Fig. 2

BRCA1 secondary splice-site mutations are enriched in ARIEL2/4 clinical trial patient samples following PARPi treatment. A BRCA1 secondary splice-site mutations increased form 1% (pre-PARPi) to 10% (post-PARPi) in patient tumor/plasma samples from the ARIEL2 and ARIEL4 clinical trials. B The BRCA1 (NM_007294.4) exon 11 donor splice-site mutations identified in these patients and the DNA sequence context are presented. *Patient 5 is an ARIEL4 chemotherapy to PARPi cross-over (XO) arm patient, not included in part A (n = 5). C-D The predicted outcomes on BRCA1 gene splicing based on these disruptions of the exon 11 donor splice site (detailed in Supplementary Table 9) were confirmed for most mutations (D) using the previously described BRCA1 minigene system, with the c.1127delA used as a primary mutation control and included in all SSM minigenes [6]. Mutations driving lower levels of ∆11/∆11q also had a reduced minigene transfection efficiencies relative to other samples (measured as BSD (blasticidin resistance gene) expression). E Summary of BRCA1 secondary events detected in Patient 2 (ARIEL2) before and after PARPi therapy, and their relative proportions in each sample (by colour). F A number of BRCA1 secondary events were also detected in the screening biopsy for platinum and rucaparib resistant Patient 3 (ARIEL2) prior to PARPi, and the number increased at end of rucaparib treatment (EOT). G In contrast, platinum resistant (4 prior lines of platinum) Patient 1 (ARIEL2) had no secondary BRCA1 events detected at first cycle of rucaparib, and had stable disease (SD) on treatment. Three secondary events were detected at cycle 12 of treatment, including a splice-site mutation c.4096G > A. H Patient 4 (ARIEL4) was partially platinum sensitive (2 prior lines) with no secondary BRCA1 events detected at cycle 1 of rucaparib. The EOT plasma sample was positive for multiple reversion events and two splice site mutations (4096 + 1G > T and 4096 + 1G > A) confirmed by minigene to alter splicing (D). I Patient 5 (ARIEL4) was platinum resistant and was enrolled in the chemotherapy arm of ARIEL4. They then crossed over (XO arm) to receive rucaparib treatment where they had stable disease. There were no secondary events detected prior to starting rucaparib, but at cycle 6 there were two BRCA1 splice-site mutations detected (c.4096 + 2 T > C and c.4093_4096 + 10del), without other reversion events. c.4096 + 2 T > C was found to drive alternative BRCA1 splicing (D). Red bold text indicates SSMs detected in patient samples (E-I), while other events presented are exonic secondary/reversion variants predicted to restore the BRCA1 reading frame