Papillary thyroid carcinoma behavior: clues in the tumor microenvironment

Abstract

The prevalence of thyroid carcinoma is increasing and represents the most common endocrine malignancy, with papillary thyroid carcinoma (PTC) being the most frequent subtype. The genetic alterations identified in PTCs fail to distinguish tumors with different clinical behaviors, such as extra-thyroidal extension and lymph node metastasis. We hypothesize that the immune microenvironment may play a critical role in tumor invasion and metastasis. Computational immunogenomic analysis was performed on 568 PTC samples in The Cancer Genome Atlas using CIBERSORT, TIMER and TIDE deconvolution analytic tools for characterizing immune cell composition. Immune cell infiltrates were correlated with histologic type, mutational type, tumor pathologic T stage and lymph node N stage. Dendritic cells (DCs) are highly associated with more locally advanced tumor T stage (T3/T4, odds ratio (OR) = 2.6, CI = 1.4-4.5, P = 5.4 × 10-4). Increased dendritic cells (OR = 3.4, CI = 1.9-6.3, P = 5.5 × 10-5) and neutrophils (OR = 10.5, CI = 2.7-44, P = 8.7 × 10-4) significantly correlate with lymph node metastasis. In addition, dendritic cells positively correlate with tall cell morphology (OR = 4.5, CI = 1.6-13, P = 4.9 × 10-3) and neutrophils negatively correlate with follicular morphology (OR = 1.3 × 10-3, CI = 5.3 × 10-5-0.031, P = 4.1 × 10-5). TIDE analysis indicates an immune-exclusive phenotype that may be mediated by increased galectin-3 found in PTCs. Thus, characterization of the PTC immune microenvironment using three computational platforms shows that specific immune cells correlate with mutational type, histologic type, local tumor extent and lymph node metastasis. Immunologic evaluation of PTCs may provide a better indication of biologic behavior, resulting in the improved diagnosis and treatment of thyroid cancer.

Keywords: RNA sequencing; immunogenomics; papillary thyroid carcinoma; thyroid cancer; tumor immunology.

Figure 1.. Distribution of immune cell infiltrate and pathologic tumor T stage (N=568).

A. CIBERSORT distribution of activated dendritic cell fractions in low T stage (T1/T2) and high T stage (T3/T4) tumors. B. TIMER distribution of dendritic cell fractions in low T stage (T1/T2) and high T stage (T3/T4) tumors. C. CIBERSORT distribution of follicular helper T cell fractions in low T stage (T1/T2) and high T stage (T3/T4) tumors.

Figure 2.. Distribution of immune cell fractions in tumors without (N0) and with (>N0) lymph node metastasis (N=568).

A. CIBERSORT distribution of resting dendritic cells in tumors without (N0) and with (>N0) lymph node metastasis. B. TIMER distribution of dendritic cells in tumors without (N0) and with (>N0) lymph node metastasis. C. TIMER distribution of neutrophils in tumors without (N0) and with (>N0) lymph node metastasis. D. CIBERSORT distribution of naïve B cells in tumors without (N0) and with (>N0) lymph node metastasis.

Figure 3.. Tumor immune dysfunction and exclusion (TIDE) analysis (N=568).

A. Overall TIDE scores and predicted tumor response to checkpoint blockade. B.TIDE dysfunction score. DS>0 indicates T cell dysfunction, DS<0 indicates functional T cells. C. TIDE T cell exclusion score. ES>0 indicates an T cell exclusive tumor, ES<0 indicates a T cell inclusive tumor. D. IHC of patient tumors, with CD8+ T cells shown in black.

Figure 4.. Immune gene expression in activated DC high PTCs (N=131).

A) Negative log₁₀ of the FDR value for pathway enrichment for terms in the top cluster from DAVID analysis of differentially expressed genes between low and high activated DC thyroid samples. Red line marks p-value of 0.05. B) Heatmap of the differentially expressed genes from DC enriched immunity signature. Green is high expression and red is low expression. Yellow marks high activated DC and blue marks low activated DC tumors. Most high activated DC samples are in the cluster with predominately low expression of the genes in the immune signature. C) Heatmap of differentially expressed genes from T_FH enriched immunity signature. Green is high expression and red is low expression. Yellow marks high T_FH and blue marks low T_FH. Most high T_FH samples are in the cluster with predominately high expression of the genes in the immunity signature. D) Ratio of LGALS3 to LAG3 in activated DC low and high samples (p=5.47 × 10⁻¹⁰, Wilcoxon rank Test). Samples that had p>=0.05 in CIBERSORT were excluded from this analysis.

Figure 5.. GAL3 expression in NIFTPs and infiltrative FVPTCs (N=502).

A. GEPIA analysis of LGALS3 expression in TCGA THCA (p<0.01). Statistical analysis performed in GEPIA is a one-way ANOVA, using disease state as a variable for calculating differential expression. B. Tumor morphology was evaluated and tumors were stained for GAL3 expression (p=0.0019), 100X magnification. C. Representative IHC images of GAL3 staining (left-negative; middle-subcapsular; right-strong/diffuse).