The Degree of Segmental Aneuploidy Measured by Total Copy Number Abnormalities Predicts Survival and Recurrence in Superficial Gastroesophageal Adenocarcinoma

Abstract Background: Prognostic biomarkers are needed for superficial gastroesophageal adenocarcinoma (EAC) to predict clinical outcomes and select therapy. Although recurrent mutations have been characterized in EAC, little is known about their clinical and prognostic significance. Aneuploidy is predictive of clinical outcome in many malignancies but has not been evaluated in superficial EAC

The degree of segmental aneuploidy measured by total copy number abnormalities predicts survival and recurrence in superficial gastroesophageal adenocarcinoma

Background: Prognostic biomarkers are needed for superficial gastroesophageal adenocarcinoma (EAC) to predict clinical outcomes and select therapy. Although recurrent mutations have been characterized in EAC, little is known about their clinical and prognostic significance. Aneuploidy is predictive of clinical outcome in many malignancies but has not been evaluated in superficial EAC. Methods: We quantified copy number changes in 41 superficial EAC using Affymetrix SNP 6.0 arrays. We identified recurrent chromosomal gains and losses and calculated the total copy number abnormality (CNA) count for each tumor as a measure of aneuploidy. We correlated CNA count with overall survival and time to first recurrence in univariate and multivariate analyses. Results: Recurrent segmental gains and losses involved multiple genes, including: HER2, EGFR, MET, CDK6, KRAS (recurrent gains); and FHIT, WWOX, CDKN2A/B, SMAD4, RUNX1 (recurrent losses). There was a 40-fold variation in CNA count across all cases. Tumors with the lowest and highest quartile CNA count had significantly better overall survival (p = 0.032) and time to first recurrence (p = 0.010) compared to those with intermediate CNA counts. These associations persisted when controlling for other prognostic variables. Significance: SNP arrays facilitate the assessment of recurrent chromosomal gain and loss and allow high resolution, quantitative assessment of segmental aneuploidy (total CNA count). The non-monotonic association of segmental aneuploidy with survival has been described in other tumors. The degree of aneuploidy is a promising prognostic biomarker in a potentially curable form of EAC. © 2014 Davison et al

Representative EGFR and MYC FISH Results.

<p>FISH to determine EGFR and MYC copy number. Gene specific probes are labeled red (EGFR and MYC) while corresponding centromere probes (CEP7 and CEP8, respectively) are labeled green. (A) Tumor cells with a near normal 1∶1 ratio of EGFR/CEP7 and approximately 2 signals from each probe per cell. (B) High level EGFR amplification. EGFR amplification was detected as clusters of numerous red fluorescent signals which is a characteristic pattern caused by high EGFR copy number (see reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079079#pone.0079079-Marx1" target="_blank">[57]</a>) with variable chromosome 7 centromere copy number. (C) Tumor cells with a near normal MYC and centromere 8 copy number. (D) High level MYC amplification characterized by numerous MYC signals per cell and a high MYC/CEP8 ratio.</p

Total CNA Count in Relation to Potential Pathologic Prognostic Variables.

<p>For categorical variables, none of the differences between groups was statistically significant when comparing across the three groups nor in pairwise comparisons with low CNA count (p-value>0.05, Fisher's exact test).</p><p>The difference in tumor size and mean number of resected lymph nodes was not significant across the three CNA count groups (p-value>0.05, Kruskal-Wallis one way ANOVA), nor in pairwise comparisons (p-value>0.05, Mann-Whitney U Test).</p

Clinical and Pathologic Characteristics of the Study Population.

<p>Clinical and Pathologic Characteristics of the Study Population.</p

Prevalence of Copy Gains and Losses in Superficial EAC in Previously Reported Candidate Genes.

<p>large deletion region.</p

Overall Survival and Time to First Recurrence for Superficial EAC Stratified by Total CNA Count.

<p>(A) Patients with intermediate total CNA counts had significantly worse overall survival than patients with low or high total CNA counts (log rank p-value = 0.032). (B) Similarly, patients with intermediate total CNA counts had significantly shorter time to first recurrence than those with low or high total CNA counts (log rank p-value = 0.010).</p

Heatmap Depicting the Correlation between Total CNA Count in Each Tumor and Total Copy Number Gains, Total Copy Number Losses and Total CNA Count by Chromosome.

<p>Cases are ordered from lowest to highest total CNA count down the left-most column. Lowest counts in each column are blue and the highest counts in each column are red as illustrated in the color scale below. In adjacent columns, the heatmap shows the correlation of total CNA count with total copy number gains, total copy number loss and total CNA count by chromosome. The heatmap illustrates that as total CNA count increases, the frequency of gains and losses increase and the frequency of CNA counts tends to increase throughout all chromosomes. Correlation coefficients for each column with total CNA count (Pearson's r) are listed below with the corresponding p-values.</p

Schematic Illustration of Copy Number Abnormalities in Two Tumors Representing High and Low CNA Count and a Spectrum of Genomic Complexity.

<p>For both A and B, the horizontal axis represents position of segments along chromosome 7 (in Mb) and the vertical axis represents estimated copy number (truncated at 16). (A) Tumor 2515 (dark grey) has a large number of independent CNAs on chromosome 7 (N = 56), including a complex copy number gains at ∼55 Mb (region of EGFR) as well as other gains and losses throughout the chromosome. (B) By contrast, tumor 2634 (light grey) has few independent copy number changes (N = 2, gain at ∼39 Mb and loss at ∼97.4 Mb). For this illustration, copy numbers in the normal range of 1.7–2.3 were assigned a value of 2.</p