To determine whether matrix metalloproteinase-9 (MMP-9) may be a useful adjunctive tool for predicting unfavorable biological behavior of papillary thyroid carcinoma (PTC) by evaluating the expression profile and proteolytic activity of MMP-9 in PTC by different techniques and correlating the findings with clinicopathological prognostic factors. Immunohistochemical localization of MMP-9 was analyzed with antibodies specific for either total or active MMP-9. Activation ratios of MMP-9 were calculated by quantifying gel zymography bands. Enzymatic activity of MMP-9 was localized by in situ zymography after inhibiting MMP-2 activity. Immunostaining of total and active MMP-9 was observed in tumor tissue and occasionally in non-neoplastic epithelium. Only active MMP-9 was significantly associated with extrathyroid invasion, lymph-node metastasis, and the degree of tumor infiltration (P<0.001, P=0.004, and P<0.001, respectively). Gelatin zymography revealed a correlation between the MMP-9 activation ratio and nodal involvement, extrathyroid invasion, and the degree of tumor infiltration. In situ zymography showed that gelatinases exerted their activity in tumor parenchymal and stromal cells. Moreover, after application of MMP-2 inhibitor, the remaining gelatinase activity, corresponding to MMP-9, was highest in cancers with the most advanced degree of tumor infiltration. This is the first report suggesting that the evaluation of active MMP-9 by immunohistochemistry and determination of its activation ratio by gelatin zymography may be a useful adjunct to the known clinicopathological factors in predicting tumor behavior. Most important, in situ zimography with an MMP-2 inhibitor for the first time demonstrated a strong impact of MMP-9 activity on the degree of tumor infiltration during PTC progression.

Objectives: Papillary thyroid carcinoma (PTC) usually has a good prognosis, but some patients develop an aggressive course of the disease, leading to a poor outcome. Vascular endothelial growth factor C (VEGF-C) and matrix metalloproteinase 9 (MMP-9) have been shown to play roles in tumor progression in various human malignancies. Methods: We analyzed VEGF-C and active MMP-9 expression profiles in PTC samples using immunohistochemistry and Western blotting. Results: Immunohistochemistry showed positive staining for VEGF-C and active MMP-9 in 83% and 57% of PTCs, respectively (n = 60), with a positive correlation between their expression levels (Spearman, P < .001). Concomitant high expression of VEGF-C and active MMP-9 correlated with the presence of lymph node metastasis (P = .005), pT status (P = .004), pTNM tumor stage (P = .005), and particularly the degree of tumor infiltration (P < .001, Fisher exact test). Densitometric analysis of Western blot bands confirmed correlation between VEGF-C and active MMP-9 expression (Wilcoxon and Spearman tests) and significant association with the clinicopathologic parameters (Mann-Whitney and Kruskal-Wallis tests). Conclusions: Association of coexpressed high levels of VEGF-C and active MMP-9 with lymphatic spreading and local invasiveness of PTC suggests their potential usefulness as predictive biomarkers of aggressive PTC behavior.

To gain a better insight into the differences in biological behaviour between papillary microcarcinoma (PMC) and clinically evident papillary thyroid carcinoma (PTC). Immunohistochemical analysis of apoptosis related molecules (Bcl-2, Bax, p53) and proliferation related marker (PCNA) in 39 archival cases of PMC and 46 cases of PTC. Bcl-2 and Bax were expressed in most PMCs and PTCs. The average Bcl-2 staining score did not differ significantly between PMCs and PTCs (p > 0.05), but the average Bax score was significantly lower in PMCs (p < 0.05). The Bcl-2/Bax ratio was significantly higher in PMCs than in PTCs (p < 0.05). The expression of p53 was similar in PMCs and PTCs, without a correlation with clinical data, but was associated with high Bax expression (p < 0.05) in these cases in both groups. Non-malignant tissue expressed only Bcl-2, but not p53 or Bax. PCNA expression was significantly lower (p < 0.05) in PMC than in PTC and positively correlated with tumour size (p < 0.05). The higher Bcl-2/Bax ratio and lower proliferative activity in PMC suggest differences from PTC in the balance between apoptosis and proliferation. However, the presence of p53 and Bax in PMC indicates malignant potential, and thus PMC should be treated with caution.