Tumor-associated lymphocytes and increased FoxP3+ regulatory T cell frequency correlate with more aggressive papillary thyroid cancer

Abstract

Context: Ten to 30% of patients with papillary thyroid cancer (PTC) develop recurrent disease and may benefit from innovative adjuvant therapies. Immune-based therapies are under investigation to treat many types of cancer. The role of the immune system in PTC is poorly understood.

Objective: We investigated whether tumor-associated lymphocytes (TAL), in the absence of background thyroiditis (LT), contribute to disease severity. We hypothesized that the type of lymphocytes associated with PTC would correlate with parameters of disease.

Design: This retrospective study analyzed archived PTC samples for the presence of TAL and/or LT. A group of patients with TAL was evaluated for lymphocyte subsets by immunohistofluorescence.

Patients and setting: One hundred PTC patients were analyzed for LT and TAL, and 10 PTC patients with TAL were assessed for lymphocyte subsets at University of Colorado Hospital.

Main outcome: We assessed correlations between disease and the presence of TAL, LT, and lymphocyte subset frequency.

Results: Patients with TAL exhibited higher disease stage and increased incidence of invasion and lymph node metastasis compared with patients without lymphocytes or with LT. CD4(+) T cell frequency correlated with tumor size (r = 0.742; P = 0.017). FoxP3(+) regulatory T cell (Treg) frequency correlated with lymph node metastases (r = 0.858; P = 0.002), and CD8 to Treg ratio correlated inversely with tumor size (r = -0.804; P = 0.007).

Conclusions: TAL and high Treg frequency in primary thyroid tumors correlates with more aggressive disease. Future prospective studies may identify Treg frequency as a predictive factor in PTC, and the suppressive effects of Treg should be considered in the design of immune-based therapies.

Figure 1

Tumor-associated LI and disease severity. Hematoxylin and eoxin stains of thyroid tissue sections from 100 PTC patients were reanalyzed for LI. A, PTC stage was compared between patients with or without LI; B and C, representative hematoxylin and eosin images are shown from patient with lymphocytic thyroiditis (B) or tumor-associated lymphocytes (C); D, samples were grouped based on the absence of lymphocytes (No LI), the presence of TAL in the absence of thyroiditis, or the presence of lymphocytes with background thyroiditis (LT), and disease stage was compared for each group; E and F, 24 patients with TAL were grouped based on lymphocyte localization and compared for tumor size (E) and the percentage of patients with invasive tumors (F, P = 0.189) or LN metastases (F, P = 0.679).

Figure 2

Lymphocyte subset analysis in PTC patients with TAL. Archived thyroid tissues from 10 PTC patients with evidence of TAL were stained for key lymphocyte markers using standard immunohistofluorescence techniques. A–C, Both peritumoral (A) and intratumoral (B and C) lymphocyte aggregates are shown at ×10 magnification; D, lymphocyte aggregates were imaged at ×60 for quantification; E, lymphocytes were also found as single cells or multicellular clusters within the tumor. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (blue). The nuclei of PTC cells are characterized by their grooved appearance and were easily distinguished from infiltrating lymphocytes (E). CD25⁺ and CD25⁻CD4⁺FoxP3⁺ Tregs were identified within lymphoid aggregates (LA) and within the tumor (PTC, F). Arrows designate thyroid tumor cells (A–D) and CD25⁻ Tregs (F). Nonspecific, noncellular staining was due to antibody interactions with residual follicular colloid proteins.

Figure 3

Correlation analysis of subset frequency and disease severity. Lymphocyte aggregates in 10 PTC samples were imaged at ×60 magnification. CD4⁺, CD8⁺, and CD4⁺FoxP3⁺ subsets were quantified, and relative frequencies were analyzed for correlation with tumor size and LN metastases. Spearman correlation coefficient (r) and P values are shown. The correlation between CD8 to Treg ratio and tumor size remains statistically significant when patient 10 is omitted (r = −0.729; P = 0.031).