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

Partial gene suppression improves identification of cancer vulnerabilities when CRISPR-Cas9 knockout is pan-lethal

Abstract Background Hundreds of functional genomic screens have been performed across a diverse set of cancer contexts, as part of efforts such as the Cancer Dependency Map, to identify gene dependencies—genes whose loss of function reduces cell viability or fitness. Recently, large-scale screening efforts have shifted from RNAi to CRISPR-Cas9, due to superior efficacy and specificity. However, many effective oncology drugs only partially inhibit their protein targets, leading us to question whether partial suppression of genes using RNAi could reveal cancer vulnerabilities that are missed by complete knockout using CRISPR-Cas9. Here, we compare CRISPR-Cas9 and RNAi dependency profiles of genes across approximately 400 matched cancer cell lines. Results We find that CRISPR screens accurately identify more gene dependencies per cell line, but the majority of each cell line’s dependencies are part of a set of 1867 genes that are shared dependencies across the entire collection (pan-lethals). While RNAi knockdown of about 30% of these genes is also pan-lethal, approximately 50% have selective dependency patterns across cell lines, suggesting they could still be cancer vulnerabilities. The accuracy of the unique RNAi selectivity is supported by associations to multi-omics profiles, drug sensitivity, and other expected co-dependencies. Conclusions Incorporating RNAi data for genes that are pan-lethal knockouts facilitates the discovery of a wider range of gene targets than could be detected using the CRISPR dataset alone. This can aid in the interpretation of contrasting results obtained from CRISPR and RNAi screens and reinforce the importance of partial gene suppression methods in building a cancer dependency map

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

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

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