Abstract
Background
Despite the increasing recognition of PD-L1 as predictor of immunotherapeutic response in various malignancies, its role and prognostic significance in thyroid cancer remain underexplored and subject to debate. This study begins to address this gap by comprehensively analyzing PD-L1 expression in papillary thyroid carcinoma (PTC) and investigating its correlation with key clinicopathological variables.
Methods
We conducted immunohistochemistry (IHC) to assess PD-L1 expression in whole-tissue sections from 121 primary papillary thyroid carcinoma (PTC) cases. We then analyzed the correlations between PD-L1 expression and various clinicopathological variables.
Results
PD-L1 expression was detected in 33.1% of papillary thyroid carcinomas (PTCs), predominantly exhibiting weak to moderate intensity. Notably, this study found no significant correlation between PD-L1 expression and various clinicopathological variables. The lack of association with traditional factors such as age, sex, histological subtype, and tumor size suggests the complex and multifaceted nature of PD-L1 regulation in PTC. Multivariate logistic regression analysis identified chronic lymphocytic thyroiditis with oncocytic metaplasia as the sole independent predictor of PD-L1 expression (P = 0.014), underlining the potential influence of the tumor microenvironment on immune checkpoint expression in PTC.
Conclusions
Our study underscores the intricate interplay between chronic lymphocytic thyroiditis with oncocytic metaplasia and PD-L1 expression in papillary thyroid carcinoma. The observed link suggests a potential avenue for therapeutic intervention using anti-PD-1/PD-L1 therapies in surgery-refractory PTC. Understanding the dynamics of immune checkpoint regulation in the context of the tumor microenvironment is crucial for devising effective treatment strategies. Future research endeavors should delve deeper into the molecular mechanisms underlying this interaction and explore its implications for patient outcomes. As the field of immunotherapy continues to evolve, our findings contribute valuable insights into the complex immunological landscape of thyroid cancer.
Keywords: PD-L1, Papillary Thyroid Carcinoma, Immunohistochemistry, Clinicopathological Features, Chronic Lymphocytic Thyroiditis, Extrathyroidal Extension, Prognosis
Introduction
Thyroid cancer (TC) stands as the most common endocrine malignancy, constituting nearly 1% of all human malignancies [1]. The global incidence of TC has shown a notable increase [2]. Papillary thyroid carcinoma (PTC) alone comprises around 90% of all thyroid cancers. While the prognosis for thyroid cancer is generally favorable, with a 98% overall 5-year survival rate [3], approximately 10% of patients with differentiated thyroid cancer develop progressive disease, 5% manifest distant metastases, and 20–30% experience disease recurrence [4]. Consequently, the identification of novel biomarkers capable of predicting prognosis and potential therapeutic response is crucial for enhancing the outcomes in patients with progressive, recurrent, and radioiodine-refractory diseases.
Programmed cell death ligand 1 (PD-L1) currently serves as a prognostic marker for melanoma, non-small cell lung carcinoma, breast carcinoma, and several other cancers [5]. The Food and Drug Administration (FDA) has approved the use of anti-PD-1/PD-L1 monoclonal antibodies for treating melanoma, non-small cell lung cancer, urothelial carcinoma, and other cancers [5]. Notably, a study by Girolami et al. [6] observed a tendency for higher PD-L1 expression in precancerous lesions of the head and neck compared to normal mucosa. The authors speculated that PD-L1 expression might represent a delicate balance between the host immune response and cancer escape ability. In a recent clinical trial (Phase 1b KEYNOTE-028), pembrolizumab monotherapy was evaluated in 22 advanced PTC and follicular thyroid carcinomas, revealing a partial response (overall response rate = 9%) in only two patients [7].
Several studies have delved into PD-L1 expression in thyroid cancer, yielding conflicting data. The frequency of PD-L1 positivity varies widely among different studies, ranging from 6.1 to 82.5% of PTC patients [8–16]. This substantial variability can be attributed to multiple factors, including differences in clinical trial design, PD-L1 immunohistochemistry (IHC) assays, primary monoclonal antibodies, interpretation, and scoring systems. These scoring systems encompass aspects such as the site of PD-L1 expression (membranous versus cytoplasmic), type of cells analyzed (tumor cells alone versus tumor plus lymphocytes), and the cut-offs defining a positive result (considering intensity and extent) [15, 16].
While only membrane staining of PD-L1 is considered positive, the optimal cut-off value for PD-L1 staining has not been definitively validated [9]. This lack of consensus could contribute to the inconsistent results reported. The prognostic value of PD-L1 expression in the thyroid gland has also been explored. Aghajani et al. [14] suggested that PD-L1 expression could identify patients with more aggressive PTC, and Chowdhury et al. [4] observed PD-L1 positivity associated with recurrence and shortened disease-free survival. In contrast, Fadia et al. [15] did not report an association between membrane PD-L1 expression and adverse prognostic factors [15]. Given the persistent uncertainty, this study analyzes PD-L1 expression in PTC and its association with clinicopathological variables.
Materials and Methods
Patients and Specimens
We conducted a cross-sectional study by reviewing the records of patients diagnosed with papillary thyroid carcinoma (PTC) who underwent thyroidectomy between 2010 and 2016 at Hospital de Clinicas de Porto Alegre, Brazil. Included patients met the following criteria: age > 18 years, availability of adequate archival material, and a histopathologic diagnosis of PTC. Patients who were retrospectively considered to have non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) were excluded from the study. Archived formalin-fixed paraffin-embedded (FFPE) tissue blocks and hematoxylin and eosin (H&E)-stained slides were retrieved and reviewed by two pathologists (V.B.S. and M.S.G.). Clinicopathological parameters were obtained from histopathological analyses and the clinical database, summarized in Table 1. In total, 121 patients (98 females and 23 males) were included in this study. Approval for this study was obtained from the Ethics Committee of Hospital de Clinicas de Porto Alegre (protocol number: 44034920.2.0000.5327).
Table 1.
Patient characteristics
| Variables | (n = 121;100%) |
|---|---|
| Age (years) range ± SD | 53.2 ± 14.5 |
| Sex – n (%) | |
| Female | 98 (81.0) |
| Male | 23 (19.0) |
| Histological subtype n (%) | |
| Classic PTC | 102 (84.3) |
| Follicular-patterned of PTC | 16 (13.2) |
| Tall cell PTC | 1 (0.8) |
| Columnar cell PTC | 2 (1.7) |
| Tumor size (cm) – median (p25-p75) | 1,5 (0.8–2.52) |
| Classification of tumor size – n (%) | |
| Microcarcinoma (≤ 1cm) | 44 (36.4) |
| Carcinoma (> 1cm) | 77 (63.6) |
| Tumor focality - n (%) | |
| Unifocal | 81 (66.9) |
| Multifocal | 40 (33.1) |
| Stage – n (%) | |
| pT1 | 82 (67.8) |
| pT2 | 27 (22.3) |
| pT3 | 11 (9.1) |
| pT4 | 1 (0.8) |
| Lymph node status – n (%) | |
| pN0 | 40 (33.1) |
| pN1 | 53 (43.8) |
| pNx | 28 (23.1) |
| Extrathyroidal extension – n (%) | 67 (55.4) |
| Lymphovascular invasion – n (%) | 49 (40.5) |
| Lymphocytic thyroiditis – n (%) | 35 (28.9) |
| Chronic lymphocytic thyroiditis alone | 21 (17.3) |
| Chronic lymphocytic thyroiditis with oncocytic metaplasia | 14 (11.5) |
| Margin Status n (%) | |
| Uninvolved | 93 (76.9) |
| Involved | 28 (23.1) |
| Total thyroidectomy n (%) | 118 (97.5) |
Histological Examination
Histological evaluation was performed on whole-tissue section slides stained with hematoxylin and eosin from 121 cases of papillary thyroid carcinoma (PTC) (Fig. 1a). The evaluation aimed to confirm the diagnosis, assess histological subtypes, and examine clinicopathologic variables, including maximum tumor size, tumor multifocality (defined as two or more foci within the thyroid), chronic lymphocytic thyroiditis (CLT) alone and with oncocytic metaplasia (characterized by dense lymphocytic infiltration, atrophy of the follicular epithelium, and oxyphilic change), extra-thyroidal extension (classified as “microscopic” and “gross”), lymphovascular invasion (vessels with tumor emboli covered by endothelium), lymph node metastasis, and surgical margins. According to the current staging system, patients were categorized based on age, using 55 years as the cut-off point, resulting in younger (age < 55 years) and older (age ≥ 55 years) groups [17]. The pT, pN, and stage groupings followed the cancer staging guidelines of the eighth edition of the American Joint Committee on Cancer [18].
Fig. 1.
a Papillary thyroid carcinoma (PTC) with papillary structures and concurrent lymphocytic thyroiditis (400x). b Placental tissue was used as a positive control for PD-L1 staining (200x). c Histiocytes surrounding PTCs exhibit PD-L1 expression (400x). d PD-L1 negative staining (200x). e PD-L1 positive staining with intensity 1+ (400x). f PD-L1 positive staining with intensity 2+(400x). g Infiltrative follicular PTC (40x). h PD-L1 diffusely positive staining with an intensity of 3 + in follicular variant PTC (200x)
Immunohistochemical Analysis
PD-L1 expression, assessed through immunohistochemistry (IHC), was performed on formalin-fixed, paraffin-embedded (FFPE) whole-tissue 3-µm sections, deparaffinized using the EZ PREP reagent. Antigen retrieval was achieved using CC1 pH 9.0 at 100 °C for 92 min. Endogenous peroxidase activity was blocked by incubation with the Optiview Peroxidase Inhibitor solution. PD-L1 staining utilized a PD-L1 rabbit monoclonal primary antibody (clone SP263; Ventana, USA) on a Benchmark Ultra Autostainer (Ventana Medical Systems, Tucson, Arizona, USA) for 16 min at 36 °C, as has shown to be an optimal antibody for use in clinical practice [19].The Optiview HQ linker and Optiview HRP multimer (Ventana) were applied for 8 min each following the manufacturer’s instructions. Slides were counterstained with Mayer´s hematoxylin and bluing reagent (Li2CO3 + Na2CO3), coverslipped, and subsequently evaluated. Placental tissue served as a positive external control (Fig. 1b), and macrophages were utilized as positive internal controls (Fig. 1c). Only cancer cells with membranous staining of any intensity were considered positive when assigned a score. Cytoplasmic and nuclear staining of the tumor cells were not considered positive and were disregarded. PD-L1 expression was evaluated by measuring staining intensity and assessing the tumor proportion score (TPS) [20]. The intensity of IHC staining was scored as 0 (negative), 1+ (weak), 2+ (moderate), or 3+ (strong). TPS represents the percentage of viable tumor cells displaying partial or complete membranous staining (intensity ≥ 1+) relative to the total number of viable tumor cells in the sample. Samples were interpreted as having no PD-L1 expression for TPS < 1%, low PD-L1 expression for TPS 1–49%, and high expression for TPS ≥ 50%. In this study, tumors were considered positive for PD-L1 if the intensity was weak (1+) and the TPS was low (1–49%), following the criteria established by Fadia et al. [15]. All stained sections were independently scored by two pathologists (V. B. S. and M. S. G.). Any discrepancies in the scoring interpretations were resolved through consensus.
Statistical Analysis
With a significance level of 5%, power of 80%, a PD-L1 prevalence of 16.4%, and an odds ratio (OR) of 8.68 for lymphocytic thyroiditis as an associated factor, as reported in the study by Fadia et al. (2020) [15], a minimum sample size of 48 patients was determined. However, when considering a prevalence of PD-L1 of 46.4% and an OR of 3.34 for staging as an associated factor, based on the study by Aghajani et al. (2018) [14], a minimum of 120 patients was calculated. The maximum sample size was included in this study, and the sample size calculation was performed using WinPEPI (programs for epidemiologists for Windows) version 11.65. Quantitative variables were described using means and standard deviations or medians and interquartile ranges, depending on the data distribution. Qualitative variables were presented with absolute and relative frequencies. Means between groups were compared using Student’s t-test, and for asymmetry, the Mann-Whitney U test was applied. Proportions were compared using Pearson’s chi-square or Fisher’s exact test. A Logistic Regression model was employed to control for confounding factors. The criterion for entering a variable into the multivariate model was a p-value < 0.20 in the bivariate analysis, and the criterion for retention in the final model was a p-value < 0.10. The significance level was set at 5%, and all analyses were conducted using the Statistical Package for the Social Sciences (SPSS) version 28.0.
Results
Patient Characteristics
The clinicopathological characteristics of 121 patients diagnosed with papillary thyroid carcinoma (PTC) are summarized in Table 1. The average age of PTC patients was 53.2 years ± 14.5 (SD), with 62 patients being ≥ 55 years old. Consistent with the known demographics, the majority (81%) of patients were female. Among the cases, 102 were diagnosed as classic PTC, 16 cases of follicular-patterned PTC, including 10 infiltrative follicular PTC and 6 invasive encapsulated follicular variants, 1 as the tall cell PTC, and 2 as the columnar cell PTC. According to the AJCC classification (8th edition), 44 of examined cases were papillary thyroid microcarcinoma. The median tumor size in our cohort was 1.5 cm. The cohort included 82 cases of stage 1, 27 of stage 2, 11 of stage 3, and 1 of stage 4 disease. Tumor multifocality was observed in 40 patients (33.1%), while extrathyroidal extension was noted in 67 patients (55.4%), including 64 cases (52,9%) with only microscopic invasion and 3 cases (2,5%) with “gross” evident extension. Lymph node metastasis was present in 53 cases (43.8%), lymphovascular invasion in 49 cases (40.5%), and positive surgical margins in 28 cases (23.1%). Total thyroidectomy (TT) was performed during the initial surgery in 118 patients (97.5%). Chronic lymphocytic thyroiditis (CLT) was identified in 35 patients (28.9%), 21 presenting with CLT alone and 14 cases demonstrating CLT with oncocytic metaplasia.
PD-L1 Immunohistochemistry Results
Overall, PD-L1 expression was identified in 40 patients (33.1%). PD-L1 positivity in papillary thyroid carcinoma (PTC) exhibited predominantly weak (1+) or moderate (2+) intensity with tumor proportion scores (TPS) less than 50% in 36 cases (Fig. 1e, f). The remaining four cases exhibited strong (3+) intensity. Among the 16 cases of follicular-patterned PTC, two showed positive staining for PD-L1. One case demonstrated strong (3+) intensity with a TPS ≥ 50% (Fig. 1g, h). In the tall cell PTC, PD-L1 positivity was observed with moderate staining (2+). The two cases of columnar cell PTC were negative for PD-L1 expression. Histiocyte staining, present in all cases and utilized as an internal control, was not considered positive (Fig. 1c).
Correlation Between PD-L1 Expression and Clinicopathologic Features
The clinicopathological characteristics of the 121 patients, stratified by tumoral PD-L1 status, are summarized in Table 2. The associations between PD-L1 expression and various factors, including age (p = 0.782), sex (p = 0.959), histological subtype (p = 0.088), tumor size (p = 0.153), multifocality (p = 1.000), stage (p = 0.679), lymph node metastasis (p = 0.397), extra-thyroidal extension (p = 0.193), lymphovascular invasion (p = 0.905), surgical margins (p = 0.729), and chronic lymphocytic thyroiditis (p = 0.085), were not found to be statistically significant.
Table 2.
The correlation between the PD-L1 expression and other clinicopathological parameters
| Variables | PD-L1 positive (n = 40) | PD-L1 negative (n = 81) | P-value |
|---|---|---|---|
| Age (years) range ± SD | 52.7 ± 13.6 | 53.5 ± 15.0 | 0.782 |
| Sex - n (%) | 0.959 | ||
| Female | 33 (82.5) | 65 (80.2) | |
| Male | 7 (17.5) | 16 (19.8) | |
| Histological subtype – n (%) | 0.088 | ||
| Classic PTC | 37 (92.5) | 65 (80.2) | |
| Follicular-patterned of PTC | 2 (5.0) | 14 (17.3) | |
| Tall cell PTC | 1 (2.5) | 0 (0.0) | |
| Columnar cell PTC | 0 (0.0) | 2 (2.5) | |
| Tumor size (cm) – median (p25-p75) | 1.6 (1.0-2.9) | 1,5 (0.8–2.5) | 0.153 |
| Classification of tumor size – n (%) | 0.104 | ||
| Microcarcinoma (≤ 1cm) | 10 (25.0) | 34 (42.0) | |
| Carcinoma (> 1cm) | 30 (75.0) | 47 (58.0) | |
| Tumor focality - n (%) | 1 | ||
| Unifocal | 27 (67.5) | 54 (66.7) | |
| Multifocal | 13 (32.5) | 27 (33.3) | |
| Stage – n (%) | 0.679 | ||
| pT1 | 25 (62.5) | 57 (70.4) | |
| pT2 | 11 (27.5) | 16 (19.8) | |
| pT3 | 4 (10.0) | 7 (8.6) | |
| pT4 | 0 (0.0) | 1 (1.2) | |
| Lymph node status – n (%) | 0.397 | ||
| pN0 | 11 (27.5) | 29 (35.8) | |
| pN1 | 21 (52.5) | 32 (39.5) | |
| pNx | 8 (20.0) | 20 (24.7) | |
| Extrathyroidal extension – n (%) | 0.193 | ||
| Yes | 26 (65.0) | 41 (50.6) | |
| No | 14 (35.0) | 40 (49.4) | |
| Lymphovascular invasion – n (%) | 0.905 | ||
| Yes | 17 (42.5) | 32 (39.5) | |
| No | 23 (57.5) | 49 (60.5) | |
| Margin – n (%) | 0.729 | ||
| Uninvolved | 32 (80.0) | 61 (75.3) | |
| Involved | 8 (20.0) | 20 (24.7) | |
| Chronic lymphocytic thyroiditis – n (%) | 0,085 | ||
| Yes, with oncocytic metaplasia | 8 (20,0) | 6 (7,4) | |
| Yes, alone | 8 (20,0) | 13 (16,0) | |
| No | 24 (60,0) | 62 (76,5) |
pT: pathological tumor category, pN: pathological lymph node category
PD-L1 Expression Between CLT Alone and CLT with Oncocytic Metaplasia
Among the 14 cases of CLT with oncocytic metaplasia, PD-L1 expression in tumor cells was observed in 8 cases (57.1%). In this subgroup, there were 11 classic PTC cases, 1 infiltrative follicular PTC, 1 invasive encapsulated follicular variant, and 1 columnar cell PTC. Notably, PD-L1 expression was present in 7 out of 11 classic PTC cases and the single case of infiltrative follicular PTC. When comparing PD-L1 expression between cases of chronic lymphocytic thyroiditis (CLT) alone and CLT with oncocytic metaplasia, it was observed that out of the 14 cases with CLT and oncocytic metaplasia, 8 (57.1%) exhibited PD-L1 expression in the background benign follicular epithelium, and 11 (78.6%) expressed PD-L1 in oncocytes, and 7 cases had overlapping expression in both oncocytic and background benign follicular epithelium. In contrast, within the CLT alone group, only 4 out of the 21 cases (19%) showed expression in the background benign follicular epithelium. Significantly, CLT with oncocytic metaplasia demonstrated a higher association with PD-L1 expression in the benign follicular epithelium (p = 0.031) (Table 3). Interestingly, PD-L1 expression was not observed in the background benign follicular epithelium of patients without CLT.
Table 3.
PD-L1 expression in background epithelium between CLT alone and CLT with oncocytic metaplasia
| Variable | PD-L1 positive in background epithelium (n = 12) |
PD-L1 negative in background epithelium (n = 23) |
P-value |
|---|---|---|---|
| CLT alone | 4 | 17 | 0,031 |
| CLT with oncocytic metaplasia | 8 | 6 |
Clinicopathological Features Affecting PD-L1 Expression
Variables with p-values less than 0.20 in the previous analysis were included in the multivariate logistic regression model. Following adjustment, chronic lymphocytic thyroiditis with oncocytic metaplasia emerged as the sole independent predictor of PD-L1 expression (p = 0.014) (Table 4). Patients with chronic lymphocytic thyroiditis and oncocytic metaplasia exhibited a 365% increased risk of PD-L1 positivity (OR = 4.65; 95% CI: 1.36–15.9).
Table 4.
Multivariate logistic analysis of clinicopathological parameters influencing PD-L1 expression
| Variables | OR | 95% CI | P-value |
|---|---|---|---|
| Extrathyroidal extension | |||
| Yes | 2.31 | 0.99–5.41 | 0.053 |
| No | 1.00 | ||
| Chronic Lymphocytic thyroiditis | |||
| Yes, with oncocytic metaplasia | 4.65 | 1.36–15.9 | 0.014 |
| Yes, alone | 1.81 | 0.63–5.23 | 0.270 |
| No | 1.00 | ||
| Tumor size (cm) | 1.17 | 0.92–1.48 | 0.192 |
OR: odds ratio, CI: confidence interval
Discussion
In our study, PD-L1 expression was identified in 33.1% of patients with papillary thyroid carcinoma (PTC). This finding contrasts with studies such as Cunha et al. [12], who reported PD-L1 expression in 82.5% of PTC, with malignant tissues showing more intense staining. Angell et al. [8] found PD-L1 overexpression in 53% of PTC cases with BRAF p. V600E. The observed variability in PD-L1 expression levels across studies can be attributed to methodological differences, including variations in processing procedures, immunohistochemistry assays, antibody clones, cut-off values, and interpretation of immunohistochemical results. Notably, in clinical trials of anti-PD-1/PD-L1 directed therapies, membranous staining, but not cytoplasmic staining, was considered in patient selection [20–22]. In our study, we interpreted membrane staining of tumor cells as positive while disregarding cytoplasmic staining [20]. Contrary to our findings, Ahn et al. [9], in their analysis of 407 thyroid cancers using tissue microarrays with the SP142 clone to define PD-L1 positivity, found expression in only 6.1% of PTC. This lower level of PD-L1 expression could be attributed to the potential limitation of tissue microarray assays in detecting intratumoral heterogeneity, which may result in missing some positive cases. Studies such as those by Rimm et al. [23] and Adam et al. [24] have highlighted differences in PD-L1 expression detected by various anti-PD-L1 antibodies, but the most commonly used clones appear to be similarly efficacious [19]. For instance, Rimm et al. [23] observed significantly less PD-L1 expression with the SP142 antibody in tumor cells and immune cells compared to other antibodies in non-small cell lung cancer. Therefore, the interpretation of PD-L1 immunohistochemical staining, especially when using SP142 and tissue microarrays in PTC, should be approached with caution.
Clinically, no significant associations were observed between PD-L1 expression and other clinicopathological variables in our study. This finding aligns with the results reported by Fadia et al. [15], who used the same SP263 clone and did not find a statistically significant association between PD-L1 expression and clinicopathological variables. In contrast, Chowdhury et al. [4] reported a high PD-L1 expression associated with aggressive variants. However, in our study, no significant association was found between PD-L1 expression and histological subtype (p = 0.088). This difference in results may be attributed to the predominant composition of classic PTC in our sample (84.3%). Although we observed PD-L1 positivity in one tall cell and two follicular-patterned PTC, this difference was not statistically significant. In contrast to our findings, An et al. [25], in their analysis of PD-L1 expression in 116 patients, found a significant correlation with lymph node metastasis. In our study, we did not observe a significant association with lymph node metastasis, despite a notable prevalence of cases with lymph node metastasis (43.8%). An et al. [25] considered cytoplasmic staining of PD-L1 as positive, which might explain the significant association between PD-L1 and lymph node metastasis in their study.
The association between papillary thyroid carcinoma (PTC) and chronic lymphocytic thyroiditis (CLT) appears more intricate than initially conceived. CLT acts both as a potential driver for carcinogenesis and as a marker of tumor immunity [26]. A hypothesis worth exploring is whether the cross-reaction of antitumor immunity with normal thyrocytes may precipitate CLT in patients with PTC who are genetically predisposed to thyroid autoimmunity [27]. In our study, CLT with oncocytic metaplasia emerged as an independent predictor of PD-L1 expression in PTC. These findings align with Fadia et al. [15], who reported significantly higher PD-L1 expression in PTC with CLT. However, in their analysis, the authors did not distinguish between CLT alone and those with oncocytic metaplasia. Chowdhury et al. [4] observed increased PD-L1 expression in benign chronic lymphocytic and Hashimoto thyroiditis, proposing that chronic inflammation may create a microenvironment enriched with cytokines such as IFN-γ, IL-1, IL-10, and IL-6, leading to PD-L1 upregulation. Similar to our results, Lubin et al. [28] demonstrated increased PD-L1 expression in PTC and non-neoplastic follicular epithelium, including metaplastic oncocytes, in the background of Hashimoto thyroiditis (HT). The authors speculated that PD-L1 expression by follicular cells in HT could represent an adaptive response, where thyroid cells lacking PD-L1 expression are eliminated by an autoimmune infiltrate, and the underlying pathology of the background influences PD-L1 expression in PTC. Our study found 14 cases of CLT with oncocytic metaplasia, with 11 cases showing PD-L1 expression in the metaplastic oncocytes. Dell’Aquila et al. [29] reported PD-L1 expression in oncocytic adenomas (67.3%) and oncocytic thyroid carcinomas (75%), suggesting that PD-L1 may serve as a potential marker for oncocytic neoplasms. Our findings hint at oncocytic cells as potential predictors of PD-L1 expression in PTC. However, larger studies are needed to assess the molecular mechanisms linking autoimmunity, oncocytic metaplasia, and PTC, paving the way for the development of new treatments and therapeutic strategies.
Extrathyroidal extension (ETE) stands as a crucial predictive factor for outcomes in papillary thyroid carcinoma (PTC) [30]. The eighth edition of the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) pathologic staging for well-differentiated thyroid cancers currently recognizes only “gross” ETE for tumor staging, under the premise that “minimal” ETE does not significantly impact survival or recurrence, leading to concerns about potential overtreatment in such cases [31]. In our study, ETE did not predict PD-L1 expression. Among our cohort, 64 cases (52.9%) exhibited “microscopic” ETE, while only 3 cases (2.5%) displayed “gross” ETE. Notably, all three cases with “grossly” ETE were PD-L1 negative. Contrary to our observations, Shi et al. [13] reported a significant association between PD-L1 expression and ETE in their analysis of 260 PTCs. However, their study did not distinguish between the types of ETE. It’s noteworthy that our sample predominantly consisted of cases in the early stage (pT1 or pT2), constituting 109 cases (90.1%). The prevalence of early-stage cases and microscopic extrathyroidal extension (ETE) in our sample may have contributed to the absence of an association between ETE and PD-L1 expression. However, the precise impact of “microscopic” and “gross” ETE on PD-L1 expression remains unclear. Future studies with larger sample sizes are warranted to validate and refine these observations and shed light on the nuanced interplay between ETE types and PD-L1 expression in PTC.”
Our study observed constitutive expression of PD-L1 in macrophages, designating them as reliable internal positive controls for our immunohistochemistry assays. This observation aligns with the recommended use of macrophages as a standard for assessing PD-L1 expression in various tissue samples. While certain studies propose a potential decrease in PD-L1 expression over time in older tissue blocks [20], our research consistently identified PD-L1 expression in macrophages across all cases, including blocks maintained for more than ten years. Further investigation, covering a diverse range of tissues and storage durations, is necessary to validate and enhance these findings. If confirmed, this insight may carry practical implications for the use of older tissue blocks in PD-L1 expression studies, providing researchers with a broader pool of archival samples for retrospective analyses.
While our study provides valuable insights, it is essential to acknowledge certain limitations that temper the generalizability of our findings. Firstly, the relatively small sample size and the retrospective nature of the study impose inherent constraints on the robustness of our conclusions. The retrospective design introduces the possibility of selection bias and limits our ability to establish causation. Additionally, our dataset primarily comprises cases in the early stages of papillary thyroid carcinoma (PTC), with a majority falling within stages I and II. This distribution may influence the generalizability of our results to more advanced stages of the disease. It is crucial to recognize that the impact of PD-L1 expression might be underestimated in our study due to predominant representation of low pathological T stage cases. Furthermore, the absence of data on disease-free survival (DFS) and overall survival (OS) is a notable limitation. The lack of survival data restricts our ability to draw conclusions about the prognostic significance of PD-L1 expression in PTC. Future studies with larger sample sizes, prospective designs, and comprehensive survival data are warranted to further elucidate the role of PD-L1 in predicting clinical outcomes of PTC, and further, the relationship of PD-L1 to underlying tumor genetics, another factor not incorporated into our data herein.
In conclusion, our study reveals that chronic lymphocytic thyroiditis with oncocytic metaplasia significantly influences PD-L1 expression in papillary thyroid carcinoma (PTC), suggesting a dynamic interplay between the tumor and its microenvironment characterized by adaptive immune resistance. The observed association raises the possibility of potential clinical benefits from anti-PD-1/PD-L1 therapy in PTC. Looking ahead, the field would benefit from additional multicenter, prospective, and longitudinal studies with larger sample sizes. Such research endeavors are essential for establishing a comprehensive understanding of the relationship between PD-L1 expression and critical clinical parameters, including disease progression, aggressive variants, recurrence, and overall survival in PTC. PD-L1’s potential role as a prognostic biomarker in thyroid cancer warrants further exploration in future investigations.
Author Contributions
All authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by Vitor Barreto Santana and Marcia Silveira Graudenz. The first draft of the manuscript was written by Vitor Barreto Santana, and all the authors commented on the previous versions of the manuscript. All authors have read and approved the final manuscript.
Funding
This study was not supported by any funding agency.
Declarations
Conflict of Interest
The authors declare that they have no conflicts of interest.
Ethical Approval
This article does not contain any studies with human participants or animals performed by any of the authors. This study was approved by the Ethics Committee of Hospital de Clinicas de Porto Alegre/CEP-HCPA - (CAAE-44034920.2.0000.5327).
Informed Consent
This study obtained IRB approval from (CEP-HCPA) and the need for informed consent was waived.
Consent for Publication
Consent for publication was not required for this study.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
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