Anaplastic and poorly differentiated thyroid carcinomas: genetic evidence of high‐grade transformation from differentiated thyroid carcinoma

Abstract Anaplastic thyroid carcinoma (ATC) is the most advanced and aggressive thyroid cancer, and poorly differentiated thyroid carcinoma (PDTC) lacks anaplastic histology but has lost architectural and cytologic differentiation. Only a few studies have focused on the genetic relationship between the two advanced carcinomas and coexisting differentiated thyroid carcinomas (DTCs). In the present study, we investigated clinicopathologic features and genetic profiles in 57 ATC and PDTC samples, among which 33 cases had concomitant DTC components or DTC history. We performed immunohistochemistry for BRAF V600E, p53, and PD‐L1 expression, Sanger sequencing for TERT promoter and RAS mutations, and fluorescence in situ hybridization for ALK and RET rearrangements. We found that ATCs and PDTCs shared similar gene alterations to their coexisting DTCs, and most DTCs were aggressive subtypes harboring frequent TERT promoter mutations. A significantly higher proportion of ATCs expressed p53 and PD‐L1, and a lower proportion expressed PAX‐8 and TTF‐1, than the coexisting DTCs. Our findings provide more reliable evidence that ATCs and PDTCs are derived from DTCs.


Introduction
Thyroid cancer is the most common endocrine tumor; its incidence has risen over the past 20 years [1].Follicular cell-derived thyroid carcinoma comprises about 95% of all thyroid malignancies, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), invasive encapsulated follicular variant of PTC, oncocytic carcinoma, high-grade follicular cell-derived nonanaplastic thyroid carcinoma [including poorly differentiated thyroid carcinoma (PDTC) and differentiated high-grade thyroid carcinoma (DHGTC)], and anaplastic thyroid carcinoma (ATC).PTC, FTC, invasive encapsulated follicular variant of PTC, oncocytic carcinoma, and DHGTC are considered differentiated thyroid carcinomas (DTCs).Most DTC types (except DHGTC) usually have an excellent prognosis.In contrast, ATC and PDTC are relatively uncommon but have more aggressive behavior.A DTC component can be identified in a subset of ATC and PDTC.
BRAF and RAS mutations are frequent in DTCs [2].Two major molecular groups of PTC, the BRAF-like group and the RAS-like group, were categorized by the cancer genome atlas group [3].For ATC and PDTC, late events such as telomerase reverse transcriptase (TERT) promoter and TP53 mutations occur frequently [4].It has been concluded that ATC and PDTC are derived from DTC through progressive accumulation of genomic alterations [5,6].Recent studies have revealed the genetic alterations in ATC and PDTC, but only limited studies with small sample sizes have compared the genetic differences between ATC and the coexisting DTC (e.g.BRAF V600E, RAS, PIK3CA, and TERT promoter) [4,[6][7][8][9][10][11][12][13][14][15].In addition, no study is about PDTC and its DTC counterpart.
In this study, we investigated the status of a panel of key biomarkers in a large series of ATC/PDTC samples with their DTC counterparts and noncancerous tissues, including expression of BRAF V600E, pan-cytokeratin (pan-CK), PAX-8, TTF-1, p53, CD8, and programmed cell death protein ligand 1 (PD-L1), mutations of KRAS/NRAS/HRAS and the TERT promoter, and rearrangements of RET and ALK.Our aim was to gain additional insights into the evolutionary relationship between the two advanced carcinomas and DTC.

Samples
All ATC and PDTC surgical samples were obtained from the Affiliated Hospital of Qingdao University between 2009 and 2022.Two certificated pathologists (HY Gu and JG Wang) reviewed the slides to re-evaluate the morphological features of ATC, PDTC (Turin consensus proposal), and coexisting DTC according to the 2022 World Health Organization criteria [15].The clinical characteristics were obtained from the archived medical records, including the patient's age, gender, TNM stage, treatment, and followup information.The study was performed in accordance with the Declaration of Helsinki and was approved by the institutional ethics committee of the Affiliated Hospital of Qingdao University.
A total of 42 ATCs and 15 PDTCs were enrolled, of which 39 ATCs and 11 PDTCs were primary tumors, 2 ATCs and 3 PDTCs were recurrences, and 1 ATC and 1 PDTC were distant metastases.DTC components were identified in 24 ATCs and 9 PDTCs, of which 31 cases had concomitant DTCs and 2 cases had prior DTCs.Immunohistochemistry (IHC) and Sanger sequencing were performed on formalin-fixed paraffin-embedded (FFPE) tissues.For DNA extraction, ATC, PDTC, and coexisting DTC areas were separately dissected on unstained sections and then scraped off under a microscope.

Immunohistochemistry
IHC stains for pan-CK (clone: AE1/AE3, Maxim Biotechnology, Fuzhou, PR China), PAX-8 (clone: EP298, Maxim Biotechnology), TTF-1 (clone: SPT24, Maxim Biotechnology), p53 (clone: MX008, Maxim Biotechnology), CD8 (clone: SP16, Maxim Biotechnology), and BRAF V600E (clone: VE1, Ventana Medical Systems, Roche, Tucson, AZ, USA) were performed using a Roche BenchMark ULTRA automated system.BRAF V600E staining with a mutation-specific monoclonal antibody is sensitive and specific for the mutation [16].PD-L1 (PD-L1 IHC 22C3 pharmDx, Agilent Technologies, Dako, Santa Clara, CA, USA) staining was performed using the Dako Autostainer Link 48 automated system.The expression of pan-CK, PAX-8, and TTF-1 was interpreted to be positive when the percentage of immunopositive tumor cells was ≥1%.A p53 mutant staining pattern was characterized by diffusely positive tumor cells (missense mutation) or complete negativity (nonsense mutation).A BRAF V600E mutant pattern was defined by diffuse cytoplasmic staining.PD-L1 expression was assessed using tumor proportion score (TPS; the percentage of tumor cells showing membranous staining) and combined positive score (CPS; the number of positive tumor cells, lymphocytes, and macrophages divided by the total number of tumor cells and multiplied by 100).PD-L1 positivity was defined as TPS ≥ 1% or CPS ≥ 1.As CD8 expression on T cells within the tumor microenvironment is often associated with the immune response against cancer cells, we further evaluated the CD8 expression levels using IHC, categorizing it into two groups: low expression (negative or ≤10% positive cells of the intratumoral inflammatory cells) and high expression (>11% positive cells of the intratumoral inflammatory cells).

Sanger sequencing
DNA extraction was performed using a QIAmp DNA FFPE Tissue Kit (Cat.No. 56404, Qiagen, Valencia, CA, USA) based on the manufacturer's instructions.RAS mutations at codons 12, 13, and 61 and TERT promoter mutations were amplified using PCR using primers listed in supplementary material, Table S1.The PCR conditions included an initial denaturation step at 94 C for 1 min, 35 cycles of 94 C denaturation for 10 s, 60 C annealing for 30 s, 72 C elongation for 1 min, and a final elongation step at 72 C for 2 min.The PCR product was sent to Sangon Biotech (Qingdao, PR China) for Sanger sequencing.The results were analyzed using Chromas 2.6.6 software (Technelysium, South Brisbane, Australia).

Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) analysis for ALK and RET rearrangements was performed on tissue microarrays using the ZytoLight SPEC ALK or RET Dual Color Break Apart Probes according to the manufacturer's instructions (ZytoVision, Bremerhaven, Germany).
At least 100 nonoverlapping cells with hybridization signals were calculated with a fluorescence microscope (Olympus BX51, Tokyo, Japan).Aberrant signals were indicated by the split of orange and green signals or isolated orange and green signals.More than 15% of tumor cells with an aberrant signal was considered as rearrangement.

Statistical analysis
Statistical analysis was performed using SPSS 19.0 (IBM, Armonk, NY, USA).The relationships between IHC expression and clinicopathologic parameters were analyzed using the chi-square or Fisher's exact tests.Survival data were calculated by the Kaplan-Meier method and compared by log-rank test.p < 0.05 (two-tailed) was considered to be statistically significant.

Clinicopathologic characteristics
The study cohort included 42 ATCs and 15 PDTCs, and the clinicopathologic characteristics are summarized in Table 1.The median age was 64.5 years for ATC and 52 years for PDTC.The female-to-male ratio was 1.3:1 for ATC and 0.9:1 for PDTC.Local extension was frequently observed, with extrathyroidal strap muscle extension in 8 ATCs and 2 PDTCs, and larynx/trachea/esophagus invasion in 28 ATCs and 7 PDTCs.Distant metastasis was found in 11 ATC and 8 PDTC patients by postoperative scintigraphy.The following sites of metastases were documented: lung (seven ATCs and four PDTCs), bone (three ATCs and three PDTCs), brain (two ATCs and one PDTC), adrenal gland (one ATC), and subcutaneous soft tissue (one ATC and one PDTC).
In pure ATCs without coexisting DTCs, the expression rates of PAX-8 and TTF-1 were slightly lower than ATCs with DTCs, but these differences were not found in PDTCs.Besides, pan-CK, p53, PD-L1, and CD8 expression patterns were similar between the two groups with and without DTC (supplementary material, Table S4 and Figures S34-S57).

BRAF V600E expression and TERT promoter and RAS mutations in ATCs/PDTCs and the coexisting DTCs
The mutation profiles of BRAF, RAS, and the TERT promoter in ATC and PDTC components were almost consistent with the coexisting DTC components.In detail, 12 cases (11 ATCs and 1 PDTC) showed positive expression for BRAF V600E in both components and 20 cases were negative in both (12 ATCs and 8 PDTCs) (Figures 3M-P and 4).Twenty-two cases (18 ATCs and 4 PDTCs) harbored TERT mutations in both ATC/PDTC and DTC components, including c.-124C>T in 21 cases and c.-146C>T in one case.Eight cases (six ATCs and two PDTCs) harbored RAS mutations in both the ATC/PDTC and DTC components, including KRAS c.35G>A (G12A, one case), NRAS c.182A>G (Q61R, four cases), and HRAS c.182A>G (Q61R, three cases, Figure 2D,E).ATC components harbored different genetic alterations from the coexisting DTCs in only three cases (Figure 4).All these mutations were validated as somatic mutations by comparing them with  In pure ATCs/PDTCs without coexisting DTC, the TERT promoter mutation rate was also lower than in tumors with DTC (supplementary material, Table S4).

ALK and RET gene fusion analysis
ALK and RET gene fusions were absent in both ATCs/ PDTCs and DTC components in all cases.

Treatment and outcomes
Treatment details were obtained in 40 patients; all patients received thyroid-stimulating hormone suppression therapy after surgery, and some received other additional treatments.In detail, four ATCs and one PDTC received external beam radiotherapy, seven PDTCs had radioactive Iodine ( 131 I) therapy, three ATCs received chemotherapy, two ATCs and two PDTCs took small molecule kinase inhibitors, and one ATC received both radiotherapy and chemotherapy.Follow-up data were available in 27 ATC patients and 13 PDTC patients, in which 27 ATCs and 9 PDTCs died of tumor progression during periods ranging from 2 months to 6.5 years.The median overall survival (OS) was 4 months for ATCs and 32 months for PDTCs.We then generated univariate analyses to investigate the impact of different clinicopathologic factors on OS patterns of ATC patients, including DTC components, treatment strategies, and molecular alterations.We found that an aberrant p53 staining pattern, which correlates with TP53 mutation, was an adverse prognostic indicator for ATC patients.However, the other variables were not associated with OS patterns of ATCs (supplementary material, Figure S58).The survival patterns of PDTC were not analyzed because of the limited sample size.

Discussion
In the present study, we have retrospectively reviewed the clinicopathologic characteristics of 42 ATCs and 15 PDTCs.We confirmed that the two advanced malignancies have aggressive biological behaviors and are frequently accompanied by DTC components.The most important finding is that ATCs and PDTCs harbored identical genetic alterations with the coexisting DTCs, although their immunohistochemical expression patterns differed.Our findings provide more evidence regarding the origin of ATCs and PDTCs and support the theory of high-grade transformation of DTCs.

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ATCs and PDTCs are considered to be derived from preexisting DTCs [4,13].In our study, DTC components could be identified in more than half of ATCs and PDTCs, and most DTCs belonged to aggressive subtypes, including tall-cell PTC, columnar PTC, and hobnail PTC.It has been reported that squamous ATCs tend to be accompanied by tall-cell PTC [17].Our findings further demonstrated that epithelioid ATCs were also frequently accompanied by tall-cell PTC and had a high BRAF mutation rate.Besides, we found that spindle ATCs seemed to have a closer relationship with FTCs and most harbored RAS mutations.We consider it possible to conclude that driver gene status depends on the cytologic features of ATCs, although the analysis of more cases is needed to investigate this further.
The genetic discrepancies between ATCs/PDTCs and coexisting DTCs were also evaluated.We found the different components of the same case usually harbored identical BRAF V600E, RAS, and TERT promoter genotypes.This finding supports the hypothesis that ATCs and PDTCs are transformed from preexisting DTCs.BRAF and RAS mutations represent early oncogenic events during DTC development [2].
Likewise, the two genes remained the main drivers in ATCs and PDTCs, but additional late events, such as TP53 and TERT promoter mutations, frequently occurred.It is generally recognized that TERT promoter mutation is relatively uncommon in DTCs but occurs in about 22% of PDTCs and 56-75% of ATCs [4,16,18,19].In our series, TERT promoter mutation was detected in 75% of ATC-associated DTCs and 44% of PDTC-associated DTCs, similar to Ragazzi et al's report [13].This finding indicates that most preexisting DTCs may have aggressive biological behaviors.TP53 mutation was found in 27% of PDTCs and 42-63% of ATCs, which was considered a genetic hallmark of advanced thyroid tumors [4,16,20].Interestingly, it was observed exclusively in ATC or PDTC areas (not DTC areas), suggesting that TP53 mutation was not inherited from preexisting DTC (acquired after transformation).In addition, ALK or RET rearrangements were not identified in our cases.Previous studies showed that oncogenic fusions are infrequent in ATCs, but are relatively common in PDTCs, with an incidence of 22% (9/41) [4].In a large series with 144 ATC cases, ALK rearrangement was found in only one case [21].It should be noted that our FISH study was performed using a tissue microarray so that the confined region and tumor heterogeneity may lead to a false negative result.The expression of pan-CK, PAX-8, and TTF-1 in ATCs was usually focal and weak.However, the retained immunopositivity could help us confirm its follicular cell nature.TTF-1 expression was lost in only a few ATCs, whereas strong in most PDTCs, which could help to distinguish ATCs from PDTCs.Of note, there were 18 pure ATCs and 6 pure PDTCs without DTC components.In pure ATCs/PDTCs without DTC components, PAX-8 expression, BRAF V600E expression, and TERT promoter mutation were less common than in those with DTC counterparts.In any case, the coexisting DTC, expression of follicular cell markers, and presence of BRAF mutation or RAS mutation are quite informative for diagnosing ATC or PDTC.The differential diagnosis includes a group of nonfollicular cell-derived malignancies, such as synovial sarcoma, malignant peripheral nerve sheath tumor, angiosarcoma, leiomyosarcoma, rhabdomyosarcoma, and melanoma.
Treatment of ATCs is mostly palliative, and the present OS statistics are quite discouraging.Surgical resection with adjuvant radiation therapy and chemotherapy may prolong survival somewhat and improve quality

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Anaplastic and poorly differentiated thyroid carcinomas of life.The median OS was only 4 months in our ATC series.In recent years, targeted therapy for ATC has also developed dramatically.A retrospective cohort study showed that targeted therapy significantly improved the OS of patients with ATC compared with those who did not receive targeted therapy [22].Inhibitors of BRAF, NTRK, RET, mTOR, CDK4/6, and other targets have been taken into clinical trials [23].For example, the FDA has approved dabrafenib and trametinib (targeting BRAF and MEK1/2) for treating individuals with BRAF V600E-mutant ATC.Besides, our ATCs showed extremely high expression of PD-L1 and a greater density of CD8+ TILs compared with DTCs and PDTCs, consistent with previous reports [24][25][26].These findings suggested that anti-PD-1/PD-L1 agents may provide clinically meaningful benefits in ATC patients.A recent clinical trial showed that ATCs responded well to PD-1 inhibition (spartalizumab), especially for those patients with PD-L1 TPS ≥ 50% [27].In addition, patients with PD-L1 TPS ≥ 50% benefited from lenvatinib and pembrolizumab treatments [28].Therefore, genetic testing is still strongly recommended for the diagnosis and treatment of ATC.
In conclusion, our results suggest that ATCs and PDTCs are follicular cell-derived aggressive thyroid carcinomas.They retain genetic alterations from the preexisting DTCs (BRAF, RAS, and TERT promoter mutation) and acquire some aggressive mutations (TP53) during development.Although the prognosis is poor and therapies have limited impact, a series of genetic changes and high expression of PD-L1 still offer optimistic hope for new targeted therapies.

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Gu et al © 2024 The Authors.The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.J Pathol Clin Res 2024; 10: e356 paired normal tissues.The detailed sequencing results of the whole cohort are described in supplementary material, Figures S1-S57.

Table 1 .
The clinicopathologic features of the patients included in the whole cohort

Table 2 .
Immunohistochemical comparison between ATC/PDTC and coexisting DTC © 2024 The Authors.The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.J Pathol Clin Res 2024; 10: e356