Killing capacity analysis of tumor-infiltrating cytotoxic lymphocytes and impact on lymph node metastasis in differentiated papillary carcinoma of thyroid with the BRAF V600E mutation.

Background: Cytotoxic lymphocytes (CLs) express potent toxins, including perforin (P) and granzyme-B (G), which brings about target cell death. The purpose of this study was to evaluate the killing capacity of tumor-infiltrating CLs by means of P and G analysis, and explore the association with lymph node metastasis in papillary carcinoma of thyroid (PTC) without Hashimoto's thyroiditis (HT).

Methods: Infiltration of lymphocytes in PTC was observed in frozen sections. Both fresh tumor tissues and paracancerous tissues with lymphocyte infiltration were collected and prepared into a single cell suspension. Flow cytometry was used to detect the percentages of CD3⁺P⁺, CD3⁺G⁺, CD8⁺P⁺, and CD8⁺G⁺ T lymphocytes (TLs) and CD16-CD56⁺P⁺ and CD16-CD56⁺G⁺ natural killer (NK) cells. Finally, we investigated differential expression of P and G in NK cells and cytotoxic T lymphocytes (CTLs) in paired tumor tissues (group T, n = 44) and paracancerous tissues (group N, n = 44) from patients with PTC with the BRAF V600E mutation. Furthermore, patients were divided into two groups according to whether cervical central lymph node metastasis (CCLNM) existed: group A (with lymph node metastases, n = 27) and group B (with nonlymph node metastases, n = 17). Patients were also divided into three groups according to the total number of positive CCLNM: group B, group C (with low-level lymph node metastases, less than 5, n = 17) and group D (with high-level lymph node metastases, no less than 5, n = 10).

Results: The percentage of CD3⁺P⁺ CTLs was significantly higher in group N than in group T (P < 0.05). The percentage of CD8⁺G⁺ CTLs was significantly higher in group T than in group N (P < 0.05). The percentages of CD3⁺G⁺, CD16-CD56⁺P⁺and CD16-CD56⁺G⁺ NK cells showed no significant difference in either group T or group N (P > 0.05). The percentages of CD3⁺P⁺ CTLs in group A and group C were significantly higher in the paracancerous tissue than in the tumor tissue (P < 0.05). The percentages of CD8⁺G⁺ CTLs in group A and group C were significantly higher in the tumor tissues than in the paracancerous tissues (P < 0.05). The percentage of CD16-CD56⁺G⁺ NK cells in group D was significantly higher in the tumor tissues than in the paracancerous tissues (P < 0.05).

Conclusions: The killing capacity of infiltrating CLs in PTC differed between tumor tissues and paracancerous tissues. In cases with CCLNM, higher expression of CD16-CD56⁺G⁺ NK cells in tumor tissues may be associated with a high risk of lymph node metastasis.