Abstract PO-106: Genomic heterogeneity and clonal dynamics of resistance evolution and metastatic progression in rectal cancer
Authors: Hirsch Daniela , Lieberich Judith , Heselmeyer-Haddad Kerstin , Ceribas Yonca , Kelly Michael , Talsania Keyur , Zhao Yongmei , Gaiser Timo , Ried Thomas 
Published: December 2020

Abstract
The standard treatment for locally advanced rectal cancer is chemoradiotherapy followed by surgery. However, patient response to pre-operative chemoradiotherapy is highly heterogeneous, ranging from complete tumor regression to no response. Over the course of disease, up to 40% of patients suffer from local relapse or the development of metachronous metastatic disease, compromising survival. It is not yet known how treatment resistance and disease progression in these patients evolve – whether from the selective outgrowth of pre-existing resistant clones during treatment or through the acquisition of additional, resistance conferring genomic alterations. We aim to delineate the genomic evolution and clonal architecture underlying treatment resistance and metastatic progression in rectal cancer based on a cohort of 42 patients, from which 98 tumor samples were collected longitudinally over the course of treatment and metastatic progression. Tumor diagnosis and treatment response assessment are routinely based on the histopathologic evaluation of formalin-fixed paraffin-embedded (FFPE) patient tissues. While formalin fixation and paraffin embedding optimally preserve histomorphology and allow long-term tissue storage at ambient temperature, they, on the other hand, lead to DNA crosslinks posing a challenge for sequencing analyses. To overcome this limitation, we have successfully developed a protocol for dissociation of FFPE tissues into single cells that are suitable for immunolabeling, flow sorting and genomic analysis by whole exome sequencing of pure tumor cell populations, single cell copy number variation (CNV) sequencing and multiplex interphase fluorescence in situ hybridization (miFISH). Our genomic analyses at the population as well as individual cell level revealed divergent aberration patterns and distinct levels of intra-tumor heterogeneity. Major tumor clones were characterized by gains of EGFR (7p11.2), MYC (8q24.21), CDX2 (13q12.2) and ZNF217 (20q13.2) along with losses of SMAD4 (18q21.2) and TP53 (17p13.1). While in some patients the clonal composition remained largely stable throughout treatment, in others major clonal shifts occurred favoring clones with TP53 loss. Our preliminary results indicate different propensities of genetically distinct tumor cell clones in therapy response and metastatic progression. Furthermore, our data show the feasibility of high-resolution clonal reconstruction from whole exome sequencing and single cell CNV sequencing data of FFPE cells.
Citation Format: Daniela Hirsch, Judith Lieberich, Kerstin Heselmeyer-Haddad, Yonca Ceribas, Michael Kelly, Keyur Talsania, Yongmei Zhao, Timo Gaiser, Thomas Ried. Genomic heterogeneity and clonal dynamics of resistance evolution and metastatic progression in rectal cancer [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 PO-106.