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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.

Differentiation of malignant mesothelioma from reactive mesothelial proliferations is a frequent diagnostic dilemma in pathology, and few ancillary tests exist to help with this differential. Loss of BRCA-associated nuclear protein 1 (BAP1) by immunohistochemistry and deletion of Cyclin-dependent kinase inhibitor 2A (CDKN2A, often referred to as "p16") by fluorescence in situ hybridization (FISH) both have excellent specificity for mesothelioma, but they have limited sensitivity: for BAP1 the sensitivity for epithelioid mesotheliomas is on the order of 60% to 70%, but for sarcomatoid mesotheliomas it is below 20%.1-5 For CDKN2A, the sensitivity is in the range of 60% to 80% in the pleural cavity, but it is considerably less in the peritoneal cavity. (1,5-7) The combination of these 2 markers improves sensitivity compared with either marker alone, but the availability of CDKN2A FISH is restricted because of the cost and expertise required. (1,6)

Methylthioadenosine phosphorylase (MTAP) is an enzyme involved in purine metabolism that plays a role in salvage of adenosine and methionine. MTAP may function as a tumor suppressor gene. (8) The gene for MTAP is located very close to CDKN2A at the 9p21.3 locus and has previously been reported to be deleted in tandem with CDKN2A in the vast majority (91%-100%) of malignant pleural and peritoneal mesotheliomas as determined by FISH. (9,10)

MTAP immunohistochemistry thus might serve as a substitute for CDKN2A FISH. Zimling et al (11) previously reported that MTAP immunohistochemistry showed loss of the protein in both malignant (65%) and benign (23%) mesothelial processes, suggesting that it may not be useful in this setting. However, recently, Hida et al (12) concluded that immunohistochemical expression of MTAP is a highly sensitive surrogate marker for 9p21 deletion, with 74% of CDKN2A FISH deleted cases also showing loss of MTAP by immunohistochemistry, and excellent specificity, with no loss of MTAP in any reactive mesothelial proliferations.

Neurofibromin 2 (NF2) is a tumor suppressor gene that has been reported previously to be lost in malignant mesotheliomas, with recent whole-exome sequencing studies showing homozygous mutations or deletions in 50% of malignant pleural mesotheliomas. (13-15) These alterations in NF2 have only been able to be assessed by FISH or next-generation sequencing approaches, with no clinically useful immunohistochemical assay. (15-17)

In this study, we aimed to assess the utility of MTAP immunohistochemistry as a first-line approach to distinguishing malignant epithelioid mesothelioma from benign reactive mesothelial proliferations, and to compare the results to those obtained with the established markers of BAP1 immunohistochemistry, and CDKN2A and NF2 FISH.


Tissue Microarray

This study was approved by the research ethics review board of the University of British Columbia (Vancouver, Canada). A tissue microarray containing 17 cases of reactive mesothelial proliferations, 20 malignant epithelioid mesotheliomas, 12 high-grade serous ovarian carcinomas, and 21 cases of adenocarcinoma of the lung was used for this study.


BAP1 and MTAP immunohistochemical staining was performed on deparaffinized 4-im sections on a Dako Omnis instrument (Agilent Technologies, Santa Clara, California). For BAP1, the BAP primary antibody clone C4 (SC-28383, Santa Cruz Biotechnology, Mississauga, Ontario, Canada) was used at a dilution of 1:50 after heat-induced epitope retrieval at pH 9.0 for 30 minutes. For MTAP, the MTAP primary antibody clone 2G4 (Abnova, Walnut Creek, California) was used at a dilution of 1:100 after heat-induced epitope retrieval at pH 9.0 for 40 minutes. Both were blocked with Dako Envision Flex Peroxidase-Blocking reagent and were visualized with Dako Envision Flex horseradish peroxidase reagent. For MTAP, loss of nuclear and cytoplasmic staining was viewed as loss; for BAP1, only loss of nuclear staining was viewed as loss (see comments in Discussion).

Fluorescence In Situ Hybridization

Dual-color FISH was performed for both CDKN2A and NF2, as previously described. (16) CDKN2A was assessed using a Spectrum Orange-labeled, locus-specific probe (Abbott Molecular, Des Plains, Illinois) with a Spectrum Green-labeled chromosome 9 centromeric probe (CEP9). Probes for NF2 assessment included a fluorescein isothiocyanate-labeled chromosome 22 centromeric (CEP22q) probe and a Texas Red-labeled, locus-specific NF2 probe (Abnova). Each core on the tissue microarray was identified, and only individual and well-delineated cells were scored; overlapping cells were excluded from the analysis. At least 60 cells were scored for each case. Each tumor was assessed by the average and the maximum numbers of copies of either CDKN2A or NF2 per cell and the average ratio of the gene to chromosome 9 enumeration probe (CEP9) and chromosome enumeration probe 22q (CEP22q) copy numbers, respectively. The case was considered to be positive for deletion if homozygous deletion or homozygous/heterozygous deletion was identified in at least 20% of nuclei. (9,16)


None of the reactive mesothelial proliferations showed loss of MTAP on immunohistochemical stain (Figure, A and B; Table 1). Loss of MTAP staining was observed in 13 of 20 epithelioid malignant mesotheliomas (65%; Figure, C and D). In 11 of the mesotheliomas, there was loss in 100% of mesothelial cells (Figure, C and D); in the 2 other cases there was loss in 50% and 75% of the mesothelial cells. These cases were counted as positive for MTAP loss (see Discussion). The remaining mesotheliomas all showed staining in 100% of the tumor cells (Figure, E and F). All cases showed staining of stromal cells or inflammatory cells that served as a positive internal control (Figure).

None of the reactive mesothelial proliferations showed loss of BAP1 by immunohistochemistry (Table 1), and 11 of 20 malignant epithelioid mesotheliomas (55%) showed BAP1 loss.

For the malignant mesotheliomas, the MTAP immunohistochemistry results were compared to CDKN2A FISH (Table 2). Overall, 12 of the 17 malignant mesotheliomas evaluable by FISH showed deletion of CDKN2A (60%). There was concordance between loss of MTAP staining and CDKN2A deletion in 14 of 17 evaluable cases (82%). A total of 2 of the discordant cases showed intact MTAP staining by immunohistochemistry with CDKN2A deletion by FISH, and 1 case showed MTAP loss without CDKN2A deletion (see below). Of the 2 cases of MTAP partial loss, 1 case was evaluable by CDKN2A FISH, and it showed CDKN2A deletion. NF2 FISH showed no case of NF2 deletion in 17 evaluable cases.

The lung adenocarcinomas had 14% (4 of 21) MTAP loss and 0% (0 of 21) BAP1 loss (Table 1). The high-grade ovarian serous carcinomas had 8% (1 of 12) MTAP loss and 0% (0 of 12) BAP1 loss (Table 1).


The differentiation of reactive from malignant mesothelial proliferations remains an important but difficult pathologic diagnosis that cannot always be made morphologically. Because benign mesothelial proliferations usually require no further treatment, and the diagnosis of pleural malignant mesothelioma carries an extremely poor prognosis even with invasive treatment, it is particularly important that any ancillary test have a very high specificity for malignant mesothelioma. A wide variety of immunohistochemical tests have been proposed in the past for this purpose, including p53, epithelial membrane antigen (EMA), desmin, insulinlike growth factor 2 mRNA-binding protein 3 (IMP-3), and glucose transporter 1 (GLUT-1), but in our view none of these is sufficiently specific to be useful. (5) However, loss of nuclear BAP1 staining immunohistochemistry and CDKN2A deletion by FISH both have reasonably good sensitivity and 100% specificity in this setting. (1-7)

The gene for MTAP is located close to the CDKN2A locus at 9p21 and had previously been reported, using FISH analysis, to be deleted in tandem with CDKN2A in most malignant mesotheliomas. (9,10) Although FISH analyses are accurate, they require specialized techniques and are relatively slow and expensive; hence, immunohistochemical staining offers a potential advantage. However, as noted in the introduction, the extant literature on MTAP immunohistochemical staining is discordant. The Zimling et al (11) study, analyzing the results as H scores, concluded that MTAP immunohistochemistry might be useful in separating benign from malignant, but in our view their reported loss rate of 23% of reactive mesothelial proliferations means that the specificity of the test is too low for diagnostic use.

More recently, however, Hida et al (12) showed reasonable sensitivity and 100% specificity for MTAP loss in diagnosing malignant mesothelioma. Hida et al (12) used staining intensity equal to or above the internal control inflammatory or stromal cells as an indication of positive staining (ie, retention) of MTAP. Although their results agree with ours in a broad sense, they differ in detail because Hida et al accepted staining of as few as 60% of reactive mesothelial cells or cells of malignant mesothelioma as an indication of retention of MTAP.12 Conversely, 21 of 23 of their mesotheliomas showed retention of MTAP in up to 32% (but usually less than 20%) of tumor cells (ie, loss rates typically greater than 80%).

We found that MTAP staining was considerably easier to interpret. A total of 7 of 20 mesotheliomas (35%) did not show any loss of MTAP at all, whereas 11 of 20 (55%) showed 100% loss, and only 2 of 20 (10%) showed partial loss, with loss values in those 2 cases still above 50%. One of the latter cases was confirmed to have CDKN2A deletion by FISH, and the other was not able to be evaluated by FISH. It should also be noted that Hida et al (12) illustrate cases in which every cell demonstrates both nuclear and cytoplasmic staining for MTAP. However, we found that nuclear staining was often focal, even when cytoplasmic staining was present in every cell (Figure, F), and we suggest that loss of cytoplasmic staining is easier to evaluate for diagnostic purposes. The fact that there was excellent (84%) concordance between MTAP loss by immunohistochemistry and CDKN2A deletion by FISH suggests that this is a reasonable approach.

In 3 of our cases there was discordance between the MTAP and CDKN2A results. The 1 case in which there was MTAP protein loss without CDKN2A deletion may be a manifestation of hypermethylation of the MTAP gene, as has been reported in other types of tumor. The 2 cases with intact MTAP staining but loss of CDKN2A by FISH are more difficult to explain but might reflect unclear transcriptional and posttranscriptional factors or technical issues in the hybridization. Hida et al (12) also found occasional discrepancies between immunostaining and FISH.

As a matter of interest, we also asked whether MTAP immunohistochemistry might be of diagnostic use in lung adenocarcinomas and high-grade serous carcinomas, tumors in which BAP1 loss is sufficiently rare (<1% of cases) that the finding of BAP1 loss is strong evidence against either diagnosis. (18,19) For comparison, 21 adenocarcinomas of the lung were stained and showed 14% MTAP loss, as were 12 high-grade ovarian serous carcinomas, which showed 8% MTAP loss. MTAP immunohistochemistry thus does not appear to be useful in either setting.

NF2 FISH has been shown to be useful as a prognostic marker in peritoneal mesotheliomas, (16) but here no deletion was seen in this group of largely pleural mesothelioma.

Overall we recommend the use of both MTAP and BAP1 immunohistochemistry as a first-line approach to separating benign from malignant mesothelial proliferations in morphologically problematic cases. In our hands this combination had a sensitivity of 90% and a specificity of 100% for the diagnosis of malignant mesothelioma.


(1.) Sheffield BS, Hwang HC, Lee AF, et al. BAP1 immunohistochemistry and p16 FISH to separate benign from malignant mesothelial proliferations. Am J Surg Pathol. 2015;39(7):977-982.

(2.) Nasu M, Emi M, Pastorino S, et al. High incidence of somatic BAP1 alterations in sporadic malignant mesothelioma. J Thor Oncol. 2015;10(4):565576.

(3.) Cigognetti M, Lonardi S, Fisogni S, et al. BAP1 (BRCAl-associated protein 1) is a highly specific marker for differentiating mesothelioma from reactive mesothelial proliferations. Mod Pathol. 2015;28(8):1043-1057.

(4.) Farzin M, Toon C, Clarkson A, et al. Loss of expression of BAP1 predicts longer survival in mesothelioma. Pathology. 2015;47(4):302-307.

(5.) Churg A, Sheffield BS, Galateau-Salle F. New markers for separating benign from malignant mesothelial proliferations: are we there yet? Arch Pathol Lab Med. 2015;140(4):318-321.

(6.) Hida T, Hamasaki M, Matsumoto S, et al. BAP1 immunohistochemistry and p16 FISH results in combination provide higher confidence in malignant pleural mesothelioma diagnosis: ROC analysis of the two tests. Pathol Int. 2016;66(10): 563-570.

(7.) Takeda M, Kasai T, Enomoto Y, et al. 9p21 deletion in the diagnosis of malignant mesothelioma, using fluorescence in situ hybridization analysis. Pathol Int. 2010;60(5):395-399.

(8.) Huang HY, Li SH, Yu SC, et al. Homozygous deletion of MTAP gene as a poor prognosticator in gastrointestinal stromal tumors. Clin Cancer Res. 2009; 15(22):6963-6972.

(9.) Krasinskas AM, Bartlett DL, Cieply K, et al. CDKN2A and MTAP deletions in peritoneal mesotheliomas are correlated with loss of p16 protein expression and poor survival. Mod Pathol. 2010;23(4):531-538.

(10.) Illei PB, Rusch VW, Zakowski MF, et al. Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas. Clin Cancer Res. 2003;9(6):2108-2113.

(11.) Zimling Z, Jrgensen A, Santoni-Rugiu E. The diagnostic value of immunohistochemically detected methylthioadenosine phosphorylase deficiency in malignant pleural mesotheliomas. Histopathology. 2012;60(6B):E96-E105.

(12.) Hida T, Hamasaki M, Matsumoto S, et al. Immunohistochemical detection of MTAP and BAP1 protein loss for mesothelioma diagnosis: comparison with 9p21 FISH and BAP1 immunohistochemistry. Lung Cancer. 2017;104(2):98-105.

(13.) Bianchi A, Mitsunaga S, Cheng J, et al. High frequency of inactivating mutations in the neurofibromatosis type 2 gene (NF2) in primary malignant mesotheliomas. Proc Natl Acad Sci USA. 1995;92(24):10854-10858.

(14.) Cheng J, Lee W, Klein MA, et al. Frequent mutations of NF2 and allelic loss from chromosome band 22q12 in malignant mesothelioma: evidence for a twohit mechanism of NF2 inactivation. Genes Chromosomes Cancer. 1999;24(3): 238-242.

(15.) Guo G, Chmielecki J, Goparaju C, et al. Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma. Cancer Res. 2015;75(2):264-269.

(16.) Singhi AD, Krasinskas AM, Choudry HA, et al. The prognostic significance of BAP1, NF2, and CDKN2A in malignant peritoneal mesothelioma. Mod Pathol. 2015;29(1):14-24.

(17.) Sheffield BS, LoretteJ, Shen Y, et al. Immunohistochemistry for NF2, LATS1/ 2, and YAP/TAZ fails to separate benign from malignant mesothelial proliferations. Arch Pathol Lab Med. 2016;140(5):391-391.

(18.) Andrici J, Jung J, Sheen A, et al. Loss of BAP1 expression is very rare in peritoneal and gynecologic serous adenocarcinomas and can be useful in the differential diagnosis with abdominal mesothelioma. Hum Pathol. 2016;51(5):915.

(19.) Owen D, Sheffield B, Ionescu D, et al. Loss of BRCA1-associated Protein 1 (BAP1) expression is rare in non-small cell lung cancer. Hum Pathol. 2017;60(2): 82-85.

Kyra B. Berg, MD; Sanja Dacic, MD; Caitlyn Miller, BA; Simon Cheung, BSc; Andrew Churg, MD

Accepted for publication June 13, 2018.

Published onlineJuly 30, 2018.

From the Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada (Dr Berg); the Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (Dr Dacic and Ms Miller); and the Department of Pathology, Vancouver General Hospital, Vancouver, British Columbia, Canada (Dr Churg and Mr Cheung).

The authors have no relevant financial interest in the products or companies described in this article.

Corresponding author: Andrew Churg, MD, Department of Pathology, Vancouver General Hospital, JPPN 1401, 910 West 10th Avenue, Vancouver, BC, Canada V5Z 1M9 (email: achurg@

Caption: Methylthioadenosine phosphorylase (MTAP) immunohistochemical staining. A and B, Reactive mesothelial proliferation. MTAP is retained in all of the mesothelial cells. Inflammatory cells and histiocytes infiltrating between fat cells serve as an internal positive control. C and D, Epithelioid malignant mesothelioma showing loss of MTAP staining in 100% of the tumor cells and showing strong staining in the inflammatory and stromal cells. E and F, Epithelioid malignant mesothelioma showing no loss of MTAP staining in the tumor cells. Note that all of the tumor cells demonstrate cytoplasmic staining, but only some show nuclear staining. This is the usual pattern observed. Positively stained stromal and inflammatory cells serve as an internal control (hematoxylin-eosin, original magnification X200 [A, C, and E]; MTAP immunostain, original magnification X200 [B, D, and F]).
Table 1. Methylthioadenosine Phosphorylase (MTAP) and BRCA-Associated
Nuclear Protein 1 (BAP1) Loss by Immunohistochemistry in Reactive
Mesothelial Proliferations, Malignant Epithelioid Mesotheliomas, and
Lung Adenocarcinomas

                            No.   MTAP      BAP1      MTAP or
                                  Loss,     Loss,     BAP1 Loss,
                                  No. (%)   No. (%)   No. (%)

Reactive mesothelial        17    0 (0)     0 (0)     0 (0)
Malignant epithelioid
  Pleural                   18    12 (67)   11 (61)   17 (94)
  Peritoneal                2     1 (50)    0 (0)     1 (50)
  Total                     20    13 (65)   11 (55)   18 (90)
Lung adenocarcinoma         21    4 (14)    0 (0)     0 (0)
High-grade serous ovarian   12    1 (8)     0 (0)     1 (8)

Table 2. Comparison of Methylthioadenosine Phosphorylase (MTAP) and
BRCA-Associated Nuclear Protein 1 (BAP1) Loss by Immunohistochemistry
(IHC) With Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) and
Neurofibromin 2 (NF2) Deletion by Fluorescent In Situ Hybridization
(FISH) (a)

                  MTAPIHC   BAP1 IHC   CDKN2A FISH   NF2 FISH

1    Pleural      Lost      Intact     Deleted       Intact
2    Pleural      Lost      Intact     Deleted       Intact
3    Pleural      Lost      Intact     Deleted       Intact
4    Pleural      Lost      Lost       --            --
5    Pleural      Lost      Lost       Deleted       Intact
6    Pleural      Intact    Lost       Intact        Intact
7    Pleural      Lost      Lost       Deleted       Intact
8    Pleural      Lost      Lost       Deleted       Intact
9    Pleural      Intact    Intact     Intact        Intact
10   Pleural      Intact    Lost       Deleted       Intact
11   Pleural      Intact    Lost       Intact        --
12   Pleural      Lost      Intact     Deleted       Intact
13   Pleural      Lost      Lost       Deleted       Intact
14   Pleural      Intact    Lost       Deleted       Intact
15   Pleural      Lost      Lost       Deleted       Intact
16   Pleural      Lost      Intact     Deleted       Intact
17   Pleural      Lost      Intact     --            --
18   Pleural      Intact    Lost       --            Intact
19   Peritoneal   Intact    Lost       Intact        Intact
20   Peritoneal   Lost      Intact     Intact        Intact

(a)--indicates cases that were not evaluable by FISH.
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Author:Berg, Kyra B.; Dacic, Sanja; Miller, Caitlyn; Cheung, Simon; Churg, Andrew
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Date:Dec 1, 2018
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