Integrative analysis of DNA copy number and gene expression in metastatic oral squamous cell carcinoma identifies genes associated with poor survival

Abstract

Background: Lymphotropism in oral squamous cell carcinoma (OSCC) is one of the most important prognostic factors of 5-year survival. In an effort to identify genes that may be responsible for the initiation of OSCC lymphotropism, we examined DNA copy number gains and losses and corresponding gene expression changes from tumor cells in metastatic lymph nodes of patients with OSCC.

Results: We performed integrative analysis of DNA copy number alterations (CNA) and corresponding mRNA expression from OSCC cells isolated from metastatic lymph nodes of 20 patients using Affymetrix 250 K Nsp I SNP and U133 Plus 2.0 arrays, respectively. Overall, genome CNA accounted for expression changes in 31% of the transcripts studied. Genome region 11q13.2-11q13.3 shows the highest correlation between DNA CNA and expression. With a false discovery rate < 1%, 530 transcripts (461 genes) demonstrated a correlation between CNA and expression. Among these, we found two subsets that were significantly associated with OSCC (n = 122) when compared to controls, and with survival (n = 27), as tested using an independent dataset with genome-wide expression profiles for 148 primary OSCC and 45 normal oral mucosa. We fit Cox models to calculate a principal component analysis-derived risk-score for these two gene sets ('122-' or '27-transcript PC'). The models combining the 122- or 27-transcript PC with stage outperformed the model using stage alone in terms of the Area Under the Curve (AUC = 0.82 or 0.86 vs. 0.72, with p = 0.044 or 0.011, respectively).

Conclusions: Genes exhibiting CNA-correlated expression may have biological impact on carcinogenesis and cancer progression in OSCC. Determination of copy number-associated transcripts associated with clinical outcomes in tumor cells with an aggressive phenotype (i.e., cells metastasized to the lymph nodes) can help prioritize candidate transcripts from high-throughput data for further studies.

Figure 1

Influence of DNA copy number on gene expression in the 20 lymph node metastatic OSCC. A. Summary of gene expression in subgroup of transcripts with different cancer-normal DNA copy number ratio (Box plot in each subgroup indicates 25^th, 50^th, and 75^th percentile of the gene expression). X-axis: subgroups with different DNA copy number ratio. Y-axis: base-2 log-transformed relative gene expression; B. Distribution of the estimated correlation coefficients between DNA copy number and gene expression. X-axis: value of the correlation coefficient; Y-axis: frequency; C. P-value distribution for the correlation coefficients.

Figure 2

Summary of genome-wide DNA copy number aberrations and concordant gene expression changes. A. Top panel: Percentage of genome DNA amplification (red) and deletion (green) of the 20 OSCC on the human autosomes. Only CNAs with tumor-normal DNA copy number ratio less than 0.7 or greater than 1.4 are counted as deleted or amplified, respectively. Bottom-panel: Manhattan plot of the negative base-10 log-transformed p-value for the correlation coefficients between DNA copy number and gene expression for all the studied genes at each loci. Line indicates the Bonferroni p-value cutoff = -log10(0.01/23,466) = 6.37; B. A close-up view of the 11q13.2-q13.3 high correlation region.

Figure 3

Correlation between DNA copy number and gene expression for genes in the high correlation region of chromosome 11q13.2-q13.3. A. Locations of the genes and the unknown transcript in the region. B. DNA copy number (x-axis, base-2 log-transformed ratio of cancer to normal) and gene expression (y-axis, base-2 log-transformed intensity) of each gene in the 20 lymph node metastatic OSCC. For genes measured by multiple probe sets, the probe set with the strongest correlation is shown.

Figure 4

ROC analysis of 2-year survival comparing AJCC stage with gene expression data. A, ROC curves for 2-year survival for models "stage," "122-PC," and "27-PC" B, ROC curves for 2-year survival for models "stage," "stage + 122-PC" and "stage + 27-PC".