scores were more variable within than between tumor types, and within each tumor type a subset of patients with elevated scores was identifiable although with different prevalence associated with each tumor type, the latter consistent with the observed clinical responsiveness to anti PD-1 blockade. Notably, TIS scores only minimally correlated with mutation load in most tumors and ranking tumors by median TIS score showed differing association to clinical sensitivity to PD-1/PD-1 ligand 1 (PD-L1) blockade than ranking of the same tumors by mutation load. The expression patterns of the TIS algorithm genes were conserved across tumor types yet appeared to be minimally prognostic in most cancers, consistent with the TIS score serving as a pan-cancer measurement of the inflamed tumor phenotype. Characterization of the prevalence and variability of TIS will lead to increased understanding of the immune status of untreated tumors and may lead to improved indication selection for testing immunotherapy agents

Additional file 1: of Pan-cancer adaptive immune resistance as defined by the Tumor Inflammation Signature (TIS): results from The Cancer Genome Atlas (TCGA)

Figure S7. Distribution of TIS scores in stage IV disease. TIS scores are shown for all TCGA patients with stage 4 disease. Cancer types are ordered by median TIS score in all patients, identical to Fig. 1. (PDF 30 kb

PAM50 breast cancer intrinsic subtypes and effect of gemcitabine in advanced breast cancer patients

<div><p></p><p><b>Background.</b> In vitro studies suggest basal breast cancers are more sensitive to gemcitabine relative to other intrinsic subtypes. The main objective of this study was to use specimens from a randomized clinical trial to evaluate whether the basal-like subtype identifies patients with advanced breast cancer who benefit from gemcitabine plus docetaxel (GD) compared to single agent docetaxel (D).</p><p><b>Material and methods.</b> From patients randomly assigned to GD or D, RNA was isolated from archival formalin-fixed, paraffin-embedded primary breast tumor tissue and used for PAM50 intrinsic subtyping by NanoString nCounter. Statistical analyses were prespecified as a formal prospective-retrospective clinical trial correlative study. Using time to progression (TTP) as primary endpoint, overall survival (OS) and response rate as secondary endpoints, relationships between subtypes and outcome after chemotherapy were analyzed by the Kaplan-Meier method, and Cox proportional hazards regression models. Data analysis was performed independently by the Danish Breast Cancer Cooperative Group (DBCG) statistical core and all statistical tests were two-sided.</p><p><b>Results.</b> RNA from 270 patients was evaluable; 84 patients (31%) were classified as luminal A, 97 (36%) luminal B, 43 (16%) basal-like, and 46 (17%) as HER2-enriched. PAM50 intrinsic subtype was a significant independent prognostic factor for both TTP (p = 0.014) and OS (p = 0.0003). Response rate was not different by subtype, and PAM50 was not a predictor of TTP by treatment arm. PAM50 was however a highly significant predictor of OS following GD compared to D (p_interaction = 0.0016). Patients with a basal-like subtype had a significant reduction in OS events [hazard ratio (HR) = 0.29, 95% confidence interval (CI) = 0.15–0.57; p_interaction = 0.0006].</p><p><b>Conclusion.</b> A significantly improved and clinically important prolongation of survival was seen from the addition of gemcitabine to docetaxel in advanced basal-like breast cancer patients.</p></div