A preoperative diagnostic test that distinguishes benign from malignant thyroid carcinoma based on gene expression

Abstract

Accurate diagnosis of thyroid tumors is challenging. A particular problem is distinguishing between follicular thyroid carcinoma (FTC) and benign follicular thyroid adenoma (FTA), where histology of fine-needle aspirates is not conclusive. It is often necessary to remove healthy thyroid to rule out carcinoma. In order to find markers to improve diagnosis, we quantified gene transcript expression from FTC, FTA, and normal thyroid, revealing 73 differentially expressed transcripts (P < or = 0.0001). Using an independent set of 23 FTCs, FTAs, and matched normal thyroids, 17 genes with large expression differences were tested by real-time RT-PCR. Four genes (DDIT3, ARG2, ITM1, and C1orf24) differed between the two classes FTC and FTA, and a linear combination of expression levels distinguished FTC from FTA with an estimated predictive accuracy of 0.83. Furthermore, immunohistochemistry for DDIT3 and ARG2 showed consistent staining for carcinoma in an independent set 59 follicular tumors (estimated concordance, 0.76; 95% confidence interval, [0.59, 0.93]). A simple test based on a combination of these markers might improve preoperative diagnosis of thyroid nodules, allowing better treatment decisions and reducing long-term health costs.

Figure 1

Relative levels of expression determined by quantitative RT-PCR in 23 samples of FTA and FTC (black bars) and in normal thyroid tissues (gray bars). Transcript levels were normalized to the average of ribosomal protein 8 and t-complex 1, which were uniformly expressed in all three thyroid SAGE libraries. Numbers 1–10 correspond to FTA and 11–23 to FTC cases described in Table 2. The statistical analysis of RT-PCR values revealed that expression of the genes DDIT3, ARG2, and ITM1 was significantly different at the 0.05 level, and expression of C1orf24 was significant at the 0.10 level.

Figure 2

Quantitative RT-PCR products of three genes with statistically significant expression differences, showing adenoma (A), FTC (C), and normal thyroid (N) tissues and thyroid carcinoma cell lines (CL). The genes DDIT3, ARG2, and ITM1 are expressed in most of the FTCs, the thyroid follicular carcinoma cell line (CL1), the papillary thyroid carcinoma cell line (CL2), and the undifferentiated thyroid carcinoma cell line (CL3), but not in normal tissue and most FTAs. Case 6 (A6) expressed ARG2 and ITM1 and was misclassified according to our class-predicted genes. Universal Human Reference RNA (UHR) was used as a control. Ribosomal protein 8 was used as a calibrator gene. The 100-bp DNA ladder (M) is shown in the far left and far right lanes. The results are shown in triplicate, and the numbers correspond to cases analyzed (Table 2). The product sizes and PCR conditions are described in Methods and summarized in Supplemental Table 1. Negative control (NC) consists of PCR mix and primers, but no template.

Figure 3

Immunohistochemical analysis of DDIT3 (A–F) and ARG2 (G–L) in paraffin-embedded sections of FTAs and FTCs. FTCs exhibited strong brown immunostaining for DDIT3 (D–F) and ARG2 (J–L). In contrast, FTAs (A–C and G–I) exhibited no immunoreactivity. The arrows in D and F show the vascular invasion in FTCs and the follicular cells that are positive for DDIT3. The arrow in L shows the normal thyroid tissue that was negative for ARG2, adjacent to tumor area that was positive for ARG2. Hematoxylin was used as a nuclear counterstain. Original magnification, ×100 for A–E and G–L, ×40 for F.