Identification of a Recurrent LMO7-BRAF Fusion in Papillary Thyroid Carcinoma

Abstract

Background: The BRAF^V600E mutation is the most common driver in papillary thyroid carcinoma (PTC) tumors. In recent years, gene fusions have also been recognized as important drivers of cancer in PTC. Previous studies have suggested that thyroid tumors with fusion genes frequently display an aggressive course. These observations prompted further exploration of gene fusions in PTC tumors. The aim was to search for previously unrecognized gene fusions using thyroid tissue samples from PTC patients.

Methods: Gene fusions were analyzed in RNA sequencing data obtained from 12 PTC tumors and paired unaffected thyroid tissue samples. Candidate fusions were further filtered and validated using reverse transcriptase polymerase chain reaction, Sanger sequencing, and fluorescence in situ hybridization. An Ohio cohort of 148 PTC tumor samples was screened for a LMO7-BRAF fusion and the BRAF^V600E mutation. Functional assays were performed to assess the LMO7-BRAF fusion.

Results: Two coding fusions (CCDC6-RET and LMO7-BRAF) were found in one tumor sample each. The novel LMO7-BRAF fusion was validated by reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization. The LMO7-BRAF fusion was a recurrent somatic alteration with a frequency of 2.0% (3/148) in PTC tumors, while the BRAF^V600E point mutation was found in 63.5% (94/148) of tumors. Enforced expression of LMO7-BRAF fusion protein stimulated endogenous ERK1/2 phosphorylation and promoted anchorage independent cell growth to an extent similar to BRAF^V600E.

Conclusions: A novel fusion gene, LMO7-BRAF, was identified in PTC tumors. The results indicate that the LMO7-BRAF fusion behaves as an oncogenic alteration. This observation expands the spectrum of fusion genes involving kinases in thyroid cancer.

Keywords: BRAFV600E; LMO7–BRAF; fusion gene; papillary thyroid carcinoma.

FIG. 1.

Validation of and screening for the LMO7–BRAF fusion in papillary thyroid carcinoma (PTC) tumors. (A) Totally, 148 tumor samples were screened; 14 samples are shown in this gel picture. Reverse transcriptase polymerase chain reaction (PCR) was performed using the forward primer in LMO7 exon 6 and the reverse primer in BRAF exon 9. Samples #2, #10, and #13 are positive for the LMO7–BRAF fusion. Sample #10 is the index sample in which the LMO7–BRAF fusion was originally identified with RNA sequencing (RNA-Seq) data. The BRAF^V600E mutation was screened in the same samples, and the results are shown. +, positive; −, negative. GAPDH was used as a control. M, molecular marker; (–), negative control for PCR. (B) Sanger sequencing chromatograph of the LMO7–BRAF fusion in sample #10. The arrow shows the breakpoint of the fusion between LMO7 (NM_001306080.1, end of exon 6) and BRAF (NM_004333.4, start of exon 9).

FIG. 2.

Detection of LMO7–BRAF with fluorescence in situ hybridization (FISH). (A) Diagram of the FISH probes (kindly provided by Obio). The dual-colored FISH probes cover the fusion partners of the LMO7 and the BRAF genes. The genomic coordinates of the fusion point in the LMO7–BRAF fusion transcript were obtained from UCSC genome browser (hg19). LMO7: chr13:76370885, forward strand; BRAF: chr7:140487384, reverse strand. (B) FISH images were obtained with tumor touch imprints from sample #10. Three types of colored signals are shown: red for the LMO7 gene, green for the BRAF gene, and yellow for the fusion between the LMO7 and the BRAF genes.

FIG. 3.

LMO7–BRAF fusion induces MAPK activation and promotes anchorage-independent cell growth. (A) Immunoblotting of lysates from HEK293T cells transfected with pcDNA3 control vector, or plasmids encoding BRAF(wt), BRAF^V600E, and LMO7–BRAF fusion. The primary antibodies to phospho-ERK1/2 (pERK), total ERK (ERK), and actin (ACTIN) were applied. (B) Soft agar transformation assay of NIH 3T3 cells. The bar graph displays the relative anchorage independent cell growth analyzed by fluorimetric assays. All values are presented as mean ± standard deviation of assays performed in triplicate. *p < 0.05, two-tailed Student's t-test.