Mismatch repair-deficient (MMRd) colorectal cancers (CRCs) have high mutation burdens, which make these tumours immunogenic and many respond to immune checkpoint inhibitors. The MMRd hypermutator phenotype may also promote intratumour heterogeneity (ITH) and cancer evolution. We applied multiregion sequencing and CD8 and programmed death ligand 1 (PD-L1) immunostaining to systematically investigate ITH and how genetic and immune landscapes coevolve. All cases had high truncal mutation burdens. Despite pervasive ITH, driver aberrations showed a clear hierarchy. Those in WNT/β-catenin, mitogen-activated protein kinase, and TGF-β receptor family genes were almost always truncal. Immune evasion (IE) drivers, such as inactivation of genes involved in antigen presentation or IFN-γ signalling, were predominantly subclonal and showed parallel evolution. These IE drivers have been implicated in immune checkpoint inhibitor resistance or sensitivity. Clonality assessments are therefore important for the development of predictive immunotherapy biomarkers in MMRd CRCs. Phylogenetic analysis identified three distinct patterns of IE driver evolution: pan-tumour evolution, subclonal evolution, and evolutionary stasis. These, but neither mutation burdens nor heterogeneity metrics, significantly correlated with T-cell densities, which were used as a surrogate marker of tumour immunogenicity. Furthermore, this revealed that genetic and T-cell infiltrates coevolve in MMRd CRCs. Low T-cell densities in the subgroup without any known IE drivers may indicate an, as yet unknown, IE mechanism. PD-L1 was expressed in the tumour microenvironment in most samples and correlated with T-cell densities. However, PD-L1 expression in cancer cells was independent of T-cell densities but strongly associated with loss of the intestinal homeobox transcription factor CDX2. This explains infrequent PD-L1 expression by cancer cells and may contribute to a higher recurrence risk of MMRd CRCs with impaired CDX2 expression. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

PDF file 325K, Additional validation of ST6GalNAc2 as a metastasis suppressor including in a spontaneous metastasis assay using the parental 4T1 cells

PDF file 299K, Additional characterization of human breast cancer cell lines; immunofluorescence analysis and lung retention assays

PDF file 32K, Supplementary Table S1 Antibody reagent list used in study

PDF file 54K, Supplementary Table S5. Tumor-specific CDK12 mutations described in publically available sequencing data

Supplementary Table 1. Expression of estrogen receptor regulated genes in patient 207 was determined using Nanostring. Data was normalized as described in the Online Methods and is presented as the Log2 ratio. Supplementary Table 2. A list of cell lines including FGFR status. Supplementary Table 3. Copy Number Variation and Gene Expression Assays.

Clinical activity of FGFR inhibitor AZD4547 in FGFR amplified breast and gastric cancer.

High-level FGFR2 amplified cell lines are highly sensitive to FGFR inhibition.

PDF file 149K, In vivo validation of 3 hits identified in the screen (Mre11a, Fen1, Wwc1)

PDF file 174K, Additional characterization of the 4T1-Luc cells in lung retention assays

PDF file 58K, Supplementary Table S4. DNA repair involvement of PARP inhibitor sensitisation genes found in the screen. PMID numbers refer to pubmed entries

Investigation of PI3K-AKT signalling in FGFR amplified cell lines.

Results of parallel siRNA screens in FGFR driven cell lines with analysis of ERK and AKT phosphorylation in and panel of FGFR driven cancer cell lines.

PDF file 120K, Supplementary figure legends and supplementary materials and methods

PDF file 107K, Inventory of supplementary material. Supplementary Table S1 listing primers and siRNAs. Legends for Supplementary Fig. S1 - S7 Legends for Supplementary Movies S1 and S2 Supplementary Figures S1 - S6 legends Supplementary Movies S1 and S2 legends

Supplementary figures and tables

MOV file 2886K, Video showing dynamic flow adhesion assays of ZR75.1 shNTC and shST6 cells on a HUVEC monolayer in the presence of the galectin-3 inhibitor GCS-100

PDF file 3292K, Supplementary Figure S1. Genome wide shRNA screen information. Supplementary Figure S2. Validation of ATAD5 as a genetic determinant of olaparib response. Supplementary Figure S3. A working model of PARP1/2 inhibitor-induced DNA repair. Supplementary Figure S4. Inhibition of CDK12 sensitises serous ovarian cancer cells to olaparib and cisplatin. Supplementary Figure S5. CDK12 silencing and the impact on expression of DNA repair proteins. Supplementary Figure S6. Targeting of CCNK sensitises serous ovarian cancer cells to olaparib and cisplatin. Supplementary Figure S7. Animal body weights from in vivo study

PDF file 543K, Supplementary Table S2 shRNA constructs with Olaparib Drug Effect Z scores <-1.96

FGFR2 expression in cancers with high-level amplification of FGFR2.