Clinicopathologic Features and Cytologic Correlation of ALK-Rearranged Papillary Thyroid Carcinoma: A Series of Eight Cases

Anaplastic lymphoma kinase (ALK) gene fusions are rare in papillary thyroid carcinoma (PTC) but may serve as a therapeutic target. This study aims to evaluate the preoperative cytologic findings and clinicopathologic features of a series of eight ALK-rearranged PTCs from our pathology archives and consultations. All cases were confirmed by ALK D5F3 immunohistochemistry and six with additional targeted RNA-based next-generation sequencing (NGS). The original fine-needle aspiration (FNA) cytology diagnosis included the Bethesda System (TBS) category II in three (37.5%), TBS III in two (25%), TBS V in two (25%), and TBS VI in one (12.5%). Six cases had available FNA cytology and were reviewed. The cytologic features showed microfollicular architecture as well as limited or reduced nuclear elongation and chromatin alterations in all six. Nuclear grooves and pseudoinclusions were absent in two cases, rarely or focally noted in three, and frequently found in one. Two cases initially diagnosed as TBS II, showing microfollicular architecture without well-developed nuclear features, were revised to TBS III (with architectural atypia only). For histologic correlations, four were infiltrative follicular variant PTCs, three as classic subtype PTC with predominant follicular growth, and one as solid/trabecular subtype PTC. All eight cases demonstrated reduced PTC nuclear features with respect to nuclear elongation and chromatin alterations compared to those typically identified in “BRAF-like” PTCs. The NGS testing revealed EML4::ALK fusion in three, STRN::ALK fusion in two, and ITSN2::ALK fusion in one. In conclusion, although ALK-rearranged PTCs have been associated with neutral gene expression profile from a BRAF-RAS scoring perspective, the “RAS-like” nuclear features were more commonly identified in this series, resulting in frequent indeterminate diagnosis of preoperative FNA.


Introduction
Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid [1].The majority of PTC harbors BRAF mutations, reported in 59.7 to 86.8% [2][3][4].In addition, kinase gene fusions are also commonly detected in about 8.5 to 15.3% of PTCs [2][3][4].Among them, the RET fusions are the most frequent, followed by NTRK and BRAF [4].Anaplastic lymphoma kinase (ALK) belongs to the gene family of receptor tyrosine kinases, which control various cell functions, such as cell proliferation and survival.ALK gene rearrangement has been reported in many tumors across various organs, including anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, and non-small cell lung carcinoma [5,6].PTCs with ALK fusion have been reported in 0-2.2% of unselected patients, 2-7.3% in pediatric and young adult patients, and up to 12.7% in patients with radiation exposure [7][8][9][10][11].Although PTC generally has a favorable prognosis, distant metastasis and disease progression can occur in a subset of patients.Due to the rarity of ALK-rearranged PTCs, their clinical behaviors were still not well-characterized.In addition, ALK fusion can serve as a potential therapeutic target given the targeted tyrosine kinase inhibitors.Therefore, identifying ALK fusion in PTCs has important clinical implications in the era of precision medicine.
The ALK-rearranged PTCs were first discovered in atomic bomb survivors in 2012 and these tumors frequently showed a peculiar solid/trabecular-like architecture [11].Since then, various histologic subtypes have been described in ALK-rearranged PTCs, including follicular variant as well as classic, solid/trabecular, diffuse sclerosing, tall cell, oncocytic, and Warthin-like subtypes [8,11].In addition, Extended author information available on the last page of the article relatively subtle nuclear atypia has also been observed [8,12].In fine-needle aspiration (FNA) cytology, subtle cytologic atypia with mild nuclear enlargement and less frequent nuclear grooves were noted [13].To the best of our knowledge, the description of cytologic and histologic findings of ALK-rearranged PTCs was mostly mentioned in separate studies.The detailed cytomorphologic features as well as cytology-histology correlations were not well reported.In this study, we investigated the cytologic features, the distribution of the Bethesda System (TBS) categories, and the histopathological characteristics of these tumors in a series of eight ALK-rearranged PTCs.

Patient Selection
This study was approved by the Institutional Review Board (IRB) of Taipei Veterans General Hospital (IRB no.: 2019-07-001BC) and Changhua Christian Hospital (IRB no.: 230902).This series consisted of cases identified from our previous studies of BRAF p.V600E wild-type PTCs, routine cases, and consultations [4,14].The BRAF p.V600E status was evaluated by a validated immunohistochemical assay (clone VE1, Spring Bioscience, Pleasanton, CA, USA).Previous study has showed ALK immunohistochemistry (IHC) with good sensitivity and specificity for detecting ALK-rearranged PTCs [8].Thus, ALK IHC (clone D5F3, Ventana, Oro Valley, AZ) was applied as the primary diagnostic tool on BRAF p.V600E wild-type PTCs in this series.The ALK IHC-positive cases passed the initial quality check for RNA were subjected to targeted RNA nextgeneration sequencing (NGS) using FusionPlex pan solid tumor v2 (ArcherDX, Boulder, CO) to identify the specific fusion partner genes.Clinical characteristics, including surgical procedure, age, sex, tumor size, TNM stage, dose of radioactive iodine therapy, and follow-up status, were recorded from the medical charts.

Cytology and Pathology Examination
The preoperative FNA specimens were prepared either by conventional or SurePath (Becton Dickinson, Franklin Lakes, NJ) methods, and were stained with Papanicolaou and/or Liu stains.The diagnosis was based on the 2023 Bethesda System for Reporting Thyroid Cytopathology, including TBS I: nondiagnostic, TBS II: benign, TBS III: atypia of undetermined significance (AUS), TBS IV: follicular neoplasm, TBS V: suspicious for malignancy, and TBS VI: malignant.The TBS III category was further divided into AUS-nuclear atypia and AUS-other [15].All available FNA slides were reviewed.Architecture features (macrofolliclular, microfollicular, and papillary patterns), nuclear features (elongation, chromatin alterations (powdering/margination), groove, and pseudoinclusion), and the amount of colloid were examined.
All available pathologic slides were reviewed.The diagnosis of PTC and histological subtypes was based on the 2022 World Health Organization classification.Histological features including proportions of growth patterns (macrofollicular, microfollicular, papillary, and solid/trabecular), nuclear features (elongation, chromatin alterations (powdering/margination), groove, and pseudoinclusion), tumor border, the presence of psammoma body, extrathyroidal extension, lymphatic invasion, vascular invasion, and background thyroid parenchyma were recorded.Tumor border was classified into three types: well-circumscribed, multinodular permeative (invasive growth in a multinodular pattern), and infiltrative (irregular and spiculated tumor invasion with desmoplastic change).

Clinical Information
Eight ALK-rearranged PTCs were identified in this study.Cases 1 to 4 were identified through ALK IHC screening on the tissue microarrays of 60 BRAF p.V600E wild-type PTCs from our previous study cohort [14].Case 5 was diagnosed as ALK-rearranged PTC in our routine practice and had been reported in our prior study [4].Cases 6 to 8 were from the corresponding author's consultation files.The clinicopathologic features are summarized in Table 1.None of the eight cases had a history of radiation exposure.Six patients underwent total thyroidectomy and two received lobectomy.The average age was 39 years (range: 17-59 years).The femaleto-male ratio was 6:2.The average tumor size was 2.2 cm (range: 0.9-4.5 cm).The pT stage included pT1a in three, pT1b in two, pT2 in one, pT3a in one, and pT4a in one.The pT4a case (case 3) showed tracheal invasion and lost to follow-up after the surgery.Six patients were subjected to lymph node dissection.Central compartment cervical lymph node metastasis (N1a) was found in two cases (cases 7 and 8) and lateral compartment cervical lymph node metastasis (N1b) was found in one case (case 2).Six patients received post-operation radioactive iodine therapy.Seven of the eight cases had no sign of disease recurrence and remained free of disease upon follow-up with a mean interval of 47.4 months (4-87 months).The ALK IHC showed diffuse moderate to strong cytoplasmic expression in all eight cases (Figs.1C and F, 2C and F).Targeted RNA NGS was successfully performed in six cases and the fusion variants included EML4::ALK in three, STRN::ALK in two, and ITSN2::ALK in one.The remaining two cases failed the test due to poor RNA quality.

Cytological Features
The cytologic features of ALK-rearranged PTCs are summarized in Table 2. Preoperative FNA was performed in all eight cases and the original diagnosis included TBS II in three, TBS III in two, TBS V in two, and TBS VI in one.The FNA of case 3 (with TBS III diagnosis) was not performed in our country and the remaining seven cases had available FNA slides for review.The FNA from case 7 (with TBS II diagnosis) showed bland follicular cells without PTC nuclear features in a lymphocytic background and very scant colloid.Thus, the findings suggested lymphocytic thyroiditis rather than PTC.It is assumed that the tumor cells were not adequately represented through the FNA and thus the case was removed for the following analysis.In the two cases (cases 2 and 6) (Fig. 1A and D) assigned initially as TBS II, the nuclear features were limited but focal microfollicular architecture was identified.Thus, the diagnosis was revised to TBS III (AUS-other, with architecture atypia only).In case 8 assigned initially as TBS III, the cytology showed focal papillary architecture and rare nuclear atypia (Fig. 3A).Therefore, we assigned the diagnosis as TBS III (AUS-nuclear atypia) after review.The final reviewed diagnosis of the six cases showed TBS III: AUS-nuclear atypia in 16.7% (1/6), TBS III: AUS-other in 33% (2/6), TBS V in 33.3% (2/6), and TBS VI in 16.7% (1/6).Microfollicular architecture was identified in all six cases.Scant papillary architecture and macrofollicular architecture were present in only one case each (case 8 and case 2, respectively).The typical PTC nuclear features of grooves and pseudoinclusions were absent in two cases (Fig. 1A and D), rarely seen in one case, focally found in two cases (Fig. 2A and D), and readily identified in one case (Fig. 4A).Nuclear elongation and chromatin alterations were limited in two cases (Fig. 1A  and D) and reduced in the remaining four cases (Figs.2A  and D, 3A, and 4A).

Pathological Features
The pathologic features of ALK-rearranged PTCs are summarized in Table 3.All eight cases demonstrated a predominant follicular growth.Four cases (case 2, 3, 7, and 8) showed a multinodular permeative tumor border and were classified as infiltrative follicular variant PTC (Figs. 3B  and 5C).One case (case 1) showed microfollicular with predominant solid/trabecular growth and was diagnosed as solid/trabecular subtype PTC (Fig. 4B).The three remaining cases (case 4, 5, and 6) showed discernible papillary architecture (Fig. 2E) that precludes infiltrative follicular variant PTC and non-invasive follicular thyroid neoplasm with papillary-like nuclear features.Thus, these three cases were diagnosed as classic subtype PTC with predominant follicular growth.Case 5 was the only one that showed

Discussion
In this study, we examined the cytologic characteristics, the distribution of the TBS categories, and the histologic correlates of a series of eight ALK-rearranged PTCs.In our series, the original FNA cytology diagnosis yielded a notable false-negative result (TBS II) in three out of eight patients.Following the review of seven cases with available FNA slides, we excluded one TBS II case with missed sampling for the final analysis.Two cases initially designated as TBS II were reclassified to TBS III due to the presence of scant microfollicles.Another case originally classified as TBS III remained the diagnosis of TBS III after review based on rare nuclear grooves and pseudoinclusions.The high rate of TBS III interpretation (50%, 3/6) in our series likely resulted from subtle nuclear features and the presence of only microfollicular pattern.Two cases (33.3%, 2/6) were diagnosed as TBS V based on reduced nuclear elongation and chromatin alterations as well as focal grooves and pseudoinclusions.Only one case (16.7%, 1/6) met the criteria for TBS VI.In a case report, Jurkiewicz et al. illustrated a STRN::ALK PTC with preoperative FNA diagnosis of TBS III, displaying focal cytological atypia with mild nuclear enlargement and occasional nuclear grooves [13].Panebianco et al. also observed attenuated nuclear features in FNA cytology of ALK-rearranged PTC and a high rate of indeterminate diagnoses (TBS III: 66%, TBS IV: 10%, TBS V: 16%) [12].The corresponding pathological features of ALKrearranged PTCs in this study showed predominant follicular growth and rare papillary growth in a subset of cases.Limited nuclear elongation and reduced chromatin alterations were also observed.Nuclear grooves and pseudoinclusions were infrequently identified and were obvious in only three of eight cases (37.5%).The findings between cytology and pathology overlapped with good correlation in our study.The terms "BRAF-like" and "RAS-like" was first applied in The Cancer Genome Atlas (TCGA) study to describe gene expression pattern in PTCs [2].TCGA study identified that ALK-rearranged PTCs had a neutral gene expression profile which was intermediate between those of BRAF p.V600E mutant tumors ("BRAF-like") and RAS mutant tumors ("RAS-like") based on their BRAF-RAS scoring system.Recently, a few studies and expert reviews have adopted "BRAF-like" and "RAS-like" designations to describe the cytomorphologic features that are most commonly associated with tumors with the BRAF p.V600E mutation (or tumors with a similar gene expression profile) as "BRAFlike" whereas tumors with a RAS mutation (or tumors with a similar gene expression profile to tumors with a RAS mutation) show "RAS-like" nuclear atypia [16][17][18].While "BRAF-like" nuclear atypia is characterized by classical chromatin margination (nuclear clearing) and heterogenous nuclear membrane irregularities resulting in intranuclear pseudoinclusions, "RAS-like" tumors are characterized by The data from our study showed that ALKrearranged tumors usually demonstrate "RAS-like" nuclear atypia rather than "BRAF-like" atypia.Interestingly, Chou et al. also described that the majority of ALK-rearranged PTCs (eight out of 14 cases) showed a predominance of follicular growth and variable nuclear atypia, which would be considered morphologically "RAS-like" [8].

Conclusion
Although ALK-rearranged PTCs have been shown to harbor a neutral gene expression profile from a BRAF-RAS scoring perspective of TCGA study, one can see tumors with "RASlike" and "BRAF-like" nuclear alterations or the combination of both in an individual case.In this current study, the "RASlike" nuclear features were more commonly identified.The  diagnostic challenges due to attenuated nuclear alterations and the absence of a discernible papillary architecture seem to result in frequent indeterminate diagnosis of preoperative FNA.The corresponding histopathologic features included a predominant follicular growth along with variable and subtle nuclear features.Recognition of these findings is essential for the identification of ALK-rearranged PTC.

CPTC
classic subtype PTC, FVPTC follicular variant PTC, TCPTC tall cell subtype PTC, OCPTC oncocytic subtype PTC, WLPTC Warthinlike subtype PTC, DSPTC diffuse sclerosing subtype PTC, STPTC solid/trabecular subtype PTC, S/T solid/trabecular, NED no evidence of disease, AWD alive with disease a Only cases of DSPTC b 126 cases of FVPTC with tumor size more than 1 cm c 16 cases of subcentimeter PTC with lymph node metastasis d 59 cases of radioactive iodine-refractory PTC e Study using RT-PCR aimed for STRN::ALK fusion f 79 cases of BRAF-mutant PTC g 262 cases of BRAF-negative PTC h PTC negative for mutations of BRAF, HRAS, NRAS, and KRAS as well as rearrangements of RET, and PAX8::PPARG i 41 cases of PTC with patients' age under 40 years old j 51 cases of PTC with patients' age under 18 years old k 93 cases of PTC with patients' age between 6 and 20 years old l 79 cases of PTC in atomic bomb survivors m 63 cases of PTC with patients' age under 18 years old with radiation exposure after Fukushima accident n Patients with radiation exposure after Chernobyl accident

Table 1
The clinicopathologic features of ALK-rearranged

Table 2
The cytologic features of ALK-rearranged papillary thyroid carcinoma TBS the Bethesda System, AUS-O atypia of undetermined significance associated with other patterns, AUS-N atypia of undetermined significance with nuclear atypia * Not evaluated due to missed sampling of the tumor

Table 4
Summary of previous studies of ALK-rearranged papillary thyroid carcinoma (PTC)