Introduction
Malignant mesothelioma (MM) is a relatively rare but highly aggressive malignant neoplasm arising from mesothelial cells of the pleura, peritoneum, pericardium, and tunica vaginalis. It is well-correlated with occupational and environmental asbestos exposure. (1,2) The incidence of MM has increased in many countries; (3) in Japan, mortality due to MM has increased since the 1990s, and is predicted to peak in the 2030s (4).
Epithelioid mesothelioma (EM) must be differentiated from reactive mesothelial hyperplasia (RMH), which is a non-neoplastic condition frequently caused by pleuritis, peritonitis, or serosal invasion of other cancers. Due to the close resemblance of EM to RMH, differentiation by routine histological observation alone can be challenging.
Various established and novel immunohistochemical markers have been utilized to distinguish EM from other malignancies (5–8) and RMH (6,9–17) Multiple potential immunohistochemical markers, including Ki-67, desmin, epithelial membrane antigen (EMA), p53, glucose transporter 1, insulin-like growth factor 2 messenger RNA binding protein-3 and BRCA1-associated protein 1 (BAP1) have been evaluated. However, despite the use of these immunohistochemical markers, the distinction between EM and RMH remains challenging in some cases.
Recently, detection of p16 (CDKN2A) homozygous deletion (p16 HD) using fluorescence in situ hybridization (FISH) has been used to differentiate MM from RMH, with 100% specificity. However, the sensitivity of this marker for pleural EM varies between 45 and 86%, while its sensitivity for peritoneal EM ranges from 14 to 41% in different laboratories (10,18–20). In our unpublished experience, p16 HD (detected by FISH) was present in 63.2% (12/19) of EM cases, but absent in all RMH cases (0/20). Although the detection of p16 HD using FISH may be considered highly specific, its sensitivity in differentiating EM from RMH is not very high. In addition, FISH analysis cannot be applied in all cases or in all pathology laboratories, given its high cost and stringent experimental requirements.
We recently reported that phorbol 12-myristate-13-acetate-induced protein-1 (PMAIP-1; Noxa) and baculoviral IAP repeat-containing 5 (BIRC5; Survivin) mRNA expression levels are significantly higher in EM than in non-neoplastic pleural tissue, and discussed the utility of anti-Noxa antibody for the distinction between EM and RMH (21). However, the utility of Survivin IHC for the differentiation of benign and malignant mesothelial proliferation has not yet been assessed.
Here, we studied the utility of Survivin and Ki-67 expressions along with the loss of BAP1 expression in distinguishing benign from malignant mesothelial proliferation.
Discussion
Accurate histopathological differentiation between MM and RMH is extremely important, not only for clinical management, but also for the appropriate operation of the public compensation system for victims of environmental and occupational asbestos exposure and their dependents. To obtain a better marker for EM, we evaluated the diagnostic utilities of Survivin, BAP1, and Ki-67 in differentiating EM from RMH. We found that the sensitivity and specificity of the nuclear Survivin labelling index following the use of a properly determined cut-off value was appropriate in distinguishing EM from RMH. The utility of Survivin IHC for the differentiation between benign and malignant mesothelial proliferation has not been reported to date. To the best of our knowledge, this is the first report evaluating the utility of Survivin IHC in differentiating EM from RMH.
Survivin is the smallest member of the inhibitor of apoptosis (IAP) family, and is expressed highly in most human foetal tissues and cancers. However, it is completely absent in terminally-differentiated tissues. Survivin functions as a regulator of both cell division and apoptosis. The function of Survivin differs according to cellular localization. Cytosolic Survivin is believed to function as an apoptotic suppressor, while nuclear Survivin is postulated to regulate cell division (24). Overexpression of Survivin is associated with tumour progression and poor prognosis in many types of human malignancies, including MM (25,26). In fact, several reports indicate that Survivin is a promising marker for the diagnosis of malignant pleural effusion (27). Survivin has also been reported to be associated with anti-tumour activity and outcomes of chemotherapy in MM, and is a new therapeutic target for the treatment of MM (28–30).
While the Survivin labelling indices of the EM cases in our study were similar to those reported by Meerang et al (25), they were significantly lower than those reported by Hmeljak et al (median, 67; mean, 63; range, 9.7–94.9; SD, 20.8) (26). This discrepancy in Survivin expression may be due to differences in staining technique, source of antibodies used for analysis, and the quantification technique. In our study, we used fully automated immunohistochemical staining utilising equipment from Roche for reproducible results. We also used commercially available antibodies from reputable sources and evaluated nuclear reactivity alone. Evaluation of nuclear reactivity was reproducible and was independently confirmed by 3 pathologists.
Several studies have determined that germline mutations in the gene for BAP1 predispose individuals to developing various tumours, including MM, cutaneous melanocytic tumours, uveal melanoma, lung adenocarcinoma, and meningioma (31). These studies suggest that germline mutations in BAP1 result in a ‘tumour predisposition syndrome’ linking BAP1 to many other cancers. Somatic mutations in the BAP1 gene have also been relatively frequently reported in MMs, uveal melanomas, and renal cell carcinomas (31). BAP1 is encoded by the BAP1 gene, which is located on the short arm of chromosome 3 (3p21). BAP1 is a deubiquitinase targeting histones and the host cell factor-1 transcriptional co-factor, and plays a role in transcriptional regulation, chromatin modulation, cell cycle regulation, and DNA repair (31,32). Several different alterations in the BAP1 gene have been described, including large deletions of exons leading to loss of the N-terminal region, or to premature protein termination, focal deletions, frameshift mutations due to insertions or deletions, splice site mutations, and base substitutions leading to nonsense and missense mutations. Frameshift mutations and missense and nonsense substitutions are the most common sequence alterations. Truncating mutations frequently result in loss of the nuclear localization signal and/or the C-terminal protein-binding domain, while missense mutations interfere with the ubiquitin hydrolase function of BAP1 (31). As the detection of these alterations in BAP1 has been made possible in recent years using immunohistochemistry (IHC), immunohistochemical detection of BAP1 loss has also been reported to be useful in distinguishing MM from RMH. However, the sensitivity of this assay in differentiating MM from RMH does not exceed 70% (10–13). Several studies indicate that the loss of nuclear BAP1 expression as assessed by IHC is closely correlated with genetic alterations in BAP1 (33–35).
In the present study, the frequency of BAP1 loss in EM was 66.2% (49/74), similar to those found in previous reports (10–13). Recently, Hida et al reported a focal heterogeneous BAP1 staining pattern in mesothelioma cases (10). However, in our study, almost all EM cases had either a uniform positive staining pattern or completely negative staining for BAP1. There were some EM cases that appeared to have focal staining for BAP1; however, careful observation of these cases under high power magnification confirmed that these focal positive cells were in fact inflammatory cells infiltrating into the mesothelioma or stromal cells. We classified such cases as cases with no loss of BAP1 expression. This may be the reason for the observed heterogeneous BAP1 staining pattern in mesothelioma. However, other reasons, such as differences in staining techniques and improper processing of the tumour, may also contribute to apparent differences between studies.
The specificity of a Survivin labelling index of over 5% and a loss of BAP1 expression was 100%. However, sensitivity of Survivin labelling index (67.7%) and loss of BAP1 expression (66.2%) alone are not sufficient for differential diagnosis. Although diagnostic accuracies of Survivin (84.8%) and BAP1 (83.6%) as single markers were inferior to that of EMA (95.5%), (21) the diagnostic accuracy of the combination of Survivin and BAP1 (Survivin-positive and/or BAP1-loss) was 95.3%, which was almost similar to EMA. Recently, Shinozaki-Ushiku et al proposed using a combination of BAP1 and enhancer of zeste homolog 2 (EZH2) expression to differentiate between MM from RMH; the sensitivity of this combination was 90%, while the specificity was absolute (36). The sensitivity (89.8%) and specificity (100%) of the combination of Survivin and BAP1 IHC in this study was comparable to those of previous reports (36).
A positive correlation between nuclear Survivin and Ki-67 labelling indices was previously reported by Meerang et al (25). We observed a similar correlation between Survivin and Ki-67 labelling indices in our study (data not shown). Although this correlation was present in both EM and RMH, it was more conspicuous in EM. Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in resting cells (G0). Therefore, Ki-67 is well known as a so-called ‘proliferation marker’, and the Ki-67 labelling index is often correlated with the clinical course of cancer (37,38). On the other hand, nuclear Survivin plays important roles in the regulation of mitosis. Survivin expression is found to be dominant only in the G2/M phase, and Survivin is known to localize to components of the mitotic spindle during the metaphase and anaphase of mitosis (39,40). Therefore, both nuclear Survivin and Ki-67 may be considered proliferation markers. We can thus explain both the high expression of Survivin and Ki-67 in EM compared to RMH, and the positive correlation between the nuclear Survivin and Ki-67 labelling indices.
Although various studies have reported the usefulness of Ki-67 IHC in differentiating EM from RMH, (14–17) it is not routinely utilized for the confirmation of mesothelioma due to its low sensitivity and specificity.
The sensitivity, specificity, and diagnostic accuracy of Ki-67 (85.1, 87.5, and 86.2%, respectively) in this study were almost the same or slightly higher compared with previous reports (14,15,17). These values were relatively high but not sufficient for differential diagnosis by single marker. However, the diagnostic accuracy of the combination of Ki-67 and BAP1 was 94.8%, which was almost the same as that of the combination of Survivin and BAP1.
We evaluated the utility of Survivin, BAP1, and Ki-67 IHC in distinguishing EM from RMH. Based on our results, ‘Survivin-positive and/or BAP1-loss’ finding strongly suggest EM, therefore we recommend the use of a combination of Survivin and BAP1. In addition, further evaluation of the Ki-67 labelling index may be useful for accurate differential diagnosis.