PD-1 and PD-L1 expression in thymic epithelial ...

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JCP Online First, published on February 8, 2018 as 10.1136/jclinpath-2017-204788 Original article

PD-1 and PD-L1 expression in thymic epithelial tumours and non-neoplastic thymus Emine Kilic Bagir,1 Arbil Acikalin,1 Alper Avci,2 Derya Gumurdulu,1 Semra Paydas3 1

Abstract Aims We explored the relationships between programmed death-1 (PD-1) and programmed deathligand 1 (PD-L1) expression and the pathological and clinical features of thymic epithelial tumours and thymic hyperplasia. Methods We evaluated PD-1 and PDL-1 expressions within epithelial and microenvironmental components in thymic epithelial tumours (n=44) and thymic hyperplasias (n=8), immunohistochemically. We Correspondence to Dr Emine Kilic Bagir, Department compared the results with demographic, clinical and of Pathology, Faculty of histopathological features of the cases. Medicine, Cukurova University, Results We found 48% epithelial expression Adana 01130, Turkey; and 82.7% microenvironment expression for eminebagir@yahoo.com PD-1 and 11.5% epithelial expression and 34.6% This study was presented as microenvironment expression for PD-L1. There a poster at the 26th National was no PD-1 expression, in either the epithelial or Pathology Congress 2–6 microenvironment, in the thymic hyperplasia group. November 2016, Antalya, PD-1 and PD-L1 positivity was more significant in thymic Turkey, and the 29th European Congress of Pathology 2–6 epithelial tumours than thymic hyperplasia. Patients with September 2017, Amsterdam, PD-1-positive microenvironments exhibited significantly The Netherlands. shorter mean estimated survival time than their negative counterparts. Received 12 September 2017 Conclusion These findings suggest that anti-PD-1 and Revised 18 December 2017 Accepted 20 January 2018 anti-PD-L1 therapies may benefit patients due to high release of PD-1 and PD-L1 in thymic epithelial tumours.

Department of Pathology, Faculty of Medicine, Cukurova University, Adana, Turkey 2 Department of Chest Surgery, Faculty of Medicine, Cukurova University, Adana, Turkey 3 Department of Medical Oncology, Faculty of Medicine, Cukurova University, Adana, Turkey

and PD-L1 in thymic neoplasms and non-neoplastic lesions. In this study, we sought to determine the relationship between PD-1/PD-L1 expression and pathological and clinical features of thymic epithelial tumours and thymic hyperplasia.

Materials and methods Patients’ characteristics

This retrospective study included 52 patients who underwent anterior mediastinal mass resection and were diagnosed at the Cukurova University Medical Faculty Surgery and Pathology Departments. Pathological diagnosis and tumour subtyping were performed according to the 2015 WHO histological classification of TETs and Masaoka-Koga staging system. We obtained data pertaining to the patient’s sex, age, presence of myasthenia gravis (MG), tumour size, surrounding organ invasion, metastatic spread and follow-up information (recurrence, progression, death). We excluded patients whose data were not available, patients where only biopsy specimens were obtained (ie, no resection) and patients without follow-up data. The study was approved by the Ethics Committee of Cukurova University.

Immunohistochemical staining Introduction

To cite: Bagir EK, Acikalin A, Avci A, et al. J Clin Pathol Epub ahead of print: [please include Day Month Year]. doi:10.1136/ jclinpath-2017-204788

Cell surface molecules that are endogenous regulators of immune responses play an important role in immunity control. These pathways are important for cancer cells and cancer microenvironments, as well as for helping tumour cells escape immune system responses.1 Programmed death-1 (PD-1) is an important inhibitor receptor that helps tumour cells escape immune system responses.2 3 T cells, B cells, macrophages and dendritic cells express programmed death-ligand 1 (PD-L1), and these proteins are also present in many tumours such as non-small cell carcinoma of the lung, renal cell carcinoma, malignant melanoma and sarcomas, and are considered to be prognostic factors.3–5 Thymic epithelial tumours (TETs) are rare neoplasms and include thymomas and thymic carcinomas. Histopathological classification is controversial because of indefinite diagnostic criteria and heterogeneous morphology.6–8 While it is possible to cure early stage disease with surgery, survival is variable in patients with unresectable tumours, which are typically treated with chemotherapy and/or radiotherapy. There are various treatment protocols for metastatic and/or recurrent disease.9 Recently, there have been a few studies on PD-1

Immunohistochemical staining was performed on 5 mm sections of formalin-fixed, paraffin-embedded tissue. We used monoclonal antibody MRQ-22 (Ventana) to detect PD-1 and CD274/ PDL1 and AM26531AF-N (Acris, Germany) were used to detect PD-L1. The visualisation system used was the BenchMark XT with enzymatic digestion (ISH protease 2; Ventana) and the iView Blue Detection Kit (Ventana). Specimens stained with anti-PD-1 and PD-L1 were scored according to intensity of cytoplasmic and/or membranous positivity as follows: 0 (no staining), 1+ (weak or equivocal staining), 2+ (moderate staining) or 3+ (strong staining). Thymic epithelial cells and microenvironments were evaluated separately. Epithelial and microenvironment positivity was considered when more than 5% of the population was stained. The cut-off value of 5% was performed based on previous studies.4 10

Statistical analysis

Analysis of categorical data was performed with χ2 and Fisher exact tests. Ordinal data analysis was performed with the Mann-Whitney U test. Survival analysis was by Kaplan-Meier and log-rank test. We grouped type B3 thymoma cases with thymic carcinoma cases for the purpose of statistical

Bagir EK, et al. J Clin Pathol 2018;0:1–5. doi:10.1136/jclinpath-2017-204788

1

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Original article Table 1 Patients’ characteristics Parameters

n

Age years (mean, range)

45.2 (17–79)

%

Sex Female

23

44.2

Male

29

55.8

4

7.7

Ex Masaoka-Koga stage I

23

52.3

IIa

6

13.6

IIb

5

11.4

III

3

6.8

IVa

4

9.1

3

6.8

IVb Tumour size (cm) (minimum–maximum)

6 (3–15)

>5

31

40.4

≤5

21

59.6

Histological type n (%) Thymoma (types A, B and AB) Type A

3

5.8

Type B1

6

11.5

Type B2

8

15.4

Type B3

6

11.5

Type AB

15

28.8

6

11.5

8

15.4

Thymic carcinoma Thymic hyperplasia Follow-up (median)

45.18±15.01

analysis, considering both conditions exhibit clinically aggressive outcomes. Statistical comparisons were made among patients with thymic hyperplasias, thymomas and the combined ‘poor prognosis’ group (thymic carcinoma+type B3 thymoma). For PD-1 epithelial component evaluation, we grouped weak (1+) and moderate (2+) cases. Likewise, for PD-L1 epithelial component evaluation, we grouped moderate (2+) and severe (3+) cases for the purpose of statistical analysis.

Results Clinical and pathological characteristics

Patients with thymoma (n=38), thymic carcinoma (n=6) and thymic hyperplasia (n=8), who had regular follow-ups, were included in the study. Twenty-nine cases (55.8%) were men and 23 were women (44.2%). The mean age was 45.2 (range, 17–79). Median follow-up in TETs was 47 months (range 15–120 months). Resection specimens were re-evaluated by two pathologists. Histopathologically, the most common type of thymoma was type AB (n=15), followed by type B2 thymoma (n=8), type B1 thymoma (n=6), type B3 thymoma (n=6) and type A thymoma (n=3). Of the six patients with thymic carcinoma, three were squamous cell carcinomas, two were well-differentiated carcinomas and one was a lymphoepithelioma-like carcinoma. Twenty-eight cases (53.8%) had MG, diagnosed within the preceding year. An additional six patients could not complete resection. Masaoka-Koga stage was performed in patients with TETs. The patients’ characteristics are presented in table 1.

Immunohistochemical PD-1 expression

We observed PD-1 expression in 48% (n=25) of epithelial component and 11.5% (n=6) of microenvironment samples. 2

Figure 1 H&E sections and PD-1, PD-L1 expression of each group.

There was no PD-1 in epithelial tumour cells or in the microenvironment samples from patients with thymic hyperplasia, whereas expression was detected in 21 of 38 patients with thymoma; 17 cases expressed PD-1 only in the epithelial cells, 4 cases expressed PD-1 in both components and 17 cases had no expression in either component. In patients with thymic carcinoma, PD-1 was expressed in both the microenvironment and epithelial cells (n=1), only in the microenvironment (n=1) and only in epithelial cells (n=3). One of the cases had no expression in either component. PD-1 expression was not statistically correlated with age, gender, Masaoka-Koga staging, tumour size and survival (data not shown). PD-1 epithelial component positivity was more significant in patients with thymoma, and thymic carcinoma+type B3 thymoma, compared with patients with thymic hyperplasia (P=0.011) (figures 1 and 2).

Immunohistochemical PD-L1 expression PD-L1 was expressed in 82.7% (n=43) of the epithelial samples and in 34.6% (n=18) of microenvironment samples. PD-L1 was expressed in seven out of eight patients with thymic hyperplasia; five patients expressed PD-L1 only in epithelial cells, two patients expressed PD-L1 in both components and one case had no expression of either component. In patients with thymoma, PD-L1 was expressed in 31 of 38 cases; 17 patients expressed PD-L1 only in epithelial cells, 14 patients expressed PD-L1 in both components and 7 patients had no expression of either component. Five of six patients with thymic carcinoma expressed PD-L1; three patients expressed PD-L1 only in epithelial cells, two patients expressed PD-L1 in both components and one case had no expression of either component. PD-L1 expression was not a statistically significant factor when compared with age, gender, Masaoka-Koga staging, tumour size and survival. However, PD-L1 epithelial component positivity was more significant in patients with thymoma and thymic carcinoma+type B3 thymoma, compared with the thymic hyperplasia group (P=0.010) (figures 1 and 2). Bagir EK, et al. J Clin Pathol 2018;0:1–5. doi:10.1136/jclinpath-2017-204788


Original article

Figure 2 Distribution of PD-1 and PD-L1 expression in all patients.

Patients with myasthenia gravis

Twenty-eight patients had MG. When we examined the histopathological subgroups of these patients who were examined, patients with MG were diagnosed with thymic carcinoma (n=1), type A thymoma (n=1), type AB thymoma (n=8), type B1 thymoma (n=4), type B2 thymoma (n=5), type B3 thymoma (n=3) and thymic hyperplasia (n=6). In patients with MG, PD-L1 and PD-1 expression was not statistically significant when compared with age, gender, Masaoka-Koga staging, tumour size and survival. The median values of age (P=0.017) and mass-patient size (P=0.031) were significantly larger in patients without, than in patients with, MG (tables 2 and 3).

Survival analysis

The mean estimated survival time of PD-1 microenvironment-positive patients was 51.8±8.3 months, while PD-1 microenvironment-negative patients had a mean estimated

survival of 109.5±6.6 months. The estimated 2-year survival probability of PD-1 microenvironment-positive patients was 60%, while that of PD-1 microenvironment-negative patients was 91.7% (P=0.004) (figure 3). We could not determine the relationship between survival and other parameters.

Discussion

In recent years, studies about immunotherapies, especially targeting PD-1/PD-L1, have become a promising treatment choice in various neoplasms. This study first documented the expression of PD-1 in thymic epithelial cells. PD-L1 and PD-1 play particularly important roles in immune system inhibition. PD-L1 expression is associated with poor prognosis in patients with breast cancer, colon cancer, lung cancer and TETs.11–13 PD-1 is an inhibitory molecule, found in activated T cells, which limits immunological activation. Two ligands (PDL-1 and PDL-2), which bind to PD-1 and enable this

Table 2 Distribution of age and tumour size in patients with and without myasthenia gravis (MG) Without MG (n=24)

MG (n=28)

Mean±SD

Median (minimum– maximum)

Mean±SD

Age

50.5±15.6

52 (17–79)

40.7±13.1

Tumour size

7.8±2.9

8 (4–15)

6.1±2.7

Median (minimum– maximum)

P

40 (20–69)

0.017

5 (3–14)

0.031

P, Mann-Whitney U test.


3


Original article Table 3 PD-1 and PD-L1 expression profile in patients with and without myasthenia gravis (MG) PD-1

PD-L1

Microenvironment

Epithelial cells

Positive n (%)

Negative n (%)

Positive n (%)

Negative n (%)

Without MG (n=24)

4 (16.7)

20 (83.3)

13 (54.2)

MG (n=28)

2 (7.1)

26 (92.9)

12 (42.9)

P

0.397

Microenvironment

Epithelial cells

Positive n (%)

Negative n (%)

Positive (1) n (%)

11 (45.8)

5 (20.8)

19 (79.2)

8 (33.3)

12 (50.0)

4 (16.7)

16 (57.1)

13 (46.4)

15 (53.6)

8 (28.6)

15 (53.6)

5 (17.9)

0.415

0.080

Positive (2–3) Negative n (%) n (%)

0.933

P, Mann-Whitney U test.

inhibition, can be found in antigen-presenting and tumour cells. Tumours with PD-L1 may bind to PD-1 in T cells, inhibiting the immunological response they elicit. The response rate observed with this monoclonal antibody may be higher in tumours with immunohistochemical evidence of PD-L1.1 14 15 In this study, we have observed that significantly more TETs (thymoma (types A, AB, B1, B2), thymic carcinoma+type B3 thymoma groups) exhibited PD-L1 and PD-1 epithelial component positivity (P=0.010; P=0.011). Previous studies on TETs have reported different prognostic effects of PD-L1 and PD-1 expression. The first study to determine the relationship between prognosis and expression was performed by Padda et al. They examined the correlation between clinical parameters and PD-L1 expression. They reported PD-L1 expression for thymoma (68%), thymic carcinoma (75%) and the microenvironment (14.8%), using tissue microarrays, in the study of 69 TETs and 17 thymic control patients. They concluded that intense staining of PD-L1 correlates with high histological grade, advanced disease stage and poor clinical outcomes.11 Similar to this study, researchers have found similar correlations between PD-L1 expression and histological grade or disease stage, but could not determine the relationship between PD-L1 expression and overall survival.9 16 17 In two separate studies by Yokoyama et al, PD-L1 expression was found in thymic carcinoma (80%) and thymoma (54%). Contrary to this, Padda et al associated lower diseasefree and overall survival with low PD-L1 expression in patients with thymic carcinomas. In the case of thymoma, high PD-L1

Figure 3 Kaplan-Meier overall survival curve for PD-1 microenvironment-positive patients. 4

expression was associated with higher tumour stage, higher histological grade, decreased disease-free survival and increased rate of recurrence, but not with overall survival or presence of MG.5 18 We found correlations with high tumour stage and high histological grade but not with PD-1 and PD-L1 expression. Weissferdt and colleagues found 61% expression of PD-L1 in thymic epithelial tumours, and they reported high expression in patients with thymoma who received neoadjuvant chemotherapy. Katsuya et al reported that both PD-1 expression and PD-L1 expression were high in patients receiving neoadjuvant chemotherapy.10 19 There is conflicting data on the prognostic effects of PD-L1 expression in TETs. We found higher expression of PD-L1 in TETs than in patients with thymic hyperplasia, but we also observed expression of PD-L1 in 34% of microenvironments. PD-L1 is expressed by lymphocytes, macrophages and/or histiocytes in tumour microenvironments, as well as in tumour cells. PD-L1 microenvironment expression suggests the presence of a negative feedback loop to protect the tumour cells from cytokines released by the tumour-reactive immunocytes, interfering with immune response processes. The mechanism of PD-L1 expression in microcellular cells is still unclear. We believe that the results and effects of expression in TETs will be elucidated by future study efforts.20–22 There are many reports that indicate that PD-1-positive T lymphocytes in the microenvironment are a good prognostic factor. However, other studies suggest PD-1 expression is associated with poor prognosis.2 18 23 Compared with PD-L1, there have been fewer examinations of PD-1 in TETs. The only study that associated increased PD-1 microenvironment positivity with poor prognosis in patients with thymic carcinoma was reported by Yokoyoma et al.18 In our study, we expected that the mean estimated survival time of patients with PD-1-positive microenvironments would be shorter than that of PD-1-negative patients (P=0.004). We found no significant correlations between expression of PD-L1 and PD-1 and age, sex, MG, tumour stage or histological grade. These results seem to suggest that PD-1 is a poor prognostic factor, similar to findings by Yokoyama et al.18 Another study found a relationship between PD-1 and higher stage in patients with thymic carcinomas, but they did not correlate with histological subtype, lymph node status or the presence of neoadjuvant chemotherapy. Researchers have not found any association between PD-1-positive and/or PD-1-negative patients and overall survival, similar to Marchevsky et al.9 10 All other studies found PD-1 in the cellular microenvironment. We observed PD-1 in epithelial components, unlike other studies. While there is no clear finding about the expression mechanism, we know that PD-1 is expressed in tumour specimens in different studies3 24 25; however, we did not find any data on this topic in our cohort of patients with thymic lesions. Bagir EK, et al. J Clin Pathol 2018;0:1–5. doi:10.1136/jclinpath-2017-204788


Original article Additionally, there was no relationship between MG and PD-L1 and PD-1 expression as in our study. However, the median age (P=0.017) and tumour dimensions (P=0.031) of patients without MG were significantly larger than patients with MG. We think that patients with MG are referred to the hospital earlier because of MG-related symptoms (eg, ptosis, diplopia, weakness in arms and legs, dysphagia). There were some limitations to our study because of the imbalance between the histopathological groups relative to the number of patients. Additionally, our follow-up time was insufficient. Future studies should examine patients receiving immunotherapy. It is possible that immunotherapy could emerge as a standard adjuvant therapy to chemotherapy.

Conclusion

It would be premature to issue a definite comment in support of these results, which exhibited increased expression of PD-1 and PD-L1 in the neoplastic developmental process. However, it is possible that immunotherapy has a role in the treatment of these patients, particularly those in advanced disease stages, as well as those with unresectable and/or recurrent thymic epithelial tumours.

Take home messages ►► Programmed death-1 (PD-1) and programmed death ligand-1

(PD-L1) positivity was more significant in thymic epithelial tumours than in thymic hyperplasia. ►► Patients with PD-1-positive microenvironments exhibited significantly shorter mean estimated survival time than PD-1negative patients. ►► There was no relationship between myasthenia gravis and PD-L1 and PD-1 expression. Handling editor Tahir S Pillay. Competing interests None declared. Ethics approval Cukurova University ethics committee. Provenance and peer review Not commissioned; externally peer reviewed. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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PD-1 and PD-L1 expression in thymic epithelial tumours and non-neoplastic thymus Emine Kilic Bagir, Arbil Acikalin, Alper Avci, Derya Gumurdulu and Semra Paydas J Clin Pathol published online February 8, 2018

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References

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