Abstract A045: Multiple roles for plasticity in metastasis and therapy resistance in long-term survivors of metastatic colorectal cancer
Authors: Berner Alison May , Gabbutt Calum , Nowinski Salpie , Househam Jacob , Trahearn Nick , Cresswell George D. , Nadhamuni Vinaya Srirangam , Kimberley Christopher , Fassan Matteo , Baker Ann-Marie , Sottoriva Andrea , Thirlwell Christina , Bridgewater John , Graham Trevor 
Published: May 2022

Abstract
Long-term survivors (LS) of metastatic colorectal cancer (mCRC) who experience multiple recurrences with resectable oligometastatic disease provide an opportunity to explore co-evolution of the tumor and immune microenvironment. We profiled 16 LS of mCRC with a median follow-up of 9.3 years and median of 3 biopsies/resections per patient (range 2-7). We performed multi-omic profiling of 56 primary and 176 metastatic samples from formalin-fixed paraffin-embedded tissue using low pass whole genome sequencing, 3’ RNA sequencing and DNA methylation arrays. A machine learning cell classifier was used to quantify immune cell and fibroblast infiltration from hematoxylin and eosin staining. Copy number profiling showed that the fraction of genome altered remained relatively stable across time and tissue type but that already-altered segments underwent progressive fragmentation. Inter-timepoint divergence of copy number alterations was significantly higher than intra-timepoint divergence, and intra-timepoint divergence was lower for liver and lung metastases than for primary tumors. Chemotherapy treatment did not significantly affect either divergence type. Differential expression and gene set enrichment analysis (GSEA) revealed common pathways dysregulated in metastases compared to primaries, including reductions in E2F (important in G1/S checkpoint) and G2M checkpoint, suggestive of the onset of senescence in metastases. Tumors underwent progressive hypomethylation over time and analysis of genes with concordant changes in promoter methylation and expression revealed dysregulation in pathways related to endocytosis, cell adhesion and migration. This suggests an important role for phenotypic plasticity in driving the metastatic phenotype. There were transient increases in the proportion of macrophages, lymphocytes and neutrophils in tumors that had undergone neoadjuvant chemotherapy in the 6 months prior to resection and slight increases in M1 macrophage activity (by GSEA) in tumors that were previously therapy naïve. There were concordant transient increases in pathways associated with immune response (MYC V1 and MTORC1), as well as xenobiotic metabolism. The latter is a known mechanism of drug resistance to both the platinum- and fluoropyridine-based therapies used in CRC. There were also more sustained increases post-chemotherapy in inflammatory and immune pathways associated with the adaptive immune response and tissue injury and repair. These findings were corroborated by concordant changes in promoter methylation. These data suggest that there is a threshold level of aneuploidy required to facilitate CRC metastasis but the migratory phenotype and adaptation to the metastatic niche are driven by plasticity. Chemotherapy induces differing short-term and long-term anti-tumor immune responses in the local microenvironment. We see evidence that mCRCs are able to mount plastic pro-survival mechanisms in response to chemotherapy.
Citation Format: Alison May Berner, Calum Gabbutt, Salpie Nowinski, Jacob Househam, Nick Trahearn, George D. Cresswell, Vinaya Srirangam Nadhamuni, Christopher Kimberley, Matteo Fassan, Ann-Marie Baker, Andrea Sottoriva, Christina Thirlwell, John Bridgewater, Trevor Graham. Multiple roles for plasticity in metastasis and therapy resistance in long-term survivors of metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A045.