# Genomic Analysis of Response to Neoadjuvant Chemotherapy in Esophageal Adenocarcinoma
## 3 Results
### Mutational Profiles Associated with Response to NAC
#### 3.2.1 Non-Responders Have More Chromosomal Instability and Unique Copy Number Alterations  
We next moved to consider chromosomal and copy number events and correlate these with mRNA expression, where possible, before considering point mutations. To investigate correlations between chromosomal instability (CIN) and response to NAC, we measured the proportion of the genome affected by copy number alteration. Non-responders exhibited a higher level of CIN as evidenced by a higher Genomic Instability Index (GII) [50] (Wilcoxon rank-sum test, \(p\) < 0.001, Figure 3A). To confirm these findings, we evaluated the CIN70 signature in matched RNA-Seq data, a gene signature whose expression was consistently correlated with total functional aneuploidy across multiple cancer types [51]. We found a higher CIN70 signature in non-responders, but this was not significant (\(p\) = 0.064), likely due to the small size of the RNA-Seq cohort. We then identified recurrently amplified or deleted regions using GISTIC2.0 [34]. In responders, a total of 3626 CNVs were detected (median 136/patient, range: 0-292) including 2961 amplifications (median 115/patient, range: 0-286) and 665 deletions per case (median 28/patient, range: 5-53). In non-responders, there were a total of 9637 CNVs (median 282/patient, range: 5-504) including 6198 amplifications (median 185.5/patient, range: 0-382) and 3439 deletions (median 99.5/patient, range: 0-239). The total CNVs and the number of amplifications and deletions were higher in non-responders (Wilcoxon rank-sum test, \(p\)-values < 0.001, 0.025 and 0.001 respectively, Figure 3B). At the chromosomal arm level, we found recurrent amplifications of chr 20q in 48% of responders (FDR < 0.1), while we identified no significant amplification in non-responders (Tables S2 and S3). Furthermore, we found 13 and 20 significant deletions of chromosomal arms in responders and non-responders, respectively. Deletion of 14p was unique to responders, and deletions of 8p, 9q, 9p, 10q, 15q, 16p, 19q, and 22q were unique to this cohort of non-responders, showing a higher level of large-scale deletion in non-responders. Figure 3: Non-responders have less stable genomes and unique patterns of copy number change in EAC driver genes. **(A)** Proportions of the genome affected by copy-number changes (Genomic Instability Index, GII). Non-responders showed a higher level of genomic instability (\(p\) = 2.5 \(\times\) 10\({}^{-13}\)). **(B)** Violin plots depicting the frequency of all amplifications and deletions in responders and non-responders. **(C)** Amplified peak regions across the genome plotted for responders vs non-responders (\(n\) = 63) using GISTIC2.0 (FDR \(<\) 0.1). Amplifications unique to each group are labeled. **(D)** Oncoplot of recurrently amplified/deleted EAC drivers among responders and non-responders identified by GISTIC2.0 (FDR \(<\) 0.1). We next looked at copy number alterations in 76 previously validated EAC driver genes [25]. We restricted our initial analysis to these 76 genes as they have been comprehensively analyzed and verified in contemporaneous and clinically relevant cohorts in addition to downstream functional biological assessment. We identified significantly amplified or deleted peaks for the two groups (FDR < 0.1, Figure 3C,D, Tables S4 and S5). Distinct focal amplifications and deletions in EAC driver genes are illustrated in Figure 3D. The responders contained two unique amplification peaks: 17q12, containing \(ERBB2\) (FDR < 0.001) and 19q12, containing \(CCNE1\) and \(TSHZ3\) (FDR = 0.003) (Table S4). These focal amplifications contrast with observed chromosome arm deletions in 19q observed in non-responders. Meanwhile, the non-responders contained more unique amplification peaks: 18q11.2 containing \(GATA6\) (FDR < 0.001); 7p11.2 containing \(EGFR\) (FDR = 0.018); 11q13.32 containing \(CCND1\) (FDR < 0.001); 12p12.1 containing \(KRAS\) (FDR < 0.001); 6q23.3 containing \(MYB\) (FDR = 0.085); and 8q24.21 containing \(MYC\) (FDR = 0.007) (Table S5).