ESR1 and cancer: To address potentially artefactual FFPE variants, 12 combinations of variant filtering strategies and statistical analysis methods (classical, liberal and strict variant quality control (QC) filtering, combined with C-T weighted and un-weighted SKAT-O and logistic regression, described in Methods) were used to identify significant gene-level mutation burden differences between cancer (BBD-ER+ and BBD-ER−) and cancer-free (BBD-control) groups (Fig. 1a; Table 2).