Clinicopathological features of two cases of ETV6-NTRK3 rearranged papillary thyroid carcinoma: a case report

Rearrangements involving the neurotrophic-tropomyosin receptor kinase (NTRK) gene family (NTRK1, NTRK2, and NTRK3) have been identified as drivers in a wide variety of human cancers. However, the association between NTRK rearranged thyroid carcinoma and clinicopathological characteristics has not yet been established. In our study, we retrospectively reviewed medical records of thyroid cancer patients and identified 2 cases with NTRK rearrangement, no additional molecular alterations were observed in either of these cases. The fusion of the rearrangement in both cases was ETV6(E4)::NTRK3(E14). By analyzing the clinicopathological features of these two cases, we found that both were characterized by multiple tumor nodules, invasive growth, and central lymph node metastases, indicating the follicular subtype of papillary thyroid carcinoma. Immunohistochemical staining profiles showed CD56-, CK19+, Galectin-3+, HBME1+. These clinicopathological features suggest the possibility of ETV6-NTRK3 rearranged thyroid carcinoma and highlight the importance of performing gene fusion testing by FISH or NGS for these patients.


Introduction
Thyroid cancer ranks as the ninth most common cancer worldwide.Despite significant advancements in the management of thyroid cancer, there remain critical challenges in its diagnosis and treatment, leading to poor overall survival rates for certain aggressive subtypes and patients with metastatic thyroid cancer.Accurate diagnosis of thyroid cancer is crucial for guiding appropriate treatment and patient management.Over the past 15 years, the significance of molecular biology in thyroid pathology has not only revolutionized the field but also underscored the intrinsic value of classical histopathology.Pathologists have long identified patterns indicative of specific molecular changes, but the incorporation of molecular tools into their toolkit has bolstered our capacity to prognosticate and predict the effectiveness of targeted treatments.In the new fifth edition of the World Health Organization (WHO) histologic classification of thyroid neoplasms, the classification of thyroid tumors has evolved based on classic histopathology and molecular pathogenesis (1).
Papillary thyroid cancer (PTC) is the most common type of thyroid cancer in China, accounting for over 90% of all thyroid cancer cases (2).The prognosis and treatment of thyroid cancer depend on the tumor type and its stage.Early diagnosis and appropriate treatment can significantly reduce mortality rates and improve prognosis.Apart from age, tumor size, and lymph node involvement, the behavior of PTC is also influenced by the morphological subtypes of genetic mutations (3).Certain subtypes, such as the tall cell subtype (4), the columnar cell subtype (5), and the recently identified hobnail subtype are associated with more aggressive behavior and poorer overall survival (6).In recent years, there has been increasing evidence of molecular alterations that correspond to these pathological subtypes.The Cancer Genome Atlas (TCGA) conducted a study on 496 PTCs, confirming the significance of mutations in BRAF and RAS genes, as well as fusion mutations in RET and NTRK1.The study also revealed correlations between morphology and genetic alterations, with BRAF V600E -positive tumors being associated with the conventional and hypercellular variants of PTC, while RAS-positive tumors were linked to the follicular variant of PTC.However, the clinicopathological association of NTRK-rearranged thyroid carcinoma has not yet been established (7).
Rearrangements involving the neurotrophic-tropomyosin receptor kinase (NTRK) gene family, namely NTRK1, NTRK2, and NTRK3, have been identified as drivers in a wide range of human cancers (8)(9)(10).These rearrangements result in NTRK gene fusions with various partner genes, which confer oncogenic potential by generating chimeric Trk proteins.These proteins constitutively activate kinase function, leading to the downstream stimulation of cellular proliferation through the RAS/RAF/MAPK pathway and PI3K-AKT signaling pathways (11,12).Larotrectinib and entrectinib, both tyrosine kinase inhibitors (TKIs), have been proven to be safe and effective treatment options for patients with NTRK fusion-positive solid tumors.Studies have shown that larotrectinib and entrectinib yield significant responsiveness and minimal primary resistance in thyroid tumors (13-17).However, due to the high cost of testing, not all patients with thyroid cancer who may have an NTRK rearrangement can be tested and treated.In this report, we present the clinicopathological data of two patients with ETV6-NTRK3, which may provide valuable insights for identifying this specific type of thyroid cancer.

Patient selection
For this study, we retrospectively reviewed medical records of thyroid cancer patients treated at our center from January 2021 to December 2022.Two patients were identified from the hospital pathology database.Inclusion criteria were: histologically confirmed thyroid cancer, availability of formalin-fixed paraffin-embedded (FFPE) tissue blocks, complete clinical and pathological data, the molecular pathologic test results were negative for BRAF mutation.Patients with a history of other malignancies, inadequate tissue specimens were excluded.This study was approved by the institutional ethics committee.

Fluorescence in situ hybridization
FISH analysis was conducted on FFPE sections using commercially available break-apart probe sets (NTRK3) from Guangzhou LBP Medicine Science & Technology Co., Ltd.(Guangzhou, PRC.).Fifty interphase nuclei were analyzed.The FFPE specimen underwent standard FISH pretreatment, hybridization, and fluorescence microscopy following specimenspecific protocols.The FISH analysis was independently evaluated by two qualified clinical cytogenetic technologists and interpreted by a board-certified (ABMGG) clinical cytogeneticist.

Clinical and staging parameters
Clinical and staging parameters are summarized in Table 1.The two patients were 32-year-old and 57-year-old women, both were euthyroid, none of the patients with available information had a family history of thyroid cancer or personal history of prior irradiation.Fine needle aspirate biopsy (FNAB) findings were not available in them.both patients underwent total thyroidectomy and a formal central compartment dissection.
The tumor size of the two cases was 1.5cm and 1.2 cm respectively, both had separate foci of papillary thyroid carcinoma and without extrathyroidal extension.Nodal metastases were noted in both patients with lymph node sampling (1/5 and 5/5 respectively); None of the patients received radioactive iodine.Both patients were free of disease at time of last follow-up (25 and 23months), satisfied with the treatment results, in good mental condition, and showed no signs of anxiety or depression.

Histological, immunophenotypic, and molecular characterization
Both cases were diagnosed as the follicular variant of papillary thyroid carcinoma (FVPTC) at the time of diagnosis.They were characterized by the presence of multiple tumor nodules, invasive growth, and central lymph node metastases.Histopathological features of the two cases including: a combination of papillary and follicular growth patterns.The tumor is composed of small-to normal-sized follicles and has nuclear features of papillary thyroid carcinoma showing enlarged nuclei, irregular nuclear membranes, and chromatin clearing and glassy nuclei (Figure 1).

Discussion
Papillary carcinoma is the predominant pathological type of thyroid cancer in the Chinese population, accounting for over 90% (2).In addition to clinical staging, the behavior of papillary thyroid carcinoma (PTC) is also influenced by its pathological subtypes or genetic variants.In our study, we retrospectively reviewed medical records of thyroid cancer patients treated at our center from January 2021 to December 2022 and identified two of the patients showed NTRK rearrangement in their tumor tissues, specifically the ETV6-NTRK3 rearrangement, both cases had the fusion of exon 4 of ETV6 to exon 14 of NTRK3, ETV6(E4)::NTRK3(E14), and were classified as follicular variant of papillary thyroid carcinoma (FVPTC).The correlation between these clinicopathological features and the molecular subtypes of ETV6-NTRK3 re a r r a n g e m e n t s s u g g e s t s t h a t p a t i e n t s wi t h s i m i l a r clinicopathological features, especially those who are negative for BRAF V600E mutation, should undergo further FISH or ETV6-NTRK3 targeted sequencing.
In our study, we applied immunohistochemical staining with CD56, HBME-1, CK19 and Galectin-3 for distinction of follicular variant of papillary carcinoma from follicular adenoma (FA) and follicular carcinoma.CD56 is a neural cell adhesion molecule (NCAM) that is normally expressed in thyroid follicular cells.Reduced expression of CD56 is associated with malignant tumors.Its expression is decreased or completely absent in papillary thyroid  carcinoma, follicular carcinoma, and anaplastic carcinoma.CK19 expression is significantly higher in classical PTC and follicular variant PTC than in follicular thyroid carcinoma (FTC) and follicular adenoma, so it can be used to distinguish between thyroid papillary carcinoma and follicular adenoma.Galectin 3 plays a role in cell apoptosis regulation and cell movement, and is involved in the progression of thyroid cancer.HBME-1 expression in follicular variant PTC is significantly higher than in follicular adenoma or follicular carcinoma.The best combination for FVPTC cases was HBME-1, CD56, CK19 and Galectin 3, with a sensitivity reaching 91.1% (27).
FISH can detect large structural variations at the DNA level and is commonly used in clinical laboratories to detect oncogenic fusions in solid tumors.In theory, split probes have sufficient sensitivity and specificity for chromosomal abnormalities, but there are practical technical considerations in interpreting split FISH assays.Shorter split lengths are difficult to distinguish from split lengths in some normal cells, which can lead to false-negative results (28).Additionally, while positive results from split probes indicate the presence of structural variations involving the probe gene, it cannot be determined whether the abnormality leads to functional transcriptional fusion.The advantages of FISH include the small amount of material required, typically only a few unstained slides (usually one unstained slide per examination), and a turnaround time of only a few days (29).
In recent years, the application of immunohistochemistry (IHC) in the detection of NTRK rearrangements has been widely discussed.Studies have observed the clinical and pathological characteristics of NTRK1/3 fusion PTC, examined the utility of pan-TRK IHC, and compared IHC with fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS).It was found that pan-TRK IHC has a sensitivity of 58.3% and a specificity of 100% for NTRK1/3 rearrangements in BRAF V600Enegative PTC.Pan-TRK IHC shows high specificity and moderate sensitivity for NTRK1/3 rearranged PTC, and should be interpreted with caution (30).
In conclusion, previous studies on NTRK-related thyroid cancers have demonstrated their molecular diversity, encompassing multiple subtypes, with papillary thyroid carcinomas (PTCs) being the most prevalent (31).Our study identified ETV6-NTRK3 as the most common fusion type in PTC among the Chinese population.The pathological and immunohistochemical characteristics indicated that the follicular variant of papillary thyroid carcinoma (FVPTC) was the predominant subtype.Both cases examined exhibited a multi-nodular histological appearance and displayed an early tendency for extensive lymphovascular dissemination.,These clinicopathological features suggest the possibility of ETV6-NTRK3 rearranged thyroid carcinoma and emphasize the importance of conducting FISH or targeted RNA next-generation sequencing (NGS) for these patients.One of the limitations of our study is its retrospective nature, as our patients did not receive TRK inhibitors to provide information on treatment response.Previous studies have demonstrated that ETV6-NTRK3 fusion tumors exhibit the highest sensitivity to entrectinib and larotrectinib, with minimal adverse effects (19).The incidence of grade 3 or 4 treatment-related adverse events, as defined by the Common Terminology Criteria for Adverse Events (CTCAE), is 15% (11,15).Therefore, correlation studies between ETV6-NTRK3 and clinicopathological characteristics are essential for identifying ETV6-NTRK3 rearranged thyroid cancer based on clinicopathological manifestations, thereby providing greater clinical benefits to these patients.

FIGURE 1
FIGURE 1 Histopathological features of ETV6-NTRK3 rearranged thyroid cancer.Tumors typically exhibited a combination of papillary and follicular growth patterns.(A) Case 1: The tumor displayed noticeable nuclear enlargement, clearing, and irregularities in the cell membrane (H&E, x200).(B) Case 2: The tumor showed follicular epithelium hyperplasia with crowded nuclei (H&E, x200).

TABLE 1
Clinical and staging parameters for ETV6-NTRK3 translocated papillary thyroid carcinoma.