Atypical Papillary Thyroid Carcinoma with Squamous Metaplasia and RET/NRAS/TERT/PIK3CA Mutations: A Case Report

Background: Papillary thyroid carcinoma (PTC) with squamous metaplasia is a relatively rare and special subtype adenocarcinoma of thyroid which involves multiple genetic changes. We reported a rare case of atypical PTC with squamous metaplasia and RET/NRAS/TERT/PIK3CA mutations which was conrmed after surgical resection pathologically. Case summary: A 2.5×2.5×2 cm 3 , smooth, hard, clear boundaries and solid nodule on the left thyroid gland was found on relevant physical examination of the patient. And then, the unilateral radical resection of thyroid carcinoma was performed after diagnosing. The tumor cells were squamous metaplasia and arranged in structures with diffuse growth pattern microscopically. At high magnication, stretched nucleus, nuclear grooves and internuclear pseudoinclusions in tumor cells were detected, and the follicular epithelium cells were atypical. Immunohistochemical staining shown strong positiveness of CK19, TTF-1, P40 and Galectin-3, partial positiveness of TG of tumor cells and negativeness of Calcitonin, which could exclude medullary thyroid carcinoma (MTC). Furthermore, rst generation sequencing of 18 PTC related genes techniques shown RET, NRAS, TERT and PIK3CA was mutated. Conclusion: Genetic detection is vital to the diagnosis of thyroid adenocarcinoma, especially for the PTC with atypical morphology or rare metaplasia are sensitive to group I


Introduction
Papillary thyroid carcinoma (PTC) with squamous metaplasia is not only a very rare and special subtype adenocarcinoma of thyroid, but also belongs to diffuse sclerosing variant papillary thyroid carcinoma (DSVPTC) which is considered a poor prognosis (1)(2)(3)(4). DSVPTC is recognized by prominent squamous metaplasia, innumerable psammoma bodies, in a background of lymphocytic thyroiditis and stromal brosis (3). To our knowledge, only dozens of relevant literature about PTC with squamous metaplasia or DSVPTC have been published in National Center for Biotechnology Information (NCBI) database so far, and mostly focus on pathologic ndings since rst authoritatively introduced at 1987 (5, 6). DSVPTC has different clinical, pathological and molecular pro les when compared to conventional PTC (7). DSVPTCs should be considered high-risk PTCs because of high propensity for tumor invasion, metastasis, relapse and mortality. Aggressiveness of DSVPTCs might be related to a different molecular pathway than that in conventional PTCs (8). On genetic analysis, the occurrence of BRAF and RAS mutations are uncommon events in DSVPTC and activation of RET/PTC rearrangements are common (9). Genetic detection is not only crucial to the diagnosis for PTC, and also used to assess the prognosis (10,11). When the morphology is atypical, genetic detection can be of great help to pathologists. Here, we used a rst generation sequencing of thyroid carcinoma containing 18 genes techniques as a support to diagnose the case of PTC with squamous metaplasia after histology and immunohistochemisty, and reported an unique typical papillary thyroid carcinoma with squamous metaplasia and RET/NRAS/TERT/PIK3CA mutations.

Case Description
This is a case of a 60-years-old male who had diagnosed with diabetes 7 years ago, treated with metformin orally, and presented with a left anterior neck mass more than 1 year ago inadvertently. No pain, redness, fever, hoarseness, dysphagia, dyspnea, excitability, irritability and hunger was found during the period, and the patient did not go to the hospital and receive any treatment. The patient noticed an enlargement of the mass recently, and then came to our hospital for further treatment. There was no abnormal carotid pulse, venous engorgement, and hepatojugular re ux on relevant physical examination.
However, a 2.5×2.5×2 cm 3 , smooth, hard, clear boundaries and solid nodule on the left thyroid gland was found. The patient had no familial genetic, psychosocial and exposure to radiation and toxins history. The mass was about 3×2×2cm 3 in size with a 0.2 cm capsule of the left thyroid. The histological characteristics were sclerosing with clear boundaries by the capsule (Fig. 3A). At low magni cation, the extensive vitri cation, calci cation, and necrosis was in the background, and the tumor cells were squamous metaplasia, scattered, and arranged in clusters with diffuse growth pattern (Fig. 3B). Besides, some tumor cells and psammoma bodies have in ltrated to the capsule (Fig. 3C). At high magni cation, the tumor cells were enlarged and shown stretched nucleus, the follicular epithelium cells of thyroid were atypical (Fig. 3D). Furthermore, the nuclear grooves (Fig. 3E) and internuclear pseudoinclusion (Fig. 3F) was detected.
The patient remained under careful observation by ultrasonic examination follow-up and treated with levothyroxine sodium tablets orally. and there was no recurrence or metastasis in the 6 months follow-up. The patient got appropriate perspective including the assessment and the episode of care in every 3 months. No adverse and unanticipated events happened during the period. Finally, the patient was satis ed with the treatment plan, process and prognosis, and will continue to follow up as prescribed by the doctors.

Discussion
DSVPTC is an uncommon variant of PTC which is macroscopically involved the thyroid extensively without forming a dominant mass, and microscopically revealed extensive brosis, squamous metaplasia and numerous psammoma bodies (12). But in this case, we macroscopically got a distinct mass with an incrassated capsule of the left thyroid.
Histopathology of DSVPTC always shows the numerous psammoma bodies, lymphoplasmacytic in ltrates with germinal centre, brous stroma, squamoid differentiation and nuclear characteristic of PTC (6, 13). In this case, the extensive vitri cation, calci cation, and necrosis was in the background, and the tumor cells were squamous metaplasia, scattered, some arranged in clusters with diffuse growth pattern. Besides, The tumor cells were atypical except the nuclear grooves and internuclear pseudoinclusion. Histology of this case seemed impossible to make an accurate diagnosis because of the morphometric atypism. Immunohistochemical staining results supported the thyroid primary adenocarcinoma, nonetheless, we still needed the genetic detection to distinguish the subtype of PTC.
BRAF mutation was signi cantly associated with increased cancer-related mortality among patients with PTC which support further investigation of the prognostic and therapeutic implications of BRAF status in PTC (14,15). Several drugs have been developed, which inhibit signaling kinases or oncogenic kinases (BRAFV600E, RET/PTC), such as those associated with platelet-derived growth factor receptor and vascular endothelial growth factor receptor (16). But in this case, BRAF mutation was not found in PTC formalin-xed, para n embedded tumor tissues.
Alterations of RET gene or protein have been found in diverse thyroid cancer subtypes, and observed in PTC, which result in RET fusion products (17). RET variant c.2071G>A (p.G691S) have been described in the general population as well as in patients with MTC and with Hirschsprung syndrome (18). MTCs produce calcitonin, measurement of which indicates the presence of tumor in at-risk individuals and the effectiveness of therapy in treated patients (19). In this case, the negativeness of Calcitonin could exclude the diagnosis of MTC, besides, there was no Hirschsprung syndrome in this patient. p.G691S also could act synergistically in the development or progression of follicular thyroid cancer (FTC) (20). But in this case, we were unable to diagnose the FTC because of the histopathology. Signi cantly, this is the rst description of a p.G691S mutation in association with DSVPTC. Furthermore, NRAS and TERT mutations are related to higher PTC aggressiveness, and are potential use in diagnostics in PTC patients (21,22). Mutational activation of PI3K signaling, through mutational activation of PIK3CA or loss of PTEN, are well described in aggressive thyroid cancer (23). In this case, we found the mutations of RET, NRAS, TERT and PIK3CA gene, it was useful for the diagnosis of PTC. Taking together, RET, NRAS, TERT and PIK3CA mutation happened on the same patient, and combined with histopathology and immunohistochemical results, it would be more reasonable to diagnose PTC although its histology was not typical. But we did not use the next generation sequencing to detect the gene mutation, that was the limitation of this case.
In summary, we presented an unusual and rare case of PTC with squamous metaplasia and RET, NRAS, TERT and PIK3CA mutation which was rst reported. Although the tumor has characteristic histological features, awareness is important for its diagnosis. Therefore, genetic detection is vital to the diagnosis of thyroid adenocarcinoma, especially for the PTC with atypical morphology or rare metaplasia.

Declarations
Ethics approval and consent to participate Informed consent was obtained in this case, and protocols were approved by the Ethics approval of Chongqing University Cancer Hospital. The patient provided informed consent for the publication of this report and any accompanying images.

Consent for publication
The patient consented all the individual person's data to publish.

Availability of data and materials
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that there is no con ict of interest.

Funding
No funding information.
Author's contributions ZL contributed to acquisition, analysis and interpretation of patient data and the drafting of the manuscript. DG and YF contributed to the acquire the ultrasonic examination data. JZ contributed to the immunohistochemistry and molecular pathological methods. QJ and HH gave the nal approval of the report. All authors read and approved the nal manuscript.