BAP1 (BRCA1-Associated Protein 1) Is a Highly Specific Marker for Differentiating Mesothelioma From Reactive Mesothelial Proliferations

Marta Cigognetti; Silvia Lonardi; Simona Fisogni; Piera Balzarini; Vilma Pellegrini; Andrea Tironi; Luisa Bercich; Mattia Bugatti; Giulio Rossi; Bruno Murer; Mattia Barbareschi; Silvia Giuliani; Alberto Cavazza; Gianpietro Marchetti; William Vermi; Fabio Facchetti


Mod Pathol. 2015;28(8):1043-1057. 

In This Article

Materials and Methods

Case Selection

Histological, cytological, and cell-block samples were collected from the pathology archives from University-Spedali Civili of Brescia, Policlinico Hospital (Modena), IRRCCS (Reggio Emilia), Angelo Hospital (Mestre), and S. Chiara Hospital (Trento). All cases had been diagnosed or reviewed by pathologists with expertise in pleuropulmonary pathology.

Histological diagnoses of cases included in the study are reported in Table 1 . Normal mesothelium, covering lung tissue (4 cases) or tuba (7 cases) from samples surgically removed for nonmesothelial-related diseases, was used as control. Benign mesothelial tumors included three abdominal benign multicystic mesotheliomas, two abdominal benign papillary mesotheliomas, three uterine, and four paratesticular adenomatoid tumors. A total of 212 biopsies of mesothelioma obtained from the same number of patients included either thoracoscopic biopsies (136 cases) or surgical specimens (76 cases) and had been removed from pleura (207 cases) or peritoneum (5 cases); in all cases, diagnosis was based on clinical and imaging data, and fully supported by both positive and negative mesothelioma immunohistochemical markers, as recommended.[8,9] A total of 42 biopsies classified as reactive mesothelial proliferation were obtained from 41 patients and were further subclassified as simple reactive mesothelial proliferation (27 cases) or atypical reactive mesothelial proliferation (15 cases), according to Churg et al.[10] These samples (40 thoracoscopic biopsies and 2 surgical specimens) were removed from pleura (38 cases) or peritoneum (4 cases); 28 pleural biopsies were performed in symptomatic patients with recurrent effusion/pleuritis and past asbestos exposure (15 cases) and/or thoracoscopic anomalies (13 cases). Clinical data from cases of reactive mesothelial proliferation are reported in Table 2 . In addition, five surgical tissue blocks were part of an extensive sampling of invasive pleural mesothelioma and were classified as atypical reactive mesothelial proliferation because of absence of stromal invasion.

Cytological and cell-block samples included 18 cases of benign mesothelial reaction associated with inflammation (15 cases) or lung adenocarcinoma (2 cases), 45 cases of mesothelioma, and 8 samples defined as atypical mesothelial cells of indeterminate nature. Data on cytological/cell-block samples are reported in Table 3 .

A large series of nonmesothelial pleural and peritoneal malignant tumors most commonly included in the differential diagnosis with mesothelioma were also enrolled in the study; they were represented by 184 cases of lung adenocarcinoma (all major subtypes), 21 cases of lung squamous cell carcinoma, 95 cases of ovarian carcinoma (all major subtypes), and 8 cases of lung epithelioid hemangioendothelioma. Lung and ovarian tumors were tissue microarrays samples (containing from two to four representative 1 mm cores for each case), whereas epithelioid hemangioendothelioma were surgical samples ( Table 4 ).

In all cases with histological or cytological diagnosis of reactive mesothelial proliferation, the clinical, radiological, and eventual subsequent histological data were collected during a follow-up period of variable duration (up to 9 years), until pleural disease resolution, frank mesothelioma diagnosis, extramesothelial diseases, or neoplastic nonmesothelial malignancy were identified. Furthermore, the Brescia Province as well as the National Mesothelioma Registries were consulted.

The study was performed in accordance with the institutional ethical board protocols of Brescia, Modena, Reggio Emilia, Mestre, and Trento hospitals.

BAP1 Immunohistochemistry

On 4-μm-thick formalin-fixed, paraffin embedded sections, BAP1 immunostain was performed upon microwave oven epitope retrieval in Tris ethylenediamine tetra-acetic acid (EDTA) buffer (pH 9.0). Sections were incubated for 60 min with anti-BAP1 mouse monoclonal antibody (clone C-4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by horseradish peroxidase-conjugated Novolink polymer (Leica Microsystem, Newcastle upon Tyne, UK). The reaction was developed using diaminobenzidine (DAB) as chromogen and sections were counterstained with hematoxylin. The same procedure was used for cytological and cell-block samples; the former were represented by slides previously stained with papanicolaou or hematoxylin–eosin, as previously described.[59]

On selected cases, especially to distinguish between mesothelial cells and associated histiocytes, double immunohistochemistry for BAP1 combined either with epithelial membrane antigen (Leica Microsystem), calretinin (Invitrogen, Carlsbad, CA, USA), or cytokeratin 5/6 (Invitrogen) as mesothelial markers and CD11c (Leica Microsystems) and CD68 (clone PGM1, Dako, Glostrup, Denmark) as histiocyte markers was performed, as previously described,[60] using the Mach 4-alkaline phosphatase (AP) detection system (Biocare Medical, Concord, CA, USA) and Ferangi Blue (Biocare Medical) or New Fucsin (Dako) as chromogens.

Only the nuclear expression of BAP1 was considered for evaluation, despite the fact that in some cases fine granular cytoplasmic positivity was also noticed. All cases contained positive controls represented by nonmesothelial BAP1-reactive cells, such as inflammatory cells, fibroblasts, pneumocytes, or endothelial cells (Figure 1). On occasional cases, nuclei of neutrophils or lymphocytes were weakly positive or negative for BAP1. The 'positivity' or 'negativity' of mesothelial cells was defined as unambiguous presence or absence of BAP1 expression in mesothelial nuclei, without percentage or intensity cutoff values.

Figure 1.

Positive BAP1 immunoreactivity in normal mesothelium and lung alveolar epithelium (a) and in a case of adenomatoid tumor (b); in a peritoneal benign papillary mesothelioma (c, hematoxylin and eosin), BAP1 is also strongly expressed by mesothelial cells (d).


FISH analysis for BAP1 gene anomalies was performed on 51 mesothelioma biopsies, 41 showing BAP1 protein loss, 10 with normally expressed protein, as well as on 6 control samples with nontumoral mesothelium. Deparaffinized tissue sections were treated with HCl 0.2 N for 20 min, subsequently with the pretreatment solution (Vysis Paraffin Pretreatment Kit; Abbott Molecular, Des Plaines, IL, USA) at 82 °C for 30 min and digested with protease I, 250 mg at 37 °C for 10 min. Samples were incubated with the BAP1/CEN3q probe (Abnova, Taipei, Taiwan) using the Hybrite system (Hibridizer, Dako) at 75 °C for 5 min for codenaturation and at 37 °C overnight for hybridization. Posthybridization stringency wash was carried on in 2 × SSC/0.3% NP-40 at 73 °C for 2 min. Finally, slides were mounted with DAPI/antifade (Vector Laboratories, Burlingame, CA, USA) and examined with the epifluorescent microscope (Nikon, Eclipse 90i). FISH images were captured at × 100 magnification and elaborated using the Genikon software (Nikon Instruments S.p.A., Italy).

The BAP1/CEN3q probe labels the chromosome 3 centromere green (G) and the BAP1 gene red (R). In normal interphase cells, two green and two red signals (2G–2R) were clearly detectable. Based on the evaluation of 60 normal nuclei in each 6 normal samples, cutoff values for gene anomalies were defined as follows: (1) 20% for homozygous deletion (at least one green without red signals, 1/2G–0R or >2G–0R), (2) 29% for heterozygous deletion (two green with a single red signal, 2G–1R, or green more numerous than red signals, G>R), and (3) 43% for chromosome 3 monosomy (a single green and red signal, 1G–1R).

Sensitivity and specificity, as well as diagnostic predictive values, were calculated using the online MedCalc statistical software (; statistical analysis of mesothelioma incidence in reactive mesothelial proliferations in relation to BAP1 expression was performed using the two-tailed Fisher's exact test.