Cell adhesion-related gene somatic mutations are enriched in aggressive papillary thyroid microcarcinomas

The incidence of differentiated thyroid carcinoma (DTC) is increasing rapidly in many countries, but its mortality remains low and stable [1, 2]. Approximately 50% of the increase in DTC is due to the identification of papillary thyroid microcarcinoma (PTMC), which is a thyroid papillary carcinoma (PTC) tumor ≤ 1 cm in diameter. Although most PTMC have an indolent clinical course, some are high-risk for aggressiveness and tumor invasion, metastasis, and patient mortality [3, 4]. Certain patient traits, such as being male, older than 45 years-of-age, having tumors > 5 mm that are multifocal with extrathyroidal extensions and lymphovascular invasion, are thought to be aggressive markers for PTMC [5, 6]. However, despite combining multiple clinical factors, the specificity and positive predictive value of these models are not sufficient for providing useful preoperative risk stratification [7]. Furthermore, many potentially important risk factors can only be determined postoperatively based on thyroid pathology [8]. Thus, molecular signatures may be used at presentation to predict whether a PTMC will be aggressive.

It was reported that the BRAF^V600E mutation is frequent in PTMC (30–67%) [9]. Recent studies suggest that BRAF^V600E-mutation-positive PTMC are more likely to be aggressive and have extrathyroidal tumor extensions, lymph node metastases, and recurrence [10], but studies suggest little use for BRAF mutation testing to stratify PTMC tumor risk as there is no correlation between BRAF-positive primary PTMCs and more aggressive or recurrent disease [11]. The discovery of TERT promoter mutations in thyroid cancer [12] were found to be useful, as 9.4% of PTC have TERT promoter mutations that are associated with older patients, higher MACIS scores and high risk of recurrence [13]. For PTMC, TERT promoter mutations are uncommon (4.7%) and not correlated with unfavorable clinical features [14].

Major genetic alterations in PTC have been evaluated with whole-exome sequencing (WES) by the Cancer Genome Atlas (TCGA) Research Network [13] and others [15]. Lately, several comprehensive and integrated analyses of thyroid cancer were reported across the Cancer Genome Atlas [16, 17]. However, to our knowledge, PTMC was not a focus of these studies. Thus, we require a better predictor for aggressive PTMC, and next-generation sequencing may accelerate clinical research in this regard by enabling analysis of genomes and identification of a gene mutation signature in aggressive PTMC.

We used WES for 16 matched PTMCs to investigate genetic alterations and biological consequences and targeted sequencing to verify these genes in additional aggressive PTMC samples. PTC may have evolved from PTMC that shared similar gene mutations. Aggressive PTMC had more mutational burdens and were enriched for specific sets of mutational genes related to cell adhesion, and this may be used to treat PTMC.

Increased DTC is thought to be tied to PTMC, which is a focus of thyroid cancer research. We lack a method for predicting aggressive PTMC, and this hinders patient prognosis and treatment. Genetic analysis yielded insights related to altered gene mutations, signaling pathways, and biological functions of PTC, but such analyses are rare. Therefore, by understanding molecular biological characteristics, we may be able to better treat PTMC. Here we offer data regarding genomic profiles of key genes related to PTMC aggressiveness.

We used next-generation sequencing to find the prevalence of mutations in a cohort of PTMC and to identify unique patterns. WES was applied to 16 paired clinical specimens of PTMC, 8 of which were invasive and metastatic. The other 8 had no invasion and metastasis (non-aggressive). First, PTMCs in our WES cohort had an 0.49 overall mutation burden, suggesting it is “quiet” like PTC in TCGA [13]. Second, the mutation frequencies of BRAF and NRAS in PTMCs were 50% and 6.2%, respectively. Our data agree with previous work to describe driver mutations of PTC in TCGA [13, 15]. Third, of the 234 mutated genes identified in PTMC, multiple biological functions and pathways altered at high frequency were associated with them. For example, regulation of small GTPase-mediated signal transduction, regulation of Ras protein signal transduction, cell adhesion and ECM-receptor interaction, hypertrophic cardiomyopathy, and focal adhesion were included. Data show several common pathways for genetic aberrations for the most mutated genes in PTMC and PTC [13, 15‐17]. Thus, we speculate that PTC evolved from PTMC because they share similar gene mutations and oncogenic signaling pathways.

Tumors arise, to a degree, from gene mutations, and the tumor mutation burden (TMB) is a measure of somatic mutations occurring in a tumor specimen. High TMB was associated with improved objective immune checkpoint inhibitor (ICPI) response and progression-free survival in lung and bladder cancer and for melanoma patients [18]. Therefore, TMB may be a genomic marker of prognosis and a predictor of treatment response [19]. To explore the relationship between TMB and aggressiveness, we analyzed differences in TMB between each group, and it was greater in the aggressive group, which is consistent with Robinson’s study regarding increased mutations for metastatic and primary tumors [20]. Therefore, the accumulation of mutations promotes PTMC aggressiveness and may be a focus of treatment.

We confirmed that BRAF was the most frequently mutated gene in PTMC and that it was associated with PTMC. Previously, BRAF mutations were associated with older age, tall-cell variants, extrathyroidal extensions, positive surgical margins, lymph-node metastasis, and stage IV disease [9, 10]. We found that BRAF^V600E was present in 8 tumor tissues (50%): 5 from the aggressive group and 3 from the non-aggressive group, indicating that BRAF mutations may not be correlated with invasion and metastasis. Because our study was limited to a small sample, validation of our preliminary data is required.

One particularly intriguing finding pertains to somatic mutations of cell-adhesion genes enriched in aggressive PTMCs. As cancer progresses and metastasizes, it often acquires new mutations. Core signaling pathways involved in nonsynonymous mutated genes (MUC5B, TNN, SSPO, PPFIA1, PCDHGA2, ITGA8, ITGA4, DCHS1, CRNN, ROCK1, RELN, LAMC2, and AEBP1) associated with cell and biological adhesion were identified in the aggressive group. Because tumor occurrence and development is dynamic and associated with adhesion, especially tumor cell migration [21], this may dictate the aggressiveness of PTMC. The targeted sequencing of the 13-gene panel in the subsequent expanded samples also verified the enrichment. Similar to ThyroSeq v3 [22], a DNA- and RNA-based next-generation sequencing assay that uses a genomic classifier (GC) to separate thyroid malignant from benign lesions, our 13-gene panel may have clinical utility for aggressive PTMC. Future research is needed with larger samples to confirm our preliminary data that cell adhesion related gene mutations can be used to predict PTMC aggressiveness and allow tailored treatment.

PTC may have evolved from PTMC due to sharing similar gene mutations, and the accumulation of such mutations promoted PTMC aggressiveness. Somatic mutations of 13 cell-adhesion genes were identified to be uniquely associated with PTMC aggression. These candidate gene mutations may offer novel diagnostic and treatment options.