Deregulated alternative splicing of CD44 messenger RNA transcripts in neoplastic and nonneoplastic lesions of the human thyroid.

CD44 is a polymorphic family of immunologically related cell surface proteoglycans and glycoproteins implicated in cell-cell and cell-matrix adhesion interactions, lymphocyte activation and homing, cell migration, and tumor metastasis. CD44 exists as a standard form and as multiple isoforms, each generated by alternative splicing of up to 10 variant exons (termed v1-v10) encoding parts of the extracellular domain. Using semiquantitative reverse transcriptase-PCR and Southern hybridization, alternative CD44 mRNA splicing was examined in 10 papillary thyroid carcinomas, 8 nodular goiters, 9 adenomas, 2 cases of thyroiditis, and 3 histologically normal thyroid controls. The amount of input cDNA for the CD44 PCRs was standardized against an internal control gene (glyceraldehyde phosphate dehydrogenase). Four papillary carcinomas showed significant overexpression of CD44 transcripts migrating between 750 and 1000 bp. These cases demonstrated reduced levels of the 482-bp standard isoform transcript. In six papillary cancers, we found a prominent v6-containing isoform at 750 bp that was present in only trace amounts in normal thyroid tissue. It is of interest that similar findings were seen in the majority of the goiters and adenomas but not in the cases of thyroiditis. These results show that deregulation of alternative CD44 splicing is a common feature of disordered thyroid follicular cell growth, both in neoplastic and nonneoplastic lesions. The data imply an important role for CD44, including CD44v6, in the pathogenesis of various thyroid lesions.

Nuclear accumulation of MDM2 protein in well-differentiated papillary thyroid carcinomas.

Papillary thyroid cancer is the most common endocrine malignancy. Of all solid cancers presenting in adults, papillary thyroid cancer generally carries the best long-term prognosis. However, very little is understood about the molecular pathogenesis of this neoplasm. We recently hypothesized that increased nuclear levels of MDM2 protein might occur in well-differentiated papillary thyroid carcinomas (Gerasimov et al., Exp. Mol. Pathol. 62, 52-62, 1995). MDM2 is known to complex with and inactive the p53 tumor suppressor protein. Since p53 inactivation by gene mutation has an established role in the pathogenesis of undifferentiated (anaplastic) thyroid carcinoma, we reasoned that abrogation of p53 function by nuclear MDM2 protein accumulation might participate in the pathogenesis of certain well-differentiated thyroid cancers such as papillary cancer. In the present report we present the first direct evidence of MDM2 protein accumulation in the nuclei of papillary thyroid carcinoma cells in a subset of tumors. Using the IF-2 monoclonal antibody, which reacts specifically with human MDM2 protein, we studied 24 well-differentiated papillary thyroid carcinomas and 26 benign lesions (nodular goiters, adenomas, thyroiditis). Nuclear staining was quantitated using the CAS computerized image analysis system. We found positive nuclear MDM2 immunoreactivity in 8 (33%) of the carcinomas. In contrast, MDM2 staining was negative in all benign lesions (P = 0.001, two-tailed Fisher exact test). Normal thyroid tissue was also negative. These data suggest that nuclear accumulation of MDM2 protein might be implicated in the pathogenesis of a subset of papillary carcinomas. Further studies to investigate this possibility are warranted.

Nuclear p53 immunoreactivity in papillary thyroid cancers is associated with two established indicators of poor prognosis.

The tumor suppressor protein, p53, protects somatic cells against the accumulation of genomic alterations. Cells harboring mutant or inactivated wild-type p53 protein are at risk for the development of genomic instability. Nuclear accumulation of p53 protein is associated with the stepwise dedifferentiation of papillary carcinoma. We asked whether nuclear p53 accumulation is associated with two known indicators of poor prognosis in papillary carcinoma. We studied 55 consecutive papillary cancers (28 from Russia, and 27 from upstate New York). Nuclear p53 immunoreactivity was assessed using a monoclonal antibody, DO-1, on Formalin-fixed paraffin-embedded specimens. The DNA index was determined by computerized image analysis of Feulgen-stained sections. Nearly all cases were well differentiated and none were associated with distant metastases or extrathyroidal invasion. All primary lesions were less than 4 cm in diameter, and almost all patients were female. Nuclear p53 immunoreactivity was associated with a high-risk group characterized by two known indicators of poor prognosis: age > 50, aneuploid DNA content, or both. In the high-risk group (N = 24) 33% of cases displayed nuclear p53 positivity, compared with only 6% in a low-risk group (N = 31) which lacked both features (P = 0.015, two-tailed Fisher exact test). Nuclear accumulation of immunoreactive p53 protein is associated with two established indicators of poor prognosis in papillary carcinoma of the thyroid. This result is consistent with the idea that aberrations in p53 function are associated with the stepwise loss of differentiation in this cancer.

Preferential expression of the cell adhesion molecule CD44 in papillary thyroid carcinoma.

CD44 is a polymorphic family of immunologically related integral membrane glycoproteins associated with cell matrix adhesion, lymphocyte activation and targeting, and tumor growth and metastasis. We studied CD44 expression in 114 formalin-fixed paraffin-embedded thyroid tumors using the A3D8 anti-human CD44 monoclonal antibody. Sixty-five of 67 papillary carcinomas (97%) strongly expressed CD44 with an intense plasma membrane pattern. Thirty-seven of these cases originated from Albany, New York, and 30 cases from Russia. Immunoreactivity was also observed in 9 of 16 follicular adenomas (56%); 4 of 8 Hurthle cell neoplasms (50%); 5 of 15 medullary carcinomas (33%); and 3 of 8 follicular carcinomas (38%). These results show that among thyroid neoplasms, papillary carcinomas preferentially display the CD44 antigen (P < or = 0.001). Nonneoplastic follicular epithelium exhibited a low to moderate level of staining. To further characterize the CD44 isoform, we tested a subset of cases with the 2F10 anti-human CD44 variant 6 monoclonal antibody, which recognizes a CD44 variant exon (CD44v6) implicated in tumor metastasis. Eleven of 11 papillary carcinomas tested were 2F10 positive, and 1 of the follicular carcinomas was positive. These results suggest the hypothesis that deregulated CD44v6 expression on the plasma membrane of papillary carcinoma cells contributes to the ability of those cells to metastasize to regional lymph nodes and then to remain dormant for years. Our results suggest that human papillary thyroid cancer will be an interesting model in which to further study the role of CD44 isoforms, particularly those containing CD44v6, in tumor metastasis and lymphatic invasion.