Abstract PR012: Genetic and immune landscape evolution defines subtypes of MMR deficient colorectal cancer
Authors: Challoner Benjamin R. , Woolston Andrew , Lau David , Buzzetti Marta , Barber Louise J. , Lund Tom , Sansano Harold B. , von Loga Katharina , Lázare-Iglesias Héctor , Begum Ruwaida , Crux Richard , Cunningham David , Chau Ian , Starling Naureen , Ruiz-Bañobre Juan , Dhillon Tony , Gerlinger Marco 
Published: May 2022

Abstract
Mismatch repair deficient (MMRd) CRCs have high mutation/neo-antigen loads, leading to high immunogenicity and good immunotherapy response rates. We reasoned that the MMRd hypermutator phenotype should also promote intratumor heterogeneity (ITH) and evolvability; and investigated the genetic and immunological co-evolution in 69 regions from 6 localized and 13 metastatic MMRd CRCs by multi-region DNA-, RNA-sequencing and immunohistochemistry. All tumors had high truncal mutation loads (median: 44 mutations in 191 sequenced genes; whole exome equivalent: 1870 mutations). A median of 16.1% mutations per region were heterogeneous, indicating pervasive ITH. Phylogenetic analyses showed that metastases had diverged before subclonal diversification of the primary tumor in 75% of assessable cases. Thus, the ability to metastasize was frequently acquired early during tumor evolution. Driver aberrations evolved with a clear hierarchy: those in the WNT and RTK-MAPK pathways and in TGFbR-family members were almost always truncal (87.0%, 86.4% and 83.7%), indicating a critical role for cancer initiation. In contrast, genetic aberrations that are known to confer immune evasion (IE) were predominantly subclonal (71.4%) and parallel evolution of IE drivers occurred in 4/6 tumors that harboured any subclonal IE driver. This substantiates immune selection pressure as the main driver of Darwinian evolution during tumor progression. These IE drivers are known to confer resistance to checkpoint-inhibitor immunotherapy. ITH therefore needs to be addressed for predictive biomarker development. We quantified CD8 T-cell infiltrates as a surrogate measure of tumor immunogenicity; distinguishing tumors with low CD8 T-cell infiltrates (mean: 3.9% T-cells of all nucleated cells) and those with high infiltrates (mean: 12.2%). T-cell infiltrates showed high ITH in the latter group. This suggested a tumor-intrinsic setpoint, accompanied by marked variability in tumors with dense infiltrates. T-cell densities did not correlate with truncal mutation loads or heterogeneity metrics, questioning how immunogenicity is regulated. Phylogenetic analysis defined three patterns of IE evolution: tumors with subclonal, with pan-tumor, or without any identifiable IE drivers. CD8 T-cell abundance was highest in tumors with subclonal IE, supporting selection pressure from high CD8 T-cell infiltrates as the proximate cause for IE evolution. Tumors with pan-tumor IE showed low CD8 T-cell infiltrates. Surprisingly, tumors without IE drivers had the lowest CD8 T-cell abundance, indicating an alternative mechanism of immune escape. Low densities of CD8 T-cells at the tumor margin and low expression of T-cell chemo-attractants suggested impaired T-cell recruitment in these. Together, we show that immune recognition is a major driver of Darwinian evolution in MMRd CRCs and that immune infiltrates and IE drivers co-evolve interdependently. Whether sensitivity to checkpoint-inhibitor immunotherapy differs between the three phylogenetic MMRd CRC subtypes needs to be assessed in clinical trials.
Citation Format: Benjamin R. Challoner, Andrew Woolston, David Lau, Marta Buzzetti, Louise J. Barber, Tom Lund, Harold B. Sansano, Katharina von Loga, Héctor Lázare-Iglesias, Ruwaida Begum, Richard Crux, David Cunningham, Ian Chau, Naureen Starling, Juan Ruiz-Bañobre, Tony Dhillon, Marco Gerlinger. Genetic and immune landscape evolution defines subtypes of MMR deficient 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 PR012.