Abstract P3-07-06: Evidence for tumor heterogeneity and clonal evolution during invasive progression in breast cancer

Abstract

Abstract Purpose: Intratumoral heterogeneity is well recognized to be an important driver of treatment resistance and metastasis. We undertook this N of three study to measure the degree of heterogeneity in three large preinvasive lesions, all with invasive components to determine the relationship between tumor heterogeneity, spatial distribution, clonal evolution, and invasive progression. Methods: We identified patients A, B, C with extensive DCIS measuring 7.5 cm, 6 cm, and 7 cm associated with 0.3 cm, 3.8cm, and 3.4 cm of an invasive component and 0, 7 and 1 positive lymph node, respectively. We sequenced the tumor sample for Case A from 32 unique blocks with precise geospatial localization; invasive cancer was identified in 3 of 32 blocks. Case B had 26 blocks sequenced with invasive cancer in 13 of 26 blocks. Case C had 23 blocks sequenced with invasive in 11 of 23 blocks. For germline reference, we sequenced DNA from an uninvolved tissue from each case. NGS libraries were made from FFPE derived DNA (20-40ng) for full exome sequencing. Variant calling was performed by GATK HaplotypeCaller, Platypus and Mutect. Identified somatic mutations were annotated with Oncotator and pathway enrichment analysis was performed with Bioconductor. To investigate the clonal evolution and progression history, phylogenetic trees were constructed in R and sub-clonal analysis was performed with Treeomics. Results: The sequence data was analyzed with Platypus, MuTect and GATK HaplotypeCaller. The somatic mutation sites were concatenated into one sequence for each sample. Both neighbor-joining trees and maximum parsimony trees were built for each case. Phylogenetic analysis and sub-clonal analysis support the multi-clonal invasion model of invasive cells, in which invasive cancer can evolve from multiple clades, either early or late in the evolutionary history, independently. Dense sampling allowed reconstruction of the temporal order of mutations that accumulated in the cell lineage of the invasive cancers. Furthermore, phylogeny and sub-clone spatial analysis revealed that distant regions may be closely genetically related and showed a weak spatial sub-clone clustering pattern, which is consistent with the predictions of Big Bang model. For driver genes, we find that except for SETD2 in Case B, the majority of driver gene mutations are sub-clonal. Somatic mutations on ATP-binding cassette (ABC) transporter pathway was found in all cases. Conclusions: Extensive sampling and sequencing of tumors yields important insights about tumor heterogeneity and tumor progression of DCIS to invasive cancer. Variable invasive propensity was identified, with foci of invasion were geospatially associated with preinvasive regions of progressively higher mutational load. Citation Format: Ding Y, Marks J, King L, Hardman T, Hall A, Mallo D, Rodrigo A, Maley C, Hwang S. Evidence for tumor heterogeneity and clonal evolution during invasive progression in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-07-06.