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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.

GEAs are aggressive tumors in which several targeted therapy trials have failed. We assessed intratumor heterogeneity (ITH) and its impact on progression and therapy failure by applying an 81-gene NGS panel and SNP array copy number aberration (CNA) analysis to multiple primary tumor (T) regions and lymph node (LN) metastases from 9 GEAs. Analysis of 39 samples found ITH in all cases. 8 chromosomally instable (CIN) GEAs predominantly evolved through CNAs, with 17-76% of the genome affected by heterogeneous CNAs. A microsatellite instable GEA showed parallel evolution and diversified exclusively through point mutations (58% ITH). This demonstrates ongoing genomic instability rather than punctuated evolution and that specific instability mechanisms impact evolutionary trajectories. LN metastases contributed more to ITH (p To assess the phenotypes established by MAPK-activating amp evolution, we analyzed 135 published primary CIN subtype GEAs. Cytolytic activity (CYT), estimating tumor immune recognition from RNA expression data, correlated with the mutation load in GEAs with EGFR, ERBB2 or MET amp (p=0.04). In contrast, CYT did not correlate with mutation load in GEAs with KRAS or ERBB3 amp (p=0.22, NRAS/ERK2: insufficient data), indicating that these specific alterations, that also recurrently evolved in LN, may enable immune evasion. Downregulation of TAP and Class I MHC genes (p Moreover, ITH of MAPK-activating amp is likely to confer resistance to upstream tyrosine kinase inhibition. We used GEA cell lines with various MAPK-activating amp (ERBB2, MET, NRAS) to investigate downstream MAPK-pw inhibition as a novel strategy to broadly target heterogeneous subclones. Growth control was incomplete with ERK- and MEK-inhibitors but the panRAF/SRC inhibitor CCT196969 was effective in all lines, suggesting that it can effectively intercept subclonal heterogeneity in GEAs. In conclusion, we identified ITH with parallel and convergent evolution in 9/9 metastatic GEAs. Distinct selection pressures in LN foster the evolution of subclonal MAPK-activating amp that decrease immunogenicity and drive evolutionary pre-adaptation to future targeted drugs that can be intercepted by panRAF/SRC inhibitors. Citation Format: Matthew N. Davies, Louise J. Barber, Georgia Spain, Filipa Lopes, Katharina von Loga, Beatrice Griffiths, Andrew Woolston, Donat Alpar, Marta Gomez, Kamil A. Lipinski, Kerry Fenwick, Zakaria Eltahir, Stefano Lise, Emese I. Agoston, Laszlo Harsanyi, Richard Marais, Andrew Wotherspoon, A Szasz, Caroline Springer, Marco Gerlinger. Lymph node metastasis evolution drives immune evasion and targeted therapy resistance in gastro-esophageal adenocarcinomas (GEAs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2017-422