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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/SNF-complex 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 MSeq-analysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies.

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 FGF10- and 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.

535 Background: The bispecific antibody CEA-TCB binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells. This triggers T cell killing of colorectal cancer cell lines expressing moderate to high levels of CEA at the cell surface (Bacac, Clin Cancer Res 2016). Patient derived organoids (PDOs) may more accurately represent patient tumors than established cell lines. Yet, determinants of CEA-TCB resistance have not been studied in PDOs. Methods: PDOs were established from biopsies of eight multidrug-resistant metastatic CRCs, GFP labelled and adapted to 2D culture. Allogenic CD8 T cells and CEA-TCB or a non-targeting control antibody were added and cancer cell killing and growth were monitored for 10 days. CEA expression of PDOs was determined by FACS. Results: CRC PDOs could be categorized into three groups based on CEA cell-surface expression: CEAhigh (n = 3), CEAlow (n = 2), and CEA heterogeneous PDOs (n = 3) that stably maintained populations of both CEAhigh and CEAlow cells, which has not previously been described in CRC cell lines. Heterogeneity of cell-surface CEA expression is common in CRC cells in patients, supporting that PDOs may better represent these tumors than established cell lines. CEAhigh cells were sensitive whereas CEAlow cells showed resistance to CEA-TCB. All PDOs with heterogeneous CEA expression were resistant to CEA-TCB, suggesting that CEA-negative cells maintain cancer cell growth. Culture of FACS sorted CEAhigh and CEAlow cells from PDOs with heterogeneous CEA expression demonstrated high plasticity of CEA expression which may contribute to rapid resistance acquisition through CEA antigen loss. Conclusions: These results suggest that cell-surface CEA expression is a major determinant of CEA-TCB sensitivity and resistance in PDOs. In addition, we identified heterogeneous CEA expression in several PDOs and demonstrated that this could confer CEA-TCB resistance in vitro. These PDO models are likely to provide insights into the mechanism of CEA loss and may inform therapeutic opportunities to counter CEA-TCB resistance. RNA-sequencing and functional experiments are ongoing to investigate this and will be presented.

Despite stratification of only RAS wt CRCs for anti-EGFR antibody (aEa) therapy, many patients (pts) do not benefit and the molecular resistance (res) landscape remains incompletely understood. In order to decipher novel res mechanisms, we treated 40 RAS wt CRCs with single-agent aEa in a prospective trial and applied exome- and RNA-Seq to biopsies (bx) taken at baseline (BL) and at progression (PD). Among 20 BL bx from tumors with primary progression, 7 showed BRAF V600E mutations (mut), 3 harbored noncanonical BRAF D594K and/or KRAS L19F/A18D mut or KRAS amplifications (amp), 1 had a MEK1 mut and 1 was ERBB2 amp. Inactivating mut of the NF1 gene, a negative regulator of RAS, were found as novel mechanisms of primary res in 2 pts. No genetic drivers of primary res could be identified in 6/20 bx. Using transcriptomic analysis, 78% of pts with prolonged clinical benefit (≥16wks) displayed the Transit-Amplifying (TA) molecular subtype (Sadanandam et al., 2013). Stem-like (SL), goblet and inflammatory subtypes predominated (75%) among primary progressors. This significant enrichment in aEa sensitive CRCs (p=0.017) validates the TA subtype as a predictive biomarker. Exome- and deep Seq of PD bx from 13 pts who acquired res after prolonged clinical benefit detected KRAS mut or amp in only 2 cases. The FGFR2 ligand FGF10 was amplified in 1 PD bx and was validated in vitro as a novel mechanism of acquired res. No mut/amp of genes to which aEa res is usually attributed (RAS/RAF, EGFR, MET, ERBB2, MEK1) were found in the remaining 10 PD bx. Ultra-sensitive circulating tumor DNA-Seq at PD in 7 of these pts detected EGFR exodomain and RAS mut, including parallel evolution of multiple mut, in 3 cases. However, comparison to truncal TP53/APC mut showed that these res drivers were confined to small subclones and could therefore not explain the bulk of res. Overall, aberrations of RAS/RAF-pathway members and regulators were less abundant as drivers of acquired aEa res in this prospective trial than in reported retrospective series. Hence, we suspected additional novel mechanisms of acquired res. Molecular subtype switching from the aEa sensitive TA subtype to the comparatively insensitive SL subtype occurred in paired BL/PD bx from 3/7 pts who progressed after prolonged aEa benefit without detectable genetic res drivers in PD bx. Subtype switching was not observed in any of 6 analyzed BL/PD pairs from primary progressors. Considering the strong association of aEa sensitivity with TA subtype at BL, these data suggest subtype switching from TA to SL as a novel mechanism of acquired aEa res. This prospective trial revealed novel genetic (NF1 mut, FGF10 amp) and likely transcriptomic (TA to SL subtype switching) mechanisms of aEa resistance in CRC. These results should enable more precise aEa therapy stratification and may open opportunities to prevent res through SL subtype targeting strategies. Citation Format: Khurum Hayat Khan, Andrew Woolston, Georgia Spain, Louise Barber, Yatish Patil, Beatrice Griffiths, Reyes GonzalezExposito, Sonia Mansukhani, Matthew Davies, Sheela Rao, David Watkins, Francesco Sclafani, Jana Suntharanathan, Clare Peckitt, Ruwaida Begum, Isma Rana, Janet Thomas, Jacqui Oates, Annette Bryant, Andrew Wotherspoon, Nicos Fotiadis, Nasir Khan, Sebastian Guettler, Katharina von Loga, Naureen Starling, Ian Chau, Anguraj Sadanandam, David Cunningham, Marco Gerlinger. Molecular subtypes and novel genetic mechanisms of primary and acquired anti-EGFR resistance in colorectal cancer in the Prospect C biomarker trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4339.