The diagnostic application of RNA sequencing in patients with thyroid cancer: an analysis of 851 variants and 133 fusions in 524 genes

Abstract

BackgroundThyroid carcinomas are known to harbor oncogenic driver mutations and advances in sequencing technology now allow the detection of these in fine needle aspiration biopsies (FNA). Recent work by The Cancer Genome Atlas (TCGA) Research Network has expanded the number of genetic alterations detected in papillary thyroid carcinomas (PTC). We sought to investigate the prevalence of these and other genetic alterations in diverse subtypes of thyroid nodules beyond PTC, including a variety of samples with benign histopathology. This is the first clinical evaluation of a large panel of TCGA-reported genomic alterations in thyroid FNAs.ResultsIn FNAs, genetic alterations were detected in 19/44 malignant samples (43 % sensitivity) and in 7/44 histopathology benign samples (84 % specificity). Overall, after adding a cohort of tissue samples, 38/76 (50 %) of histopathology malignant samples were found to harbor a genetic alteration, while 15/75 (20 %) of benign samples were also mutated. The most frequently mutated malignant subtypes were medullary thyroid carcinoma (9/12, 75 %) and PTC (14/30, 47 %). Additionally, follicular adenoma, a benign subtype of thyroid neoplasm, was also found to harbor mutations (12/29, 41 %). Frequently mutated genes in malignant samples included BRAF (20/76, 26 %) and RAS (9/76, 12 %). Of the TSHR variants detected, (6/7, 86 %) were in benign nodules. In a direct comparison of the same FNA also tested by an RNA-based gene expression classifier (GEC), the sensitivity of genetic alterations alone was 42 %, compared to the 91 % sensitivity achieved by the GEC. The specificity based only on genetic alterations was 84 %, compared to 77 % specificity with the GEC.ConclusionsWhile the genomic landscape of all thyroid neoplasm subtypes will inevitably be elucidated, caution should be used in the early adoption of published mutations as the sole predictor of malignancy in thyroid. The largest set of such mutations known to date detects only a portion of thyroid carcinomas in preoperative FNAs in our cohort and thus is not sufficient to rule out cancer. Due to the finding that variants are also found in benign nodules, testing only GEC suspicious nodules may be helpful in avoiding false positives and altering the extent of treatment when selected mutations are found.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0849-9) contains supplementary material, which is available to authorized users.

Molecular Variants and Their Risks for Malignancy in Cytologically Indeterminate Thyroid Nodules

“…To identify presurgical thyroid FNA specimens with AUS/ FLUS or SFN/FN cytology that underwent molecular testing and resection, we performed a PubMed search for studies published between January 1, 2009, and December 31, 2018 (see Supplementary Data S1 for the search keywords and search parameters). The resulting 540 abstracts were reviewed, and 113 publications that potentially met the inclusion criteria described below were combined with an additional 18 publications (9,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) identified from bibliographies or personal files. These 131 publications underwent a detailed review and data extraction by at least 2 reviewers ( Fig.…”

“…While our study highlights the limited data available on the association of most variants and fusions to predict cancer among cytologically ITNs, data are beginning to emerge linking genomic alterations with specific types of neoplasms, their behavior, routes of metastasis, and prognosis (7)(8)(9). Data to support the independent prognostic value of genomics are currently sparse, however, and randomized controlled studies based on the presence of a specific variant have not been performed to demonstrate clinical utility for a variant-based treatment plan.…”

“…Initial studies included single genomic alterations, then small panels of several genes, and most recently numerous alterations among many genes (7)(8)(9)(10)(11)(12). Despite discoveries of specific genetic variants and fusions in thyroid cancer specimens, many of these studies are not necessarily applicable to the preoperative evaluation of cytologically indeterminate thyroid nodules (ITNs) as they evaluated postsurgical histological rather than preoperative FNA specimens.…”

Targetable gene fusions identified in radioactive iodine refractory advanced thyroid carcinoma

“…Moleculelevel barcoding (or unique molecule identifier tagging) and sample-level barcoding (also known as index tagging) are both incorporated during Archer MBC ligation. In the first and second PCR a specific primer set was used to cover relevant exons in 34 genes including ALK (exon 5′; 2, 4, 6, 10, 16-23, (intron19)), AXL (exon 3′; [18][19][20], BRAF (exon 5′; 7-11, exon 3′; 7, 8, 10), CCND1 (exon 5′; 1-4, exon 3′; 1, 2, 4), FGFR1 (exon 5′; 2, 8-10, 17, exon 3′; 17), FGFR2 (exon 5′; 2, 5, 7-10, exon 3′; 17), FGFR3 (exon 5′; 3, 5, 8-10, exon 3′; 17, (intron17)), MET (exon 5′; 2, 4-6, 13, 14, 16, 17, 21, exon 3′; 2), NRG1 (exon 5′; 1-3, 6), NTRK1 (exon 5′; 2, 4, 6, 8, 10-13), NTRK2 (exon 5′; 5,7,9,[11][12][13][14][15][16][17], NTRK3 (exon 5′; 4, 7, 10, 13-16), PPARG (exon 5′; 1-3, 5), RAF1 (exon 5′; [4][5][6][7][9][10][11][12], RET (exon 5′; 2,4,6,8,[9][10][11][12][13][14], ROS1 (exon 5′; 2, 4, 7, 31-37) and THADA (exon 3′; 24-30, 36, 37). This method enables to detect known gene fusions as well as novel gene fusion partners.…”

“…BRAF and RAS genes) as well as gene fusions (e.g. RET-PTC 1-12 and NTRK) leading to activation of the mitogen-activated protein kinase (MAPK) pathway are crucial for tumorigenesis and progression in thyroid tumors (6,7,8,9,10). These mutations are almost always mutually exclusive (6).…”