The killing capacity analysis of tumor infiltrating cytotoxic lymphocytes and impact on lymph node metastasis in differentiated papillary carcinoma of thyroid with BRAF V600E mutation

Abstract

Abstract Background: Cytotoxic lymphocytes(CLs) express potent toxins,including the 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 perforin and granzyme-B analysis,and explore the association with lymph node metastasis in papillary carcinoma of thyroid (PTC) without Hashimoto's Thyroiditis(HT). Methods: Infiltration of lymphocyts in PTC were observed by frozen section.Both fresh tumor tissues and paracancerous tissues with lymphocytes infiltration were respectively collected and made into signal cell suspension. Flow cytomtry was used to detect the percentage of CD3+P+,CD3+G+,CD8+P+,CD8+G+ T lymphocytes(TLs),and CD16-CD56+P+,CD16-CD56+G+ natural killer(NK) cells.Finally we investigated the differential expression of perforin and granzyme B of NK cells and cytotoxic T lymphocytes (CTLs) in paired tumor tissues (group T,n=44) and paracancerous tissues(group N,n=44) from patients of PTC with BRAF V600E mutation . Further more patients were divided into two groups according to whether exist cervical central lymph node metastasis(CCLNM):group A (with lymph node metastases, n=27) and group B(with non-lymph node metastases,n=17).Patients were 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 the group N than that of group T(P<0.05). The percentage of CD8+G+ CTLs was significantly higher in the group T than that of group N(P<0.05). The percentage of CD3+G+,CD8+P+CTLs, CD16-CD56+P+and CD16-CD56+G+ NK cells showed no significant difference in both group T and group N(P>0.05).The percentage of CD3+P+ CTLs in group A and group C were significantly higher in the paracancerous tissue than that of tumor tissue(P<0.05).The percentage of CD8+G+ CTLs in group A and group C were significantly higher in the tumor tissues than that of paracancerous tissues(P<0.05).The percentage of CD16-CD56+G+ NK cells in group D was significantly higher in the tumor tissues than that of 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 associate with high risk of lymph node metastasis.