Differential nuclear and cytoplasmic expression of PTEN in normal thyroid tissue, and benign and malignant epithelial thyroid tumors

Abstract

Germline mutations in PTEN (MMAC1/TEP1) are found in patients with Cowden syndrome, a familial cancer syndrome which is characterized by a high risk of breast and thyroid neoplasia. Although somatic intragenic PTEN mutations have rarely been found in benign and malignant sporadic thyroid tumors, loss of heterozygosity (LOH) has been reported in up to one fourth of follicular thyroid adenomas (FAs) and carcinomas. In this study, we examined PTEN expression in 139 sporadic nonmedullary thyroid tumors (55 FA, 27 follicular thyroid carcinomas, 35 papillary thyroid carcinomas, and 22 undifferentiated thyroid carcinomas) using immunohistochemistry and correlated this to the results of LOH studies. Normal follicular thyroid cells showed a strong to moderate nuclear or nuclear membrane signal although the cytoplasmic staining was less strong. In FAs the neoplastic nuclei had less intense PTEN staining, although the cytoplasmic PTEN-staining intensity did not differ significantly from that observed in normal follicular cells. In thyroid carcinomas as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. The cytoplasmic staining was more intense than the nuclear staining in 35 to 49% of carcinomas, depending on the histological type. Among 81 informative tumors assessed for LOH, there seemed to be an associative trend between decreased nuclear and cytoplasmic staining and 10q23 LOH (P = 0.003, P = 0.008, respectively). These data support a role for PTEN in the pathogenesis of follicular thyroid tumors.

Figure 1.

Western analysis of whole-cell protein lysates from thyroid cancer cell lines using the anti-PTEN monoclonal antibody 6H2.1 (top panel) and using the anti-α-tubulin antibody as a control (bottom panel). NPA-87 and K-1, which are two PTC lines, and WRO-82–1, a FTC line, have endogenous PTEN. FTC-133 is a FTC line that is PTEN null. Lane 1, NPA-87; lane 2, K-1; lane 3, FTC-133; lane 4, FTC-133 transfected with empty vector; lane 5, FTC-133 transfected with vector containing PTEN; lane 6, WRO-82–1. The same membrane was used for Western blot with 6H2.1 as well as anti-tubulin antibody.

Figure 2.

Immunohistochemical analysis of normal thyroid, FA, FTC, PTC, and UTCs using the anti-PTEN monoclonal antibody 6H2.1. A: Normal thyroid tissue (magnification, ×20). Note strong (+++) to moderate (++) nuclear and weak (+) cytoplasmic staining in the follicular epithelial cells. B: Follicular thyroid adenoma (magnification, ×20). In this particular sample, the intensity of nuclear PTEN staining in the majority of follicular cells is strong (graded +++ to ++); the intensity of cytoplasmic staining is weak (+), not greatly different from that of normal epithelial cells. Note the strong staining intensity of the endothelial cells that serve as positive controls (←). C: FTC (magnification, ×20) with mainly weak (+) nuclear staining. D: PTC (magnification, ×20) with weak (+) nuclear staining. E: UTC (magnification, ×20) with absent (−) nuclear and cytoplasmic staining. Note the intense immunostaining in the endothelial cells, which serve as an internal positive control (←). F: PTC (magnification, ×20) with absent (−) nuclear immunostaining but moderate (++) cytoplasmic staining. G: Same PTC (magnification, ×40). Note the completely absent (−) staining in the nucleus compared to the cytoplasm. H: UTC (magnification, ×10) with heterogeneous PTEN immunostaining pattern. Islands of immunopositivity interspersed among large areas of immunonegativity. I: Same UTC (magnification, ×20). Note that the immunopositive cells are small and round whereas the immunonegative cells are large with large pleomorphic nuclei.

Figure 3.

Distribution of PTEN immunostaining intensity in and cytoplasm in thyroid samples (50 normal thyroid tissues, 55 thyroid adenomas, 28 FTC, 37 PTC, and 26 UTC). Strong ▪, moderate ▩, weak ▤, and absent staining □. NTT, normal thyroid tissue. The y-axis represents percentage of samples with various intensities of nuclear (top) or cytoplasmic (bottom) staining.