Molecular heterogeneity of clinically diverse cases of follicular thyroid carcinoma in next-generation sequencing: case report

INTRODUCTION

Thyroid cancer is the most common endocrine malignancy globally and its incidence has tripled over the past 3 decades.^1-3 Follicular thyroid carcinoma (FTC) is the second most common histological type after papillary thyroid cancer (PTC) followed by medullary thyroid carcinoma (MTC), poorly differentiated thyroid carcinoma (PDTC), and anaplastic thyroid carcinoma (ATC). FTC is classified into three subtypes.⁴ The encapsulated angioinvasive (eaFTC) and minimally invasive (miFTC) subtypes are often associated with an excellent prognosis. Current treatment strategies with surgical extirpation of loco-regional disease, TSH suppressive thyroxine, and 131-I radioactive iodine (RAI) therapy have shown favorable outcomes. Whereas, the widely invasive (wiFTC) subtype has a propensity for distant metastases through the hematogenous spread, refractoriness to RAI therapy, and a higher morbidity and mortality rate. Thus, patients harboring FTC have clinically diverse courses. It is likely that besides histological grade, certain gene alterations could contribute to the aggressive biological behavior of the tumor. However, there is a paucity of data on the genomic characteristics of FTC. In particular, India lacks comprehensive genomic data on FTC. Moreover, the clinical relevance of genomic characteristics of FTC is insufficiently studied. The molecular mechanisms underlying FTC cancer initiation and progression are poorly understood. Hence, it is pertinent to evaluate the molecular profile of FTC and correlate it with the diverse clinical courses to facilitate informed treatment decisions. Recent developments in molecular technology have led to a better understanding of tumor biology. Next-Generation Sequencing (NGS) is a high-throughput sequencing technology that allows thousands of genes of interest to be analyzed simultaneously. Hence, we investigated the genomic and transcriptomic landscape of follicular thyroid carcinoma using the NGS illumina Novaseq 6000 platform in two patients with diverse clinical courses.

CASE REPORT

Case 1

A 21-year-old woman presented with a progressive neck swelling of 6 months duration and occasional dysphagia. There was no high-risk feature including childhood exposure to ionizing radiation, family history of cancer, rapid increase in size, or voice change. Neck ultrasound showed a hypoechoic solid nodule about 3×2 cm in the right lobe of the thyroid gland and no significant lymphadenopathy. Cytology suggested nodular colloid goiter, Bethesda category II. Total Thyroidectomy (TT) was carried out. Surprisingly, histopathology revealed minimally invasive follicular thyroid carcinoma (Figure 1). The postoperative baseline thyroglobulin level was 12 ng/mL and she received 72 mCi of 131-I radioactive iodine therapy. 131-I post-therapy whole-body scan revealed no residual thyroid lesion nor any evidence of distant metastasis. The patient receives suppressive thyroxine therapy under regular follow-up.

Case 2

A 59-year-old man presented with multiple lytic lesions of the spine and incidental thyroid nodule. Computed Tomography-guided biopsy of the vertebral lesion revealed follicular thyroid cells suggesting distant metastasis from the thyroid primary. Neck ultrasound showed a highly vascular solid hypoechoic nodule 1×2 cm in the left lobe of the thyroid and guided-cytology revealed follicular thyroid neoplasm-Bethesda IV. A provisional diagnosis of thyroid malignancy with distant vertebral metastases was made and TT was planned in a multidisciplinary tumor board. He was a known case of systemic hypertension for the past 15 years on regular medications including two classes of antihypertensives (Tab Telmisartan 40 mg/day, tab amlodipine 5 mg/day) and anti-platelet medication-Tab. Aspirin 75 mg/day. Echocardiography revealed left ventricular hypertrophy and diffuse global hypokinesia. The ejection fraction was 58%. Given malignancy and worsening bone pain, the patient was subjected to surgery under general anesthesia ASA class III after adequate blood pressure control and withdrawing aspirin for 7 days. On induction of anesthesia, blood pressure rose from 140/90 mmHg to as high as 210/110 mmHg. Intraoperative hemodynamics were stabilized, and TT was performed. Given the absence of cervical lymphadenopathy, lymphadenectomy was not performed. The extubation and postoperative recovery were uneventful. Histopathological Examination (HPE) of the surgical specimen revealed wiFTC (Figure 2). The patient was referred to a nuclear physician for 131-I radioactive iodine therapy and 131-I post-therapy whole body scan. The patient received TSH suppressive thyroxine therapy. However, the patient developed cor-pulmonale and died of respiratory failure within 2 months after surgery.

Targeted whole exome sequencing

For DNA & RNA samples

Live thyroid tissue samples (roughly 0.5×0.5 cm) harvested during thyroid surgery were immediately immersed in RNA-protect solution, transported, and stored at -80℃ for targeted exome sequencing at MedGenome, Bengaluru, India under stringent conditions.

DNA/RNA extraction & sample QC

DNA was extracted for all samples using Qiagen DNeasy Mini Kit (Qiagen, Cat# 69504). The DNA samples were quantified using DNA Assay BR (Invitrogen, Cat# Q32853). DNA purity was checked using QIAxpert and DNA integrity was checked on 1% agarose gel. RNA was extracted by using Trizol Method from the above samples. The RNA samples were quantified using Qubit RNA BR Assay (Invitrogen, Cat# Q10211). RNA purity was checked using QIAxpert, and RNA integrity was checked using RNA Screentapes (Agilent, Cat# 5067-5576).

DNA/RNA library preparation and sequencing protocol

Custom targeted library preparation for Illumina was carried out. DNA or cDNA samples were processed and enriched using gene specific probes for final DNA library. The libraries were then checked for fragment size distribution on Tape Station using D1000 DNA Screentapes (Agilent, Cat# 5067-5582) or on Fragment Analyzer using HS NGS Fragment Kit (1-6000bp) (Agilent, Cat# DNF-474-1000) before loading on Illumina Novaseq 6000 instrument to generate ~250X, 150bp paired-end reads. Bioinformatic analyses were done and compared against reference sequence databases available at NCBI library.

QIAseq tumor mutation burden panel summary

QIAseq tumor mutation burden (TMB) panel by Qiagen is a UMI-based panel for estimating mutation load in a sample. The analysis is done using CLC Genomics. Samples are analyzed after alignment using QIAseq Tumor Mutation Burden (TMB) panel (1.3 Mb) which covers 486 genes. The panel just not targets the hotspot regions but the entire CDS for these genes in general.

Targeted exome sequencing

The total data generated for each sample ranges from 5 Gb to 6.1 Gb. The overall alignment and the passed alignment percentage (alignment to hg38) in all the samples are above 90%, respectively. The average panel depth for each sample ranges from 391X to 2294X. The sequence read of both the FTC samples has been processed and archived in the SRA database of NCBI library with BioProject accession number PRJNA901638. Raw data analysis is summarized (Table 1). The distribution of overall pass-ontarget variants and non-synonymous annotated variants are given in Table 2 and Table 3 respectively.

DNA-based genomic analysis in 2 FTC cases revealed unique somatic mutations in BRAF, NTRK, and ALK genes (Table 4). Case 1 exhibited (i) RET mutation - Arg982Cys and (ii) NTRK1 mutation – His604Tyr which were not seen in case 2. Whereas, case 2 harbored a unique BRAF mutation ch7:140734758T>A, which was not seen in case 1. However, the classic BRAF V600E nor the rarer BRAF K601E mutants, which are associated with PTC were not detected. Mutations observed in ALK and NTRK2 genes were common in both cases. RNA-based transcriptomic analysis revealed gene rearrangements including RAF1-CHSY3, TMBIM1-NOTCH2, and MED12-IRF2BPL in Case 2 (Table 5).

DISCUSSION

Our NGS detected novel gene rearrangements including RAF1-CHSY3, TMBIM1-NOTCH2 and MED12-IRF2BPL in the lethal FTC case but none in case 1. Little information exists on these novel fusions. However, studies have shown the RAF-MEK1/2-ERK cascade is the key signal transduction pathway, dysregulated in thyroid carcinogenesis, in keeping with our observations. RAF1 (rapidly accelerated fibrosarcoma) has been shown to promote the survival of thyroid cancer cells independent of ERK pathway activation, offering new perspectives on targeted therapy.^5,6

Oncofetal chondroitin sulfate, is exclusively expressed in the placenta, and chondroitin sulfate synthase 3 (CHYS3) overexpression is associated with poor prognosis in many solid cancers and is a promising target for immunotherapy.⁷ Transmembrane Bax Inhibitor Protein Motif (TMBIM) 1 encodes for an evolutionarily conserved hydrophobic protein family which regulates calcium homeostasis and apoptosis.^8,9 Dysregulation of TMBIM protein has been implicated in cancer progression. Notch Signaling cascade regulates cell fate decision, proliferation, and differentiation of vast array of cells. Constitutive activation of NOTCH signaling by point mutations or chromosomal translocation is involved in many human cancers including T-cell leukemia, pancreatic and breast cancer.¹⁰ Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes for a protein that acts as transcriptional activators.¹¹ Mutations of Mediator of RNA polymerase II transcription subunit 12 homolog (MED12) have been reported in 15% of PDTC patients with fatal outcomes, and are associated with tumor virulence.^12,13 It is plausible that the accumulation of these fusion genes could have contributed to the fatal outcome of FTC case 2. However, Classical RAS mutations and PAX8-PPARγ gene rearrangements common in FTC were not detected in our patients, which could possibly be attributed to ethnic and geographic variations. Previous reports have shown that PAX8-PPARγ gene rearrangements are uncommon in FTC among Asian population, corroborating with our findings.^14,15 Nevertheless, unique somatic mutations involving ALK, NTRK1 and NTRK2 genes were identified in both cases of FTC. A unique BRAF mutation, ch7:140734758T>A was identified in lethal FTC case 2, who had an advanced histopathological profile and systemic metastases. This somatic mutation has been associated with cardiomyopathy.

Likewise, our patient developed cardiomyopathy, cor-pulmonale and died of respiratory failure. Moreover, the patient harbored multiple gene fusions. Studies have shown that fusion-positive thyroid cancer exhibited heightened tumor virulence with higher rates of mortality.^16,17 Whereas, miFTC case 1 without fusion genes displayed a less aggressive course. Studies have shown that mutation burden was associated with a worse prognosis, independent of histological classification.⁴ Hence, it is likely that besides the clinicopathological profile, the molecular heterogeneity of FTC could contribute to the diverse course. The genomic landscape of FTC is less well-described. Recent studies have demonstrated highly heterogenous mutations involving oncogenes (MDM2, FLI1), transcription factors and repressors (MITF, FLI1, ZNF331), epigenetic enzymes (KMT2A, NSD1, NCOA1, NCOA2), and protein kinases (JAK3, CHEK2, ALK) in FTC cases.^18,19

A few studies have demonstrated FTC-specific driver genes including DICER1, EIF1AX, KDM5C, NF1, PRDM1, PTEN, and TP53 mutations and fusion genes involving THADA.⁴ However, these mutations were not detected in our patients. Nevertheless, the unique somatic mutations present in our FTC patients have not been reported previously as pathogenic in the Cancer Genome Atlas (TCGA), Catalogue of Somatic Mutations in Cancer gene census (COSMIC), and BROCA-curated thyroid cancer susceptibility genes (tests.labmed.washington.edu/BROCA).^20,21 Thus, the molecular profile of FTC is highly heterogeneous, potentially attributing to clinicopathological and ethnic diversities. Fusion-positive FTCs are likely to have an aggressive course with a higher mortality rate. The study highlighted that NGS application can facilitate the identification of distinctive molecular signatures in the subset of patients with advanced, refractory, and progressive diseases who may benefit from newer targeted therapies.

CONCLUSION

In conclusion, our study utilizing Next-Generation Sequencing in follicular thyroid carcinoma revealed a heterogenous molecular profile, diverging from Western literature by the absence of RAS mutation and PAX-PPARG. Nevertheless, unique somatic mutations in BRAF, NTRK, and ALK genes, akin to those in other Asian populations signify the geographic and ethnic diversity. Novel gene rearrangements including RAF1-CHSY3, TMBIM1-NOTCH2, and MED12-IRF2BPL identified in the lethal FTC case, are potential drivers of aggressive disease behavior. These molecular insights highlighted the importance of NGS in identifying therapeutic targets for personalized therapies, particularly in advanced FTC cases, thus aiding prognosis and targeted therapy development. Further research is crucial to validate these observations and enhance our understanding of FTC biology and treatment strategies.

Funding: Indian Council of Medical Research extramural grant

Conflict of interest: None declared

Ethical approval: Not required

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