Gene expression profiling of papillary thyroid carcinoma identifies transcripts correlated with BRAF mutational status and lymph node metastasis

Abstract

Purpose: To identify papillary thyroid carcinoma (PTC)-associated transcripts, we compared the gene expression profiles of three Serial Analysis of Gene Expression libraries generated from thyroid tumors and a normal thyroid tissue.

Experimental design: Selected transcripts were validated in a panel of 57 thyroid tumors using quantitative PCR (qPCR). An independent set of 71 paraffin-embedded sections was used for validation using immunohistochemical analysis. To determine if PTC-associated gene expression could predict lymph node involvement, a separate cohort of 130 primary PTC (54 metastatic and 76 nonmetastatic) was investigated. The BRAF(V600E) mutational status was compared with qPCR data to identify genes that might be regulated by abnormal BRAF/MEK/extracellular signal-regulated kinase signaling.

Results: We identified and validated new PTC-associated transcripts. Three genes (CST6, CXCL14, and DHRS3) are strongly associated with PTC. Immunohistochemical analysis of CXCL14 confirmed the qPCR data and showed protein expression in PTC epithelial cells. We also observed that CST6, CXCL14, DHRS3, and SPP1 were associated with PTC lymph node metastasis, with CST6, CXCL14, and SPP1 being positively correlated with metastasis and DHRS3 being negatively correlated. Finally, we found a strong correlation between CST6 and CXCL14 expression and BRAF(V600E) mutational status, suggesting that these genes may be induced subsequently to BRAF activation and therefore may be downstream in the BRAF/MEK/extracellular signal-regulated kinase signaling pathway.

Conclusion: CST6, CXCL14, DHRS3, and SPP1 may play a role in PTC pathogenesis and progression and are possible molecular targets for PTC therapy.

Figures

Figure 1

Relative expression levels (RE) of selected transcripts in 71 thyroid samples as determined by qPCR. Tissue histology consists of 28 PTCs (15 classical and 13 follicular variant), 17 FTCs, 12 FTAs and 14 normal adjacent tissues. Transcripts were normalized to the average of two control genes (QPC and S8) and RE was calculated as described in material and methods. The RE data were log transformed before the application of statistical test (ANOVA with Bonferroni correction). The results are presented as mean of log transformed data with 95% CI (confidence interval). Significant differences were observed to CST6, CXCL14, DHRS3 and BCAN.

Figure 2

Immunohistochemical analysis of CXCL14 in paraffin-embedded sections of thyroid tumors. A-D, PTCs exhibited a strong staining in epithelial cancer cells while no staining was observed in non-tumoral tissues. E-D, FTC and FTA respectively exhibited no immunoreactivity. The arrow in PTC shows negative staining in stromal cells and normal adjacent follicular cells. Original magnification is x 100 (C, E and F), x 200 (A and D) and x 400 (B).

Figure 3

Relative expression levels (RE) determined by qPCR in 76 samples of nonmetastatic and 54 metastatic primary PTCs. Transcripts were normalized to the average of two control genes (QPC and S8) and RE was calculated as described in material and methods. The RE values were log transformed before the application of statistical analysis (Student’s t-test). The results are presented as mean of log transformed data with 95% CI (confidence interval).

Figure 4

BRAF status and qPCR analysis in metastatic and non-metastatic PTCs. RE- Relative expression levels determined by qPCR were calculated as described in figure 3. Association between BRAF status and gene expression was calculated using Student’s t-test. CST6 and CXCL14 were associated with the presence of BRAF V600E mutation.

Figure 5

Representative results from qPCR analysis in NPA and WRO thyroid carcinoma cell lines. A) qPCR products were visualized on an agarose gel. Each experiment was performed in triplicate. Transcripts were normalized to the average of S8 and RE was calculated as described in material and methods. B) The RE values were log transformed before the application of statistical analysis (Student’s t-test; P=0.0047). The results are presented as mean of the two triplicates from two independent experiments.