BRAFV600E/pTERT double mutated papillary thyroid cancers exhibit immune gene suppression

Abstract

Introduction: BRAFV600E mutation (BRAF^mut) is common in papillary thyroid cancer (PTC), and most patients have an excellent outcome. However, a TERT-promoter mutation (pTERT^mut) in the presence of BRAF^mut (BRAF^mutpTERT^mut) has been demonstrated to confer a more aggressive behavior to PTC. Lymphocytic infiltration is often present in PTC. In this study, we sought to decipher the relationship between the BRAF and pTERT mutations and immune gene dysregulation in tumor samples from a cohort of 147 samples of PTC.

Methods: The abundance of 770 immune gene transcripts was determined by multiprex capture/detection and digital counting of mRNA transcripts using the NanoString nCounter^® PanCancer Immune Profiling Panel.

Results: We identified 40 immune transcripts differentially expressed in BRAF^mutpTERT^mut vs BRAF^mutpTERT wildtype (pTERT^wt) (P<0.05). Transcripts induced by BRAF^mut alone were significantly repressed in BRAF^mutpTERT^mut samples, such as genes expressed by lymphoid cells, antigen-presenting cells, and cytotoxic cells, including chemokines, cytokines, checkpoint control proteins, interferon downstream markers, TNF superfamily proteins and BMP markers. A validation analysis using 444 samples from The Cancer Genome Atlas (TCGA) PTC dataset yielded similar results. Deconvolution analysis confirmed differences in the immune cell populations such as increased presence of M2 macrophages in the BRAF^mutpTERT^mut Mayo cohort and a lower abundance of M1 macrophages in the BRAF^mutpTERT^mut TCGA cohort compared to BRAF^mutpTERT^wt. Most of the immune gene pathways were enriched in the BRAF^mutpTERT^wt tumors in both Mayo and TCGA cohorts but not in BRAF^mutpTERT^mut. BRAF^mutpTERT^wt had higher stromal lymphocytes infiltration as compared to BRAF^wtpTERT^wt tumors, corroborating the transcriptomic findings.

Discussion: To our knowledge this is the first report of a potential link between TERT and the immune microenvironment, offering an explanation for the aggressive nature of BRAF^mutpTERT^mut PTC.

Keywords: BRAF mutation V600 E; TERT promoter mutation (pTERT); immune genes; lymphocytic infiltration; papillary thyroid cancer (PTC).

Figure 1

Differential gene expression as a function of BRAF and TERT mutational status. (A) BRAF and TERT mRNA abundance in relationship to the presence of BRAFV600E and TERT promoter mutations. (B) The TERT promoter mutations are associated with repression of CALCA, CCL21, strong induction of FN1, CXCL17, LCN6 and LCN10, and seems to have no effect on CHIT1 and ITGAX expression in BRAFV600E mutated tumors. (C) Immune genes whose expression is repressed in the presence of TERT promoter mutation but appears unaltered by the presence of BRAFV600E.

Figure 2

Panels (A-E). Differential gene expression of immune gene subsets. (A) Lymphoid markers. (B) Cytotoxic cell markers. (C) Antigen presentation cell markers. (D) Myeloid markers. (E) Checkpoint Control I/IO drug targets. Panel (F). Differentially expressed transcripts in 5 BRAF^wt samples harboring the TERT promoter mutation vs BRAF^wtpTERT^wt.

Figure 3

Differential expression of genes involved in signaling activity: (A) Interferon (IFN) downstream markers. (B) Tumor necrosis factor/TNF/AP1 signaling. (C) Bone morphogenetic proteins/transforming growth factor beta (BMP/TGFB).

Figure 4

Differential gene expression of immune genes in the TCGA samples compared to the Mayo Clinic cohort. (A) We identified 40 immune genes significantly differentially expressed (p<0.05) in both the Mayo Clinic cohort (black bars) and in the TCGA samples (purple bars). Panels (B-F). Differential gene expression of immune gene subsets: (B) T cell markers; (C) NK/cytotoxic cell markers; (D) antigen presenting cell markers; (E) CCL21 and CCR7 and (F) checkpoint genes.

Figure 5

Pathway analysis of Mayo (red bars) and TCGA (gray bars) BRAF^mutpTERT^wt vs BRAF^mutpTERT^mut samples. * indicates p-value <0.05.

Figure 6

Deconvolution analysis of (A) Mayo and (B) TCGA BRAF^mutpTERT^wt vs BRAF^mutpTERT^mut sample cohorts. p-value <0.05 was considered statistically significant. ¥, mean of absolute fraction of activated dendritic cells in BRAF^mutpTERT^wt, 0.00006; &, mean of absolute fraction of activated dendritic cells in BRAF^mutpTERT^mut, 0.00137.