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

Disclosures

Mod Pathol. 2015;28(8):1043-1057. 

In This Article

Discussion

By analyzing a large cohort of clinical samples, this study establishes that BAP1 expression by immunohistochemistry represents a biomarker of excellent clinical utility for the diagnosis of malignant mesothelioma. Although expressed in all benign mesothelial lesions, BAP1 protein was lost in a large proportion of mesotheliomas, especially with epithelioid (128/184, 70%) and biphasic (9/15, 60%) features. BAP1 loss was also seen in sarcomatoid and desmoplastic mesothelioma, although with lower frequency (2/13, 15%). Remarkably, BAP1 protein loss was paralleled by homozygous deletion of the BAP1 locus in the vast majority of BAP1-negative tumors (31/41, 76%). Our results show the high specificity (100%) of BAP1 loss for mesothelioma diagnosis, in keeping with data recently obtained on a small series of tissue microarray mesothelial lesions;[39] in contrast with the latter study, however, sensitivity was much higher (66% versus 27%), probably because of the higher number of epithelioid/biphasic mesothelioma cases included in the present study.

The use of BAP1 immunostain can be particularly useful in the differential diagnosis between mesothelioma and reactive mesothelial proliferations that can be challenging in several instances[9–11,61] and especially on small tissue samples where stromal invasion cannot be properly evaluated. In this differential scenario, the role of immunohistochemistry is very controversial, as the results obtained using several markers (e.g., desmin, epithelial membrane antigen, p53, IMP3, GLUT-1, CD146, CD147, p16) were poorly reproducible among studies, with marked variability in sensitivity and specificity.[13–38] This lack of reproducibility might depend on preanalytical issues[62] or, alternatively, on data interpretation (eg, definition of the biomarker cutoff values). In contrast, the evaluation of BAP1 immunostain is straightforward and protein loss or retention is easily recognizable, therefore requiring no threshold values. BAP1-negative tumor cells were also easily recognizable in mesothelioma cases showing heterogeneity. Notably, in one of the cases, FISH analysis confirmed heterogeneity at the genomic level.

BAP1 stain in reactive mesothelial proliferations was of great support in the identification of malignancy, as five cases originally defined as simple or atypical reactive mesothelial proliferation and lacking protein expression were diagnosed having mesothelioma within a time period ranging from 2 to 104 weeks. An additional fifth case classified as atypical reactive mesothelial proliferation of the peritoneum was from a patient who died 4 years later with multiple abdominal metastases; unfortunately, no further biopsies were performed in this case. Overall, BAP1 negativity in cases of reactive mesothelial proliferation had a 100% positive predictive value for mesothelioma development, whereas BAP1 positivity had a negative predictive value of 90%.

Interestingly, the reactivity of BAP1 (including BAP1 loss) was highly concordant between the invasive component and the surface mesothelial layer, independently from its morphological features and degree of atypia. This indicates that, in a fraction of mesotheliomas, loss of BAP1 protein might represent an early and irreversible event anticipating full mesothelial transformation. With this in mind, biopsies including only BAP1-negative surface mesothelium should lead to immediate reevaluation to exclude invasive mesothelioma. A similar conclusion was made by Hwang et al[63] who applied FISH for the CDKN2A gene in 18 mesotheliomas and found homozygous deletion in 6/18 cases in both invasive tumor and the corresponding surface mesothelial proliferation.

In addition to its relevant role in distinguishing mesothelioma from reactive mesothelial proliferations, BAP1 stain also showed utility in the differential of mesothelioma from most common pleural and peritoneal mimickers, such as lung and ovary carcinomas, with specificity and sensitivity of 99/70% and 100/70%, respectively.

Malignant mesothelioma often presents with recurrent serous effusions and cytology of the pleural fluid represents the initial diagnostic procedure. Unfortunately, the diagnostic sensitivity is extremely variable with a high rate of false-negative cases, the latter finding partially explained by the broad morphologic overlap between reactive and malignant mesothelial cells.[64,65] Accordingly, the International Mesothelioma Interest Group established that effusion cytology has limited usefulness for a definitive diagnosis of mesothelioma.[65] Immunohistochemical markers used on histological samples to differentiate between benign and malignant mesothelial proliferation have also been used in cytology with similar unsatisfactory results.[28–36] The present study shows that the BAP1 profile of mesothelial cells is also easily identifiable on effusions and cell blocks. Benign mesothelial cells were invariably positive for BAP1, whereas 64% of mesotheliomas showed loss of protein; in equivocal cases by morphology, BAP1 negativity on mesothelial cells had a 100% positive predictive value for the diagnosis of mesothelioma: all six samples containing mesothelial cells negative for BAP1 were associated with a histological diagnosis of BAP1-negative mesothelioma.

BAP1 gene loss of function has been previously detected in cases of sporadic mesothelioma and related to different and sometimes coexisting mechanisms, including homozygous/heterozygous deletions or sequence mutations.[54,56,58,66] The possibility of posttranslational mechanism has also been considered, as loss of protein has been occasionally associated with preserved BAP1 transcript.[54] In this study FISH analysis of BAP1 gene showed that 32 out of 41 BAP1-negative mesotheliomas revealed biallelic (31/41, 76%) or monoallelic (1/41, 2%) deletion of the BAP1 locus; in other studies performed on mesothelioma cohorts not selected on the basis of BAP1 expression, gene deletions were detected with lower frequency.[54,56,66] Interestingly, BAP1 homozygous deletion was also identified in 1 out of 10 cases of mesothelioma expressing BAP1 protein; it is conceivable to retain that in this single case the gene region transcribing the epitope recognized by anti-BAP1 antibody is uncovered by the BAP1 probe. Taken together, these data suggest that BAP1 FISH analysis can support immunohistochemistry in confirming the diagnosis of mesothelioma.

A striking difference in the percentage of mesothelioma cases showing BAP1 loss was found between epithelioid/biphasic and sarcomatoid/desmoplastic mesothelioma (69% versus 15%). These data are in keeping with those reported by Yoshikawa et al,[56] whereas Bott et al[54] did not find correlation between gene anomalies and histologic mesothelioma variants. It is known that epithelioid and sarcomatoid mesotheliomas show different antigen expression, with many mesothelial markers being frequently negative in sarcomatoid subtype.[67–73] A recent gene expression profile analysis applied to a large series of pleural mesothelioma cell lines and tissue samples identified two mesothelioma subgroups. Although sarcomatoid and desmoplastic mesotheliomas belonged to a single group associated with poorer prognosis, epithelioid mesotheliomas were either included in the sarcomatoid/desmoplastic group or fell in a distinct subgroup with a better prognosis. Interestingly, in this study mutation analysis of BAP1, CDKN2A, CDKN2B, NF2, and TP53 was also performed and showed that the subgroup with better prognosis exhibited more frequent BAP1 genetic variants.[74] This observation is in keeping with the data obtained by Arzt et al[57] who found that lack of BAP1 protein expression in mesothelioma is associated with better survival.

The role of BAP1 mutations during mesothelial transformation is poorly understood. In contrast to sporadic or familiar atypical Spitz tumors where BAP1 mutations are not sufficient to induce malignant transformation,[52] in mesothelial cells their occurrence is invariably associated with malignancy. A recent study in mice showed that the heterozygous deletion of BAP1 increased the susceptibility to mesothelioma induced by chronic exposure to asbestos; interestingly, transformed mesothelial cells acquired a second hit and showed biallelic loss of BAP1 gene.[75] BAP1 haploinsufficiency, however, is not sufficient to promote spontaneous tumorigenesis in the host, as no mesotheliomas or other malignancies were observed in unexposed mice during 87 weeks of surveillance.

In conclusion, this study shows that BAP1 protein is frequently lost in malignant mesothelioma, especially of epithelioid/biphasic subtype (69%). This marker has an absolute specificity (100%) in the distinction between benign and malignant mesothelial proliferations, whereas sensitivity is lower. Nevertheless, in cases with uncertain diagnosis the identification of loss of BAP1 protein in mesothelial cells should prompt to immediately reevaluate the patient with additional biopsies or close follow-up. BAP1 loss might be useful in mapping tumor extent and planning surgical resection, especially in peritoneal mesothelioma where prognosis may depend on surgical radicality.[76] Finally, despite the fact that loss of protein likely depends more commonly on somatic events involving the BAP1 gene, the identification of mesothelioma with BAP1-negative phenotype might guide to perform BAP1 germline testing in family members.

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