Software

We employed primer sets that amplify the entire kinase domains of PRKD1, PRKD2 and PRKD3. The primers pairs were designed as previously described^{10 (link)} (Supplementary Figure 1 and Supplementary Table 1) and the specificity was also tested using in a previously described in silico method^{19 (link)} (https://genome.ucsc.edu/cgi-bin/hgPcr). PCR amplification of 10ng of genomic DNA was performed using the AmpliTaq 360 Master Mix Kit (Life Technologies, Norwalk, CT) on a Veriti Thermal Cycler (Life Technologies). The thermocycling protocol consisted of an initial incubation step of 95°C for 5 min and then 35 cycles of 95°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, and one final extension step of 72°C for 10 min. PCR fragments were purified with ExoSAP-IT (Affymetrix, Santa Clara, CA), and the sequencing reactions were performed on an ABI 3730 capillary sequencer using ABI BigDye Terminator chemistry (v3.1, Life Technologies) according to the manufacturer’s instructions. Sequences of the forward and reverse strands were analyzed using MacVector software (MacVector, Inc, Cary, NC).^{10 (link)} All analyses were performed in duplicate.

We employed primer sets that amplify the entire coding region of TP53 and the hotspot mutation sites in exons 9 and 20 of the PIK3CA gene. The selection of these two genes was based on the observation that in large whole exome sequencing studies of TNBCs, TP53 and PIK3CA were found to be the most frequently mutated genes in these tumours.^{33 (link),35 (link)} We designed the primer pairs as described previously^{45 (link)} (for the primer sets used, please see Table S1). PCR amplification of 10 ng of genomic DNA was performed using the AmpliTaq 360 Master Mix Kit (Life Technologies) on a Veriti Thermal Cycler (Life Technologies). The thermocycling protocol consisted of an initial incubation step of 95°C for 5 min and then 35 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 30 s and one final extension step of 72°C for 10 min. PCR fragments were purified with ExoSAP-IT (Affymetrix, Santa Clara, CA, USA), and the sequencing reactions were performed on an ABI 3730 capillary sequencer using ABI BigDye Terminator chemistry (version 3.1; Life Technologies), according to the manufacturer’s instructions. Sequences of the forward and reverse strands were analysed using MacVector software (MacVector, Inc., Cary, NC, USA).^{45 (link)} All analyses were performed in duplicate. Insertions and deletion were annotated manually.