Purpose: Previous efforts to develop transcriptional markers of chemotherapy sensitivity in TNBC had limited success due to the heterogeneity of this disease. The purpose of this study was to identify genomic differences between extremely chemotherapy sensitive and highly chemotherapy resistant TNBC through whole exome sequencing and to assess measures of genomic heterogeneity as predictive markers.
Methods: Twenty nine cases were selected from a prospectively collected cohort of fine needle aspiration specimens obtained before preoperative chemotherapy (MDACC) to represent two extreme response cohorts including pathologic complete response (pCR, N=17) or extensive residual cancer (eRD, N=12). DNA was extracted from specimens stored in RNAlater, exomes were captured using NimbleGen SeqCap EZ Exome Library preparation and paired-end sequencing of 75 base pair fragments was performed on Illumina HiSeq 2000. Alignment and variant calling were performed with BWA and GATK haplotype caller. Variants were filtered against the 1000 Genomes and TCGA normal breast samples to identify candidate somatic variants. Fisher-exact test was used to identify variant genes associated with sensitivity to chemotherapy. We calculated overall mutational load and normal-adjusted clonal entropy of driver mutations as measures of genome heterogeneity. The chi-squared test was used to compare differences in mutational spectra and genome heterogeneity between the two response groups.
Results: The mean coverage was over 150X and 93% of target regions had > 20X coverage. The number of non-silent COSMIC mutations was similar in tumors from the two response groups (pCR: 63, range 49-82; eRD: 59, range 43-78) as well as the number of novel non-silent mutations (eRD: 223, range 113-388; pCR: 192, range 125-293). Gene level aggregation of variants identified 4 genes (MUC21, SLCO5A1, LRBA, STNE1) with response-associated mutational patterns. However, mutations were non recurrent and p-values were modest, ranging from 0.04 to 0.005. We observed greater overall mutational load and subclonal heterogeneity (clonal entropy of cancer related mutations) associated with eRD compared to pCR. Both measures suggest that higher genomic DNA diversity is associated with chemotherapy resistance. However, some genes (BRCA1 and MKI67) had higher mutational load (sum of minor allele frequencies per gene) associated with pCR compared to eRD. In general, a higher proportion of C>T transition (P=0.011) and lower A>G transition (P=0.028) was associated with eRD. The same mutational spectrum shift was previously described in the comparison of trunk and branch driver events suggesting that eRD tumors may undergo heterogeneous branched evolution.
Conclusion: We observed greater genomic diversity and distinctive mutational spectra in original pre-treatment samples of TNBC that were associated with extensive residual disease compared to pCR. Our analysis suggests that broad measures of genomic diversity may serve as markers of resistance to chemotherapy.
Citation Format: Tingting Jiang, Weiwei Shi, William F Symmans, Charles Li, James Platt, Rosanna Lau, Vikram B Wali, Richard Lifton, Lajos Pusztai, Christos Hatzis. Broad exonic DNA diversity is associated with resistance to taxane-FAC chemotherapy in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-2.