Multiparameter DNA content analysis identifies distinct groups in primary breast cancer

Background: Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique. Methods: The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox's Regression (CR) analysis. Results: Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76-1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI =1.18-1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI..1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and multiploid tumours' (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% 10%) independently associated with poor overall survival (P=0.027, CR). Conclusion: Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.