Follicular thyroid tumors with the PAX8-PPARgamma1 rearrangement display characteristic genetic alterations

Abstract

Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) gamma1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARgamma1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARgamma target genes were up-regulated in PAX8-PPARgamma-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARgamma expression profile presented a putative PPARgamma-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARgamma and as members of novel molecular pathways involved in the development of thyroid carcinomas.

Figure 1

Hierarchical clustering of thyroid tumors. Classification is determined using the 1859 filtered features, with an agglomerative algorithm based on Pearson correlation coefficient. The figure represents a hyperbolic lens view of the classification tree. The red plot is the root of the clustering tree, the blue plots are classification nodes, and the brown plots represent the samples (NT, non tumoral tissues; t(2;3), presence of PAX8-PPARγ translocation in the sample).

Figure 2

Profile comparison and ontology analysis. Distribution of the ontology functions of the genes differentially expressed between normal tissues and FTCs with or without the PAX8-PPARγ translocation. Each gene was associated to an ontology cluster determined by using three gene description databases (FatiGO, Panther Ontology, and On Line Mendelian Inheritance In Man). The percentage value on the horizontal axis corresponds to the proportion of genes belonging to an ontology cluster compared with all genes of N/T+ set or N/T− set. Percentage on the left of axis (green) corresponds to down-regulated genes, whereas percentage on the right of axis (red) corresponds to up-regulated genes.

Figure 3

Clustering of tumors presenting or not the PAX8-PPARγ1 translocation using the T−/T+ gene set. The clustering is based on the T−/T+ set of genes determined by ANOVA. Red or green color scale represented respectively up- and down-regulation of genes in comparison with the reference. Each line corresponds to a gene and each column to a tumor (t(2;3), presence of the PAX8-PPARγ translocation in the samples). The HUGO gene symbol is associated to the average fold change value ratio between FTCs with or without the translocation.

Figure 4

Validation of gene expression in follicular thyroid tumors. Box plots for PPARγ, PAX8A, ACAA1, ANGPTL4, HBP17, FGD3, TGFA, PDE8, CRABP1, ESM1, and DCN gene expression as measured by real-time quantitative PCR normalized with four housekeeping genes. Gene expression levels are reported according to the diagnosis (N, normal; FTA, hypofunctioning FTAs; ATC, anaplastic thyroid carcinomas). For tumors without the translocation, the box shows the limits of the middle half of the data, and the line inside the box represents the median. Whiskers are drawn to represent the standard span of the 5th/95th percentile for all. Circles correspond to tumors with PAX8-PPARγ1 translocation. Horizontal barswith * or ** represent significant P values between N and FTC, and vertical barswith * or ** represent significant P values between tumors with or without the translocation.

Figure 5

PPARγ, ACAA1, and FGD3 immunostaining in thyroid carcinomas presenting the PAX8-PPARγ translocation. PPARγ immunostaining is presented at magnification ×100 (A) and ×200 (B). A strong positivity is observed in the nuclei of tumor cells (long arrows). Nuclei of endothelial cells are not stained (small arrows). For ACAA1 immunostaining at magnification ×100 (C), numerous positive intracytoplasmic vacuoles are observed in the majority of tumor cells. For ACAA1 immunostaining at magnification ×400 (D), positive large vacuoles are present in the apical part of tumor cells (long arrows); smaller vacuoles are indicated by small arrows (L, lumen of tumor follicle). FGD3 immunostaining is presented at magnification ×100 (E) and ×400 (F). Heterogeneity of the staining is observed in the tumor follicle. FGD3 localizes at the lateral membrane (long arrows), and no staining is observed at basal or apical membrane in translocated tumors.