Abstract
The EIF1AX gene mutations have been recently found in papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC). The prevalence of these mutations in other types of thyroid cancers and benign nodules is unknown. In this study, we analyzed the occurrence of EIF1AX mutations in exons 2, 5, and 6 of the gene in a series of 266 thyroid tumors and hyperplastic nodules by either Sanger or next-generation sequencing (ThyroSeq v.2). In addition, 647 thyroid fine-needle aspiration (FNA) samples with indeterminate cytology were analyzed. Using surgically removed samples, EIF1AX mutations were detected in 3/86 (2.3%) PTC, 1/4 (25%) ATC, 0/53 follicular carcinomas, 0/12 medullary carcinomas, 2/27 (7.4%) follicular adenomas, and 1/80 (1.3%) hyperplastic nodules. Among five mutation-positive FNA samples with surgical follow-up, one nodule was PTC and others were benign follicular adenomas or hyperplastic nodules. Overall, among 33 mutations identified, A113_splice mutation at the intron 5/exon 6 splice site of EIF1AX was the most common. All four carcinomas harbored A113_splice mutation and three of them had one or more coexisting mutations, typically RAS. All PTC carrying EIF1AX mutations were encapsulated follicular variants. In summary, this study shows that EIF1AX mutations occur not only in thyroid carcinomas, but also in benign nodules. The most common mutation hotspot is the A113_splice, followed by a cluster of mutations in exon 2. When found in thyroid FNA samples, EIF1AX mutations confer ~20% risk of cancer; the risk is likely to be higher in nodules carrying a A113_splice mutation and when EIF1AX coexists with RAS mutations.
Introduction
Thyroid cancer is the most common endocrine cancer and its incidence is increasing in the USA and other countries due to improved detection of indolent thyroid cancers by sensitive imaging techniques and possibly due to other factors (Davies & Welch 2006, Brito et al. 2013, Jung et al. 2014). Cytological diagnosis of thyroid nodules using cells collected by fine-needle aspiration (FNA) is the most reliable way of cancer diagnosis, but a significant proportion of FNA samples is placed in indeterminate cytological category, preventing optimal patient management (Haugen et al. 2016). Advances in sequencing technology coupled with better understanding of the molecular mechanisms of thyroid tumorigenesis have led to increasing use of genomic testing for clinical management of patients with thyroid nodules (Adeniran et al. 2006, Cantara et al. 2010, Nikiforov et al. 2011). Recently, The Cancer Genome Atlas (TCGA) study on a large cohort of papillary thyroid carcinomas (PTC) reported mutations of the EIF1AX gene in these tumors (Cancer Genome Atlas Research 2014c).
The EIF1AX gene located on chromosome X and codes for an eukaryotic translation initiation factor 1A (eIF1A). The protein promotes 43S complex formation by stabilizing the binding of the ternary complex eIF2-GTP-methionyl-initiator tRNA to the 40S subunit and, together with other translation initiation factor is involved in a sophisticated scanning system responsible for accurately locating the proper start codon on the mRNA in eukaryotes (Pestova et al. 2001, Olsen et al. 2003, Fekete et al. 2005, Hinnebusch 2014). eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, with sequences between 32 and 95 amino acids constituting the RNA-binding domain that are homologous to IF1. Additional domains in eIF1, absent in IF1, are a helical domain adjacent to the RNA-binding fold and long and highly charged unstructured N- and C-terminal tails (NTT, CTT). The CTT is primarily negatively charged but contains a few hydrophobic residues at the very end that were proposed to be involved in protein–protein interactions (Fekete et al. 2007, Saini et al. 2010). Mutations of residues on the RNA-binding surface of eIF1A are reported to cause defects in proper 43S and 48S preinitiation complex formation (Battiste et al. 2000). Prior to TCGA study of PTC, mutations in EIF1AX gene were reported in uveal melanomas (Martin et al. 2013, Ewens et al. 2014). A recent study also reported EIF1AX mutations in anaplastic thyroid carcinomas (ATC) (Kunstman et al. 2015). However, the prevalence of these mutations in other common types of thyroid cancer, such as follicular carcinoma, and in benign thyroid nodules remains unknown. Furthermore, histopathologic characteristics of thyroid tumors harboring EIF1AX mutations are not well characterized. The aim of this study was to determine the prevalence of EIF1AX mutations in all major types of thyroid tumors and benign nodules, spectrum of EIF1AX mutation, and histopathologic characteristics of thyroid lesions harboring EIF1AX mutations.
Materials and methods
Study cases
This study was approved by the University of Pittsburgh Institutional Review Board. Two groups of samples were studied. The first group included 266 surgical specimens (formalin-fixed paraffin-embedded and snap-frozen tissue samples) that were used to study the prevalence of EIF1AX mutations in various types of thyroid nodules. The second group included 647 consecutive fine-needle aspiration (FNA) samples with indeterminate cytology genotyped during a 2-month time interval.
Histologic review
Glass slides of surgical resection of all cases positive for EIF1AX mutation were retrieved from the archives of the Department of Pathology, University of Pittsburgh Medical Center and reviewed to confirm histopathologic diagnosis and tumor variant, and to assess for other salient features.
Molecular analysis
Testing for EIF1AX mutations in exons 2, 5, and 6 was performed using Sanger sequencing or next-generation sequencing panel (ThyroSeq v2) as previously described (Nikiforov et al. 2014). In addition to EIF1AX, the panel included the analysis of point mutations in the hotspots of 13 other thyroid cancer-related genes (AKT1, BRAF, NRAS, HRAS, KRAS, PTEN, TP53, GNAS, CTNNB1, RET, PIK3CA, TERT, and TSHR) and gene fusions in the RET, BRAF, NTRK1, NTRK3, ALK, PPARG, and THADA genes. For some samples, Sanger sequencing was performed on ABI13130 (Applied Biosystems) for exons 2, 5, and 6 of EIF1AX gene. For targeted next-generation sequencing analysis, the custom primers were designed using a Life Technologies design tool to generate a pool of primers for amplification of genomic regions of interest. Library concentration and amplicon size were determined using an Agilent BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). Next, multiplexed barcoded libraries were enriched by clonal amplification using emulsion PCR on Ion Sphere particles (Ion PGM Template OT2 200 Kit, Thermo Fisher Scientific) and loaded on an Ion 318 Chip. Massively parallel sequencing was carried out on a Personal Genome Machine sequencer (Ion Torrent) using the Ion PGM Sequencing 200 Kit version 2 according to the manufacturer’s instructions.
Results
First, the prevalence of EIF1AX mutation was assessed in a series of surgically resected thyroid nodules representing all common types of thyroid cancer. Among 86 surgical PTC samples, two specimens were positive for EIF1AX mutations, resulting in an overall frequency of 2.3%. Both mutations were a splice-site mutation located in the inton 5/exon 6 junction of EIF1AX gene (A113_splice). Analysis of eight dedifferentiated carcinomas (including four poorly differentiated carcinomas and four anaplastic carcinomas) identified an A113_splice mutation in one ATC. Analysis of 53 follicular thyroid carcinomas (FTC), which included 22 oncocytic (Hürthle cell) variants of follicular carcinomas, did not reveal EIF1AX mutations. Similarly, no EIF1AX mutation was identified in 12 medullary carcinomas. Screening of benign thyroid nodules detected EIF1AX mutations in two (7.4%) follicular adenomas (FA) and one (1.3%) hyperplastic thyroid nodules (Table 1).
Prevalence of EIF1AX mutations in thyroid nodules.
| Histological type | Total cases | EIF1AX mutation positive (%) |
|---|---|---|
| Papillary thyroid carcinoma | 86 | 2 (2.3) |
| Follicular thyroid carcinoma | 53 | 0 |
| Poorly differentiated thyroid carcinoma | 4 | 0 |
| Anaplastic thyroid carcinoma | 4 | 1 (25) |
| Medullary thyroid carcinoma | 12 | 0 |
| Follicular adenoma | 27 | 2 (7.4) |
| Hyperplastic nodule | 80 | 1 (1.3) |
Next, the analysis was performed on 647 consecutive thyroid FNA cytology samples with indeterminate cytology, of which 27 (4.2%) were found to be positive for EIF1AX mutations. Of the EIF1AX-positive cytology specimens, 63% had a diagnosis of atypia of undetermined significance/follicular lesion of undetermined significance, 30% had a diagnosis of follicular neoplasm/suspicious for follicular neoplasm, and 4% had a diagnosis of suspicious for malignancy. Follow-up thyroidectomy specimens were available in 5 of the 27 samples. Histopathologically, the EIF1AX-positive nodules yielded the diagnosis of PTC in one case, follicular adenoma in three cases, and hyperplastic thyroid nodule in one case. Out of 27 thyroid nodules positive for EIF1AX mutations in FNA samples, 21 nodules had no coexisting mutations, whereas 6 nodules had additional mutations. Of those, four nodules showed presence of a coexisting NRAS codon 61 mutation, one sample had a HRAS codon 61 mutation, and one had a GNAS mutation. Of these six cases, one nodule harboring EIF1AX and NRAS mutation was surgically excised to reveal the encapsulated follicular variant of PTC.
Overall, in the two sample groups, 33 EIF1AX mutations were identified (Table 2). All mutations were single nucleotide substitutions that were clustered in two areas of the gene, either in codons 6–15 located in the unstructured NTT (54%) or codon 113 corresponding to the unstructured CTT (46%) of the EIF1AX (Fig 1). Codon 113 was split by intron 5 and harbored the most common EIF1AX mutation identified in 15 (46%) of EIF1AX-positive cases. These were splice-site mutations affecting one of the two consensus intronic nucleotides (c.338-1A and c.338-2G) located in the splice site of intron 5/exon 6. The second most common mutation, found in eight (24%) cases, affected codon 9, leading to the substitution of glycine by arginine, aspartic acid, or valine. EIF1AX codon 13 missense mutations were identified in four (12%) cases and resulted in the amino acid substitution of arginine to leucine, aspartic acid, or proline. Less common mutations affected codons 8, 10, and 15, all being missense mutations, while a single case showed a splice-site mutation at codon 6, which was split by intron 1.
Spectrum of EIF1AX mutations found in thyroid nodules. Distribution of EIF1AX mutations within various domains of eukaryotic IF1A protein (144 amino acids) identified in various types of thyroid lesions. X-axis denotes the number of mutations. Green circles denotes missense mutations and red circles denotes splice-site mutations. A full colour version of this figure is available at http://dx.doi.org/10.1530/ERC-16-0043.
Citation: Endocrine-Related Cancer 23, 4; 10.1530/ERC-16-0043
Location and type of EIF1AX mutations found in thyroid nodules.
| Mutated codon | Frequency (%) | Mutation variants | Coexisting mutations |
|---|---|---|---|
| Codon 113 | 15 (46) | A113_splice | NRAS, HRAS, TP53 |
| Codon 9 | 8 (24) | G9R, G9D, G9V | |
| Codon 13 | 4 (12) | R13L, R13P, R13D | NRAS, GNAS |
| Codon 8 | 2 (6) | G8R, G8V | – |
| Codon 15 | 2 (6) | G15D | |
| Codon 10 | 1 (3) | K10N | |
| Codon 6 | 1 (3) | G6_splice | – |
A total of 11 cases that harbored EIF1AX mutation were available for histopathology review (Table 3). The size of these thyroid nodules ranged from 1.3 to 6.0 cm (mean, 2.9 cm) and histopathologic diagnoses included PTC in three cases, ATC in one case, follicular adenoma in five cases, and hyperplastic thyroid nodule in five cases (Table 3).
Clinicopathological characteristics of thyroid nodules harboring EIF1AX mutations and coexisting mutations.
| Case number | Diagnosis | Age | Gender | Nodule size (cm) | EIF1AX mutation type | Coexisting mutation |
|---|---|---|---|---|---|---|
| 1 | EFVPTC | 34 | M | 5.1 | A113_splice | NRAS |
| 2 | EFVPTC | 72 | M | 6.0 | A113_splice | NRAS |
| 3 | EFVPTC | 69 | M | 1.5 | A113_splice | – |
| 4 | ATC | 71 | F | 6 | A113_splice | NRAS, TP53, TERT |
| 5 | FA | 46 | F | 2.5 | K10N | – |
| 6 | FA | 45 | F | 2.9 | G9R | – |
| 7 | FA | 69 | F | 1.8 | G9V | – |
| 8 | FA | 77 | F | 1.3 | A113_splice | – |
| 9 | FA | 68 | F | 3.5 | A113_splice | – |
| 10 | HN | 63 | M | 4.3 | G9R | – |
| 11 | HN | 53 | F | 2.7 | G9R | – |
EFVPTC, encapsulated follicular variant of papillary thyroid carcinoma; HN, hyperplastic nodule.
All three mutation-positive PTCs carried the same EIF1AX A113_splice mutation. Two of these tumors also harbored a coexisting mutation of NRAS. The tumor nodules ranged in size from 1.5 to 6.0 cm. Microscopically, they had a complete thin or thick capsule and follicular growth pattern characteristic of the encapsulated follicular variant of PTC (Fig 2). The neoplastic follicles in each nodule varied in size with abundant colloid and were lined by cells showing moderate to prominent nuclear features of PTC. A tumor with isolated EIF1AX mutation showed focal capsular invasion (Fig. 2), whereas other tumors showed no tumor capsule or vascular invasion. None of the PTCs carrying the EIF1AX mutation had extrathyroidal extension or lymph node metastasis.
Histopathologic features of papillary thyroid carcinomas positive for EIF1AX mutations. Encapsulated follicular variant of papillary thyroid carcinoma with well-defined capsule and mixed microfollicular and normofollicular growth pattern (A) and capsular invasion (B). The neoplastic follicular cells show enlarged, overlapping, irregularly shaped nuclei (C) and strong and diffuse membranous immunoreactivity for HBME-1 (D). A, hematoxylin and eosin (H&E) stain ×20; B, H&E, ×100; C, H&E, ×40; D, HBME-1 immunostain, ×100.
Citation: Endocrine-Related Cancer 23, 4; 10.1530/ERC-16-0043
One ATC case was positive for the EIF1AX A113_splice mutation found in combination with NRAS Q61R, TERT C228T, and TP53 E180* stop codon mutation. Microscopic examination of the excised tumor revealed a combination of epithelioid, spindle cell, and multinucleated giant cell growth patterns and large areas of necrosis. No well-differentiated component was observed.
Five FA were found to harbor EIF1AX mutations, including A113_splice site (×2), G9R/V (×2), and K10N. The nodules ranged in size from 1.3 to 3.5 cm, had a thin to thick capsule, and typically a mixture of small- to normal-sized follicles (Fig 3A and B). Two adenomas were of conventional type and one showed oncocytic (Hürthle cell) morphology. Two additional EIF1AX-positive nodules were diagnosed as hyperplastic nodules. Both of them harbored a G9R EIF1AX mutation. The nodules had a thin capsule and were composed of predominantly large follicles with abundant colloid (Fig. 3C and D).
Histopathologic features of EIF1AX-positive benign thyroid nodules. (A, B) A follicular adenomas surrounded by a thin fibrous capsule (A) and showing small- to normal-sized follicles lined by cells with round, uniform nuclei and dark chromatin. (A, H&E, ×20; B, H&E, ×400). (C, D) Hyperplastic thyroid nodule with a thin capsule and a mixture of normal- and large-sized follicles with abundant colloid. (C, H&E, ×20; D, H&E, ×400). A full colour version of this figure is available at http://dx.doi.org/10.1530/ERC-16-0043.
Citation: Endocrine-Related Cancer 23, 4; 10.1530/ERC-16-0043
Discussion
The results of this study confirm the presence of EIF1AX mutations in PTC and ATC and demonstrate for the first time that these mutations also occur in benign thyroid nodules. Furthermore, we show that EIF1AX mutations may coexist with other driver mutations such as RAS and TP53, which is found more frequently in malignant nodules.
The frequency of EIF1AX mutations in PTC found in this study is comparable to that reported by TCGA (Cancer Genome Atlas Research 2014c), indicating that EIF1AX mutations are reproducibly found in 1–2% of these tumors. Most of the EIF1AX-mutated PTCs (5/6 cases; 83%) in TCGA study were registered as follicular variants (Gao et al. 2013). The sixth PTC case also showed follicular components, but with predominant classic PTC features explained by the presence of BRAF V600E mutation in addition to KRAS Q61K and EIF1AX mutations. Similarly, in our study all three PTCs were follicular variants, and all were encapsulated. This indicates that the follicular variant of PTC, typically encapsulated, is a typical phenotype of EIF1AX-mutated PTC.
Since the follicular variant of PTC is known to share genetic alterations with other follicular-patterned thyroid tumors, that is, FTC and FA (Nikiforov & Nikiforova 2011), it was important to understand if EIF1AX mutations also occur in FTC and FA. Our analysis revealed no EIF1AX mutations in any of 53 FTC, but identified them in 7% of FA. Even though a complete absence of EIF1AX mutations in FTC cannot be ruled out, these results suggest that the prevalence of EIF1AX mutations in FTC is likely to be low. The finding of these mutations in FA raises a theoretical possibility that EIF1AX-mutated FA have a propensity to progress to the follicular variant of PTC rather than to FTC.
EIF1AX mutations appear to frequently coexist with other mutations, particularly RAS. In this study, co-occurrence of NRAS mutation was observed in three of four EIF1AX-mutated cancers, but not in any benign thyroid nodules. Similarly, combination of EIF1AX and RAS mutations was observed in previously reported PTC and ATC (Cancer Genome Atlas Research 2014c, Kunstman et al. 2015), and in other types of epithelial cancer and melanomas (Cerami et al. 2012, Gao et al. 2013, Cancer Genome Atlas Research 2014a, b). This raises a possibility that EIF1AX mutations alone are not sufficient for full transformation, but requires other mutations, particularly RAS, for progression to overt malignancy.
We identified a total of 33 samples harboring EIF1AX mutations that were clustered at exon 2 and intron 5 splice site. The two consensus nucleotides within intron 5 (c.338-1A, c.338-2G) immediately upstream to the exon 6 splice site is the most common and recurrent hotspot for mutations. Of interest, four out of six thyroid nodules carrying this mutation were cancers in our study. Similar hotspot splice site mutation was also reported in two of six PTCs in TCGA study and two of four ATCs by Knutsman et al. (Cancer Genome Atlas Research 2014c, Kunstman et al. 2015, Chai et al. 2016). The EIF1AX A113_splice mutation affects the splicing of EIF1AX transcripts at the boundary of the helical domain within the C-strand and the highly charged unstructured CTT downstream involved in protein–protein interactions (Saini et al. 2010). Residues in the NTT and CTT of eIF1A act in opposite manner on scanning and start codon recognition and mutations in both NTT and CTT residues have been reported to impair preinitiation complex (PIC) assembly (Fekete et al. 2005, Saini et al. 2010). Nevertheless, an exact functional significance of codon 113 splice-site mutation that preserves RNA-binding domain, but altering the C-strand and CTT domains remains unclear.
Other common hotspot for EIF1AX mutations is exon 2, which codes for the unstructured NTT domain. Codon 9 mutations are most commonly affected at this site. Mutations involving codons 13, 10, 15, and intron 1/exon 2 splice site were also identified in this study, and those have not been previously reported in thyroid tumors. Mutations in exon 2 coding for the NTT segments were found to promote an open scanning conducive conformation of PIC leading to leaky scanning, but these mutations were located between residues 17–21 of the gene (Fekete et al. 2005, Saini et al. 2010). In this study, all nodules carrying a mutation affecting exon 2 were benign. However, exon 2 mutations were found to occur in four PTC in the TCGA study (Cancer Genome Atlas Research 2014c). Overall, the available data suggest that the EIF1AX p.A113_splice mutation confers a higher risk of cancer in a given nodule than the exon 2 mutations.
It is important to stress that not all patients with thyroid nodules underwent surgery, and therefore the histologic outcomes for those nodules were not available. This represents a limitation of the study.
In summary, we report here the occurrence of EIF1AX mutations not only in thyroid cancer, but also in benign thyroid nodules, and demonstrate that phenotypically these mutations are associated with the encapsulated follicular variant of PTC and benign follicular-pattern nodules. Furthermore, the presence of A113_splice mutation, particularly in combination with RAS mutation, is more frequently associated with thyroid cancer than isolated EIF1AX mutations or mutations located at other hotspots of the gene.
Declaration of interest
Y E N is a consultant for Quest Diagnostics. All other authors have nothing to disclose.
Funding
This work was supported in part by the NIH grant CA88041, funds from the University of Pittsburgh Cancer Institute and University of Pittsburgh Medical Center, and by the Richard A and Leslie A Snow Fund for Thyroid Cancer Research.
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