Highlights d NF1 and non-canonical KRAS and BRAF aberrations associate with cetuximab resistance d Genetic resistance drivers are absent in most biopsies that acquired resistance d Stromal remodeling is an alternative non-genetic mechanism of cetuximab resistance d Cetuximab-mediated immune modulation may sensitize CRCs to immunotherapy

BackgroundPatient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment.MethodsFour microsatellite stable PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analyzed by MS.ResultsWe identified an average of 9936 unique peptides per PDO which compares favorably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. In contrast, computational HLA binding prediction estimated that 304 mutations could generate neoantigens. One hundred ninety-six of these were located in expressed genes, still exceeding the number of MS-detected neoantigens 65-fold. Treatment of four PDOs with IFNγ upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFNγ-inducible genes. HLA class II presented peptides increased dramatically with IFNγ treatment. MEK-inhibitor treatment showed no consistent effect on HLA class I or II expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment.ConclusionsOnly 3 out of 612 non-silent mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

Mismatch repair deficient (dMMR) gastro-oesophageal adenocarcinomas (GOAs) show better outcomes than their MMR-proficient counterparts and high immunotherapy sensitivity. The hypermutator-phenotype of dMMR tumours theoretically enables high evolvability but their evolution has not been investigated. Here we apply multi-region exome sequencing (MSeq) to four treatment-naive dMMR GOAs. This reveals extreme intratumour heterogeneity (ITH), exceeding ITH in other cancer types >20-fold, but also long phylogenetic trunks which may explain the exquisite immunotherapy sensitivity of dMMR tumours. Subclonal driver mutations are common and parallel evolution occurs in RAS, PIK3CA, SWI/SNFcomplex genes and in immune evasion regulators. MSeq data and evolution analysis of single region-data from 64 MSI GOAs show that chromosome 8 gains are early genetic events and that the hypermutator-phenotype remains active during progression. MSeq may be necessary for biomarker development in these heterogeneous cancers. Comparison with other MSeqanalysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies.

Background The T cell bispecific antibody cibisatamab (CEA-TCB) binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells, which triggers T cell killing of cancer cell lines expressing moderate to high levels of CEA at the cell surface. Patient derived colorectal cancer organoids (PDOs) may more accurately represent patient tumors than established cell lines which potentially enables more detailed insights into mechanisms of cibisatamab resistance and sensitivity. Methods We established PDOs from multidrug-resistant metastatic CRCs. CEA expression of PDOs was determined by FACS and sensitivity to cibisatamab immunotherapy was assessed by co-culture of PDOs and allogeneic CD8 T cells. Results PDOs could be categorized into 3 groups based on CEA cell-surface expression: CEA hi ( n = 3), CEA lo ( n = 1) and CEA mixed PDOs ( n = 4), that stably maintained populations of CEA hi and CEA lo cells, which has not previously been described in CRC cell lines. CEA hi PDOs were sensitive whereas CEA lo PDOs showed resistance to cibisatamab. PDOs with mixed expression showed low sensitivity to cibisatamab, suggesting that CEA lo cells maintain cancer cell growth. Culture of FACS-sorted CEA hi and CEA lo cells from PDOs with mixed CEA expression demonstrated high plasticity of CEA expression, contributing to resistance acquisition through CEA antigen loss. RNA-sequencing revealed increased WNT/β-catenin pathway activity in CEA lo cells. Cell surface CEA expression was up-regulated by inhibitors of the WNT/β-catenin pathway. Conclusions Based on these preclinical findings, heterogeneity and plasticity of CEA expression appear to confer low cibisatamab sensitivity in PDOs, supporting further clinical evaluation of their predictive effect in CRC. Pharmacological inhibition of the WNT/β-catenin pathway may be a rational combination to sensitize CRCs to cibisatamab. Our novel PDO and T cell co-culture immunotherapy models enable pre-clinical discovery of candidate biomarkers and combination therapies that may inform and accelerate the development of immuno-oncology agents in the clinic. Electronic supplementary material The online version of this article (10.1186/s40425-019-0575-3) contains supplementary material, which is available to authorized users.

Anti-epidermal growth factor receptor (EGFR) antibodies (anti-EGFR-Ab) are effective in a subgroup of patients with metastatic colorectal cancer (CRC). We applied genomic and transcriptomic analyses to biopsies from 35 RAS wild-type CRCs treated with the anti-EGFR-Ab cetuximab in a prospective trial to interrogate the molecular resistance landscape. This validated transcriptomic CRC-subtypes as predictors of cetuximab benefit; identified novel associations of NF1-inactivation and non-canonical RAS/RAF-aberrations with primary progression; and of FGF10and non-canonical BRAF-aberrations with AR. No genetic resistance drivers were detected in 64% of AR biopsies. The majority of these had switched from the cetuximab-sensitive CMS2-subtype pretreatment to the fibroblast-and growth factor-rich CMS4-subtype at progression. Fibroblast supernatant conferred cetuximab resistance in vitro, together supporting subtype-switching as a novel mechanism of AR. Cytotoxic immune infiltrates and immune-checkpoint expression increased following cetuximab responses, potentially providing opportunities to treat CRCs with molecularly heterogeneous AR with immunotherapy.