CD66b+ Tumor-Infiltrating Neutrophil-like Monocytes as Potential Biomarkers for Clinical Decision-Making in Thyroid Cancer

Abstract

Background and Objectives: Thyroid nodules are a common endocrine disorder, with 10-15% exhibiting malignancy. Accurate differentiation of malignant and benign nodules is crucial for optimizing treatment outcomes. Current diagnostic tools, such as the Bethesda classification and fine-needle aspiration biopsy (FNAB), are limited in sensitivity and specificity, particularly in indeterminate cases. Tumor-infiltrating immune cells (TIICs) in the tumor microenvironment (TME) play a significant role in thyroid cancer progression. CD66b⁺ neutrophil-like monocytes constitute a novel subset of myeloid cells that are implicated in the modulation of anti-tumor immune responses, but their role in thyroid cancer remains unclear. Materials and Methods: Peripheral blood and thyroid nodule tissue samples were obtained from 24 patients with papillary thyroid carcinoma, and from 10 patients who underwent surgery for symptoms of tracheal compression due to benign thyroid nodules. Myeloid cell populations were assayed by flow cytometric immunophenotyping with CD45, HLA-DR, CD14, and CD66b. The data were statistically analyzed with the clinical properties of the patients. Results: The neutrophil-like monocytes, which were determined as HLA-DR⁺CD14⁺CD66b⁺ cells, found in the circulation (11.9 ± 2.4% of total mononuclear immune cells) of the patients with papillary thyroid carcinoma, were significantly elevated (p < 0.001). Accordingly, these cells were more frequently detected in tumor tissues (21.1 ± 2.1% of total tumor-infiltrating immune cells) compared to non-tumor thyroid tissues (p = 0.0231). The infiltration levels of neutrophil-like monocytes were significantly higher in malignant nodules as well as in the peripheral blood of the papillary thyroid carcinoma patients compared to the samples obtained from the patients with benign nodules. The tumor tissues exhibited increased immune cell infiltration and harbored CD66b-expressing neutrophil-like HLA-DR⁺CD14⁺ monocytic cells, which indicates an inflammatory milieu in malignant thyroid cancer. Conclusions: This study identifies neutrophil-like monocytes as a potential biomarker for differentiating malignant and benign thyroid nodules. Elevated levels of this novel subtype of immune cells in malignant tissues suggest their role in tumor progression and their utility in enhancing diagnostic accuracy. Incorporating these findings into clinical practice may refine surgical decision-making and improve outcomes through personalized diagnostic and therapeutic strategies, particularly for radioiodine-refractory thyroid cancer.

Keywords: CD66+ monocytes; cancer diagnosis; monocyte subtypes; neutrophil; thyroid cancer.

Figure 1

Identification of neutrophil-like monocytes via flow cytometry. FSC-A (Forward Scatter-Area) and SSC-A (Side Scatter-Area), which are the parameters used for the size and granularity of the cells. CD45 is used as a common leucocyte marker. Monocytes were gated as CD14⁺HLA-DR⁺ cells amongst the CD45⁺ total leukocytes. CD14 and HLA-DR positivity were useful for eliminating conventional CD66b⁺ granulocytes and other immune cells, such as T, B, and NK cells. Red boxex and arrows indicate the gated populations.

Figure 2

Percentages of total leukocytes (a), total monocytes (b), and neutrophil-like monocytes (c) determined in peripheral blood (PB), non-tumor thyroid tissue (nTm), and malignant tumor tissue (Tm) from thyroid cancer patients. (* p < 0.005, ** p = 0.004, **** p = 0.0001. ns indicates nonsignificant.)

Figure 3

Neutrophil-like-monocyte frequency in thyroid cancer patients with different pathologies in peripheral blood and tumor samples. (a) Percentage of CD66b⁺ CD14⁺ monocytes determined in peripheral blood from the patients with benign and malignant thyroid pathologies. Both peripheral blood and tumor. (b) Representative flow cytometry results of CD14⁺CD66b⁺ monocytes benign and malignant patients’ peripheral blood. (c) Percentile amount of CD14⁺CD66b⁺ monocytes in benign and malignant patients’ tumor tissue. (d) Representative flow cytometry results of CD14⁺CD66b⁺ monocytes benign and malignant patients’ peripheral blood. Red and black boxes indicate gated populations. (** p = 0.004, *** p = 0.0005).