Georgia State University/University of Nottingham RNA-sequencing study (ICART1) of pretreatment malignant samples from patients with primary operable invasive triple-negative breast cancer
The goal of the RNA-sequencing experiment was to identify differentially expressed genes, ontologies, and pathways based on cell cycle traverse rate (CCTR) in pre-treatment malignant samples from patients with primary operable invasive triple-negative breast cancer (TNBC). TNBC patients belonged to the well-characterized Nottingham Primary Breast Carcinoma series, the database of which was prospectively maintained for another study, although its design accommodates the present retrospective analysis. Most TNBCs exhibited high Nottingham grade and about half of patients exhibited positive lymph nodes. All patients received chemotherapy with cyclophosphamide-methotrexate-5-fluoruracil after surgery (radical mastectomy or breast-conserving surgery). Stained slides used for quantitation of Ki67 index (KI) and mitotic index (MI) were independently scored by two pathologists blinded to clinical annotation, and the scores were averaged. Cell cycle traverse rate (CCTR) was defined as the ratio of the percentage of mitotic cells to the percentage of Ki67-positive cells in full-face formalin-fixed paraffin-embedded sections from primary tumors. To estimate percentages of mitotic cells for each sample, the average mitotic counts for each mitotic score category were first determined, followed by a conversion into percentage after accounting for sample cellularity. Separate slides from formalin-fixed paraffin-embedded malignant samples were used for RNA-sequencing based on the availability of sufficient tissue for analysis. Only tissue from pre-marked tumor areas in these slides were used for RNA-sequencing. RNA was extracted from deparaffinized tissue and first- and second-strand synthesis were performed using commercial kits. Pair-read sequencing was performed using the Illumina HiSeq2500 instrument. The Human Genome Assembly GRCh38.P10 was used as the reference genome for mapping sequence reads. Transcript expression was quantified using Salmon. Differentially expressed genes, ontologies, and pathways were determined based on mean CCTR as well as the optimal CCTR to predict breast cancer-specific survival.