Introduction

Oncology Letters

1792-1074 1792-1082

D.A. Spandidos

10.3892/ol.2021.12814

OL-0-0-12814

Articles

PD-L1 expression and immune cells in anaplastic carcinoma and poorly differentiated carcinoma of the human thyroid gland: A retrospective study

Cameselle-García

Soledad

1 Abdulkader-Sande

Sámer

2 Sánchez-Ares

María

2 Rodríguez-Carnero

Gemma

3 Garcia-Gómez

Jesús

1 Gude-Sampedro

Francisco

4 5 Abdulkader-Nallib

Ihab

2 5 Cameselle-Teijeiro

José Manuel

2 5

1Department of Medical Oncology, University Hospital Complex of Ourense, Galician Healthcare Service, 32005 Ourense, Spain 2Department of Pathology, Clinical University Hospital of Santiago de Compostela, Health Research Institute of Santiago de Compostela, Galician Healthcare Service, 15706 Santiago de Compostela, Spain 3Department of Endocrinology and Nutrition, Clinical University Hospital of Santiago de Compostela, Health Research Institute of Santiago de Compostela, Galician Healthcare Service, 15706 Santiago de Compostela, Spain 4Department of Epidemiology, Clinical University Hospital of Santiago de Compostela, Health Research Institute of Santiago de Compostela, Galician Healthcare Service, 15706 Santiago de Compostela, Spain 5School of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

Correspondence to: Dr José Manuel Cameselle-Teijeiro, Department of Pathology, Clinical University Hospital of Santiago de Compostela, Health Research Institute of Santiago de Compostela, Galician Healthcare Service, Travesía Choupana s/n, 15706 Santiago de Compostela, Spain, E-mail: josemanuel.cameselle@usc.es

07 2021

24 05 2021

22 1

553

09032021 04052021

2021

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Anaplastic thyroid carcinoma (ATC) and poorly differentiated thyroid carcinoma (PDTC) have limited treatment options, and immune profiling may help select patients for immunotherapy. The prevalence and relevance of programmed death-1 ligand (PD-L1) expression and the presence of immune cells in ATC and PDTC has not yet been well established. The present study investigated PD-L1 expression (clone 22C3) and cells in the tumor microenvironment (TME), including tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs) and dendritic cells, in whole tissue sections of 15 cases of ATC and 13 cases of PDTC. Immunohistochemical PD-L1 expression using a tumor proportion score (TPS) with a 1% cut-off was detected in 9/15 (60%) of ATC cases and 1/13 (7.7%) of PDTC cases (P=0.006). PD-L1 expression in TILs was limited to the ATC group (73.3 vs. 0% in ATC and PDTC, respectively). In the ATC group, the TPS for tumor positive PD-L1 expression revealed a non-significant trend towards worse survival, but no difference was observed when investigating PD-L1 expression in TILs and TAMs. In addition to increased PD-L1 expression, all ATC cases exhibited significantly increased CD3⁺ and CD8⁺ T cells, CD68⁺ and CD163⁺ macrophages, and S100⁺ dendritic cells compared with the PDTC cases. Loss of mutL homolog 1 and PMS1 homolog 2 expression was observed in one ATC case with the highest PD-L1 expression, as well as in the only PDTC case positive for PD-L1. Notably, the latter was the only PDTC case exhibiting positivity for p53 and a cellular microenvironment similar to ATC. The current results indicated that PD-L1 expression was frequent in ATC, but rare in PDTC. In addition to PD-L1, the present study suggested that microsatellite instability may serve a role in both the TME and the identification of immunotherapy candidates among patients with PDTC.

ATC PDTC PD-L1 TME microsatellite instability immunotherapy

Instituto de Salud Carlos III, State Research Agency and Ministry of Science and Innovation (Spain), with the participation of European FEDER funds

ISCIII-PI19/01316

The present study was supported in part by grant no. ISCIII-PI19/01316 from Instituto de Salud Carlos III, State Research Agency and Ministry of Science and Innovation (Spain), with the participation of European FEDER funds.

Introduction

Thyroid cancer (TC) is the most common endocrine malignant tumor, ranking ninth in incidence for both sexes and representing the fifth most prevalent cancer in women (1). Most TCs are derived from follicular cells and are classified into three main groups: a) differentiated thyroid carcinoma (DTC) which includes papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC) and Hürthle (oncocytic) cell carcinoma (HCC); b) poorly differentiated thyroid carcinoma (PDTC); and c) anaplastic thyroid carcinoma (ATC) (2).

PTC and FTC are the most common types of TC, have a low overall mortality and are generally curable by surgery with or without radioiodine treatment (3). ATCs are rare undifferentiated cancers with no effective therapy, having almost 100% disease-specific mortality (4–6). PDTCs show limited evidence of follicular cell differentiation and are morphologically and behaviorally intermediate between DTC and ATC (2,7,8). PDTC generally shows a poor response to radioiodine treatment and has an overall 5-year survival rate of about 65%, thus necessitating more effective therapies (2,8–10). Even slight amounts of PDTC areas (≥10%) in a DTC affect the prognosis significantly (11). Some HCCs are also classified as PDTCs and have a worse prognosis than usual HCCs (12,13).

Treatment of ATC must be carried out by a multidisciplinary team generally requiring the combination of surgery (as complete as possible), radiotherapy (intensity-modulated radiotherapy) and conventional chemotherapy using doxorubicin or taxanes (docetaxel or paclitaxel) usually with cisplatin or carboplatin (4–6). Because these treatments are generally not sufficient, newer therapies in development include single or multi-tyrosine kinase inhibitors (MKIs), vascular disruptors, and immunotherapy (6). Regarding MKIs, the Food and Drug Administration have approved the combined treatment with dabrafenib and trametinib for patients with locally advanced or metastatic ATC and somatic BRAF^V600E mutation (14). Lenvatinib has demonstrated an acceptable safety profile for Japanese patients with unresectable thyroid tumors including ATC, regardless of mutational status (15). Other therapeutic options could include everolimus when somatic mutations have been detected in the PI3K/mTOR pathway (16,17), imatinib when overexpression of PDGF receptors are detected in advanced ATC (18), and immune checkpoint inhibitor (ICI) drugs targeting PD-1 or PD-L1 (6,19).

Programmed death ligand 1 (PD-L1), also designated as CD274 or B7-H1, is expressed on activated lymphocytes, dendritic cells, macrophages, healthy cells of different tissues and some tumor cells (20,21). Programmed cell-death 1 (PD-1, CD279) is a glycoprotein normally expressed by T lymphoid cells and macrophages. The binding of PD-1 to its ligands PD-L1 and PD-L2 inhibits cytotoxic CD8⁺ T cell immune response and facilitates the escape of the immune response to cells expressing these ligands (20,21). Blockade of PD-L1 was effective in inhibiting ATC in an in vivo model (BALB/c nude mice with human ATC cells) (22). Using an immunocompetent mouse model of orthotopic ATC, the combination of a BRAF^V600E inhibitor and anti-PD1/PD-L1 antibody dramatically improved mouse survival with a tumor reduction along with an increase in cytotoxic CD8⁺ T cells, NK cells and M1-polarized tumor-associated macrophages (23). Additional studies in a mouse model of orthotopic ATC confirmed that increased efficacy in reducing tumor size and improving survival using an anti-PD1/PD-L1 checkpoint inhibitor combined with an MKI (lenvatinib) was associated with a modification of ATC microenvironment (24). Indoleamine 2,3-dioxygenase (IDO1) has been associated with an altered tumor immune response; therefore IDO1 inhibitors are being investigated in clinical trials in combination with other ICIs (25).

A phase Ib proof-of-concept study of the anti-PD-1 antibody pembrolizumab in patients with advanced, PD-L1-positive PTC or FTC evidenced antitumor activity in a minority of patients treated (26). Another phase 2 trial of pembrolizumab combined with chemoradiotherapy showed good initial tolerance and effectiveness in locoregional disease control but resulted in disappointing survival outcomes (27). A retrospective study of twelve ATC patients, however, showed that in a subset of patients with ATC, pembrolizumab may be an effective salvage therapy added to kinase inhibitors (lenvatinib, dabrafenib + trametinib, trametinib alone) at the time of progression of these drugs, encouraging the incorporation of immunotherapy in patients with ATC (28). Another recent phase II clinical trial to evaluate the efficacy and tolerability of a humanized monoclonal antibody that binds PD-1 and blocks its interaction with PD-L1 (spartalizumab) was carried out in forty-two ATC patients (19). Interestingly, this study evidenced a tumor response of 52.1% in the PD-L1-positive population and that response was independent of BRAF mutational status (19). Several prospective studies are being conducted using ICIs for the treatment of patients with ATC (6), and an improvement in the understanding of the immune microenvironment and the immune biomarkers associated with these highly aggressive thyroid tumors is highly relevant.

Thus, the aim of this study was to evaluate the expression of PD-L1 and the tumor microenvironment in a series of ATCs and PDTCs. We investigated the immunohistochemical expression of PD-L1 in tumor cells, tumor-infiltrating lymphocytes (TILs) and tumor associated macrophages (TAMs), along with the phenotypes of TILs, TAMs and other cells.

Materials and methods <sec> <title>Clinicopathological features

We retrospectively analyzed the clinicopathological data of a series of 26 patients who had undergone total or partial thyroidectomy for ATC and/or PDTC at the Clinical University Hospital of Santiago de Compostela (CHUS) and at the University Hospital Complex of Ourense (CHOU), Spain. The inclusion criteria included having a sufficient excess of tumor tissue fixed in neutral, phosphate-buffered, 10% formalin and included in paraffin blocks for additional immunohistochemical studies. Fifteen patients had ATC and 11 patients had PDTC; PDTC areas coexisted with ATC in two patients and these were also included in the study. The patients consisted of 8 (30.7%) men and 18 (69.2%) women. The age of patients ranged from 47 to 77 years with a mean age of 64.53±11.29 years (range 47–83) for ATC patients and 73.5±6.63 years (range 55–87) for PDTC patients (P=0.018). The patients were followed from the time of histopathological diagnosis until death or the moment of the last clinical follow-up, and clinical data were retrieved from electronic medical records. The original hematoxylin-eosin (H&E) slides from all cases were reviewed by two expert thyroid pathologists (IA-N and JMC-T) and of each case, a paraffin block containing sufficient representative tumor tissue was selected for the additional immunohistochemical analyses of the present study. Tumors were classified according to the criteria of the latest World Health Organization (WHO) thyroid tumor classification (2). All tissue samples were provided by the Biobank of CHUS, integrated in the Spanish National Biobank Network. The study was performed in accordance with the declaration of Helsinki (and subsequent ratifications) and approved by the Santiago-Lugo Medical Research Ethics Committee (code: 2019/275). Written informed consent was obtained.

Immunohistochemical study

Paraffin-embedded tumor tissue sections were stained with H&E, and the immunohistochemical analyses were also performed on 4µm-thick paraffin tissue sections with a peroxidase-conjugated labeled dextran polymer (EnVision FLEX/HRP; Dako), using 3,3′-diaminobenzidine as the chromogen (GC80611-2; Dako). The primary antibodies and the conditions of use (clone, concentration, antigenic recovery treatment and manufacturer) were as follows: Thyroglobulin (TG) (code GA509, polyclonal, ready-to-use, pH 6; Dako); calcitonin (code GA515, polyclonal, ready-to-use, pH 6; Dako); PD-L1 (code GE006, clone 22C3, PD-L1 IHC 22C3 pharmDx, ready-to-use, pH 6; Dako), CD3 (code IR503, polyclonal, ready-to-use, pH 9; Dako); CD4 (code IR649, clone 4B12, ready-to-use, pH 9; Dako); CD8 (code IR623, clone C8/144B, ready-to-use, pH 9; Dako); CD20 (code IR604, clone L26, ready-to-use, pH 9; Dako); CD68 (code IR613, clone PGM1, ready-to-use, pH 9; Dako); CD163 (code CM353AK, clone 10D6, dilution 1:100, pH 9; Biocare Medical); S100 protein (code IR504, polyclonal, ready-to-use, pH 9; Dako); p53 (code IR616, clone DO-7, ready-to-use, pH 9; Dako); MLH1 (code IR079, clone E605, ready-to-use, pH 9; Dako); MLH2 (code IR085, clone FE11, ready-to-use, pH 9; Dako); MSH6 (code IR086, clone EP49, ready-to-use, pH 9; Dako); and PMS2 (IR087, clone EP51, ready-to-use, pH 9s; Dako). Non-tumorous thyroid tissue adjacent to carcinoma (for thyroglobulin, MLH1, MSH2, MSH6 and PMS2), a medullary thyroid carcinoma (for calcitonin), a lung adenocarcinoma (for PD-L1), a colon cancer (for p53), and normal lymphoid tissue of palatal tonsil (for CD3, CD4, CD8, CD20, CD68, CD163, and S100) have been used as positive controls. Non-immune rabbit and mouse serum samples were substituted for the primary antibodies as negative control samples.

All immunostains were evaluated simultaneously by 2 investigators (IA-N and JMC-T) using a double-head optical microscope (BX41TF; Olympus) until consensus was reached. Only PD-L1 membranous staining of viable tumor cells was evaluated and the tumor proportion score (TPS) represented the percentage of PD-L1-positive tumor cells relative to all viable tumor cells. A cutoff score ≥1% was used to define PD-L1 positivity. PD-L1 membranous staining was also scored in TILs and TAMs; stromal immune cells distant from the tumor were excluded. Membranous staining for CD4, CD8, and CD163, membranous/cytoplasmic staining for CD3, CD20, CD68, and cytoplasmic/nuclear staining for S100 were scored as 0 (negative), 1 (positive ≤10% of inflammatory cells), 2 (between 11 and 49%) and 3 (≥50%). Only nuclear staining was considered positive for MLH1, MSH2, MSH6 and PMS2. For p53, diffuse and strong nuclear staining (so-called ‘block staining’) and complete loss of staining (‘null’ phenotype) were considered positive (associated with mutations), while scattered, often weak nuclear positivity was considered a negative (‘wild-type’) normal p53 staining pattern.

Statistical analysis

The chi-square and Fisher's exact tests were, where appropriate, used to investigate the association between categorical variables. Student's t-test was used to evaluate the statistical significance of the difference in the means between the two groups. Survival analysis of differences between groups (ATC vs. PDTC and PD-L1+ vs. PD-L1-) was performed using the Kaplan-Meier method with the log-rank test. Differences with P<0.05 were considered statistically significant. All statistical analyses were performed using R statistical software (version 4.0.3).

Results

The main clinicopathological and immunohistochemical characteristics of the study are shown in Tables I and II. In the ATC group (n=15), 60% of the patients were women and 40% were men. Of the 15 ATC cases, 9 (60%) tumors were morphologically subclassified as giant cell pattern, 3 (20%) as spindled cell pattern, and 3 (20%) as epithelial (squamoid) cell pattern (Table I). In the PDTC group (n=11), 81.8% of the patients were women and 18.2% were men. Survival data appear in Table I and Fig. 1. Significant differences in survival were found when comparing ATC and PDTC (P=0.0001); while 86.6% of patients with ATC had died from the tumor after a mean follow-up of 5 months (range 0.1–16), only 54.5% of the patients with PDTC (without anaplastic component) had died after a mean follow-up of 84.22 months (range 3.3–237) (Fig. 1).

The epithelial non-tumorous thyroid tissue adjacent to PDTCs and ATCs showed positivity for thyroglobulin, MLH1, MSH2, MSH6 and PMS2, but negativity for calcitonin, p53, PD-L1, CD3, CD4, CD8, CD20, CD68 and S100 (Figs. S1–S4). All ATC tumors were negative for thyroglobulin (Fig. 2B) and calcitonin while all PDTC tumors were positive for thyroglobulin but negative for calcitonin. In the immunohistochemical evaluation of PDTC areas present in the thyroid tumors of two patients who had ATC, both tumor types were evaluated independently as PDTCs and ATCs. Heterogeneous (focal or multifocal) positivity for PD-L1 in tumor cells was detected in 60% of ATC cases, in which the percentage of positive tumor cells ranged from 15 to 90% (Tables I and II). Positivity for PD-L1 was detected in all cases of ATC with epithelial cell pattern and also in different cases with other ATC patterns (Figs. 2–4). PD-L1expression in ATC tumor cells was significantly higher than that in PDTC cases (60% vs. 7.7%, P=0.006) (Table II). The only case of PDTC with PD-L1 positive tumor cells (35%) was the one case of oncocytic cells, a poorly differentiated Hürthle cell carcinoma (case 19) (Fig. 3). In the ATC group, a slightly higher number of cases showed positivity for PD-L1 in TILs than in tumor cells (73.3% vs. 60%), but neither PD-L1-positive TILs nor TAMs were observed in the PDTC group (P=0.000) (Figs. 2–4). Using TPS, PD-L1-positive ATC patients showed a trend for worse survival than those with PD-L1-negative tumors (Fig. 1B), although the differences were not significant (P=0.15). No difference in relation to survival was found when comparing the expression of PD-L1 in ATC-TILs or TAMs (P=0.81) (Fig. 1C).

Significant differences were found in the expression of lymphocytic markers in ATC and PDTC (Tables I and II). All ATC cases showed CD3 and CD8 positive cells as well as a higher percentage of CD4 and CD20 positive cells, while no positivity for CD4 and CD20 was detected in PDTC cases; when comparing the expression of all these lymphoid markers in ATC and PDTC, the differences were significant (Table II). All ATCs showed a higher and significant percentage of CD68, CD163 and S100 positive cells compared to the group of PDTCs (Table II) (Figs. 2,3 and 5). While 11/15 (73.3%) of the ATCs showed an abnormal pattern of p53 protein expression, all PDTC cases except the poorly differentiated Hürthle cell carcinoma case (7.7%) showed a p53 wild-type (i.e., normal) staining pattern (P=0.001). Expression of DNA mismatch repair (MMR) proteins was detected in all cases except in one single case with negativity for MLH1 and PMS2 in the ATC areas and concomitant positivity for MLH1 in PDTC areas, as well as in another case of PDTC (case 19) with a complete loss of nuclear MLH1 and PMS2 expression (Fig. 3).

Discussion

The immunohistochemical expression of PD-L1 is sometimes a prerequisite for the establishment of checkpoint inhibitor therapy and has prognostic value in several types of malignant tumors (21,29). Based on the encouraging results of trials involving immunotherapy in the management of ATC (25), we have investigated PD-L1 expression and tumor microenvironment (TME) in a series of 15 ATCs and 13 PDTCs.

Thyroid carcinogenesis arises and progresses gradually as a result of the accumulation of various genetic and epigenetic alterations (2,30,31). There are early driver mutations shared by more than one category of thyroid tumors (31,32) and DTCs have a relatively low level of somatic mutations compared to cancers from other sites (33,34). The rate of mutations, however, is higher in the PDTC group than in the DTC group (35,36) and is much higher in the ATCs (37,38). This model of tumor progression is also supported, as in cases 6 and 15 of our series, by the coexistence of differentiated and less-differentiated areas in some thyroid tumors (39,40). The existence of cases of patients with DTC whose tumor recurrence and/or metastasis included a PDTC and/or an ATC (41) also supports this model; the possibility of two independent tumors, however, cannot be excluded. The mean age of patients with TC increases in relation with the loss of tumor differentiation (2), further supporting the sequential model of carcinogenesis. The older mean and median age of the 13 patients with PDTC in our series contrasted with the findings of other studies (42,43), but this could be attributed to biases due to pathological diagnostic criteria and/or the limited number of cases. The tumors in our series were classified according to the WHO (2), applying the Turin criteria (and algorithm) (7), which by definition exclude PTCs. Other groups, such as the Memorial Sloan Kettering Cancer Center (MSKCC) group, consider PDTC to be any carcinoma with follicular cell differentiation that shows fresh tumor necrosis and/or presence of ≥5 mitoses/10 high power microscopic fields (×400), thus also including PTC-derived cases (42,44). Given that PTC is more common in young adults, the exclusion of PTCs with a BRAF-like signature from the group of PDTCs in our series could explain the differences (31,32). These different diagnostic criteria (PDTC-Turin vs. PDTC-MSKCC), do, in fact, explain the variations in the prevalence of ‘early’ genomic alterations in PDTC (RAS mutations vs. BRAF^V600E mutations, respectively) (36,45).

In our series we found immunohistochemical positivity for PD-L1 in tumor cells (TPS) of more than half of the ATCs (60%) but in only one case of PDTC (7.7%). Similarly, PD-L1 expression was detected in TILs and TAMs from the ATC group (73.3%) but not in the PDTC group, probably related to the higher mutational load of ATCs (37,38). A few studies reported the expression of PD-L1 in TCs. PD-L1 immunoexpression was found in 6.1 to 82.5% of PTCs (46,47), including cases of papillary thyroid microcarcinoma (48) and cases with simultaneous chronic thyroiditis and BRAF mutation (29,49). In PTCs a significant association between PD-L1 expression and BRAF^V600E mutation has been reported in several studies (48,49). Positivity for PD-L1 (clone E1L3N) and IDO1 was detected in 7/28 (25%) and 2/28 (7.1%) respectively of a series of PDTCs (50). Positivity for PD-L1 using different antibodies (clones SP142, 5H1, E1L3N, SP142, E1L3N, 22C3, 22C3, SP263 and SP263 respectively) was detected in 2/9 (22.2%) (47), 3/13 (23.1%) (51), 14/49 (28.6%) (52), 6/8 (75%) (53), 13/16 (81.2%) (54), 1/1 (100%) (55), 1/1 (100%) (56), 1/1 (100%) (22), 1/1 (100%) (57), 10/10 (100%) (28) of the ATCs but in none of the 6 (0%) PDTCs studied by Ahn et al (47). Mutation load or tumor mutational burden (TMB) represents the amount of somatic coding, base substitution and indel mutations per megabase of genome studied in each tumor, and high TMB is considered a new biomarker of sensitivity to ICIs (34). It has been postulated that a greater number of mutations would imply more neoantigens and consequently more targets for activated immune cells, which is why these tumors are good candidates for immune checkpoint inhibitor therapy (21,58–60). In human carcinogenesis, high TMB is mainly related to germline or somatic alterations in the DNA MMR complex (61–63), related to defects in genes involved in the recognition and removal of errors during lagging- and leading-strand DNA replication such as POLD1 and POLE genes, respectively (64–66), and/or related to loss of function mutations in the TP53 gene (67), and other alterations (68,69). ATCs are tumors with a high mutational load (37), usually accompanied by mutations of the TP53 gene (31,37), which in our series was detected in 73.3% of ATCs. In the tumor of one of the patients in our series (case 15) (Fig. 5), the ATC areas showed the highest positivity for PD-L1, positivity for p53 and loss of expression of MLH1 and PMS2, while the areas of PDTC showed negativity for PD-L1, a wild type pattern for p53 and conservation of the protein expression of the MMR genes. On the other hand, in the other tumor combining anaplastic and poorly differentiated areas (case 6) (Fig. 4), the expression of PD-L1 was limited to the ATC areas without detecting alterations related to p53 or the MMR genes in any of the different tumor areas. Interestingly, the only PDTC case with PD-L1 expression (case 19) (Fig. 3), also showed positivity for p53, but loss of MLH1 and PMS2 expression, fitting with the proposed hypermutator role of the MLH mismatch-repair genes reported in ATCs (37). In colorectal cancer, a significantly higher expression of PD-L1 was detected in tumors exhibiting MMR deficiency and BRAF mutation (40); this also agrees with the data from our series since BRAF mutation is commonly an early event in the progression from conventional (BRAF-like) PTC to ATC. BRAF^V600E mutation does not participate, however, in the development of the PDTC defined by the Turin criteria (31).

Our study evidenced significant differences in TME between the ATC and PDTC groups (Table II). Similar to our data, a recent study using fluorescence multiplex immunohistochemistry found significant differences between ATC and advanced DTC in relation to PD-L1 expression and lymphoid infiltration (70). We found an exclusive presence of CD3⁺ CD8⁺ (cytotoxic) T lymphoid population and S100⁺ dendritic cells infiltrating the tumor cells, with a minority of helper (CD4⁺) T lymphocytes at the interface of the tumor/normal thyroid tissue in all ATCs but in only one case (case 19) of PDTC. The role of TILs in PTC is controversial since its association with both a favorable (71) and an unfavorable (72,73) prognosis has been described. More specifically, a protumorigenic role of FoxP3⁺ regulatory T cells (Treg) in DTC has been demonstrated while CD8⁺ T cells develop an antitumor function (73,74). In fact, exhausted PD-1⁺ CD8⁺ T lymphocytes appeared to be a marker of bad prognosis and immunosuppression in DTC (73,74). In PTCs, S100⁺ dendritic cells were more numerous when compared to normal thyroid tissue (75), and CD1a⁺ dendritic cell density was associated with better disease-free survival (71). Significant differences between ATCs and PDTCs were also observed in our series in relation to tumor infiltration by TAMs using a histiocytic pan-marker (CD68) as well as CD163, a member of a scavenger receptor cysteine-rich superfamily restricted to the monocyte/macrophage lineage (histiocytic differentiation). Our data confirmed previous observations indicating the over-representation of pro-tumor TAMs in ATC and in advanced DTCs (71). Our images also strongly support the existence of a very dense network of interconnected CD163⁺ macrophages in direct contact with intermingled cancer cells that may trap cells in the tumor stroma (76), where they correlate with poorer survival (71). Compared with type 1 inflammatory macrophages, type 2 suppressor macrophages (CD163⁺) promote invasiveness of human TC cell lines (77) and have also been associated with a worse prognosis in cancers of other locations (78,79). Our findings are consistent with a relationship between high TMB of ATC, expression of neoantigens and changes in the immune system (PD-L1 expression and changes in TME).

In a recent meta-analysis the association of PD-L1 and disease-free survival (DFS) remained strong in PTC when compared with dedifferentiated thyroid carcinomas (ATC and PDTC) in which a significant association with PD-L1 was not confirmed (20). In another PDTC series, PD-L1 expression was significantly associated with tumor size and multifocality as well as with a non-significant trend towards older age, metastasis, an increased number of CD8⁺ T cells and decreased disease-free and overall survival (50). In our series we found a significantly higher expression of PD-L1 in TILs and TAMs in ATC group when compared to the same expression in the PDTC group (P=0.0001). We also found a trend towards worse survival in ATC with PD-L1 expression in tumor cells, but the figures did not reach statistical significance, probably due to the limited number of cases (Fig. 1B). No trend, however, was evidenced when evaluating PD-L1 expression in TILs and TAMs. A meta-analysis of non-medullary TC comprising 721 positive studies showed an association between PD-L1 expression and disease recurrence (49). PD-L1 up-regulation has recently been associated with poor disease-specific survival in patients with ATC or advanced DTC (43). This association between PD-L1 expression and a poor prognosis has also been confirmed in other tumors from different locations such as head and neck (80), breast (81), kidney (81,82), urothelial carcinoma (83), non-small cell lung cancer (84), colorectal cancer (85), and other solid tumors (81,86).

Although this study has several limitations such as its retrospective nature and the limited number of the sample, which could lead to selection biases, there are invaluable data in relation to immunotherapy. In fact, we can conclude that PD-L1 expression and tumor infiltration by CD3⁺CD8⁺ T lymphocytes, S100⁺ dendritic cells and CD68⁺CD163⁺ macrophages is common in ATC but rare in PDTC. Our findings also suggest that microsatellite instability may also play a role in both TME as well as in the identification of immunotherapy candidates among PDTC patients.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

SCG, IAN, GRC, JGG and JMCT designed the study. SCG, GRC and JGG collected the clinical data. JMCT and GRC are responsible for confirming the authenticity of the data. SAS and MSA performed the staining. IAN and JMCT evaluated the immunohistochemical data. FGS performed the statistical analysis. SCG, IAN and JMCT wrote the manuscript draft. SCG, SAS, MSA, GRC, JGG, FGS, IAN and JMCT contributed to critical revision of the manuscript for important intellectual content. JMCT obtained financial support. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The study was approved by the Santiago-Lugo Medical Research Ethics Committee (Santiago de Compostela, Spain; approval no. 2019/275) and written informed consent was obtained.

Patient consent for publication

Not applicable.

Authors' information

SCG, ORCID 0000-0002-2678-7230; SAS, ORCID 0000-0003-3576-1953; MSA, ORCID 0000-0003-0098-4161; GRC, ORCID 0000-0002-2015-7369; JGG, ORCID 0000-0002-5372-2467; FGS, ORCID 0000-0002-9681-1662; IAN, ORCID 0000-0003-1247-2331; and JMCT, ORCID 0000-0002-5516-8914.

Competing interests

The authors declare that they have no competing interests.

Abbreviations

ATC

anaplastic thyroid carcinoma

DTC

differentiated thyroid carcinoma

FTC

follicular thyroid carcinoma

HCC

Hürthle (oncocytic) cell carcinoma

ICI

immune checkpoint inhibitor

MKIs

multi-tyrosine kinase inhibitors

MMR

mismatch repair

PD-L1

programmed death ligand 1

PDTC

poorly DTC

PTC

papillary thyroid carcinoma

TAMs

tumor-associated macrophages

thyroid cancer

TILs

tumor infiltrating lymphocytes

TMB

tumor mutational burden

TME

tumor microenvironment

TPS

tumor proportion score

WHO

World Health Organization

References 1

Bray

Ferlay

Soerjomataram

Siegel

Torre

Jemal

Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries

CA Cancer J Clin683944242018

10.3322/caac.21492

30207593

Lloyd

Osamura

Klöppel

Rosai

WHO Classification of Tumours of Endocrine Organs

IARC

Lyon

2017

Luster

Aktolun

Amendoeira

Barczyński

Bible

Duntas

Elisei

Handkiewicz-Junak

Hoffmann

Jarząb

European Perspective on 2015 American Thyroid Association Management Guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: Proceedings of an Interactive International Symposium

Thyroid297262019

10.1089/thy.2017.0129

30484394

Smallridge

Ain

Asa

Bible

Brierley

Burman

Kebebew

Lee

Nikiforov

Rosenthal

American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer

Thyroid22110411392012

10.1089/thy.2012.0302

23130564

Haddad

Nasr

Bischoff

Busaidy

Byrd

Callender

Dickson

Duh

Ehya

Goldner

NCCN Guidelines Insights: Thyroid Carcinoma, Version 2.2018

J Natl Compr Canc Netw16142914402018

10.6004/jnccn.2018.0089

30545990

De Leo

Trevisan

Fugazzola

Recent advances in the management of anaplastic thyroid cancer

Thyroid Res13172020

10.1186/s13044-020-00091-w

33292371

Volante

Collini

Nikiforov

Sakamoto

Kakudo

Katoh

Lloyd

LiVolsi

Papotti

Sobrinho-Simoes

Poorly differentiated thyroid carcinoma: The Turin proposal for the use of uniform diagnostic criteria and an algorithmic diagnostic approach

Am J Surg Pathol31125612642007

10.1097/PAS.0b013e3180309e6a

17667551

Ghossein

Poorly differentiated thyroid carcinoma

Semin Diagn Pathol372432472020

10.1053/j.semdp.2020.03.003

32360274

Akaishi

Kondo

Sugino

Ogimi

Masaki

Hames

Yabuta

Tomoda

Suzuki

Matsuzu

Prognostic impact of the Turin criteria in poorly differentiated thyroid carcinoma

World J Surg43223522442019

10.1007/s00268-019-05028-5

31098668

Walczyk

Kopczyński

Gąsior-Perczak

Pałyga

Kowalik

Chrapek

Hejnold

Góźdź

Kowalska

Histopathology and immunohistochemistry as prognostic factors for poorly differentiated thyroid cancer in a series of Polish patients

PLoS One15e02292642020

10.1371/journal.pone.0229264

32092093

Dettmer

Schmitt

Steinert

Haldemann

Meili

Moch

Komminoth

Perren

Poorly differentiated thyroid carcinomas: How much poorly differentiated is needed?

Am J Surg Pathol35186618722011

10.1097/PAS.0b013e31822cf962

21989341

Dettmer

Schmitt

Steinert

Moch

Komminoth

Perren

Poorly differentiated oncocytic thyroid carcinoma-diagnostic implications and outcome

Histopathology60104510512012

10.1111/j.1365-2559.2012.04188.x

22348590

Bai

Baloch

Samulski

Montone

LiVolsi

Poorly differentiated oncocytic (Hürthle cell) follicular carcinoma: An institutional experience

Endocr Pathol261641692015

10.1007/s12022-015-9367-6

25898815

Wang

Zafereo

Dadu

Ferrarotto

Busaidy

Ahmed

Gule-Monroe

Williams

Sturgis

Complete Surgical resection following neoadjuvant dabrafenib plus trametinib in BRAF^600E-mutated anaplastic thyroid carcinoma

Thyroid29103610432019

10.1089/thy.2019.0133

31319771

Takahashi

Tahara

Ito

Tori

Kiyota

Yoshida

Sakata

Yoshida

Safety and effectiveness of lenvatinib in 594 patients with unresectable thyroid cancer in an all-case post-marketing observational study in Japan

Adv Ther37385038622020

10.1007/s12325-020-01433-8

32676927

Hanna

Busaidy

Chau

Wirth

Barletta

Calles

Haddad

Kraft

Cabanillas

Rabinowits

Genomic correlates of response to everolimus in aggressive radioiodine-refractory thyroid cancer: A phase II study

Clin Cancer Res24154615532018

10.1158/1078-0432.CCR-17-2297

29301825

Harris

Hanna

Chau

Rabinowits

Haddad

Margalit

Schoenfeld

Tishler

Barletta

Nehs

Everolimus in anaplastic thyroid cancer: A case series

Front Oncol91062019

10.3389/fonc.2019.00106

30863722

Lee

Urba

Koenig

Sisson

Giordano

Worden

A phase II study of imatinib in patients with advanced anaplastic thyroid cancer

Thyroid209759802010

10.1089/thy.2010.0057

20718683

Capdevila

Wirth

Ernst

Ponce Aix

Lin

Ramlau

Butler

Delord

Gelderblom

Ascierto

PD-1 Blockade in Anaplastic Thyroid Carcinoma

J Clin Oncol38262026272020

10.1200/JCO.19.02727

32364844

Guan

Lim

Mekhail

Chang

Programmed death ligand-1 (PD-L1) expression in the programmed death receptor-1 (PD-1)/PD-L1 blockade: A key player against various cancers

Arch Pathol Lab Med1418518612017

10.5858/arpa.2016-0361-RA

28418281

You

Shang

Sun

Liu

Jiang

Mechanistic insight of predictive biomarkers for antitumor PD-1/PD-L1 blockade: A paradigm shift towards immunome evaluation (Review)

Oncol Rep444244372020

10.3892/or.2020.7643

32627031

Cantara

Bertelli

Occhini

Regoli

Brilli

Pacini

Castagna

Toti

Blockade of the programmed death ligand 1 (PD-L1) as potential therapy for anaplastic thyroid cancer

Endocrine641221292019

10.1007/s12020-019-01865-5

30762153

Gunda

Gigliotti

Ndishabandi

Ashry

McCarthy

Zhou

Amin

Freeman

Alessandrini

Parangi

Combinations of BRAF inhibitor and anti-PD-1/PD-L1 antibody improve survival and tumour immunity in an immunocompetent model of orthotopic murine anaplastic thyroid cancer

Br J Cancer119122312322018

10.1038/s41416-018-0296-2

30327563

Gunda

Gigliotti

Ashry

Ndishabandi

McCarthy

Zhou

Amin

Lee

Stork

Wirth

Anti-PD-1/PD-L1 therapy augments lenvatinib's efficacy by favorably altering the immune microenvironment of murine anaplastic thyroid cancer

Int J Cancer144226622782019

10.1002/ijc.32041

30515783

Moretti

Menicali

Nucci

Guzzetti

Morelli

Puxeddu

Therapy of endocrine disease Immunotherapy of advanced thyroid cancer: From bench to bedside

Eur J Endocrinol183R41R552020

10.1530/EJE-20-0283

32449696

Mehnert

Varga

Brose

Aggarwal

Lin

Prawira

de Braud

Tamura

Doi

Piha-Paul

Safety and antitumor activity of the anti-PD-1 antibody pembrolizumab in patients with advanced, PD-L1-positive papillary or follicular thyroid cancer

BMC Cancer191962019

10.1186/s12885-019-5380-3

30832606

Chintakuntlawar

Yin

Foote

Kasperbauer

Rivera

Asmus

Garces

Janus

Liu

A phase 2 study of pembrolizumab combined with chemoradiotherapy as initial treatment for anaplastic thyroid cancer

Thyroid29161516222019

10.1089/thy.2019.0086

31595822

Iyer

Dadu

Gule-Monroe

Busaidy

Ferrarotto

Habra

Zafereo

Williams

Gunn

Grosu

Salvage pembrolizumab added to kinase inhibitor therapy for the treatment of anaplastic thyroid carcinoma

J Immunother Cancer6682018

10.1186/s40425-018-0378-y

29996921

Girolami

Pantanowitz

Mete

Brunelli

Marletta

Colato

Trimboli

Crescenzi

Bongiovanni

Barbareschi

Eccher

Programmed Death-Ligand 1 (PD-L1) is a potential biomarker of disease-free survival in papillary thyroid carcinoma: A systematic review and meta-analysis of PD-L1 immunoexpression in follicular epithelial derived thyroid carcinoma

Endocr Pathol312913002020

10.1007/s12022-020-09630-5

32468210

Bangaraiahgari

Panchangam

Puthenveetil

Mayilvaganan

Bangaraiahgari

Banala

Karunakaran

Is there adenoma-carcinoma sequence between benign adenoma and papillary cancer of thyroid: A genomic linkage study

Ann Med Surg (Lond)606957002020

10.1016/j.amsu.2020.11.069

33318795

Volante

Lam

Papotti

Tallini

Molecular pathology of poorly differentiated and anaplastic thyroid cancer: What do pathologists need to know

Endocr Pathol3263762021

10.1007/s12022-021-09665-2

33543394

Soares

Póvoa

Melo

Vinagre

Máximo

Eloy

Cameselle-Teijeiro

Sobrinho-Simões

Molecular pathology of Non-familial follicular epithelial-derived thyroid cancer in adults: From RAS/BRAF-like tumor designations to molecular risk stratification

Endocr Pathol3244622021

10.1007/s12022-021-09666-1

33651322

Jung

Kim

Jung

Park

Kim

Liu

Bae

Lee

Kim

Lee

Chung

Mutational burdens and evolutionary ages of thyroid follicular adenoma are comparable to those of follicular carcinoma

Oncotarget769638696482016

10.18632/oncotarget.11922

27626165

Chalmers

Connelly

Fabrizio

Gay

Ali

Ennis

Schrock

Campbell

Shlien

Chmielecki

Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden

Genome Med9342017

10.1186/s13073-017-0424-2

28420421

Cancer Genome Atlas Research Network

Integrated genomic characterization of papillary thyroid carcinoma

Cell1596766902014

10.1016/j.cell.2014.09.050

25417114

Landa

Ibrahimpasic

Boucai

Sinha

Knauf

Shah

Dogan

Ricarte-Filho

Krishnamoorthy

Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers

J Clin Invest126105210662016

10.1172/JCI85271

26878173

Kunstman

Juhlin

Goh

Brown

Stenman

Healy

Rubinstein

Choi

Kiss

Nelson-Williams

Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing

Hum Mol Genet24231823292015

10.1093/hmg/ddu749

25576899

Riesco-Eizaguirre

Santisteban

Endocrine Tumours: Advances in the molecular pathogenesis of thyroid cancer: Lessons from the cancer genome

Eur J Endocrinol175R203R2172016

10.1530/EJE-16-0202

27666535

Capdevila

Mayor

Mancuso

Iglesias

Caratù

Matos

Zafón

Hernando

Petit

Nuciforo

Early evolutionary divergence between papillary and anaplastic thyroid cancers

Ann Oncol29145414602018

10.1093/annonc/mdy123

29648575

Ragazzi

Torricelli

Donati

Ciarrocchi

de Biase

Tallini

Zanetti

Bisagni

Kuhn

Giordano

Coexisting well-differentiated and anaplastic thyroid carcinoma in the same primary resection specimen: Immunophenotypic and genetic comparison of the two components in a consecutive series of 13 cases and a review of the literature

Virchows Arch4782652812021

10.1007/s00428-020-02891-9

32683537

Cameselle-Teijeiro

Rodríguez-Pérez

Celestino

Eloy

Piso-Neira

Abdulkader-Nallib

Soares

Sobrinho-Simões

Hobnail variant of papillary thyroid carcinoma: Clinicopathologic and molecular evidence of progression to undifferentiated carcinoma in 2 cases

Am J Surg Pathol418548602017

10.1097/PAS.0000000000000793

28009606

Ibrahimpasic

Ghossein

Shah

Ganly

Poorly Differentiated carcinoma of the thyroid gland: Current status and future prospects

Thyroid293113212019

10.1089/thy.2018.0509

30747050

Yoo

Song

Lee

Hwang

Kim

Jung

Kim

Cho

Won

Integrative analysis of genomic and transcriptomic characteristics associated with progression of aggressive thyroid cancer

Nat Commun1027642019

10.1038/s41467-019-10680-5

31235699

Hiltzik

Carlson

Tuttle

Chuai

Ishill

Shaha

Shah

Singh

Ghossein

Poorly differentiated thyroid carcinomas defined on the basis of mitosis and necrosis: A clinicopathologic study of 58 patients

Cancer106128612952006

10.1002/cncr.21739

16470605

Gerber

Schad

Hartmann

Springer

Zechner

Musholt

Targeted next-generation sequencing of cancer genes in poorly differentiated thyroid cancer

Endocr Connect747552018

10.1530/EC-17-0290

29133385

Cunha

Marcello

Morari

Nonogaki

Conte

Gerhard

Soares

Vassallo

Ward

Differentiated thyroid carcinomas may elude the immune system by B7H1 upregulation

Endocr Relat Cancer201031102013

10.1530/ERC-12-0313

23193072

Ahn

Kim

Jang

Kim

Choe

Shin

Hahn

Comprehensive screening for PD-L1 expression in thyroid cancer

Endocr Relat Cancer24971062017

10.1530/ERC-16-0421

28093480

Chowdhury

Veyhl

Jessa

Polyakova

Alenzi

MacMillan

Ralhan

Walfish

Programmed death-ligand 1 overexpression is a prognostic marker for aggressive papillary thyroid cancer and its variants

Oncotarget732318323282016

10.18632/oncotarget.8698

27086918

Aghajani

Graham

McCafferty

Shaheed

Roberts

DeSouza

Yang

Niles

Clinicopathologic and prognostic significance of programmed cell death ligand 1 expression in patients with non-medullary thyroid cancer: A systematic review and meta-analysis

Thyroid283493612018

10.1089/thy.2017.0441

29455638

Rosenbaum

Gigliotti

Pai

Parangi

Wachtel

Mino-Kenudson

Gunda

Faquin

PD-L1 and IDO1 are expressed in poorly differentiated thyroid carcinoma

Endocr Pathol2959672018

10.1007/s12022-018-9514-y

29372535

Sun

Yang

Lee

de las Morenas

Anaplastic thyroid cancer: Outcome and the mutation/expression profiles of potential targets

Pathol Oncol Res216957012015

10.1007/s12253-014-9876-5

25588542

Zwaenepoel

Jacobs

De Meulenaere

Silence

Smits

Siozopoulou

Hauben

Rolfo

Rottey

Pauwels

CD70 and PD-L1 in anaplastic thyroid cancer-promising targets for immunotherapy

Histopathology713573652017

10.1111/his.13230

28383817

Bastman

Serracino

Zhu

Koenig

Mateescu

Sams

Davies

Raeburn

McIntyre

JrHaugen

French

Tumor-Infiltrating T cells and the PD-1 checkpoint pathway in advanced differentiated and anaplastic thyroid cancer

J Clin Endocrinol Metab101286328732016

10.1210/jc.2015-4227

27045886

Chintakuntlawar

Rumilla

Smith

Jenkins

Foote

Kasperbauer

Morris

Ryder

Alsidawi

Hilger

Bible

Expression of PD-1 and PD-L1 in anaplastic thyroid cancer patients treated with multimodal therapy: Results from a retrospective study

J Clin Endocrinol Metab102194319502017

10.1210/jc.2016-3756

28324060

Kollipara

Schneider

Radovich

Babu

Kiel

Exceptional response with immunotherapy in a patient with anaplastic thyroid cancer

Oncologist22114911512017

10.1634/theoncologist.2017-0096

28778959

Aghajani

Cooper

McGuire

Jeffries

Saab

Ismail

de Souza

Bray

Fazekas de St Groth

Niles

Roberts

Pembrolizumab for anaplastic thyroid cancer: A case study

Cancer Immunol Immunother68192119342019

10.1007/s00262-019-02416-7

31637475

Stenman

Hellgren

Jatta

Hysek

Zemmler

Altena

Nilsson

Bränström

Zedenius

Juhlin

Metastatic anaplastic thyroid carcinoma in complete remission: Morphological, molecular, and clinical work-up of a rare case

Endocr Pathol3177832020

10.1007/s12022-020-09606-5

32016808

Snyder

Makarov

Merghoub

Yuan

Zaretsky

Desrichard

Walsh

Postow

Wong

Genetic basis for clinical response to CTLA-4 blockade in melanoma

N Engl J Med371218921992014

10.1056/NEJMoa1406498

25409260

Uram

Wang

Bartlett

Kemberling

Eyring

Skora

Luber

Azad

Laheru

PD-1 Blockade in Tumors with Mismatch-Repair Deficiency

N Engl J Med372250925202015

10.1056/NEJMoa1500596

26028255

Mehnert

Panda

Zhong

Hirshfield

Damare

Lane

Sokol

Stein

Rodriguez-Rodriquez

Kaufman

Immune activation and response to pembrolizumab in POLE-mutant endometrial cancer

J Clin Invest126233423402016

10.1172/JCI84940

27159395

Duval

Hamelin

Mutations at coding repeat sequences in mismatch repair-deficient human cancers: Toward a new concept of target genes for instability

Cancer Res62244724542002

11980631

Peltomäki

Role of DNA mismatch repair defects in the pathogenesis of human cancer

J Clin Oncol21117411792003

10.1200/JCO.2003.04.060

Mensenkamp

Vogelaar

van Zelst-Stams

Goossens

Ouchene

Hendriks-Cornelissen

Kwint

Hoogerbrugge

Nagtegaal

Ligtenberg

Somatic mutations in MLH1 and MSH2 are a frequent cause of mismatch-repair deficiency in Lynch syndrome-like tumors

Gastroenterology146643646 e82014

10.1053/j.gastro.2013.12.002

24333619

Briggs

Tomlinson

Germline and somatic polymerase ε and δ mutations define a new class of hypermutated colorectal and endometrial cancers

J Pathol2301481532013

10.1002/path.4185

23447401

Church

Briggs

Palles

Domingo

Kearsey

Grimes

Gorman

Martin

Howarth

Hodgson

DNA polymerase epsilon and δ exonuclease domain mutations in endometrial cancer

Hum Mol Genet22282028282013

10.1093/hmg/ddt131

23528559

Domingo

Freeman-Mills

Rayner

Glaire

Briggs

Vermeulen

Fessler

Medema

Boot

Morreau

Somatic POLE proofreading domain mutation, immune response, and prognosis in colorectal cancer: A retrospective, pooled biomarker study

Lancet Gastroenterol Hepatol12072162016

10.1016/S2468-1253(16)30014-0

28404093

Petitjean

Mathe

Kato

Ishioka

Tavtigian

Hainaut

Olivier

Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: Lessons from recent developments in the IARC TP53 database

Hum Mutat286226292007

10.1002/humu.20495

17311302

Lawrence

Stojanov

Polak

Kryukov

Cibulskis

Sivachenko

Carter

Stewart

Mermel

Roberts

Mutational heterogeneity in cancer and the search for new cancer-associated genes

Nature4992142182013

10.1038/nature12213

23770567

Poulos

Wong

Ryan

Pang

Wong

JWH

Analysis of 7,815 cancer exomes reveals associations between mutational processes and somatic driver mutations

PLoS Genet14e10077792018

10.1371/journal.pgen.1007779

30412573

Ahn

Jin

Song

Ryu

Song

Kim

Shong

Jeon

Kim

Immune profiling of advanced thyroid cancers using fluorescent multiplex immunohistochemistry

Thyroid3161672021

10.1089/thy.2020.0312

32611231

Ferrari

Fallahi

Galdiero

Ruffilli

Elia

Ragusa

Paparo

Patrizio

Mazzi

Varricchi

Immune and inflammatory cells in thyroid cancer microenvironment

Int J Mol Sci2044132019

10.3390/ijms20184413

31500315

French

Weber

Fretwell

Said

Klopper

Haugen

Tumor-associated lymphocytes and increased FoxP3+ regulatory T cell frequency correlate with more aggressive papillary thyroid cancer

J Clin Endocrinol Metab95232523332010

10.1210/jc.2009-2564

20207826

French

Kotnis

Said

Raeburn

McIntyre

JrKlopper

Haugen

Programmed death-1+ T cells and regulatory T cells are enriched in tumor-involved lymph nodes and associated with aggressive features in papillary thyroid cancer

J Clin Endocrinol Metab97E934E9432012

10.1210/jc.2011-3428

22466343

Severson

Serracino

Mateescu

Raeburn

McIntyre

JrSams

Haugen

French

PD-1+Tim-3+ CD8+ T lymphocytes display varied degrees of functional exhaustion in patients with regionally metastatic differentiated thyroid cancer

Cancer Immunol Res36206302015

10.1158/2326-6066.CIR-14-0201

25701326

Hilly

Koren

Raz

Rath-Wolfson

Mizrachi

Hamzany

Bachar

Shpitzer

The role of s100-positive dendritic cells in the prognosis of papillary thyroid carcinoma

Am J Clin Pathol13987922013

10.1309/AJCPAKYDO56NKMYZ

23270903

Caillou

Talbot

Weyemi

Pioche-Durieu

Al Ghuzlan

Bidart

Chouaib

Schlumberger

Dupuy

Tumor-associated macrophages (TAMs) form an interconnected cellular supportive network in anaplastic thyroid carcinoma

PLoS One6e225672011

10.1371/journal.pone.0022567

21811634

Fang

Shen

Cai

Huang

Wei

Fei

Chen

Guan

Wang

Tumor-associated macrophages promote the metastatic potential of thyroid papillary cancer by releasing CXCL8

Carcinogenesis35178017872014

10.1093/carcin/bgu060

24608042

Zhang

Sun

Wang

Zhao

A high M1/M2 ratio of tumor-associated macrophages is associated with extended survival in ovarian cancer patients

J Ovarian Res7192014

10.1186/1757-2215-7-19

24507759

Yuan

Hsiao

Chen

Liu

Hong

Chen

Yang

Opposite effects of M1 and M2 macrophage subtypes on lung cancer progression

Sci Rep5142732015

10.1038/srep14273

26399191

Wang

Prognostic value of programmed cell death ligand 1 expression in patients with head and neck cancer: A systematic review and meta-analysis

PLoS One12e01795362017

10.1371/journal.pone.0179536

28604812

Wang

Liu

Prognostic significance of PD-L1 in solid tumor: An updated meta-analysis

Medicine (Baltimore)96e63692017

10.1097/MD.0000000000006369

28471952

Wang

Feng

Liu

Clinicopathological and prognostic value of programmed death ligand-1 (PD-L1) in renal cell carcinoma: A meta-analysis

Int J Clin Exp Med814595146032015

26628942

Powles

Walker

Andrew Williams

Bellmunt

The evolving role of PD-L1 testing in patients with metastatic urothelial carcinoma

Cancer Treat Rev821019252020

10.1016/j.ctrv.2019.101925

31785413

Pan

Clinicopathological and prognostic significance of programmed cell death ligand1 (PD-L1) expression in patients with non-small cell lung cancer: A meta-analysis

J Thorac Dis74624702015

25922726

Siraj

Parvathareddy

Annaiyappanaidu

Haqawi

Al-Rasheed

AlManea

AlHussaini

Al-Dayel

Al-Kuraya

PD-L1 expression is associated with deficient mismatch repair and poor prognosis in middle eastern colorectal cancers

J Pers Med11732021

10.3390/jpm11020073

33530623

Walter

Herrmann

Schnitzbauer

Zeuzem

Hansmann

Peveling-Oberhag

Hartmann

PD-L1 expression in extrahepatic cholangiocarcinoma

Histopathology713833922017

10.1111/his.13238

28419539

Figure 1.

Survival and PD-L1 expression in patients with ATC and PDTC. (A) Kaplan-Meier analysis showing significant differences in survival between patients with ATC and PDTC. (B) Patients with PD-L1-positive ATC showed a trend for worse survival than those with PD-L1-negative tumors, although the difference was not significant. (C) No difference in survival was observed when comparing positive PD-L1 expression with negative PD-L1 expression in ATC lymphocytes and macrophages. ATC, anaplastic thyroid carcinoma; PDTC, poorly differentiated thyroid carcinoma; PD-L1, programmed death ligand 1.

Figure 2.

Immunohistochemical staining of ATC tissues. (A) H&E staining of ATC with epithelial pattern. (B) All ATC cases were negative for TG; positively stained follicles (arrows) were normal cells entrapped by the tumor cells (internal control). (C) Strong positivity for PD-L1 was detected in some ATC cases. (D) CD3⁺ lymphocytes were found in all ATC cases. (E) CD4⁺ lymphocytes (arrows) were found exclusively surrounding the tumor (asterisks). (F) CD8⁺ lymphocytes, (G) CD68⁺ and (H) CD163⁺ macrophages, and (I) S100⁺ dendritic cells were found in all ATC cases. Positivity for (J) p53, (K) MLH1 and (L) PMS2 was a common finding in ATC. Magnification, ×400. ATC, anaplastic thyroid carcinoma; H&E, hematoxylin and eosin; TG, thyroglobulin; PD-L1, programmed death ligand 1; MLH1, mutL homolog 1; PMS2, PMS1 homolog 2.

Figure 3.

Poorly differentiated thyroid carcinoma (Hürthle cell variant; case 19). (A) H&E staining. (B) Strong positivity for PD-L1 (arrows). (C) Tumor cells were negative for MLH1, but positivity was observed in stromal cells, endothelial cells (arrows) and lymphocytes (arrowheads) (internal control). (D) Strong positivity for p53. Magnification, ×400. H&E, hematoxylin and eosin; PD-L1, programmed death ligand 1; MLH1, mutL homolog 1.

Figure 4.

ATC with poorly differentiated areas (case 6). (A) Transition between poorly differentiated areas (arrows) and ATC areas (magnification, ×200). Higher magnification shows (B) a poorly differentiated area with a focus of necrosis (asterisks) and (C) another ATC area. (D) Poorly differentiated areas were negative for PD-L1, with (E) positivity for PD-L1 only observed in ATC areas. MLH1 protein expression was detected in both (F) poorly differentiated and (G) ATC areas. (H) A higher density of CD8⁺ lymphocytes was observed in ATC areas than in poorly differentiated areas. (I) CD163⁺ macrophages were only found in the ATC areas of the tumor. Magnification, ×400. ATC, anaplastic thyroid carcinoma; H&E, hematoxylin and eosin; PD-L1, programmed death ligand 1; MLH1, mutL homolog 1.

Figure 5.

ATC with poorly differentiated areas (case 15). (A) Transition between poorly differentiated areas (asterisks) and ATC areas (arrows) (magnification, ×200). Morphological differences between (B) poorly differentiated and (C) ATC areas at a higher magnification (magnification, ×400). (D) PD-L1 expression was only detected in the ATC areas (arrows), but not in the poorly differentiated component (asterisks) (magnification, ×200). (E) ATC cells showed strong and diffuse positivity for PD-L1 (magnification, ×400). MLH1 expression at magnification (F) ×200 and (G) ×400 was found in the poorly differentiated areas (asterisk), but not in the ATC areas (arrows). CD163⁺ macrophages were not detected in (H) the poorly differentiated areas, but were heavily detected in (I) the ATC areas (magnification, ×400). ATC, anaplastic thyroid carcinoma; H&E, hematoxylin and eosin; PD-L1, programmed death ligand 1; MLH1, mutL homolog 1.

Table I.

Clinicopathological and immunohistochemical findings in 15 cases of ATC and 13 cases of PDTC in 26 patients.

Case no.	Age, years	Sex	Diagnosis	Follow-up (months)	PD-L1 TPS, %	PD-L1 TILs, %	CD3	CD3	CD8	CD20	CD68	CD163	S100	p53	MLH1	MSH2	MSH6	PMS2
1	73	F	ATC, Sc	DOD (11.2)	0	0	3	0	1	1	3	3	2	B	+	+	+	+
2	83	F	ATC, Gc	DOD (0.13)	15	20	3	1^a	3	0	3	3	1	B	+	+	+	+
3	67	M	ATC, Sc	DOD (5.5)	0	1	3	0	1	0	3	3	3	WT	+	+	+	+
4	75	M	ATC, Gc	DOD (1)	0	0	3	1^a	2	1	3	3	1	N	+	+	+	+
5	76	F	ATC, Ec	DOD (0.03)	35	5	3	1^a	3	0	3	3	1	WT	+	+	+	+
6^b	73	M	ATC, Sc	DOD (1.56)	30	3	3	0	3	0	3	3	1	WT	+	+	+	+
7	68	F	ATC, Gc	DOD (0.36)	15	15	3	1^a	3	0	3	3	1	B	+	+	+	+
8	47	M	ATC, Gc	DOD (16)	0	5	3	1^a	3	1	3	3	1	WT	+	+	+	+
9	47	F	ATC, Ec	DOD (2.73)	18	0	3	0	2	1	3	3	1	B	+	+	+	+
10	60	F	ATC, Gc	DOD (6)	33	0	3	0	2	0	3	3	1	B	+	+	+	+
11	71	M	ATC, Gc	DOD (1.6)	0	1	3	0	3	1	3	3	1	B	+	+	+	+
12	67	F	ATC, Ec	DOD (1.67)	90	10	3	2^a	2	1	3	3	1	B	+	+	+	+
13	54	F	ATC, Gc	DOD (7)	30	20	3	1^a	2	1	3	3	2	N	+	+	+	+
14^c	52	M	ATC, Gc	AWD (14)	0	10	2	1^a	2	0	3	3	1	B	+	+	+	+
15^b	55	F	ATC, Gc	AWD (7)	90	80	3	1^a	3	0	3	3	2	B	−	+	+	−
15^b	55	F	PDTC	AWD (7)	0	0	1	0	0	0	0	1	0	WT	+	+	+	+
6^b	73	M	PDTC	DOD (1.56)	0	0	1	0	1	0	0	0	0	WT	+	+	+	+
16	72	F	PDTC	AAW (237)	0	0	0	0	0	0	0	0	0	WT	+	+	+	+
17	75	M	PDTC	DOD (12.6)	0	0	0	0	0	0	0	0	0	WT	+	+	+	+
18	68	F	PDTC	DAD (148.2)	0	0	1	0	1	0	0	0	0	WT	+	+	+	+
19	71	F	PDTC, Oc	DOD (149.88)	35	0	2	0	2	0	2	2	1	B	−	+	+	−
20	78	F	PDTC	DOD (99.24)	0	0	1	0	1	0	1	1	1	WT	+	+	+	+
21	73	F	PDTC	DOD (5.52)	0	0	1	0	1	0	1	1	0	WT	+	+	+	+
22	80	F	PDTC	DOD (3.36)	0	0	1	0	2	0	1	1	1	WT	+	+	+	+
23	75	F	PDTC	DAD (58.32)	0	0	0	0	0	0	1	1	0	WT	+	+	+	+
24	87	F	PDTC	DOD (29.16)	0	0	0	0	0	0	1	1	1	WT	+	+	+	+
25	63	F	PDTC	AWD (99.24)	0	0	1	0	1	0	1	1	1	WT	+	+	+	+
26	67	M	PDTC	AWD (84)	0	0	1	0	0	0	1	1	1^a	WT	+	+	+	+

Positivity exclusively in lymphoid population surrounding the tumor

These tumors combined areas of ATC and PDTC

This was the only case with a previous diagnosis as a differentiated thyroid carcinoma (Hürthle cell carcinoma diagnosed 26 months earlier). M, male; F, female; ATC, anaplastic thyroid carcinoma; PDTC, poorly differentiated thyroid carcinoma; Sc, spindle cell sarcoma-like pattern; Gc, giant cell pattern; Ec, epithelial cell pattern; Oc, oncocytic variant (Hürthle cell PDTC); DOD, dead of disease; AWD, alive with disease; AAW, alive and well; DAD, dead from another disease; TPS, tumor proportion score; TILs, tumor-infiltrating lymphocytes; 1, ≤10% positivity of inflammatory cells; 2, 11–49% positivity; 3, ≥50% positivity; B, ‘block’ staining; N, ‘null’ phenotype; WT, wild-type; PD-L1, programmed death ligand 1; MLH1, mutL homolog 1; MSH2, mutS homolog 2; MSH6, mutS homolog 6; PMS2, PMS1 homolog 2.

Table II.

Comparison of clinicopathological and immunohistochemical data between 15 cases of ATC and 13 cases of PDTC in 26 patients^a.

Characteristics	ATC	PDTC	P-value
Age, years			0.018
Median	67	73
Mean ± SD (range)	64.53±11.29 (47–83)	73.55±6.63 (55–87)
Sex, n/total (%)			0.395
Male	6/15 (40.0)	2/11 (18.2)
Female	9/15 (60.0)	9/11 (81.8)
PD-L1
TPS	9/15 (60.0)	1/13 (7.7)	0.006
TILs	11/15 (73.3)	0/13 (0.0)	<0.001
CD3	15/15 (100.0)	9/13 (69.2)	0.035
CD4	9/15 (60.0)	0/13 (0.0)	0.001
CD8	15/15 (100.0)	7/13 (53.8)	0.005
CD20	7/15 (46.7)	0/13 (0.0)	0.007
CD68	15/15 (100.0)	8/13 (61.5)	0.013
CD163	15/15 (100.0)	9/13 (69.2)	0.035
S100	15/15 (100.0)	6/13 (46.2)	0.001
p53	11/15 (73.3)	1/13 (7.7)	0.001

Among the 26 patients, coexistence of ATC and PDTC areas was observed in the tumors of 2 patients. ATC, anaplastic thyroid carcinoma; PDTC, poorly differentiated thyroid carcinoma; TPS, tumor proportion score; TILs, tumor-infiltrating lymphocytes; PD-L1, programmed death ligand 1.