Lymphocytic infiltrates in thyroid cancer may suggest a
Lymphocytic infiltrates in thyroid cancer may suggest a background of chronic lymphocytic thyroiditis. Thyroiditis is characterized by a cellular immune response with dense lymphocytic infiltration of the thyroid gland, as well as by a humoral immune response leading to the production of autoantibodies against thyroid antigens. Recently, a meta-analysis investigated the correlation between papillary thyroid cancer and histologically proven Hashimoto\'s thyroiditis. The results revealed that thyroiditis was more frequently observed in papillary thyroid cancer than in benign thyroid diseases, and cancer patients with thyroiditis had a longer duration of recurrence-free survival. This seemingly paradoxical phenomenon deserves further investigation, because it may provide insight into the immune dysregulation involved in both disorders. In the present study, we examined the data of The Cancer Genome Atlas (TCGA) project in an attempt to define the relationship between lymphocyte infiltrates and clinical and molecular presentations.
Materials and methods Patient characteristics and preprocessed gene potassium phosphate monobasic data of patients with papillary thyroid cancer were downloaded from Genomic Data Commons (https://gdc.cancer.gov/) in August 2016. The mRNA expression profile was obtained by Illumina HiSeq 2000 RNA sequencing version 2 level 3 data, and expressed as RNA-Seq by Expectation Maximization (RSEM) values. The information on new tumor event, disease status, and patient mortality was updated to incorporate the latest follow-up. Patients who had missing transcriptome profiling or who lacked tumor-infiltrating lymphocyte data were excluded from the analysis. Finally, a total of 497 patients were included in the study. The percentage of tumor-infiltrating lymphocytes of the primary tumor was significantly skewed, ranging from to 30% (mean 1.5%, median 0%). There were no tumor-infiltrating lymphocytes in 257 (52%) of the cases. We used the X-tile software (Yale University, New Haven, CT, USA) to determine an optimal cut-off point. For categorical variables, the chi-square test, Fisher\'s exact test, or the Cochran-Armitage trend test was used to determine the difference between groups. For continuous variables, unpaired two-tailed Student\'s t-test was performed. Overall survival and recurrence-free survival were compared using Kaplan-Meier curves and log-rank tests. The level of statistical significance was chosen as P < 0.05. To identify transcriptome changes in association with lymphocyte infiltration, the gene expression profile of the tumors with tumor-infiltrating lymphocytes less or more frequent than the cut-off were thoroughly compared. Median RSEM values of all gene expression data obtained from RNA sequencing were calculated; then, genes with a median value of less than 4.1374 (the first quartile) were excluded to avoid any variation caused by low-level expressions. The expression levels of remaining genes were compared one by one between the two groups using Wilcoxon rank sum tests. Genes with a P value of less than 0.01 (differentially expressed genes) were subjected to functional enrichment analyses. Spearman\'s rank correlation test was used for correlation studies. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation of differentially expressed genes was analyzed using Web-based Gene Set Analysis Toolkit (WebGestalt, http://www.webgestalt.org/) and the Database for Annotation, Visualization and Integration Discovery (DAVID, https://david.ncifcrf.gov/). In WebGestalt, the minimum number of genes for enrichment was set at 5, and the significance analysis was performed using the hypergeometric test with the significance level set at P = 0.01. In DAVID, the parameters were set to their default values.
Results In the present study, nearly one-half of the tumors were absent for lymphocyte infiltration. The cut-off of lymphocyte infiltration was determined at 1%. In total, 258 (52%) patients had tumor-infiltrating lymphocytes less than 1%, while 239 (48%) patients had tumor-infiltrating lymphocytes greater than or equal to 1%. The characteristics of the two groups are listed in Table 1. Papillary thyroid cancer with infiltrating lymphocytes ≥1% was associated with classical histologic features, multifocality, and lymph node metastasis. However, there was no difference in TNM stage or the frequency of BRAF mutation between the two groups. As shown in Fig. 1, papillary thyroid cancer with infiltrating lymphocytes ≥1% was associated with a better overall survival (log-rank P = 0.018), but there was no difference in recurrence-free survival (log-rank P = 0.851). Histological subtype did not influence overall survival (log-rank P = 0.419) or recurrence-free survival (log-rank P = 0.274).