PVALB, a new Hürthle adenoma diagnostic marker identified through gene expression

Abstract

Context: A better means to accurately identify malignant thyroid nodules and to distinguish them from benign tumors is needed. We previously identified markers for detecting thyroid malignancy, with sensitivity estimated at or close to 100%. One lingering problem with these markers was that false positives occurred with Hürthle cell adenomas (HCA) which lowered test specificity.

Methods: To locate accurate diagnostic markers, we profiled in depth the transcripts of a HCA and a Hürthle cell carcinoma (HCC). From 1146 differentially expressed genes, 18 transcripts specifically expressed in HCA were tested by quantitative PCR in a wide range of thyroid tumors (n = 76). Sensibility and specificity were calculated using receiver operating characteristic (ROC). Selected markers were further validated in an independent set of thyroid tumors (n = 82) by immunohistochemistry. To define the panel that would yield best diagnostic accuracy, these markers were tested in combination with our previous identified markers.

Results: Seventeen of the 18 genes showed statistical significance based on a mean relative level of expression (P < 0.05). KLK1 (sensitivity = 0.97) and PVALB (sensitivity = 0.94) were the best candidate markers. The combination of PVALB and C1orf24 increased specificity to >97% and maintained sensitivity for detection of carcinoma.

Conclusion: We identified tumor markers that can be used in combination for a more accurate preoperative diagnosis of thyroid nodules and for postoperative diagnosis of thyroid carcinoma in tumor sections. This improved test would help physicians rapidly focus treatment on true malignancies and avoid unnecessary treatment of benign tumors, simultaneously improving medical care and reducing costs.

Figure 1

Thyroid SAGE data analysis. A, Dendogram of four short thyroid libraries. All transcripts presenting reliable (not ambiguous) tags have been used to construct the dendrogram. Genes from thyroid libraries contain less than 2% of ambiguous tags. B, The clustering algorithm delineates patterns in the expression of all tags among all four libraries. Each row represents a tag, whereas each column corresponds to a SAGE library sample. The absolute abundance of the SAGE tag in the library (SAGE tag number) correlates with the intensity of the color (green, not present; intense red, highly abundant). This analysis reflects the relationship between the libraries based on their gene expression levels. AU, Approximately unbiased; BP, bootstrap probability.

Figure 2

Graphs showing results of qPCR for transcripts evaluated in the HCA vs. malignant groups. The results are expressed as mean of log transformed RE values [95% confidence interval (CI)]. The HCA group included 13 adenomas. The malignant group included 10 HCC, 14 FTC, and 10 follicular variant of thyroid carcinomas. As expected, transcripts are expressed in HCA while their expression is reduced (or absent) in most of malignant tumors. Of these transcripts, PVALB and KLK1 distinguish HCA and malignant class with higher specificity and sensitivity, 0.94 and 0.97, respectively (as detailed in Table 2).

Figure 3

Graphs showing results of qPCR for ATF5, KLK4, SH3BGRL, and TSPAN3 transcripts evaluated in the HCA vs. malignant groups. The results are expressed as mean of log transformed RE values [95% confidence interval (CI)]. All transcripts are expressed in HCA while their expression is reduced (or absent) in most of malignant tumors. Specificity and sensitivity are detailed in Table 2.

Figure 4

Immunohistochemical analysis of PVALB in paraffin-embedded sections of thyroid samples. HCAs exhibited strong brown immunostaining for PVALB. In contrast, FTCs, HCCs, and negative control exhibited no immunoreactivity. Normal thyroid (NT) tissue is negative for PVALB, adjacent to tumor area that was positive for PVALB. In this study, PVALB was best in detecting HCA in 22 of 23 samples. Only 2 of 43 minimally invasive malignancies stained positive.

Figure 5

Immunohistochemical analysis of KLK1 in paraffin-embedded sections of thyroid samples. HCAs exhibited strong brown immunostaining for KLK1. In contrast, FTC, most of HCC, and negative control exhibited no immunoreactivity. Adjacent normal thyroid (NT) tissue is negative for KLK1 at the same time as the tumor area is positive for PVALB.

Figure 6

The predicted best combination based on two antibody-based tests (PVALB and C1orf24) in paraffin-embedded thyroid samples and their application in nodules classified as indeterminate on FNA cytology. HN, Hyperplastic nodule.