Abstract P2-06-01: Breast-to-breast metastasis can cause hormone-receptor positive/triple negative bilateral synchronous tumors
Authors: Schwab RB , Bao L , Pu M , Crain B , Dai Y , Nazareth LV , Matsui H , Wallace AM , Hasteh F , Harismendy O , Frazer KA , Parker BA , Messer K 
Published: August 2014

Abstract
Background: Prior work suggests that synchronous bilateral breast cancers may be clonal, with one tumor a metastasis, although prior techniques lacked resolution to prove this relationship. We used deep whole exome and shallow whole genome sequencing to compare bilateral tumors in two cases. In both cases, tumors were invasive and node negative with one tumor ER+/PR+/HER2− (HR+) lobular and the other triple negative (TN) ductal. Case 1 is a 75-year-old African American woman and Case 2 a 70-year-old white woman. With 44 and 12 months of follow up, respectively, neither patient has recurred.
Methods: Agilent SureSelect All Exon 50Mb Target Enrichment Kits were used for exome capture. Paired-end sequencing was performed with 200 base pair reads on the Illumina HiSeq 2000. Sequencing depth was targeted to cover 80% of the genome at 100x for three tumors with 70% cellularity, 200x for one tumor with 40% cellularity and 30x for germline. Tumor and germline exome results were compared to identify high confidence somatic single nucleotide variants (HC SNV). HC SNV's were called using GATK and stringent custom filtering to avoid false positives resulting from unrecognized germline single nucleotide polymorphisms. For each tumor pair, we define a clonality likelihood score (CLS) as the ratio of the number of HC SNV called at the same site and with the same alternate base in both tumors, to the total number of sites with an HC SNV called in either tumor. For comparison we analyzed the called SNV data from The Cancer Genome Atlas (TCGA) for exome sequenced HR+ or TN breast cancers.
Results: In Case 1, of 102 HC SNVs called in either tumor, 82 were shared, for a CLS of 80.3%. Additionally, 11 shared SNVs were synonymous, consistent with clonality. Lastly the non-shared HC SNVs were asymmetrically found in the TN tumor, consistent with clonal evolution during metastasis. Copy number analysis (CNA) showed Case 1 to have a deletion in 6q, including the ESR1 gene, unique to the TN tumor.
To assess significance of the CLS, we found three primary/metastatic clonal pairs in the TCGA to serve as positive controls. To serve as negative controls, from 357 ER+ and 46 TN primary TCGA tumors, we formed a total of 16,422 independent ER+/TN pairs. For the 3 clonal TCGA pairs, the CLS values were 39.3%, 58.5% and 60.0%. Most of the independent TCGA pairs had a CLS of zero (98.5%), with a maximum CLS of 2.8%. As the CLS for Case 1 lies above maximum observed CLS among 16,422 independent tumor pairs, we reject the hypothesis that this tumor pair is independent, at p &lt; 0.0001. For Case 2, of 222 HC SNV sites, 5 were shared for a CLS of 2.3%, consistent with independence.
Conclusion: Somatic single nucleotide mutations identified by exome sequencing found that the two tumors in Case 1 share &gt;80% of SNVs, consistent with clonal evolution of metastasis. The two tumors from Case 2 have few shared SNVs, consistent with independent origin. CNA results were consistent. This is the first clonality analysis reported from deep sequencing of phenotypically discordant synchronous bilateral breast cancers, and demonstrates that next-generation sequencing can distinguish clonal from independent tumor pairs with high confidence.
Funding: The Breast Cancer Research Foundation
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-06-01.