Abstract PR02: Inferring the evolutionary dynamics of ductal carcinoma in situ through multi-regional sequencing and mathematical modeling
Authors: Ryser Marc D. , Sorribes Inmaculada C. , Greenwald Matthew , Wu Ethan , Hall Allison , Mallo Diego , King Lorraine M. , Hardman Timothy , Simpson Lunden , Maley Carlo C. , Marks Jeffrey R. , Shibata Darryl , Hwang E. Shelley 
Published: December 2020

Abstract
Introduction. The natural history of preinvasive breast cancer, or ductal carcinoma in situ (DCIS) remains poorly understood. Overcoming this gap would allow risk-appropriate treatment for patients diagnosed with DCIS. We used a multiregional sequencing approach in combination with mathematical modeling to characterize the evolutionary dynamics of DCIS initiation and progression. Methods. We analyzed a cohort of 18 patients diagnosed with DCIS, either with (n=9) or without (n=9) synchronous invasive cancer. Based on whole exome sequencing, tumor-specific mutation panels were constructed, each targeting 29-75 mutations (median: 60). From each tumor, and using selective ultraviolet radiation fractionation (SURF), we microdissected small spots (encompassing 1-3 duct cross-sections) from 3-4 spatially separated microscope sections (mean slide separation: 1.25cm, range: 0.34-6.0cm). Spots were spatially registered and genotyped based on targeted sequencing of the tumor-specific mutation panels. For each tumor, we performed unsupervised clonal deconvolution of the spot genotypes (CloneFinder) and constructed phylogenetic subclone trees. To quantify the spatial patterns of subclonal mutations, we introduced a dispersion index (DI), ranging from low (DI=0%) to high (DI=100%). To provide a spatio-temporal context for the heterogeneity patterns we developed a family of stochastic mathematical models of DCIS initiation and progression. Thereby, we embedded the evolutionary dynamics of tumor cell expansion in the branching topology of mammary ductal trees. Results. A total of 485 microdissected spots (median per tumor: 23, range: 10-50) were spatially registered and sequenced (median depth: 9,000x). All tumors were multiclonal, containing a median of 5 subclones (range: 2-14). Surprisingly, the correlation between spatial and genomic distances of spots was low. Individual subclones were diffusely dispersed across tumors. DCIS with synchronous DCIS and invasive cancer (mixed DCIS) had a higher mutation dispersion (DI=84.7%) than those without (pure DCIS, DI=70.5%; p=0.03, Wilcoxon rank-sum test). Mixed DCIS also had a higher fraction of spots containing more than one subclone than pure DCIS (median: 30.4% vs 0%, p=0.03). Among 7 mixed DCIS with invasive spots, 5 showed evidence of multiclonal invasion, that is more than one invading subclones were found in both in situ and invasive regions of the tumor. Mathematical modeling analyses show that the observed spatial patterns of genetic heterogeneity are consistent with a single expansion of mixing subclones across the ductal tree architecture. Conclusions. Our findings provide novel insights into the early growth and invasion dynamics of DCIS lesions. Further, we identified potential evolutionary markers for the delineation between indolent (pure) and aggressive (mixed) DCIS. This constitutes an important step towards identification of patients with low-risk DCIS who could be appropriately managed with less aggressive treatment.
Citation Format: Marc D. Ryser, Inmaculada C. Sorribes, Matthew Greenwald, Ethan Wu, Allison Hall, Diego Mallo, Lorraine M. King, Timothy Hardman, Lunden Simpson, Carlo C. Maley, Jeffrey R. Marks, Darryl Shibata, E. Shelley Hwang. Inferring the evolutionary dynamics of ductal carcinoma in situ through multi-regional sequencing and mathematical modeling [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR02.