Introduction: In most cases, the diagnostic workup of pleural mesotheliomas (MPMs) starts with cytological examination of pleural effusion, but histology is needed to confirm the diagnosis. The introduction of BAP1 and methylthio-adenosine phosphorylase (MTAP) immunohistochemistry has become a powerful tool to confirm the malignant nature of mesothelial proliferations also in cytological specimens. The objective of this study was to determine the concordance of BAP1, MTAP, and p16 expression between cytological and histological samples of patients with MPM. Methods: Immunohistochemistry of BAP1, MTAP, and p16 was performed on cytological samples and compared with the corresponding histological specimen of 25 patients with MPM. Inflammatory and stromal cells served as positive internal control for all three markers. In addition, samples of 11 patients with reactive mesothelial proliferations served as an external control group. Results: Loss of BAP1, MTAP, and p16 expression was found in 68%, 72%, and 92% of MPM, respectively. Loss of MTAP was associated with loss of p16 expression in all cases. Concordance of BAP1 between cytological and corresponding histological samples was 100% (kappa coefficient 1; p = 0.008). For MTAP and p16, kappa coefficient was 0.9 (p = 0.01) and 0.8 (p = 0.7788), respectively. Conclusions: Concordant BAP1, MTAP, and p16 expression is found between cytological and corresponding histological samples, indicating that a reliable diagnosis of MPM can be made on cytology only. Of the three markers, BAP1 and MTAP are most reliable in distinguishing malignant from reactive mesothelial proliferations.

Pleural mesothelioma (MPM) is a neoplastic process arising from the mesothelial cell layer lining the parietal and visceral pleura of the lung. The main cause of MPM is exposure to asbestos [1]. Clinical manifestations of MPM often include dyspnoea and thoracic pain. In 80% of patients, the disease is associated with pleural effusion [2]. To determine the nature of the lesion, the diagnostic workup frequently starts with cytological examination of pleural effusion [3, 4]. Because invasion cannot be assessed on cytology, transthoracic histological biopsies are required to confirm the diagnosis of MPM [5, 6].

Homozygous deletion of CDKN2A is one of the most frequent genetic alterations in MPM, occurring in 45–86% of epithelioid MPM and is associated with a poor prognosis [6, 8]. The CDKN2A locus encodes p16 protein and resides on chromosome 9p21 in proximity to a cluster of genes harbouring CDKN2B and methylthio-adenosine phosphorylase (MTAP). Homozygous CDKN2A deletion can be detected using fluorescence in situ hybridization (FISH) with high specificity but moderate sensitivity [6]. However, use of CDKN2A FISH is expensive, requires special technical skills, and has high turnaround times [9]. In previous published work, concordance between p16 immunohistochemistry and CDKN2A FISH is poor with a sensitivity of 58.1% and specificity of 85%, whereas MTAP correlates with p16-FISH with a specificity of 100% [6].

Besides CDKN2A alterations, mutations of the BAP1 gene are a frequent finding in MPM. BAP1 is a deubiquitinating enzyme which plays a role in cellular proliferation and growth inhibition. Benign mesothelial tumours always express BAP1, whereas MPM show loss of BAP1 in up to 70% of MPM with an epithelioid morphology. It should be emphasized that a subset of MPM, especially the sarcomatoid subtype, expresses BAP1 in almost 100% of cases [10]. Because of its high specificity, loss of BAP1 protein is virtually diagnostic for malignancy [10].

The introduction of new techniques including CDKN2A FISH and more recently immunohistochemical staining for BAP1 and MTAP have become reliable tools to confirm the malignant nature of a mesothelial proliferation in both cytological and histological specimens [11]. The objective of this study was to determine the concordance between BAP1, MTAP, and p16 immunoreactivity in cytological and histological samples of MPM.

Patients and Samples

Paired histological and cytological samples obtained from 25 patients between 2008 and 2014 were retrieved from the archives of our pathology department. For each case, the most representative cytological and histological samples were selected. In cases with more than one cytological specimen (3 cases), the sample closest to the date of histological sampling was selected. The mesothelial origin of the cell proliferation was confirmed using calretinin (Ventana, prediluted clone SP65) and CK5/6 (Ventana, prediluted clone D5/16B4). BerEp-4 (Ventana, 1:150, clone BS14) was used to exclude metastatic adenocarcinoma. Desmin staining (Ventana, 1:20, clone D33) was applied to differentiate reactive from neoplastic mesothelial proliferations. Cytological samples of 11 patients with reactive mesothelial proliferations served as controls.

Cell Block Preparation and Immunostaining

Effusions were processed according to standard protocols, and cell blocks were prepared as described previously [12, 13]. Immunohistological staining was performed on 2 μm thick formalin-fixed cell and tissue blocks. For p16 and MTAP, antigen retrieval using CC1-buffer (cell conditioning solution) was applied for 48 min and 64 min, respectively. For BAP1, an H2-buffer for 90 min was used. In the second step, incubation with the corresponding antibody (anti-p16INK4a [clone E6H4, Ventana Medical Systems prediluted], MTAP [ab126770 clone EPR6893, Abcam 1:500], and BAP1 [sc-28383 clone C-4, Santa Cruz 1:200]) was performed. After immunostaining, samples were counterstained with haematoxylin II. An OptiView DAB IHC Detection Kit from Ventana (p16 and MTAP) and BondRefine HRP Kits (BAP1) were used to visualize p16, MTAP, and BAP1 proteins. Inflammatory and stromal cells served as positive internal control for all three markers. In addition, samples of 11 patients with reactive mesothelial proliferation were selected as an external positive control.

Staining of BAP1, p16, and MTAP in the critical cells was compared with the internal positive control, such as lymphocytes and macrophages. Loss of p16 nuclear and/or cytoplasmatic staining was considered to be negative. Strong nuclear and/or cytoplasmatic staining for MTAP was interpreted as positive, whereas complete loss of cytoplasmic labelling was considered negative. Because of the variable expression of MTAP, staining was regarded negative when staining intensity was weaker than the internal positive control. Loss of nuclear BAP1 expression was considered as negative.

Statistical Analysis

Statistical analyses were performed using R statistical software (RStudio version 1.4.1103). Fisher’s test and Cohen’s kappa were used to evaluate the correlation between histological and cytological samples. Results were considered significant if p < 0.05. The sensitivity and specificity were calculated by comparing the cytological MPM cases with the control group.

Clinicopathological Characteristics

The cohort included 21 male and 4 female patients (mean age of 67; range 50–88 years). Mean age of the control subjects was 67 (range 30–93 years) and included 7 males and 4 females. Based on microscopic examination of cytological samples (22 pleural effusions and 3 ascites) and their corresponding histological samples, 21 cases were diagnosed as epithelioid and 4 cases as biphasic MPM.

BAP1, p16, and MTAP Immunohistochemistry

The control group samples as well as the stromal and inflammatory cells of the tumour samples showed a preserved nuclear staining for BAP1, p16, and MTAP (Fig. 1). Loss of p16 labelling was observed in 92% of the cases. In total, 72% were considered as MTAP negative. All MTAP-negative samples were also negative for p16 (Fig. 2, 3). Of all cases, 68% showed loss of BAP1 expression (Fig. 4).

Fig. 1.

Preserved MTAP and BAP1 expression in a reactive mesothelial proliferation (magnification ×400). a Haematoxylin and eosin. b BAP1. c MTAP. d P16 expression only in a few positive cells (arrows).

Fig. 1.

Preserved MTAP and BAP1 expression in a reactive mesothelial proliferation (magnification ×400). a Haematoxylin and eosin. b BAP1. c MTAP. d P16 expression only in a few positive cells (arrows).

Close modal
Fig. 2.

Cluster of malignant mesothelial cells with preserved BAP1 expression and loss of MTAP protein expression (magnification ×400). a Haematoxylin and eosin. b BAP1 positive cell (arrow). c MTAP deficient cell (arrow). d p16.

Fig. 2.

Cluster of malignant mesothelial cells with preserved BAP1 expression and loss of MTAP protein expression (magnification ×400). a Haematoxylin and eosin. b BAP1 positive cell (arrow). c MTAP deficient cell (arrow). d p16.

Close modal
Fig. 3.

Malignant pleural mesothelioma with loss of BAP1 and MTAP expression (magnification ×400). a Haematoxylin and eosin. b BAP1. c MTAP deficient cells (arrow) and internal positive control (arrowhead). d p16.

Fig. 3.

Malignant pleural mesothelioma with loss of BAP1 and MTAP expression (magnification ×400). a Haematoxylin and eosin. b BAP1. c MTAP deficient cells (arrow) and internal positive control (arrowhead). d p16.

Close modal
Fig. 4.

Malignant pleural mesothelioma with loss of BAP1, preserved MTAP expression, and retention of p16 expression in part of the tumour cells (magnification ×400). a Haematoxylin and eosin. b BAP1. c MTAP. d p16 expression.

Fig. 4.

Malignant pleural mesothelioma with loss of BAP1, preserved MTAP expression, and retention of p16 expression in part of the tumour cells (magnification ×400). a Haematoxylin and eosin. b BAP1. c MTAP. d p16 expression.

Close modal

Correlation between Cytological and Corresponding Histological Samples

Results of the cytological and corresponding histological samples are summarized in Table 1. A perfect match between the corresponding samples was found in all but one sample (96%). In one case, a discordant expression of MTAP and p16 was found, where loss of MTAP and p16 was found in the cytological specimen but preserved expression of both markers in the histological sample.

Table 1.

Contingency Table

Negative CytoPositive Cyto
BAP1 
 Negative Histo 17 
 Positive Histo 
MTAP 
 Negative Histo 17 
 Positive Histo 
p16 
 Negative Histo 22 
 Positive Histo 
Negative CytoPositive Cyto
BAP1 
 Negative Histo 17 
 Positive Histo 
MTAP 
 Negative Histo 17 
 Positive Histo 
p16 
 Negative Histo 22 
 Positive Histo 

HAP1 shows a complete concordance. MTAP shows a concordance except for one case. P16 shows a concordance except for one case. Histo, histological sample; Cyto, cytological sample.

A significant correlation between cytology and histology was found for BAP1 (p = 0.008) and MTAP (p = 0.01) with kappa coefficient of 1 (CI: 1 to 1) and 0.9049 (CI: 0.7232254–1), respectively (Table 2). The kappa coefficient for p16 was 0.7788 (CI: 0.3644298–1; p = 0.01). The Fisher’s test showed a significant p value for all three markers (BAP1: 0.008, MTAP: 0.01, p16: 0.01) (Table 2).

Table 2.

Kappa coefficient and Fisher’s test

Kappa coefficientKappa confidence intervalFisher’s test (p value)
BAP1 1–1 0.008 
MTAP 0.904 0.723–1 0.01 
p16 0.778 0.364–1 0.01 
Kappa coefficientKappa confidence intervalFisher’s test (p value)
BAP1 1–1 0.008 
MTAP 0.904 0.723–1 0.01 
p16 0.778 0.364–1 0.01 

BAP1 and MTAP have a better kappa coefficient than p16.

Sensitivity and Specificity of BAP1, MTAP, and p16 in Cytological Samples

Compared to samples of the control subjects, both BAP1 and MTAP immunohistochemistry showed a specificity of 100% (Table 3), whereas the specificity of p16 was poor (0%). Sensitivity of BAP1 and MTAP was 68% and 72%, respectively. By combining BAP1 and MTAP, sensitivity increased to 84%.

Table 3.

Sensitivity and specificity

SensitivitySpecificity
BAP1 0.68 
MTAP 0.72 
p16 0.92 
Combination of BAP1 and MTAP 0.84 
Combination of p16 and MTAP 0.92 
SensitivitySpecificity
BAP1 0.68 
MTAP 0.72 
p16 0.92 
Combination of BAP1 and MTAP 0.84 
Combination of p16 and MTAP 0.92 

The combination of BAP1 and MTAP shows the best results regarding sensitivity and specificity.

In the present study, we showed good correlation of BAP1, MTAP, and p16 protein expression between cytological and histological specimens.

Consistent with previous observations, BAP1 showed the best results regarding concordance between cytology and histology [14, 17]. BAP1 is a robust marker that is either positive or negative in all tumour cells which explains the high specificity of this marker in differentiating malignant from reactive mesothelial proliferations. In comparison to MTAP, BAP1 loss in tumour cells is easier to identify because of the retained expression in the reactive bystander cells and the lack of background staining. However, the use of BAP1 is limited because of low sensitivity with retained expression being observed in approximately 45% of all and in 30% of the epithelioid MPM [14, 16, 18]. Consistent with previous published observations, we found loss of BAP1 in 68% of epithelioid and biphasic MPM. Because epithelioid and biphasic MPM are more frequently associated with a pleural effusion than their sarcomatoid counterpart, cytological examination including BAP1 immunohistochemistry is sufficient to reach a reliable diagnosis of MPM in up to 70% of cases. This also means that in many patients, histological confirmation is not required.

In contrast to BAP1, interpretation of MTAP is more difficult due to its variable nuclear and cytoplasmatic staining. Weak cytoplasmatic staining is observed in MPM with a deletion of the MTAP gene [19]. Preserved cytoplasmatic or nuclear staining may be difficult to interpret, resulting in interobserver variability [19]. Nevertheless, Chapel et al. [19] demonstrated an excellent interobserver agreement between pathologists in MTAP immunostaining for all mesothelial proliferations (kappa: 0.85) with a good interlaboratory reproducibility. In addition, MTAP expression correlated with CDKN2A gene status determined by FISH with a sensitivity and specificity of 78% and 96%, respectively [19]. Especially, comparison of immunoreactivity between tumour cells and internal controls, such as stromal and inflammatory cells, is strongly recommended to obtain an optimal interpretation of this marker. In our own experience, concordant p16 protein expression is associated with retained expression of MTAP and as such helpful to prevent false negative interpretation of MTAP immunoreactivity. In our cohort, MTAP expression showed a good concordance between cytological and histological samples in all but one case. In contrast to BAP1, heterogeneity for MTAP is more frequent in MPM with part of the tumour cell population showing preserved MTAP expression next to MTAP-deficient cells, suggesting that BAP1 is an early event in MPM pathogenesis. Heterogeneity of MTAP expression might explain the discordance of the histological and cytological results observed in one sample [19].

Similar to MTAP, p16 shows a heterogenous staining pattern with loss or co-expression of both markers in the same tumour cells. The concordant loss of MTAP and p16 points to a common mechanism of inactivation of the CDKN2A and MTAP genes. Deletion of CDKN2A is associated with a co-deletion of MTAP in all cases, resulting in concordant loss of p16/MTAP protein expression [6, 20]. In accordance with previous published observations, we have also found that isolated MTAP deletion without CDKN2A co-deletion does not occur [19]. Expression of p16 was frequently characterized by positive staining of some but not all cells in the entire sample as well as in the internal control. This might be explained by the fact that p16 functions as a cell cycle regulator and therefore is not constantly expressed [6, 21]. Although p16 showed a good agreement between the results of cytological and histological samples, p16 immunohistochemistry is often inconclusive and less reliable than MTAP and BAP1. Compared to specimens of control subjects, immunohistochemistry of p16 showed a good sensitivity but poor specificity. Instead of p16, we recommend immunohistochemical staining for MTAP or CDKN2A FISH. However, p16 immunohistochemistry may help better distinguish MTAP-positive from -negative cases.

In summary, BAP1, MTAP, and p16 showed a very good correlation between corresponding cytological and histological samples. Of the three markers, combined use of BAP1 and MTAP is most reliable in distinguishing malignant from reactive mesothelial proliferations with good sensitivity and 100% specificity and serves as a useful ancillary tool for differentiating malignant mesothelial from reactive proliferations on cytopathologic specimens.

All procedures performed in tasks involving human participants were in accordance with the ethical standards of the Institutional and/or National Research Committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Only archival tissue was used. Ethics application was approved by the Cantonal Ethics Committee, Zürich (KEK-ZH-Nr 2014-0604). Written informed consent was obtained from all participants.

The authors declare that they have no competing interests.

There are no funding sources.

Vera Amacher: investigation, formal analysis, and writing – original draft. Peter Karl Bode: supervision, validation, and writing – review and editing. Holger Moch and Daniela Lenggenhager: writing – review and editing. Bart Vrugt: investigation, supervision, validation, and writing – review and editing.

1.
Sinn K, Mosleh B, Hoda MA. Malignant pleural mesothelioma: recent developments. Curr Opin Oncol. 2021 Jan;33(1):80–6.
2.
Bösch D. Lunge und Atemwege. Berlin: Springer; 2014.
3.
Husain AN, Colby TV, Ordóñez NG, Allen TC, Attanoos RL, Beasley MB, et al. Guidelines for pathologic diagnosis of malignant mesothelioma 2017 update of the consensus statement from the international mesothelioma interest group. Arch Pathol Lab Med. 2018 Jan;142(1):89–108.
4.
Opitz I, Scherpereel A, Berghmans T, Psallidas I, Glatzer M, Rigau D, et al. ERS/ESTS/EACTS/ESTRO guidelines for the management of malignant pleural mesothelioma. Eur J Cardiothorac Surg. 2020 Jul 1;58(1):1–24.
5.
Henderson DW, Reid G, Kao SC, van Zandwijk N, Klebe S. Challenges and controversies in the diagnosis of mesothelioma: Part 1. Cytology-only diagnosis, biopsies, immunohistochemistry, discrimination between mesothelioma and reactive mesothelial hyperplasia, and biomarkers. J Clin Pathol. 2013 Oct;66(10):847–53.
6.
Hida T, Hamasaki M, Matsumoto S, Sato A, Tsujimura T, Kawahara K, et al. Immunohistochemical detection of MTAP and BAP1 protein loss for mesothelioma diagnosis: comparison with 9p21 FISH and BAP1 immunohistochemistry. Lung Cancer. 2017 Feb;104:98–105.
7.
Krasinskas AM, Bartlett DL, Cieply K, Dacic S. CDKN2A and MTAP deletions in peritoneal mesotheliomas are correlated with loss of p16 protein expression and poor survival. Mod Pathol. 2010 Apr;23(4):531–8.
8.
Galateau-Salle F, Churg A, Roggli V, Travis WD; World Health Organization Committee for Tumors of the Pleura. The 2015 world health organization classification of tumors of the pleura: advances since the 2004 classification. J Thorac Oncol. 2016 Feb;11(2):142–54.
9.
Churg A, Nabeshima K, Ali G, Bruno R, Fernandez-Cuesta L, Galateau-Salle F. Highlights of the 14th international mesothelioma interest group meeting: pathologic separation of benign from malignant mesothelial proliferations and histologic/molecular analysis of malignant mesothelioma subtypes. Lung Cancer. 2018 Oct;124:95–101.
10.
Cigognetti M, Lonardi S, Fisogni S, Balzarini P, Pellegrini V, Tironi A, et al. BAP1 (BRCA1-associated protein 1) is a highly specific marker for differentiating mesothelioma from reactive mesothelial proliferations. Mod Pathol. 2015 Aug;28(8):1043–57.
11.
Kinoshita Y, Hida T, Hamasaki M, Matsumoto S, Sato A, Tsujimura T, et al. A combination of MTAP and BAP1 immunohistochemistry in pleural effusion cytology for the diagnosis of mesothelioma. Cancer Cytopathol. 2018 Jan;126(1):54–63.
12.
Carter J, Miller JA, Feller-Kopman D, Ettinger D, Sidransky D, Maleki Z. Molecular profiling of malignant pleural effusion in metastatic non-small-cell lung carcinoma. The effect of preanalytical factors. Ann Am Thorac Soc. 2017 Jul;14(7):1169–76.
13.
Rodriguez EF, Jones R, Gabrielson M, Santos D, Pastorello RG, Maleki Z. Application of the international system for reporting serous fluid cytopathology (ISRSFC) on reporting pericardial effusion cytology. Acta Cytol. 2020;64(5):477–85.
14.
Carbone M, Yang H. Mesothelioma: recent highlights. Ann Transl Med. 2017 Jun;5(11):238.
15.
Wang LM, Shi ZW, Wang JL, Lv Z, Du FB, Yang QB, et al. Diagnostic accuracy of BRCA1-associated protein 1 in malignant mesothelioma: a meta-analysis. Oncotarget. 2017 Sep 15;8(40):68863–72.
16.
Galani V, Varouktsi A, Papadatos SS, Mitselou A, Sainis I, Constantopoulos S, et al. The role of apoptosis defects in malignant mesothelioma pathogenesis with an impact on prognosis and treatment. Cancer Chemother Pharmacol. 2019 Aug;84(2):241–53.
17.
Yoshimura M, Kinoshita Y, Hamasaki M, Matsumoto S, Hida T, Oda Y, et al. Highly expressed EZH2 in combination with BAP1 and MTAP loss, as detected by immunohistochemistry, is useful for differentiating malignant pleural mesothelioma from reactive mesothelial hyperplasia. Lung Cancer. 2019 Apr;130:187–93.
18.
Berg KB, Dacic S, Miller C, Cheung S, Churg A. Utility of methylthioadenosine phosphorylase compared with BAP1 immunohistochemistry, and CDKN2A and NF2 fluorescence in situ hybridization in separating reactive mesothelial proliferations from epithelioid malignant mesotheliomas. Arch Pathol Lab Med. 2018 Dec;142(12):1549–53.
19.
Chapel DB, Schulte JJ, Berg K, Churg A, Dacic S, Fitzpatrick C, et al. MTAP immunohistochemistry is an accurate and reproducible surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant pleural mesothelioma. Mod Pathol. 2020 Feb;33(2):245–54.
20.
Illei PB, Rusch VW, Zakowski MF, Ladanyi M. Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas. Clin Cancer Res. 2003 Jun;9(6):2108–13.
21.
Chiosea S, Krasinskas A, Cagle PT, Mitchell KA, Zander DS, Dacic S. Diagnostic importance of 9p21 homozygous deletion in malignant mesotheliomas. Mod Pathol. 2008 Jun;21(6):742–7.