Difficulties of Preoperative Diagnosis of Cribriform Morular Thyroid Carcinoma

Background Cribriform morular thyroid carcinoma has been recently renamed in the 2022 WHO classification as a thyroid tumor of uncertain histogenesis. The epidemiologic, pathological, and pathophysiological characteristics distinguish it from papillary thyroid carcinoma (PTC). Preoperative genetic testing plays a role in facilitating the differential diagnosis. Methods This report presents a confirmed case of cribriform morular thyroid carcinoma. Initially, fine-needle aspiration cytology suggested a diagnosis of PTC. However, a genetic analysis did not reveal the typical mutations associated with follicular-cell-derived neoplasms. Results A 31-year-old woman was found to have a thyroid nodule at the left lobe measuring 11.8 × 10.2 × 12.4 mm. Ultrasonography indicated a hypoechoic, solid nodule with regular margins. Cytology revealed a papillary structure of tall cells, leading to a PTC diagnosis. Nevertheless, the genetic analysis failed to detect mutations such as BRAF V600E, NRAS Q61R, NRAS Q61K, HRAS Q61R, or HRAS Q61K mutation or the fusion of CCDC6-RET, NCOA4-RET, PAX8-PPARG, ETV6-NTRK3, TPM3-NTRK1, IRF2BP2-NTRK1, or SQSTM1-NTRK1 in the aspirated follicular cells. The patient subsequently underwent total thyroidectomy with central lymph node dissection. Pathological examination revealed a cribriform pattern of spindle-shaped cells with morular areas. Immunohistochemical staining showed positive results for β-catenin and TTF-1, except in the morular regions, and negative results for PAX8, thyroglobulin, and BRAF (clone VE1). The diagnosis was confirmed to be cribriform morular thyroid carcinoma. Conclusion Significant cytological similarity exists between PTC and cribriform morular thyroid carcinoma. Preoperative genetic analysis is important to differentiate these two diseases. Cribriform morular thyroid carcinoma can be differentiated from common follicular-cell-derived tumors by the absence of typical mutations; the presence of nuclear and cytoplasmic expressions of β-catenin; the presence of TTF-1, except in morular areas; and the absence of thyroglobulin.


Introduction
In 2022, the World Health Organization (WHO) updated the pathologic classifcation of thyroid neoplasms.In this new classifcation, cribriform morular thyroid carcinoma has been designated as thyroid tumors of uncertain histogenesis [1].Previously, this tumor has been considered a variant of papillary thyroid cancer (PTC).However, the absence of common mutations associated with thyroid follicular cells and the lack of expression for thyroglobulin distinguish this disease from usual PTC.Te pathogenesis of cribriform morular thyroid cancer involves the Wnt/ β-catenin pathway, often associated with potential germline or sporadic APC mutations, and/or with functionally equivalent genes such as CTNNB1 [2,3] and AXIN1 [4].
Cribriform morular thyroid carcinoma is a relatively rare form of thyroid cancer, and it is more commonly reported in East Asian populations.Its prevalence is approximately 1.4% based on data from a case series in Taiwan [5].Te age of onset for this type of cancer tends to be comparatively lower, with the peak incidence occurring in the third decade of life (mean age: 26 years), which contrasts with PTC [4,6].Additionally, a female predominance was observed, with a female-to-male ratio of approximately 61 : 1 [4] and with an unclear association of radiation exposure [7].In cytological fndings, a signifcant similarity existed between PTC and cribriform thyroid carcinoma such as the presence of ground-glass nuclei and distinctive nuclear clearing or a papillary architecture featuring columnar or spindle cells [8].
It would be challenging to make a preoperative diagnosis of cribriform morular thyroid carcinoma.We describe a case of cribriform morular thyroid carcinoma presented as the fne-needle cytological diagnosis of PTC with absence of preoperative genetic testing of common mutations or fusions.

Case Presentation
Our case is of a 31-year-old woman who had no underlying disease.She discovered a thyroid nodule during a routine health examination and presented at our hospital for further investigation.She did not have history of radiation exposure.She had no history of colonic polyps or a family history of familial adenomatous polyposis (FAP) or thyroid cancer.She worked as an ofce lady.She never smoked, consumed alcohol, chewed betel nuts, or used any illegal substances.Laboratory tests indicated normal levels of thyroidstimulating hormone and free T4, but the anti-thyroidperoxidase antibody levels were higher than average (548 IU/ mL, reference range: <34 IU/mL).Tyroid ultrasonography revealed a heterogeneous echo texture in the thyroid gland and a solid, hypoechoic, wider-than-tall nodule measuring 11.8 × 10.2 × 12.4 mm in size with a smooth margin and no calcifcation (Figure 1(a)).According to the hypoechoic and solid features mentioned above, the Tyroid Imaging Reporting and Data System score was 4. Fine-needle aspiration cytology revealed papillary growth of tall and spindleshaped cells with elongated nuclei and an increased nucleusto-cytoplasm ratio (Figure 1(b)).Te cytology was classifed under Bethesda category VI, and the diagnosis was PTC.To screen for common genetic alterations, we employed the TyroSCAN Cancer Diagnostics Kit (Quak BioTechnology).Te analysis of the targeted genes did not reveal the presence of mutations such as BRAF V600E, NRAS Q61R, NRAS Q61K, HRAS Q61R, or HRAS Q61K mutations nor fusions of CCDC6-RET, NCOA4-RET, PAX8-PPARG, ETV6-NTRK3, TPM3-NTRK1, IRF2BP2-NTRK1, or SQSTM1-NTRK1 in the aspirated samples.In our diferential diagnosis, we considered cribriform morular thyroid cancer and the tall cell variant of PTC.No abnormal lymph node was observed in thyroid ultrasonography or neck computed tomography fndings.Subsequently, the patient underwent total thyroidectomy and central lymph node dissection.A pathological examination revealed a mixed pattern consisting of cribriform and morular features in the stratifed tall cells, along with evidence of angioinvasion and capsular invasion.Immunohistochemical staining indicated a positive result for β-catenin (cytoplasmic and nuclear expression) but negative results for thyroglobulin, PAX8, and BRAF (clone VE1).Te result for TTF-1 was also positive, except in the morular regions (Figure 2).Te fnal diagnosis was cribriform morular thyroid carcinoma measuring 1.2 cm in the maximal length without extrathyroidal extension, lymph node invasion, or distant metastasis, and it was categorized as stage I, pT1bN0M0.Te patient had a smooth postoperative recovery and was discharged without any complications.

Discussion
Tis case report proposes the diagnostic value of preoperative genetic testing with all-negative results in thyroid nodules classifed as malignant (Bethesda VI)-PTC.A recent mutational study of PTCs conducted by targeted RNA-based next-generation sequencing using a FusionPlex Pan Solid Tumor v2 panel in Taiwan identifed 86.8% (481/554) incidence of BRAF V600E mutation, 1.4% (8/554) of cribriform morular thyroid carcinoma, 11.2% (62/554) BRAF-negative PTCs that are mostly included in the 12-panel mutation of TyroSCAN except for 5 cases of BRAF fusions, 2 cases of KRAS Q61K, 1 case of ALK fusion, 1 case of FGFR1 fusion, and 9 cases of not detected mutation [5].Terefore, cribriform morular thyroid carcinoma would be considered as potential diagnosis in fne-needle cytology class VI with all-negative results of TyroSCAN genetic testing of thyroid nodules.
Molecular testing may be adopted to determine the malignancy risk associated with thyroid nodules.Among these tests, the detection of BRAF V600E mutation is highly specifc for identifying PTC.When combined with thyroid ultrasonography or cytology, BRAF V600E detection signifcantly enhances the diagnostic sensitivity for PTC [9,10].Preoperative use of molecular analyses has been demonstrated to accurately identify most papillary cell lesions, medullary thyroid cancer, and parathyroid tumors [11].Tis information can be invaluable in planning the extent of surgical interventions particularly for nodules classifed as Bethesda category V and VI [12,13].Nevertheless, molecular testing is still difcult to distinguish benignity from malignancy in follicular and oncocytic lesions [11].

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Case Reports in Endocrinology In terms of pathological features, cribriform morular thyroid carcinoma typically presents grossly with encapsulation and the formation of divided lobules inside.Microscopically, various growth patterns may be observed, including papillary, follicular, or cribriform ones.Te tumor cells are typically cuboidal, oval, or tall and exhibit prominent cytoplasm, hyperchromatic nuclei, and pseudostratifcation.At times, the tumor cells aggregate and form squamoid morules.Te squamoid morules may have nuclear clearing without features of PTC [14].Invasion into blood vessels and the capsule has been occasionally reported.Te presence of psammoma bodies is rare [4].Te Ki-67 index that is not required for diagnosis of cribriform morular thyroid cancer may show high despite excellent prognosis [4,15].Regarding immunohistochemistry, β-catenin expression is prominent in the nuclei or cytoplasm of cells in cribriform morular thyroid carcinoma, whereas in PTC, β-catenin is mainly expressed on plasma membranes [3,7].Furthermore, morular areas of cribriform morular thyroid carcinoma often lack TTF-1 expression but have CDX2, CD5, and CK5.Te cribriform regions also exhibit aberrant progesterone and estrogen receptor expression [1].On the other hand, PAX8 expression may not be negative.Some case reports revealed positive PAX8 in immunohistochemistry of the tumor cells [14,16].Additionally, more than 30% of cribriform morular thyroid carcinoma tumors harbor somatic APC mutations, whereas up to 50% of cancer cells harboring somatic APC mutations can be found in patients with germline APC aberrations.In terms of the underlying mechanisms, the APC protein is associated with glycogen synthase kinase 3β (GSK3β), AXIN, and casein kinase 1α (CK1α).Tis complex plays a crucial role in tightly regulating the cytoplasmic levels of β-catenin.Mutations in the APC gene lead to the formation of a truncated APC protein that lacks the binding site for β-catenin.Consequently, β-catenin is unable to undergo proteasomal degradation.Te stabilized β-catenin is then translocated into the cell nuclei, where it binds to LEF/TCF proteins.Tis interaction results in the constitutive expression of MYC, CCND1 (cyclin D1), Axin2, and DKK1 and leads to increased cell proliferation and dediferentiation [4].Echegoyen-Silanes et al. also identifed two somatic mutations (c.3428_3429insA, p. (Tyr1143Ter) and c.3565del, p. (Ser1189Hisfs * 76) of APC in a 29-year-old female patient.Tey postulated that cribriform morular thyroid carcinoma might stem from endodermal progenitors, exhibiting an intestine-like phenotype due to prematurely terminated cell diferentiation.[16].Moreover, in cribriform morular thyroid carcinoma, other genetic alterations have been reported, including RET/PTC rearrangements, PIK3CA mutations, and RAS gene mutations.Tese genetic alterations can function as upstream efectors in the WNT/ β-catenin pathway [17].
Te clinical features of cribriform morular thyroid carcinoma may be associated with FAP due to APC mutations.In a Japanese case series, approximately 39% of patients with cribriform morular thyroid carcinoma were found to have FAP.Tese patients with FAP tended to present with multiple thyroid tumors compared with those with the sporadic form of the disease.Among the patients with FAP, patients who initially had thyroid tumors tended to have larger tumors compared with those who initially presented with colonic polyps.Moreover, the clinical course of cribriform morular thyroid carcinoma is generally indolent, with a low mortality rate [18].Lymph node extension occurs in only approximately 10% of patients, and distant metastases are uncommon (6%), typically occurring in older patients [4,6].When distant metastases do occur, they often involve the lungs, bones, and brain [4].In terms of the ultrasonic fndings of cribriform morular thyroid carcinoma, the lesions are frequently described as solid, hypoechoic, oval to circular, heterogeneous, and circumscribed, typically lacking calcifcations or a hypoechoic halo [19].Management of cribriform morular thyroid carcinoma typically follows a similar approach to that of PTC.Total thyroidectomy is usually considered sufcient for efective  found that the 5-year and 20-year survival rates for 11 patients suspected to have cribriform morular thyroid carcinoma were 90% and 77%, respectively [22].Te diseaserelated mortality in these cases was 2%, which is similar to the 2.5% reported in classical PTC cases [6].Table 1 summarizes the diferences in several clinical and pathological features between classical PTC and cribriform morular thyroid carcinoma.
In conclusion, cribriform morular thyroid carcinoma was seldom reported in Taiwan.Making the correct preoperative diagnosis is difcult based on fne-needle aspiration cytology.Tis case underscores the importance of preoperative molecular analysis in achieving an accurate diagnosis.Te defnitive diagnosis, however, is histopathological, based on the recognition of its peculiar tumor growth pattern together with nuclear and cytoplasmic positivity for beta-catenin.

Figure 1 :Figure 2 :
Figure 1: (a) Tyroid ultrasonography images depicting a solid, hypoechoic nodule with a well-defned border on the left lobe.(b) Fine-needle aspiration cytology fndings revealing numerous spindle-shaped tall cells with a densely packed structure and an enlarged nucleus-to-cytoplasm ratio.

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[21]0]eports in Endocrinology disease control, with or without lymph node dissection.In cases of local invasion, external beam radiotherapy can be considered as a treatment option.Furthermore, screenings for colonic polyps and congenital hypertrophy of the retinal pigment epithelium must be performed to exclude germline APC mutations and association with FAP[6,20].Te overall prognosis for individuals with cribriform morular thyroid carcinoma is generally favorable.Te reported recurrence rate is approximately 8.5%, which is lower than the 16.1% recurrence rate observed in classical PTC[21].Perrier et al.