# Genomic Analysis of Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma
## 4 Discussion  
In this study we analyzed whole-genome sequencing data from endoscopic biopsies prior to neoadjuvant chemotherapy in EAC and compared the genomes of responders to non-responders to identify potential genomic determinants of response. We comprehensively profiled CNVs, SNVs and mutational signatures in a cohort powered to identify differences between responders and non-responders. We detected distinct mutational characteristics of EAC between responders and non-responders across the spectrum from large-scale chromosomal alterations to point mutations. Our work characterizes pre-existing genomic alterations that have potential as biomarkers for resistance or sensitivity to NAC. We found that responders have higher mutational burden, in agreement with a previously published study [26]. Using a neoantigen prediction pipeline, we predicted that an increased mutational burden could lead to more abundant neoantigen recognition in responders. This could serve to bolster anti-tumor immunity as observed in the mutagenic subset of EACs reported previously [24]. Unfortunately, this study was not powered to resolve differences in NAC response between mutational signature subtypes. Although we reliably identified these mutational subtypes, there was not a clear distinction in this cohort. Consistently, we found that non-responders had impaired DNA damage response pathways and had more frequent driver gene mutations and genomic instability, despite having a lower mutation burden. The presence of an immune response related to DNA damage is known to improve survival outcomes and might contribute to this effect, as neoadjuvant therapies are genotoxic and known to stimulate anti-tumor immunity [54]. Our data suggest that responders are dominated by point mutations, whereas non-responders display more copy number changes. Non-responders displayed a unique pattern of copy number changes characterized by chromosome arm deletions and an increased burden of copy number altered segments. This is consistent with an analysis of mutational landscapes in a pan-cancer dataset (not including EAC), which suggests that tumors are dominated by either mutations or copy number changes, but never both [55]. The extremes of this spectrum are occupied by genomically unstable tumors, such as those observed in our cohort of non-responders. Genomic instability has been linked to a suppressed anti-tumor immunity during immunotherapy in gastric cancer [56], whereas in non-small cell lung cancer and melanoma a higher mutation burden is linked to greater neoantigen burden [57] and an improved clinical response to immunotherapy [58; 59; 60]. Taken together, this suggests that responders may be more likely to benefit from immunotherapy than non-responders and warrants further investigation. The unique patterns of copy number changes in driver genes have important implications for treatment of chemoresistant EAC patients. These unique amplifications and deletions included potentially druggable signaling axes in EAC, and we found that MYC signaling, RTK-RAS and cell cycle pathways were preferentially mutated in non-responders. This has implications for future clinical trials, as profiling driver mutations prior to neoadjuvant treatment, such as _EGFR_, _CDKN2A_ and _CCND1_ copy number changes, would be an effective strategy to aid clinical decision-making. This would aid in the identification of patients unlikely to respond to NAC, allowing alternative or concurrent targeted therapies to be considered to exploit these pathway alterations. Among altered pathways, we highlight cell cycle regulation as a vulnerability in non-responders. G1/S-phase checkpoint genes were disrupted, with _CCND1_ and _CDK6_ amplification as well as deletion of the _CDK4/6_ inhibitors _CDKN2A_ and _CDKN2B_. Abnormal expression of CDKs and their partner cyclins is widely reported in esophageal cancer [61; 62; 63], and _CCND1_ amplification and nuclear expression have been shown to correlate negatively with survival [64; 65].