Expression of programmed death-ligand 1 in common endocrine tumors: A potential marker for newer therapeutic target

INTRODUCTION

Immune checkpoints play a pivotal role in the regulation of the immune system, primarily aimed at maintaining self-tolerance, which prevents the immune system from attacking host cells indiscriminately.^[1] These complex pathways involve two distinct classes of checkpoint molecules. One class activates the immune response, while the other class inhibits the activation of the immune system. Notably, Cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and (programmed death-ligand 1 [PDL-1]) are pivotal components in the orchestrating of T-cell responses.^[2,3] Programmed cell death protein 1 receptors on T cells interact with PDL-1, which causes their suppression, allowing tumor cells to escape immune-mediated destruction.^[4-6] This immune evasion mechanism has been extensively studied in various cancers, including non-small cell lung cancer, melanoma, and renal cell cancer.^[7-9] PDL-1 has garnered significant attention as an emerging diagnostic marker for immunotherapeutic targets in these tumors. Immune checkpoint inhibitors targeting the PD-1/PDL-1 axis have shown benefits in clinical settings and have revolutionized cancer treatment.^[10-14] While PDL-1 inhibitors have demonstrated efficacy in several advanced and unresectable cancers, the potential role of PD-1/PDL-1 in endocrine neoplasms is relatively unexplored, with fewer publications. We know that a subset of endocrine neoplasms is also untreatable by conventional therapy, especially when reached to advance stage. Therefore, it is imperative to initiate preliminary work on the expression of PDL-1 in endocrine tumors. Understanding the role of PDL-1 in endocrine tumors can provide novel insights into their biology and microenvironment and highlight new avenues for cancer treatment.^[15] We aimed to check the expression of PDL-1 in endocrine tumors in various subtypes of carcinoma of thyroid, parathyroid adenoma (PTA), pituitary adenoma (PA), and pheochromocytoma (PC)/paraganglioma (PG).

MATERIALS AND METHODS

A cross-sectional study was conducted on cases spanning between January 2015 and August 2020 in the Department of Pathology and Lab Medicine, All India Institute of Medical Sciences, Bhopal, bearing ethics committee approval number IHEC-LOP/2019/MD0038. The patient’s demographic and clinical details were noted. Formalin-fixed paraffin-embedded blocks of histologically proven cases of PA/pituitary neuroendocrine tumors, papillary carcinoma of the thyroid (PTC), medullary carcinoma of thyroid (MTC), PTA, and PC/PG (c) were included in analysis with PDL-1. Fifty-four cases of endocrine organ tumors were included in the study: twenty-two cases of PA; twenty cases of PTC; two cases of MTC; seven cases of PTA; and three cases of PC/PG. Furthermore, PA was further studied for hormonal immunohistochemistry; growth hormone (GH), prolactin (PL), thyroid-stimulating hormone (TSH), and adrenocorticotropic hormone (ACTH). VENTANA BenchMarkXT, an automated immunostainer, was used to perform PDL-1 (PathSitu B7H1P clone) immunohistochemistry (IHC) in all cases. This In vitro diagnostics (IVD)-approved clone is a transmembrane protein and stains accordingly. VENTANA PD-L1 (SP263) Assay antibody-positive tissue blocks used as positive control during validation. The PDL-1 positivity is determined using tumor proportion score (TPS), the percentage of viable tumor cells showing partial or complete membrane staining at intensity. The specimen is considered to have PDL-1 if the tumor cutoff for positivity of PDL-1 is >1%. The PDL-1 staining is also recorded in tumor-infiltrating lymphocytes (TIL).

RESULTS

The present study analyzed the expression of PD-L1 in various endocrine and neuroendocrine tumors in our department from January 2015 to August 2020. The study included 20 cases of PTC, 2 cases of MTC, 7 cases of PTA, 22 cases of PA, and 3 cases of PG/PC.

Papillary carcinoma of thyroid

A total of 20 PTCs (5 males and 15 females) were studied. The mean age of the participants was 40.6 years (ages ranging from 24 to 65 years). Seventeen had a classic type of PTC (85%), while three had a follicular variant of PTC (15%); 16 were unifocal, and four were multifocal tumors. Among the 20 cases evaluated, 17 had stage I PTC, while in 3 participants, staging of PTC could not be assessed. Thirteen PTC has surrounding normal thyroid parenchyma with mild lymphocytic infiltration to the Hashimoto component. PDL-1 expression was positive in 10 PTC (50%) [Figure 1]. Among five men, 2 cases showed PDL-1 expression (40%), and among 15 females, 8 showed PDL-1 expression (53.33%). Out of 4 multifocal tumors, 1 showed expression of PDL-1 (25%), whereas out of 16 unifocal tumors, 9 showed PDL-1 expression (56%). One out of 3 follicular variants of PTC showed expression of PDL-1 (33.33%). Nine out of 17 classical variants showed expression of PDL-1 (52%). A total of 6 cases showed TIL. All showed PDL-1 expression. Among them, 3 cases showed PDL-1 expression for both tumor cells and TIL [Figure 2].

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Figure 1:

Papillary carcinoma of the thyroid. (a) Hematoxylin and Eosin stained (b) Moderate cytoplasmic and membranous programmed death-ligand-1 expression in tumor cells (×40).

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Figure 2:

Papillary carcinoma of the thyroid. (a) Hematoxylin and Eosin stained (b) Programmed death-ligand-1 in expression in lymphocytes (c) Faint cytoplasmic programmed death-ligand-1 expression in tumor cells (×40).

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Ninety-five percent of participants with PTC were >55 years of age. There was no significant difference in PDL-1 expression between age groups >55 years and ≤55 years or between males and females as analyzed by Fisher’s exact test. Six cases expressed TIL in >55 years of age, and all were (100%) positive for PDL-1 expression. No significant difference in PDL-1 expression was seen between classic and Follicular variants of PTC. Almost 30% of both tumor types expressed TIL, and 100% of these were PDL-1 positive.

PDL-1 expression was not significantly different between unifocal versus multifocal PTC as analyzed by the Fisher’s exact Test. All unifocal PTC with TIL show PDL-1 expression [Table 1].

Table 1: PDL-1 in papillary thyroid carcinoma; PDL-1 in medullary carcinoma of thyroid.

Variables	PDL-1 papillary thyroid carcinoma				PDL-1 in medullary carcinoma of thyroid
	Tumor		Lymphomononuclear cells		Variables	Tumor		Lymphomononuclear cells
	Positive	Negative	Positive	Negative		Positive	Negative	Positive	Negative
Age					Gender
≤55 year	1	Male	0	0	Male	0	0	0	0
>55 year	9	Female	6	0	Female	2	0	1	0
Gender					Multifocality
M	2	With	2	0	With	0	0	0	0
F	8	Without	4	0	Without	2	0	1	0
Tumor type					Bilateral distribution
Conventional	9	Present	5	0	Present	1	0	0	0
Follicular	1	Absent	1	0	Absent	1	0	1	0
Focality					Capsular invasion
Unifocal	9	Present	6	0	Present	0	0	0	0
Multifocal	1	Absent	0	0	Absent	2	0	1	0

PDL-1: Programmed death-ligand 1

Medullary carcinoma of the thyroid

A total of two cases of MTC were studied. Both cases showed PDl-1 expression. One of the two cases had a Hashimoto component [Figure 3]. Both the tumors were at stage IVa and sporadic in nature. One of the cases was bilateral. No multifocality or capsular invasion cases were studied [Table 1].

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Figure 3:

Medullary carcinoma of the thyroid. (a) Hematoxylin and Eosin stained (H & E) (b) Moderate cytoplasmic and membranous programmed death-ligand (PDL-1) expression in tumor cells (c) H & E showing lymphoid aggregate (d) Focal moderate cytoplasmic PDL-1 expression in lymphocytes (×20).

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Parathyroid adenoma

A total of seven cases (5 males and 2 females) of PTA were studied. The mean age of participants was 39.8 years (range 25–58 years). The diameter of the adenomas ranged from 1.5 to 6 cm (mean, 3.14 cm), and mitosis ranged from 0 to 2/10 HPF. None of the tumors exhibited necrosis, atypical mitosis, or prominent nucleoli [Table 2]. PDL-1 expression was seen in 57.1% of cases [Figure 4].

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Figure 4:

Parathyroid adenoma: (a) Hematoxylin and Eosin stained (b) Membranous and cytoplasmic programmed death-ligand-1 (PDL-1) expression in tumor (×40).

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Pituitary adenoma

A total of 22 cases of PA (14 males and 8 females) were studied with a mean age of 43.3 years (range 10–64 years). A case of pituitary hyperplasia was identified and was deleted from the analysis. The mitosis ranged from 0 to 2/10 HPF. A total of 18/22 cases were clinically nonfunctional, and four were functional. One case was a recurrence. IHC was done using GH, PL, TSH, and ACTH hormones for further characterization. Out of 22 cases, 14 cases express positivity to GH/PL/TSH/ACTH (63.64%) indiviually and or in combination; 9 cases were positive for PL (co-expression of GH in 2 cases, TSH in 1 case, ACTH in 3 cases); 4 cases were positive for TSH (co-expression of PL in 1 case, GH in 1 case). [Figure 5 and 6]. ACTH positivity is seen in 5 cases with PL co-expression in 2. Eight were negative for the hormone expression (36.36%). Of the 22 cases, 18 cases were positive for PDL-1 expression (81%). A total of 7/8 females and 11/14 males expressed PDL-1. Among 18/22 non-functioning adenomas, 14 expressed PDL-1 (77.8%); all 4 functioning adenomas (1 recurrent) expressed PDL-1 (100%). Among 14 cases positive for either and/or GH/PL/TSH/ACTH, 12 cases expressed PDL-1 (75%). The expression of PDL-1 in PL positive was 100%, GH 100%, ACTH 100%, and TSH 75% [Table 2].

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Figure 5:

Case of lactotroph: (a) Hematoxylin and Eosin stained (b) immunohistochemistry for prolactin shows diffuse cytoplasmic positivity (c) Membranous diffuse PDL-1 expression in tumor cells (×40).

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Figure 6:

Case of lactotroph with thyroid-stimulating hormone(TSH) co-expression: (a) Hematoxylin and Eosin stained (b) Immunohistochemistry (IHC) for prolactin shows cytoplasmic positivity (c) IHC for TSH shows cytoplasmic positivity (d) Moderate cytoplasmic and membranous programmed death-ligand-1 expression in tumor cells (×20).

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Table 2: PDL-1 expression in paraganglioma/pheochromocytoma, pitutary adenoma and parathyroid adenoma

Paraganglioma/Pheochromocytoma			Pituitary Adenoma			Parathyroid Adenoma
Variables	Positive	Negative	Variables	Positive	Negative	Variables	Positive	Negative
Gender			Gender			Gender
Male	0	2	Female	7	4	Male	2	3
Female	0	1	Male	11	1	Female	2	0
Age			Age			Age
<40	0	3	<60	17	1	≤50y	4	2
≥40	0	0	≥60	1	3	>50y	0	1
Disease site			Classification			Diameter
Adrenal	0	1	Non functioning	14	4	≤1.8cm	1	0
Extra Adrenal	0	2	Functioning			>1.8cm	3	3
Diameter			Prolactin secreting	2	0	Mitosis
<5cm	0	2	Growth hormone secreting	2	0	<1/10HPF	3	2
≥5cm	0	1	Tumor size			≥1/10HPF	1	1
Tumour necrosis			<3	14	4	Atypical mitosis
Absent	0	3	≥3	4	0	With	0	0
Present	0	0	Mitosis			Without	4	3
Capsular invasion			<1/10HPF	17	4	Necrosis
Absent	0	3	≥10/HPF	1	0	With	0	0
Present	0	0				Without	4	0
Vascular invasion						Nucleoli
Absent	0	3				With	0	0
Present	0	0				Without	4	3

HPF: High power field

Paraganglioma/pheochromocytoma

Three cases (2 males and 1 female) of PC/PG were studied. The age of participants ranged from 24 to 39 years. The tumor studied had no necrosis, capsular invasion, or TIL, and all cases were negative for PDL-1 expression [Tables 2, 3 and Figure 7].

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Figure 7:

Pheochromocytoma: (a) Hematoxylin and Eosin stained (b) Programmed death-ligand-1 expression is absent in tumor cells (×40).

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Table 3: PDL-1 Expression in immune rich endocrine tumor.

Tumors	PDL-1 in tumor		PDL-1 in tumor infiltrating lymphocytes
	Positive	Negative	Positive	Negative
Papillary carcinoma of thyroid	3	3	6	0
Medullary carcinoma of thyroid	1	0	1	0
PDL-1 Expression in immune poor endocrine tumors
Pheochromocytoma (PC)/Paraganglioma (PL)	Positive		Negative
Pituitary adenoma	18		4
Papillary carcinoma of thyroid	7		7
Medullary carcinoma of thyroid	1		0
Parathyroid adenoma	4		3
Paraganglioma/pheochromocytoma	0		3

PDL-1: Programmed death-ligand 1

DISCUSSION

PDL-1 serves as a valuable diagnostic and prognostic biomarker for guiding targeted therapies in several cancers, including lung, urinary bladder, stomach, ovarian carcinoma, and melanoma.^[7,16-18] The difference in the expression of PDL-1 in the tumors with or without TIL underscores its importance in the immune escape mechanism and microenvironment.^[19-22] While the therapeutic use of PD/PDL-1 expression in some tumors is well established, its role in the context of endocrine organ tumors is still less explored. An endocrine organ neoplasm encompasses a diverse group with variable degrees of aggressive behavior, thus prompting interest in assessing PDL-1 expression across the various subsets of endocrine organ neoplasm. Our study is particularly pertinent as the existing literature, especially within the Indian setting, is sparse.

In the present study, we aimed to bridge this gap by evaluating the Pathnsitu clone, a full-length protein (as per the manufacturer’s insert) that has not been studied previously in detail. Consequently, there exists a compelling need to investigate this particular full-length protein, especially in less-reported subsets of neoplasms. Therefore, the present study addressed the spectrum of endocrine neoplasms with different aggressive behaviors. The results of this study may lay the foundation for future studies on different clones of variable length and specific sites of protein and further mRNA-based and in vitro laboratory studies.

Our current study revealed that PDL-1 expression is more functional than nonfunctional PA., which is concordant with a study conducted by Wang et al. highlighting the potential role of immunotherapy in aggressive and recurrent functional adenomas.^[23]

In PTC, we observed that PDL-1 expression occurred in 50% of cases and 100% of TIL. This study was concordant with the study conducted by Chowdhury et al.,^[24] French et al.^[25] and Sekino et al.^[26] with recent work of. Furthermore, PDL-1 expression was seen in 100% cases of MTC as well as in TIL, mirroring findings by Bi et al.^[27] Bi et al. study also demonstrated a significant correlation between PDL-1 positivity and distant metastasis at the surgery.^[27] These findings suggest that PDL-1 positivity may serve as a marker for identifying patients who could possibly benefit from anti-PDL-1 therapies.

Regarding PTA, 57% of cases expressed PDL-1, albeit in <10% of the cells within these positive cases, except for one case. Notably, the age of participants and the size of the tumor did not correlate with PDL-1 expression, consistent with Pan et al. findings, warranting further investigation into its biology.^[28]

In our current study, all cases of PC/PG and TIL were negative for PDL-1 expression. These findings deviate from the studies conducted by Pinato et al.^[29] and Yasuhiro et al.^[30] The observed disparity between our study and previous researchers may be attributed to a smaller number of PC/PG cases examined. Nevertheless, these tumors may also rely on different immune evasion mechanisms, which are independent of the PDL-1 pathway.

The differential expression of PDL-1 across various endocrine tumors highlights the heterogeneity of the tumor microenvironment in these tumors. The association of PDL-1 expression in TILs in PTC and functional subtypes of PA may emphasize the importance of the immune escape mechanism in these tumors. PA and PTC with a higher percentage of PDL-1 expression may benefit from targeted immunotherapies, while tumors with negligible expression may require alternative treatments. There are several ways PDL-1 scoring is performed (combined proportion score [CPS] or TPS, etc.) and it depends on clones, especially when used as companion diagnostics. Most studies, however, considered >1% in tumor cells as cutoff.^[31] In the present study, the standard TPS score (>1%) is considered for positivity, and this is widely used in almost all kind of tumors. The cancer types in which CPS is standardized, the thresholds of <1%, 1% to 9%, 10% to 19%, and more than or equal to 20% are often used.^[16] Due to the absence of CPS-specific thresholds currently in the literature, unlike many other tumors, these cutoff values may be adopted in endocrine tumors if used for other study designs with a clinical trial with immunotherapy. In the present study, the positive TPS is considered as >1%.^[32,33]

Our study is a preliminary study into the previously relatively uncharted territory of endocrine neoplasm. However, this study also has its limitations, including the small sample size for a few spectrums of tumors. Higher expression in MTC, PA, or absence in PC/PG is most likely due to the limitation of sample size in comparison to other studies. However, this is the first study from India on endocrine neoplasms and PDL-1 expression, laying the foundation for further studies and the development of future therapeutic targets. The clinical perspective of immunotherapy in endocrine tumors was recently described by Anna Angelousi in 2024. The reviewers also understand that immunotherapy revolutionized advanced cancer care, but its application in various endocrine tumors is rather limited. The mainstay is restricted when the conventional therapies have failed. Anti-PD-L1 agent pembrolizumab was checked in the progressive adrenocortical carcinoma with a limited response rate. In the phaeochromocytoma/PG group, it is substantially low. Interestingly, in our study, the Pheochromocytoma (PC)/Paraganglioma (PL) is negative for PDL-1. When pembrolizumab was used in combination with tyrosine kinase inhibitors showed higher efficacy in differentiated thyroid carcinoma. However, progressive medullary thyroid cancer shows lower response to therapy. The immunotherapy has been used in parathyroid carcinoma, showing limited antitumor effect. We did not encounter any parathyroid cancer, but adenomas show positivity for PDL-1.^[34]

CONCLUSION

This study highlights that PA/pituitary neuroendocrine tumors, PTC, and PTA show heterogeneous expression of PDL-1, while its expression is absent in PC/PG. PDL-1 IHC can be used as a therapeutic marker in advanced and unresectable endocrine organ tumors. These findings pave the way for more targeted and personalized use of anti-PDL-1 therapies. However, further research, multi-institutional validation, and clinical trials assessing the efficacy of anti-PDL-1 are necessary to provide deeper insight for potential immunotherapy targets.