Abstract PD3-4: Reliability of whole exome sequencing for assessing intratumor heterogeneity from breast tumor biopsies
Authors: Shi Weiwei , Chagpar Anees , Jiang Tingting , Lannin Donald R , Killelea Brigid , Horowitz Nina , Lim Raymond , Platt James , Ng Charlotte KY , Wali Vikram B , Weigelt Britta , Reis-Filho Jorge S , Hatzis Christos , Pusztai Lajos 
Published: May 2015

Abstract
BACKGROUND: False positive findings introduced by analytical noise in sequencing and bioinformatics pipelines constitute a challenge for accurate massively parallel sequencing (MPS). Reports of intratumor genomic heterogeneity based on MPS rarely estimate the impact of false positive mutation calls. The purpose of this study was to measure apparent genomic heterogeneity in different regions of the same tumor and to assess the technical noise in variant calling in replicate sequencing of the same DNA.
METHODS: Three anatomically distinct biopsies were obtained from 3 different regions of 11 breast cancers (33 samples) including 6 low/intermediate grade, estrogen receptor (ER)-positive and 5 high-grade, triple-negative (TNBC) cancers. DNA from 8 different biopsies was split and independently processed on different days to obtain technical replicates. The NimbleGen SeqCap EZ Exome Library preparation method was used for exome capture and paired-end sequencing of 75 base pair fragments was performed on Illumina HiSeq 2000. Read alignment and variant calling were performed with BWA and GATK haplotype caller. Concordance in variant calls and in minor allele frequencies (MAF) was assessed in the 3 biopsies of the same tumor and 8 technical replicates. We adjusted for uneven sequence coverage and analyzed known germline variants from dbSNP, known cancer related variants from COSMIC separately from novel variants (i.e. not previously reported in dbSNP or COSMIC). We estimated intratumor genomic heterogeneity of genes after removing alterations identified in areas where mapping is difficult and variant calls that had low analytical reliability in the technical replicates.
RESULTS: The mean coverage was over 150X and &gt; 90% of target regions had ≥ 20X coverage. We validated the specificity (98.2%) and sensitivity (86.7%) of the variant calling pipeline on the GIAB reference data. The concordance for germ line SNPs and variants in COSMIC in technical replicates was 94.9% and 92.7%, respectively. Novel variants had very low concordance, 55.9%, in technical replicates. The concordance between MAF estimates from the technical replicates was high (0.974, 0.957 and 0.969 for single nucleotide variations, insertions and deletions, respectively). The concordance for germline SNPs and COSMIC variants in pairwise comparisons of biopsies from the same tumor was 93.2% and 91.1%. For known variants, lower concordance was observed in TNBC (88.3%-98.5%) compared to ER-positive tumors (93.6%-98.8%, P&lt;0.05) indicating greater intratumor heterogeneity. We identified variants in a small number of genes (DNAH9, PPM1E, and MAP3K1) that were called inconsistently in most technical replicates, even after excluding low mappability regions. We assessed intratumor heterogeneity in the triplicate biopsies, after excluding the technically unreliable variants. On average, two different biopsies from the same tumor shared 14272 +/-1379 common variants and differed in 816 +/- 416 variants.
CONCLUSION: We observed heterogeneity to be slightly greater in high-grade, ER-negative compared to low-grade, ER-positive breast cancers. Differences in variants observed in multiple biopsies of the same tumor are only slightly greater than those expected by technical noise alone.
Citation Format: Weiwei Shi, Anees Chagpar, Tingting Jiang, Donald R Lannin, Brigid Killelea, Nina Horowitz, Raymond Lim, James Platt, Charlotte KY Ng, Vikram B Wali, Britta Weigelt, Jorge S Reis-Filho, Christos Hatzis, Lajos Pusztai. Reliability of whole exome sequencing for assessing intratumor heterogeneity from breast tumor biopsies [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 PD3-4.