Honors Program


Date of this Version

Spring 4-23-2022

Document Type



Hajric, K. 2022. Investigating Immune Profiles in Differentiated Thyroid Cancer by Multiplex Immunofluorescence. Undergraduate Honors Thesis. University of Nebraska-Lincoln.


Copyright Kemal Hajric 2022.


BACKGROUND: As the most common endocrine malignancy in the United States (U.S.), differentiated thyroid cancer (DTC) accounts for 3.8% of all cancers in the U.S., with roughly 10% of cases progressing to distant metastatic DTC, which is associated with a poor five-year survival outcome despite conventional management, including surgery and radioactive iodine ablation. Recently, novel immunotherapies have garnered attention as a viable therapeutic resource for patients with advanced DTC. However, the response to therapy has been variable and unpredictable, which may be associated with an immune suppressive circulating phenotype. Nonetheless, the intra-tumoral immune infiltrate remains to be elucidated, demonstrating a critical need to address the gap in understanding in order to better prognosticate the disease.

OBJECTIVE: To identify and compare tumor-infiltrating immune markers with those present in the adjacent normal thyroid tissue, and collate these immune infiltrates with tumor characteristics.

EXPERIMENTAL DESIGN: Twenty-nine adult tissue samples containing tumor and stromal regions were collected from patients with DTC. The samples were analyzed using multiplex immunofluorescence (MxIF) with antibodies against cell-surface molecules CD56, PD-1, PD-L1, FOXP3, CD3, CD8, CD4, CD45, CD68, CD163, iNOS, HLA-DR, CD33, and CD19. 17 of the specimens were analyzed using HALO and a positive threshold was assigned based on review by a trained researcher.

RESULTS: In evaluating the immune profiles, important differences in the immune infiltrates between different stages of the cancer were observed. Generally, immune checkpoints PD-1 and PD-L1 were highly expressed within the tumor, despite variability in lymphocyte infiltration. Tumor from patients with distant metastases demonstrated higher immune suppressive cell infiltration (T regulatory cells, macrophages and PD-L1 positive cells) as compared to the localized tumor, indicating their importance as potential predictive biomarkers for the aggressiveness of thyroid cancer.

CONCLUSION: Immune profiling demonstrated significant differences between tumor and adjacent healthy regions, particularly in terms of PD-1 and PD-L1 expression and lymphocyte infiltration, indicating that higher intratumor infiltration of T regulatory cells, macrophages and PD-1/PD-L1 positive cells may be associated with advanced thyroid cancer. Therefore, the data demonstrates the efficacy of MxIF in gathering valuable information regarding the tumor microenvironment, which will have major implications in guiding the selection of patients for immunotherapy.