Integrative analysis of genome-wide gene copy number changes and gene expression in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) represents a genomically unstable cancer type with extensive copy number aberrations. The relationship of gene copy number alterations and subsequent mRNA levels has only fragmentarily been described. The aim of this study was to conduct a genome-wide analysis of gene copy number gains and corresponding gene expression levels in a clinically well annotated NSCLC patient cohort (n = 190) and their association with survival. While more than half of all analyzed gene copy number-gene expression pairs showed statistically significant correlations (10,296 of 18,756 genes), high correlations, with a correlation coefficient >0.7, were obtained only in a subset of 301 genes (1.6%), including KRAS, EGFR and MDM2. Higher correlation coefficients were associated with higher copy number and expression levels. Strong correlations were frequently based on few tumors with high copy number gains and correspondingly increased mRNA expression. Among the highly correlating genes, GO groups associated with posttranslational protein modifications were particularly frequent, including ubiquitination and neddylation. In a meta-analysis including 1,779 patients we found that survival associated genes were overrepresented among highly correlating genes (61 of the 301 highly correlating genes, FDR adjusted p<0.05). Among them are the chaperone CCT2, the core complex protein NUP107 and the ubiquitination and neddylation associated protein CAND1. In conclusion, in a comprehensive analysis we described a distinct set of highly correlating genes. These genes were found to be overrepresented among survival-associated genes based on gene expression in a large collection of publicly available datasets

Inconsistent results in the analysis of ALK rearrangements in non-small cell lung cancer

Background: Identification of targetable EML4-ALK fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients. Although fluorescence in situ hybridization (FISH) is regarded as the gold standard for detection of ALK rearrangements, ALK immunohistochemistry (IHC) is often used as screening tool in clinical practice. In order to unbiasedly analyze the diagnostic impact of such a screening strategy, we compared ALK IHC with ALK FISH in three large representative Swedish NSCLC cohorts incorporating clinical parameters and gene expression data. Methods: ALK rearrangements were detected using FISH on tissue microarrays (TMAs), including tissue from 851 NSCLC patients. In parallel, ALK protein expression was detected using IHC, applying the antibody clone D5F3 with two different protocols (the FDA approved Ventana CDx assay and our in house Dako IHC protocol). Gene expression microarray data (Affymetrix) was available for 194 patients. Results: ALK rearrangements were detected in 1.7% in the complete cohort and 2.0% in the non-squamous cell carcinoma subgroup. ALK protein expression was observed in 1.9% and 1.5% when applying the Ventana assay or the in house Dako protocol, respectively. The specificity and accuracy of IHC was high (>99%), while the sensitivity was between 69% (Ventana) and 62% (in house Dako protocol). Furthermore, only 67% of the ALK IHC positive cases were positive in both IHC assays. Gene expression analysis revealed that 6/194 (3%) tumors showed high ALK gene expression (≥6AU) and of them only three were positive by either FISH or IHC. Conclusion: The overall frequency of ALK rearrangements based on FISH was lower than previously reported. The sensitivity of both IHC assays was low, and the concordance between the FISH and the IHC assays poor, questioning current strategies to screen with IHC prior to FISH or completely replace FISH by IHC

Impact of gene copy number and gene expression on survival.

<p>The plot shows the number of probe sets in relation to the AUC as a measure of survival association for gene copy number (red) and gene expression values (blue) in the complete set of NSCLC (A), adenocarcinomas (B) and squamous cell cancer (C). Considering all 39,788 matched copy number/RNA pairs it is evident that RNA levels exert a higher prognostic impact than gene copy number in the total group and in adenocarcinomas, while a similar influence of RNA levels and gene copy number is seen in squamous carcinomas. The red and blue strokes at the x-axis represent genes with highest externally centered correlation coefficients (ECCC>0.7, FDR adj. p<0.05).</p

Correlation of gene copy number and corresponding gene expression values.

<p>Density analysis of the externally centered correlation coefficient (ECCC) log transformed (blue) mRNA expression values for all NSCLC cases (A), adenocarcinomas (B) and squamous cell cancer cases (C). The line forming a Gauss curve illustrates correlation values of randomly generated data. Randomization was achieved by permutation of gene copy number/mRNA pairs.</p

Gene copy number changes of non-small cell lung carcinomas.

<p>Copy number (CN) call frequencies are given for all NSCLC cases (A), adenocarcinomas (B) and squamous cell cancer (C). The frequency plots (upper graphs) give the proportion of cases with loss and gain for each chromosomal position. Positions of selected genes (ERBB4, FHIT, APC, EGFR, MET, PTEN, LRP1, ERBB2, CCNE1) were highlighted in red.</p

Correlation between gene copy number and corresponding gene expression values over all chromosomes.

<p>(A) The modified Manhattan plot gives the median (red line) of the externally centered correlation coefficient (ECCC) and the 5% and 95% quantile (black lines) along all chromosomal positions. The black dots indicate the probe sets with highest ECCC (ECCC>0.7, FDR adj. p<0.05). The triangles show the position of hotspot regions. High correlation between copy number variation and gene expression is not randomly distributed over the genome but mostly occurs within specific chromosomal regions (hotspots). (B) Example of the hotspot region on chromosome 1p35-1p34 comprising 185 genes (gray bars) with 17 highly correlating genes (red bars). Each dot represents the ECCC of a probe set based on the analysis of all 190 NSCLC cases.</p

Top 25 probe sets with highest ECCC of gene copy number levels and mRNA expression for tumors from 190 NSCLC patients.

<p>Top 25 probe sets with highest ECCC of gene copy number levels and mRNA expression for tumors from 190 NSCLC patients.</p

Highly correlating genes and association with survival.

<p>The meta-analysis of 10 NSCLC cohorts revealed that 70 of 440 highly correlating genes were associated with survival. For illustration three of the 70 genes are shown: CCT2, NUP107 and CAND1. The correlation between copy number and gene expression is shown by scatter plots (left). Survival time is visualized by Kaplan Meier plots (two panels in the middle). Patients were dichotomized at the 75% percentile for copy number (middle, left) and gene expression (middle, right). The forest plots illustrate the results of the meta-analysis including nine independent data sets and the Uppsala cohort (right).</p