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Expression profiling of circulating miRNAs in Luminal A breast cancer

ABSTRACT: Breast cancer is a common disease with distinct tumor subtypes which can be phenotypically characterized by estrogen receptor, progesterone receptor and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression. Altered miRNA expression has been demonstrated in a variety of cancer states to date presenting the potential for exploitation as cancer specific biomarkers. Blood presents an attractive medium biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting. Blood samples were prospectively collected from consenting patients with Luminal A breast cancer (n=10) and controls (n=10). RNA was extracted, reverse transcribed and subjected to microarray analysis (n=10 Luminal A; n=10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Ethical approval was granted by the Clinical Research Ethics Committee, Galway Roscommon University Hospital Group. Written informed consent was obtained from all study participants. Blood samples were prospectively collected from 20 women; this included 10 consecutive women with a new diagnosis of Luminal A breast cancer and 10 healthy control participants. Luminal A status was confirmed with immunohistochemistry and fluorescence in situ hybridization (FISH). The blood samples for the healthy control group were collected from women residing in the same catchment area as the cancer cases. These women had no personal history of malignancy and no current inflammatory or infectious condition. Venous non-fasting whole blood samples were collected in BD vacutainers ® containing 18mg dipotassium EDTA anticoagulant (BD-Plymouth, PL6 7BP, UK). Total RNA was extracted from blood (1ml) using TRI Reagent BD (Molecular Research Centre, Inc). RNA concentration and integrity were evaluated by NanoDrop spectrophotometry (NanoDrop ND-1000 Technologies Inc., DE, USA) and Agilent Bioanalyzer RNA 6000 NanoChip Kit Series II (Agilent Technologies, Germany) analysis, respectively. MiRNA microarray profiling Expression profiling of circulating miRNAs was performed using TaqMan miRNA arrays and assays in accordance with the manufacturer’s instructions (Taqman Low Density Array Human microRNA Card A and Card B, Applied Biosystems, Foster City, CA, USA). In short, total RNA was reverse transcribed using Megaplex primer pool A (Applied Biosystems) which contained sequence-specific primers for 381 specific miRNAs plus 3 controls (pool A). An additional panel of 384 miRNAs (381 miRNAs and 3 controls, pool B) was performed on a subset of 4 cancers and 4 controls. Real-time quantitative PCR was performed for 667 miRNAs, using A and B microfluidic cards, each containing primers and probes for 381 specific miRNAs plus 3 controls and thermal-cycled on an Applied Biosystems 7900HT instrument. The TLDA cards contain three endogenous controls (RNU44, RNU48 and MammU6 which is repeated four times on each card). Each card also contains a negative control, an assay unrelated to any human species, ath-miR-159a.

ORGANISM(S): Homo sapiens

SUBMITTER: Ball Graham

PROVIDER: S-ECPF-GEOD-46355 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Breast cancer is a common disease with distinct tumor subtypes which can be phenotypically characterized by estrogen receptor, progesterone receptor and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression. Altered miRNA expression has been demonstrated in a variety of cancer states to date presenting the potential for exploitation as cancer specific biomarkers. Blood presents an attractive medium biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting. Blood samples were prospectively collected from consenting patients with Luminal A breast cancer (n=10) and controls (n=10). RNA was extracted, reverse transcribed and subjected to microarray analysis (n=10 Luminal A; n=10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Ethical approval was granted by the Clinical Research Ethics Committee, Galway Roscommon University Hospital Group. Written informed consent was obtained from all study participants. Blood samples were prospectively collected from 20 women; this included 10 consecutive women with a new diagnosis of Luminal A breast cancer and 10 healthy control participants. Luminal A status was confirmed with immunohistochemistry and fluorescence in situ hybridization (FISH). The blood samples for the healthy control group were collected from women residing in the same catchment area as the cancer cases. These women had no personal history of malignancy and no current inflammatory or infectious condition. Venous non-fasting whole blood samples were collected in BD vacutainers M-BM-. containing 18mg dipotassium EDTA anticoagulant (BD-Plymouth, PL6 7BP, UK). Total RNA was extracted from blood (1ml) using TRI Reagent BD (Molecular Research Centre, Inc). RNA concentration and integrity were evaluated by NanoDrop spectrophotometry (NanoDrop ND-1000 Technologies Inc., DE, USA) and Agilent Bioanalyzer RNA 6000 NanoChip Kit Series II (Agilent Technologies, Germany) analysis, respectively. MiRNA microarray profiling Expression profiling of circulating miRNAs was performed using TaqMan miRNA arrays and assays in accordance with the manufacturerM-bM-^@M-^Ys instructions (Taqman Low Density Array Human microRNA Card A and Card B, Applied Biosystems, Foster City, CA, USA). In short, total RNA was reverse transcribed using Megaplex primer pool A (Applied Biosystems) which contained sequence-specific primers for 381 specific miRNAs plus 3 controls (pool A). An additional panel of 384 miRNAs (381 miRNAs and 3 controls, pool B) was performed on a subset of 4 cancers and 4 controls. Real-time quantitative PCR was performed for 667 miRNAs, using A and B microfluidic cards, each containing primers and probes for 381 specific miRNAs plus 3 controls and thermal-cycled on an Applied Biosystems 7900HT instrument. The TLDA cards contain three endogenous controls (RNU44, RNU48 and MammU6 which is repeated four times on each card). Each card also contains a negative control, an assay unrelated to any human species, ath-miR-159a.

Project description:BackgroundHeterogeneity of immune gene expression patterns of luminal breast cancer (BC), which is clinically heterogeneous and overall considered as low immunogenic, has not been well studied especially in non-European populations. Here, we aimed at characterizing the immune gene expression profile of luminal BC in an Asian population and associating it with patient characteristics and tumor genomic features.MethodsWe performed immune gene expression profiling of tumor and adjacent normal tissue in 92 luminal BC patients from Hong Kong using RNA-sequencing data and used unsupervised consensus clustering to stratify tumors. We then used luminal patients from The Cancer Genome Atlas (TCGA, N = 564) and a Korean breast cancer study (KBC, N = 112) as replication datasets.ResultsBased on the expression of 130 immune-related genes, luminal tumors were stratified into three distinct immune subtypes. Tumors in one subtype showed higher level of tumor-infiltrating lymphocytes (TILs), characterized by T cell gene activation, higher expression of immune checkpoint genes, higher nonsynonymous mutation burden, and higher APOBEC-signature mutations, compared with other luminal tumors. The high-TIL subtype was also associated with lower ESR1/ESR2 expression ratio and increasing body mass index. The comparison of the immune profile in tumor and matched normal tissue suggested a tumor-derived activation of specific immune responses, which was only seen in high-TIL patients. Tumors in a second subtype were characterized by increased expression of interferon-stimulated genes and enrichment for TP53 somatic mutations. The presence of three immune subtypes within luminal BC was replicated in TCGA and KBC, although the pattern was more similar in Asian populations. The germline APOBEC3B deletion polymorphism, which is prevalent in East Asian populations and was previously linked to immune activation, was not associated with immune subtypes in our study. This result does not support the hypothesis that the germline APOBEC3B deletion polymorphism is the driving force for immune activation in breast tumors in Asian populations.ConclusionOur findings suggest that immune gene expression and associated genomic features could be useful to further stratify luminal BC beyond the current luminal A/B classification and a subset of luminal BC patients may benefit from checkpoint immunotherapy, at least in Asian populations.

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Expression profiling of circulating miRNAs in Luminal A breast cancer

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