Characterization of Immune Infiltrate Along the Leading Edge of Differentiated Thyroid Cancer.

Background: Although the impact of tumor-immune infiltrate has been reported on differentiated thyroid cancer (DTC) behavior, the expression of immune checkpoints [programmed cell death protein 1 (PD-1) and its ligand (PD-L1)] alone has not been able to predict response to immunotherapies. We aimed to identify tumor-infiltrating immune cells and checkpoints associated with DTC. Methods: We performed multiplex immunofluorescence on deparaffinized thyroid tissue collected at thyroidectomy from 17 adults with DTC to characterize the tumor immune microenvironment for leukocytes (CD45+), T cells (CD3+), T regulatory cells (Tregs) (CD3+FOXP3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), macrophages (CD68+), M2 macrophages (CD68+CD163+), M1 Macrophages (CD68+ inducible nitric oxide synthase [iNOS]+), and immune checkpoints PD-1 and PD-L1. We compared the mean percentage expression of immune markers between tumor and adjacent thyroid tissue from the same patient by paired t-test and performed spatial analysis along the tumor's leading edge. Results: Immune checkpoints PD-1 and PD-L1 showed a significant increase in expression intratumorally as compared to adjacent thyroid tissue (p < 0.05). A higher trend for M2 macrophages was observed intratumorally compared to adjacent tissue. Along the leading edge, PD-L1 expression correlated negatively with CD45 and positively with CD163 intratumorally. On exploratory analysis, there was a nonsignificant trend for higher FOXP3 but less CD8 and iNOS expression in tumor from DTC with (n = 3) versus without distant metastases (n = 14). There was a nonsignificant trend for higher CD58 and iNOS expression in DTC with (n = 7) than without thyroiditis (n = 10). Conclusions: Higher tumoral PD-1 and PD-L1 expression indicate their role in DTC occurrence. A trend for more Tregs and M2 macrophages but less M1 macrophages intratumorally in patients with distant metastatic DTC, suggests their potential role as prognostic biomarkers. Future studies with larger sample sizes are needed to compare various clinicopathologic severities to harness tumor microenvironment for cancer prognostication and therapy.

Relaxin-2 is a novel biomarker for differentiated thyroid carcinoma in humans.

Relaxin's role in differentiated thyroid cancer (DTC) has been suggested but its characterization in a large clinical sample remains limited. We performed immunohistochemistry for relaxin-2 (RLN2), CD68 (total macrophages), CD163 (M2 macrophages) on tissue microarrays from 181 subjects with non-distant metastatic DTC, and 185 subjects with benign thyroid tissue. Mean pixels/area for each marker was compared between tumor and adjacent tissue via paired-t test and between DTC and benign subjects via t-test assuming unequal variances. RNA qPCR was performed for expression of RLN2, RLN1, and RXFP1 in cell lines. Amongst 181 cases, the mean age was 46 years, 75 % were females. Tumoral tissue amongst the DTC cases demonstrated higher mean expression of RLN2 (53.04 vs. 9.79; p < 0.0001) compared to tumor-adjacent tissue. DTC tissue also demonstrated higher mean expression of CD68 (14.46 vs. 4.79; p < 0.0001), and CD163 (23.13 vs. -0.73; p < 0.0001) than benign thyroid. These markers did not differ between tumor-adjacent and benign thyroid tissue groups; and amongst cases, did not differ by demographic or clinicopathologic features. RLN1 and RXFP1 expression was detected in a minority of the cell lines, while RLN2 was expressed by 6/7 cell lines. In conclusion, widespread RLN2 expression in DTC tissue and most cell lines demonstrates that RLN2 acts in a paracrine manner, and that RLN1 and RXFP1 are probably not involved in thyroid cancer cell signaling. RLN2 is a biomarker for thyroid carcinogenesis, being associated with but not secreted by immunosuppressive macrophages. These findings will guide further investigations for therapeutic avenues against thyroid cancer.