Abstract 422: Lymph node metastasis evolution drives immune evasion and targeted therapy resistance in gastro-esophageal adenocarcinomas (GEAs)
Authors: Davies Matthew N. , Barber Louise J. , Spain Georgia , Lopes Filipa , Loga Katharina von , Griffiths Beatrice , Woolston Andrew , Alpar Donat , Gomez Marta , Lipinski Kamil A. , Fenwick Kerry , Eltahir Zakaria , Lise Stefano , Agoston Emese I. , Harsanyi Laszlo , Marais Richard , Wotherspoon Andrew , Szasz A , Springer Caroline , Gerlinger Marco 
Published: July 2017

Abstract
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&lt;0.01) than any anatomic location within T. Further, subclonal aberrations that activate the Mitogen Activated Protein Kinase-pathway (MAPK-pw), including ERBB3, ERK2, KRAS and NRAS amplifications (amp) and NRAS mutations, were detected in LN metastases from 4/8 CIN GEAs. Subclonal MAPK-activating amp were enriched in LN (p=0.019) compared to T regions that only exhibited a single subclonal MET amp. Convergent evolution of LN subclones across several GEAs suggests that selection pressures differ systematically between LN and T ecosystems.
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&lt;0.05) in KRAS or ERBB3 amp GEAs suggested impaired antigen processing and presentation as the mechanisms driving T cell immune evasion.
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