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Improving the Accuracy of Mesothelioma Diagnosis in China

Open AccessPublished:December 19, 2016DOI:https://doi.org/10.1016/j.jtho.2016.12.006

      Abstract

      Introduction

      In the Western world, malignant mesothelioma (MM) is most prevalent in the pleura of older males who have been professionally exposed to asbestos. Information about MM from rapidly industrializing countries such as China is minimal. There is concern that a proportion of MM diagnoses in China may be incorrect because most Chinese physicians do not have experience diagnosing this rare cancer. We recently reported an unusually high incidence of peritoneal MM among eastern Chinese female patients. Here, we review the accuracy of MM diagnoses in China and provide suggestions to improve the accuracy of diagnosis.

      Methods

      We reviewed 92 pathological diagnosis of MM in 2002–2015 from two reference centers in the province of Zhejiang in eastern China. We performed a large set of immunohistochemistry analyses to increase the reliability of the diagnosis.

      Results

      We confirmed the MM diagnosis in 12 of 34 of the pleural tumors (35.3%), in 38 of 56 of the peritoneal tumors (67.9%), and in two of two of the MMs of the tunica vaginalis (100%). MMs were characterized by tumor cells showing nuclear Wilms tumor 1 and calretinin staining and by strong membranous staining for cytokeratin CAM5.2. The results of staining for the epithelial markers carcinoembryonic antigen, thyroid transcription factor-1, MOC31, BerEP4, p63, p40, paired box 8, ER and PR were negative. BRCA1 associated protein 1 nuclear staining was lost in percentages similar to what has been reported for samples from Western countries.

      Conclusions

      Our findings suggest that MM—especially in its pleural localization—is often misdiagnosed in eastern China. Identifying pitfalls and possible solutions in the pathological diagnosis of MM will affect both the standard of care and research in China.

      Keywords

      Introduction

      Malignant mesothelioma (MM) is an aggressive cancer with a dismal prognosis and limited therapeutic options and is usually associated with exposure to asbestos and other carcinogenic mineral fibers.
      • Napolitano A.
      • Carbone M.
      Malignant mesothelioma: time to translate?.
      Because of this strong association with asbestos, in Western countries MM is most often diagnosed in the parietal pleura of older males professionally exposed to asbestos.
      • Napolitano A.
      • Carbone M.
      Malignant mesothelioma: time to translate?.
      There are very few reports in the English literature about MM in China.
      • Luo S.
      • Liu X.
      • Mu S.
      • Tsai S.P.
      • Wen C.P.
      Asbestos related diseases from environmental exposure to crocidolite in Da-yao, China. I. Review of exposure and epidemiological data.
      • Cai S.X.
      • Zhang C.H.
      • Zhang X.
      • Morinaga K.
      Epidemiology of occupational asbestos-related diseases in China.
      • Gao Z.
      • Hiroshima K.
      • Wu X.
      • et al.
      Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women: analysis of 28 cases in southeast China.
      The incidence of MM in China has been estimated at 1.5 cases per million population,
      • Jie H.
      • Wanqing C.
      Chinese Cancer Registry Annual Report.
      which is much lower than in other countries. In addition to the English literature, we reviewed 68 papers representing all of the scientific manuscripts on MM in China that have been published in the Chinese literature (Supplementary Table 1). These papers reported a total of 1959 MM diagnoses, 293 of which were associated with asbestos exposure (approximately 15%), compared with more than 70% in most of the world.
      • Napolitano A.
      • Carbone M.
      Malignant mesothelioma: time to translate?.
      This low incidence of asbestos-related MM was unexpected because the use of asbestos in China has not been restricted until recently. Amphibole asbestos was banned in 2002; however, the use of chrysotile asbestos continues,
      • Cai S.X.
      • Zhang C.H.
      • Zhang X.
      • Morinaga K.
      Epidemiology of occupational asbestos-related diseases in China.
      • Courtice M.N.
      • Lin S.
      • Wang X.
      An updated review on asbestos and related diseases in China.
      although it was banned in selected products and industries such as auto brakes (1999), household appliances (2005), shipbuilding (2007), and mural material (2010). Asbestos mining and asbestos factories are still present in specific provinces and municipalities in China, such as in the province of Zhejiang in eastern China.
      We recently discovered that in China MMs are surprisingly more frequent in young women (peritoneal localization), and we confirmed that many of these women were not exposed to asbestos.

      Mao W, Zhang X, Guo Z, et al. Association of asbestos exposure with malignant mesothelioma incidence in eastern China [e-pub ahead of print]. JAMA Oncol. https://www.ncbi.nlm.nih.gov/pubmed?term=Association+of+Asbestos+Exposure+With+Malignant+Mesothelioma+Incidence+in+Eastern+China&TransSchema=title&cmd=detailssearch, accessed January 12, 2017.

      These findings are puzzling and their reliability is largely linked to a correct diagnosis. Pathological diagnosis of MM is aided by immunohistochemistry (IHC) and it requires a correct interpretation of the results. We reviewed all the pleural and peritoneal malignancies that were diagnosed as MM during the years 2002–2015 in two medical centers in Zhejiang Province—one that sees predominantly non–asbestos-exposed patients and the other that sees predominantly asbestos-exposed patients (see later).

      Materials and Methods

      Specimen Collection

      We collected the tissue slides used to diagnose 92 putative MMs in the Zhejiang Cancer Center Hospital (ZJCC), which is the largest university cancer hospital in the province of Zhejiang and located in the city of Hangzhou, and in the Yuyao People’s Hospital (YPH), which is located 112 kilometers from Hangzhou in the town of Yuyao, where there is a florishing asbestos textile industry. There were a total of 92 diagnoses of MM: 34 in the pleura, 56 in the peritoneum, and two in the tunica vaginalis. Of the confirmed diagnoses of MM, 28 were from ZJCC and 24 were from YPH. Demographic and epidemiological information on this set of samples has been presented elsewhere.

      Mao W, Zhang X, Guo Z, et al. Association of asbestos exposure with malignant mesothelioma incidence in eastern China [e-pub ahead of print]. JAMA Oncol. https://www.ncbi.nlm.nih.gov/pubmed?term=Association+of+Asbestos+Exposure+With+Malignant+Mesothelioma+Incidence+in+Eastern+China&TransSchema=title&cmd=detailssearch, accessed January 12, 2017.

      This retrospective study was approved by the Zhejiang Cancer Hospital Ethical Committee and the Zhejiang Academy of Medical Sciences Ethical Committee.

      Pathological Review

      All initial diagnoses of MM were supported by some, at times limited, IHC. The histopathological review of these MMs—and of the available IHC results—was conducted jointly by pathologists from the ZJCC (Z. G.) and from the University of Hawaii Cancer Center (M. C.) with extensive experience in MM diagnosis. These specimens were obtained from tissue biopsies and fine-needle biopsies. A set of IHC analyses was ordered on the basis of histological appearance, sex, and location of the tumor (for example, ER and PR stains were ordered only for peritoneal malignancies in women), and final diagnoses were made concurrently by Z. G. and M. C.

      IHC

      When tissue was available, biopsy specimens were stained for calretinin, Wilms tumor 1 (WT1), D2-40, MOC31, BerEP4, CAM5.2, thyroid transcription factor-1 (TTF-1), napsin A, CD15, paired homeobox 8 (PAX8), and BRCA1 associated protein 1 (BAP1), and for ER and PR in peritoneal tumors. These immunostains help differentiate MM from other malignancies.
      • Ordonez N.G.
      The diagnostic utility of immunohistochemistry in distinguishing between epithelioid mesotheliomas and squamous carcinomas of the lung: a comparative study.
      • Addis B.
      • Roche H.
      Problems in mesothelioma diagnosis.
      • Husain A.N.
      • Colby T.V.
      • Ordonez N.G.
      • et al.
      Guidelines for pathologic diagnosis of malignant mesothelioma: a consensus statement from the International Mesothelioma Interest Group.
      • Comin C.E.
      • Novelli L.
      • Cavazza A.
      • Rotellini M.
      • Cianchi F.
      • Messerini L.
      Expression of thrombomodulin, calretinin, cytokeratin 5/6, D2-40 and WT-1 in a series of primary carcinomas of the lung: an immunohistochemical study in comparison with epithelioid pleural mesothelioma.
      • Carbone M.
      • Shimizu D.
      • Napolitano A.
      • et al.
      Positive nuclear BAP1 immunostaining helps differentiate non-small cell lung carcinomas from malignant mesothelioma.
      In addition, those cases in which the differential diagnosis included squamous cell carcinoma (SCC) were stained also for CK5/6, p63, and p40. As a control for these IHC stains, we stained 10 primary lung adenocarcinomas in parallel: 10 primary lung SCCs and 15 primary carcinosarcomas (lung [5], esophagus [1], mediastinum [1], breast [1], pancreas [2], cervix [2], liver [1], stomach [1], and duodenum [1]). These cancer types were chosen because they often represent a diagnostic challenge in the differential diagnosis with MM.
      • Ordonez N.G.
      The diagnostic utility of immunohistochemistry in distinguishing between epithelioid mesotheliomas and squamous carcinomas of the lung: a comparative study.
      • Addis B.
      • Roche H.
      Problems in mesothelioma diagnosis.
      • Husain A.N.
      • Colby T.V.
      • Ordonez N.G.
      • et al.
      Guidelines for pathologic diagnosis of malignant mesothelioma: a consensus statement from the International Mesothelioma Interest Group.
      • Comin C.E.
      • Novelli L.
      • Cavazza A.
      • Rotellini M.
      • Cianchi F.
      • Messerini L.
      Expression of thrombomodulin, calretinin, cytokeratin 5/6, D2-40 and WT-1 in a series of primary carcinomas of the lung: an immunohistochemical study in comparison with epithelioid pleural mesothelioma.
      • Carbone M.
      • Shimizu D.
      • Napolitano A.
      • et al.
      Positive nuclear BAP1 immunostaining helps differentiate non-small cell lung carcinomas from malignant mesothelioma.
      IHC was performed with the antibodies listed in Supplementary Table 2.

      IHC for BAP1 and INO80

      IHC analysis of BAP1 protein expression was performed as previously described by using anti-BAP1 antibody (C-4 [Santa Cruz Biotechnology, Dallas, TX]).
      • Testa J.R.
      • Cheung M.
      • Pei J.
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      • Carbone M.
      • Ferris L.K.
      • Baumann F.
      • et al.
      BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs.
      • Nasu M.
      • Emi M.
      • Pastorino S.
      • et al.
      High incidence of somatic BAP1 alterations in sporadic malignant mesothelioma.
      IHC analysis of INO80 was performed by using anti-INO80 antibody (LS-B11393 [LSBIo, Seattle, WA]).

      Statistical Analyses

      Comparisons among proportions were carried by using Fisher’s exact test or the chi-square test. Sensitivity, specificity, and predictive values for each immunostain were calculated by comparing MM with all other non-MM malignancies. A p value less than 0.05 was deemed significant. All statistical analyses were carried with GraphPad Prism, version 7.00 (GraphPad Software, La Jolla, CA).

      Results

      Diagnostic Confirmation of MM Related to Tumor Location

      There were a total of 92 MM diagnoses during the years 2002–2015 in our two reference centers, ZJCC and YPH. We confirmed the diagnosis of MM in 12 of 34 of the pleural tumors (35.3%) (10 in women and two in men), in 38 of 56 of the peritoneal tumors (67.9%) (32 in women and six in men), and in two of two of the tumors of the tunica vaginalis (100%) (Table 1). Diagnostic confirmation of MM was statistically more frequent for peritoneal tumors than for pleural tumors (Fisher’s exact test, p < 0.01). No definitive conclusion could be drawn on tumors of the tunica vaginalis given their rarity overall and in our sample set. From these results, we can conclude that pleural rather than peritoneal MM is most often misdiagnosed.
      Table 1Benign and Malignant Conditions Misdiagnosed as MM
      CategorynTotal
      Tumors originally diagnosed as MM92
      Confirmed diagnoses of MM52
       Pleura12 of 34
       Peritoneum38 of 56
       Tunica vaginalis2 of 2
      Other, non-MM lesions, pleura22
       Metastatic carcinomas of lung3
       Metastatic renal cell carcinoma1
       Poorly differentiated carcinoma, unknown primary6
       Thymic carcinoma1
       Fibrous tumor of pleura1
       Synovial sarcoma1
       Chronic pleuritis1
       Inadequate specimens
      Specimens with extensive necrosis and/or few tumor cells and/or inadequate stains to differentiate between MM, carcinoma, etc.
      8
      Other, non-MM lesions, peritoneum18
       Metastatic carcinomas, ovary4
       Solitary fibrous tumor2
      High grade sarcoma peritoneum1
       Carcinosarcoma, kidney1
       Inadequate specimens
      Specimens with extensive necrosis and/or few tumor cells and/or inadequate stains to differentiate between MM, carcinoma, etc.
      10
      MM, malignant mesothelioma.
      a Specimens with extensive necrosis and/or few tumor cells and/or inadequate stains to differentiate between MM, carcinoma, etc.

      Sensitivity and Specificity of IHC for Diagnosis of MM

      For 28 of 52 of the cases diagnosed as MM and for 19 of the 40 tumors in which the diagnosis of MM was not confirmed, the paraffin blocks used to make the initial diagnosis were available to perform additional IHC studies. As expected,
      • Ordonez N.G.
      The diagnostic utility of immunohistochemistry in distinguishing between epithelioid mesotheliomas and squamous carcinomas of the lung: a comparative study.
      • Addis B.
      • Roche H.
      Problems in mesothelioma diagnosis.
      • Husain A.N.
      • Colby T.V.
      • Ordonez N.G.
      • et al.
      Guidelines for pathologic diagnosis of malignant mesothelioma: a consensus statement from the International Mesothelioma Interest Group.
      • Comin C.E.
      • Novelli L.
      • Cavazza A.
      • Rotellini M.
      • Cianchi F.
      • Messerini L.
      Expression of thrombomodulin, calretinin, cytokeratin 5/6, D2-40 and WT-1 in a series of primary carcinomas of the lung: an immunohistochemical study in comparison with epithelioid pleural mesothelioma.
      compared with non-MM tumors, MM cells were characterized by strong membranous staining for cytokeratin (CK) CAM5.2 (Fisher’s exact test, p < 0.01) and nuclear staining for calretinin (Fisher’s exact test, p < 0.0001) and WT1 (Fisher’s exact test, p < 0.0001) (Table 2). The results of staining for the epithelial markers CEA, TTF-1, MOC31, BerEP4, PAX8, ER, and PR were negative in MM (ER/PR stains were used only in peritoneal tumors) (Supplementary Table 3). Full results on sensitivity, specificity, and predictive values of each of the mesothelial IHC markers of MM are shown in Supplementary Table 4.
      Table 2Immunostains with Mesothelial Markers
      Positive ImmunostainsCAM5.2%p ValueCalretinin%p ValueWT1%p Value
      MM34 of 369447 of 529040 of 4982
      Non-MM26 of 4065<0.0114 of 4035<0.00014 of 4010<0.0001
      Carcinosarcoma
      Poorly differentiated biphasic tumors; three showed focal calretinin positivity in the epithelial component, and four showed WT1 cytoplasmic staining but no nuclear staining.
      14 of 1688ns5 of 1631<0.00011 of 166<0.0001
      Lung adenocarcinoma10 of 10100ns0 of 100<0.00010 of 100<0.0001
      Squamous cell lung cancer0 of 100<0.00017 of 10
      Focal positivity.
      70ns0 of 100<0.0001
      Ovarian adenocarcinoma2 of 450<0.052 of 4
      Focal positivity.
      50ns3 of 4
      Focal positivity.
      75ns
      WT1, Wilms tumor 1; MM, malignant mesothelioma; ns, not significant.
      a Poorly differentiated biphasic tumors; three showed focal calretinin positivity in the epithelial component, and four showed WT1 cytoplasmic staining but no nuclear staining.
      b Focal positivity.
      On the basis of our results, the chance of finding, for example, a poorly differentiated carcinosarcoma positive for all three major positive markers of MM (CAM5.2, calretinin, and WT1) is predicted from our results to be approximately 1%, whereas approximately 70% of MMs are expected to have these IHC characteristics (Fisher’s exact test, p < 0.0001, with the probability of positivity for each marker considered an independent event). From these results, we can conclude that the regular use of all three positive MM IHC markers, CAM5.2, calretinin, and WT1, would significantly improve the accuracy of MM diagnosis in China.

      Identification of MM mimickers

      The 40 malignancies (both of the pleura and peritoneum) in which we ruled out the initial diagnosis of MM are shown in Table 1. These included metastatic carcinomas from various organs, including the lung (TTF-1–positive, Napsin A–positive, and MOC31 positive), ovary (BerEP4 positive, MOC31 positive, PAX8 positive, ER positive, and PR positive), and kidney (CD15 positive and PAX8 positive); primary fibrous tumors (CD34 positive); high-grade sarcomas (CK negative); renal carcinosarcoma (PAX8 positive); and one case of chronic pleuritis. We could not make a definitive diagnosis in six CK-positive cases, which we diagnosed as poorly differentiated carcinomas because of their undifferentiated histological appearance and lack of reactivity for any MM marker. For these six cases, the paraffin block was not available for additional IHC studies. In 18 additional cases (inadequate specimens, eight from the pleura and 10 from the peritoneum [see Table 1]), we could not confirm the diagnosis of MM because these were tiny needle biopsy specimens in which invasion by malignant cells could not be identified and/or IHC data were conflicting and no paraffin block was available to perform additional studies. Figures 1 and 2 and Supplementary Figure 1 show hematoxylin and eosin staining and IHC results representative of these 92 biopsy specimens. Figure 1 shows representative hematoxylin and eosin staining and immunostain results in a case in which we confirmed the diagnosis of pleural epithelial MM compared with a case that we rediagnosed as lung adenocarcinoma, Figure 2 shows a case that we confirmed as biphasic pleural MM compared with a case that we rediagnosed as renal carcinosarcoma metastatic to the pleura, and Supplementary Figure 1 shows a case that we confirmed as sarcomatoid MM compared with a case that we rediagnosed as primary fibrous tumor of the pleura.
      Figure thumbnail gr1
      Figure 1Immunohistochemistry results for epithelial malignant mesothelioma (MM) and lung adenocarcinoma. (Left column) Epithelial MM positive for calretinin, Wilms tumor 1 (WT1), D2-40, and CAM5.2 and negative for CD15 and thyroid transcription factor-1 (TTF-1). (Right column) Lung adenocarcinoma positive for CAM5.2, CD15, and TTF-1 and negative for calretinin, WT1, and D2-40. Original magnification of main portion of all slides, ×100; original magnification of insets at right lower corner of each slide, ×400. HE, hematoxylin and eosin.
      Figure thumbnail gr2
      Figure 2Immunohistochemistry results for biphasic malignant mesothelioma (MM) and carcinosarcoma of the kidney, with both sets of slides showing a clear cell component. (Left column) Biphasic MM positive for calretinin and D2-40 and negative for Wilms tumor 1 (WT1), CAM5.2, CD15, and thyroid transcription factor-1 (TTF-1). (Right column) Carcinosarcoma of the kidney positive for CD15 and paired box 8 (PAX-8); negative for calretinin, WT1, and D2-40; and negative/weakly positive for CAM5.2. Original magnification, ×400. HE, hematoxylin and eosin.

      BAP1 and INO80 Staining

      A total of 35 adenocarcinomas, SCCs, and primary carcinosarcomas stained positive for nuclear BAP1 (Supplementary Fig. 2 and Table 3). Strong nuclear staining was detected in nearly 100% of the tumor cells in all these tumors (except for two adenocarcinomas in which some tumor nodules contained cells showing BAP1 nuclear staining and some areas contained tumor nodules that were BAP1 negative, possibly representing tumor subclones that had lost BAP1 expression).
      Table 3BAP1 Staining
      HistologyBAP1
      n%p Value vs.

      Total MM
      Total MM8 of 2236
       Epithelial3 of 1718
       Biphasic3 of 267
       Sarcomatoid2 of 2100
      Total lung cancers20 of 20100<0.0001
       Adenocarcinoma10 of 10
      All tumors showed homogeneous BAP1 nuclear staining in 100% of tumor cells, except for two lung adenocarcinomas in which positivity was seen in about 70% to 80% of tumor cells, whereas some areas contained nodules of tumor cells that entirely lacked BAP1 staining.
      100
       Squamous cell carcinoma10 of 10100
      Carcinosarcomas15 of 15100<0.0001
      Total non-MM35 of 35100<0.0001
      BAP1, BRCA1 associated protein B1; MM, malignant mesothelioma.
      a All tumors showed homogeneous BAP1 nuclear staining in 100% of tumor cells, except for two lung adenocarcinomas in which positivity was seen in about 70% to 80% of tumor cells, whereas some areas contained nodules of tumor cells that entirely lacked BAP1 staining.
      Different results were obtained in parallel immunostains of MM biopsy specimens (Fig. 3): of the 17 epithelial type MMs, two showed diffuse nuclear BAP1 positivity, one was focally positive, and 14 were negative. Of the three biphasic MMs, one was BAP1 positive, one negative, and one showed focal positivity in the spindle component only, indicating either polyclonality
      • Comertpay S.
      • Pastorino S.
      • Tanji M.
      • et al.
      Evaluation of clonal origin of malignant mesothelioma.
      or that in spite of the morphological appearance, the spindle component was benign/reactive and that this was an epithelial-type MM.
      • McGregor S.M.
      • Dunning R.
      • Hyjek E.
      • Vigneswaran W.
      • Husain A.N.
      • Krausz T.
      BAP1 facilitates diagnostic objectivity, classification, and prognostication in malignant pleural mesothelioma.
      • Cigognetti M.
      • Lonardi S.
      • Fisogni S.
      • et al.
      BAP1 (BRCA1-associated protein 1) is a highly specific marker for differentiating mesothelioma from reactive mesothelial proliferations.
      The two sarcomatoid MMs were BAP1 positive. In summary, we found that lack of BAP1 nuclear staining was preferentially associated with MM (Fisher’s exact test, p < 0.0001). Consistently, lack of nuclear staining appears to be rare in lung cancer.
      • Carbone M.
      • Shimizu D.
      • Napolitano A.
      • et al.
      Positive nuclear BAP1 immunostaining helps differentiate non-small cell lung carcinomas from malignant mesothelioma.
      In this series 35 of 35 lung cancers showed positive BAP1 nuclear staining.
      Figure thumbnail gr3
      Figure 3Expression of BRCA1 associated protein 1 (BAP1) and INO80 in malignant mesothelioma (MM). (Top row) MM, epithelial type, showing negative nuclear staining for BAP1 and for Ino80. (Middle row) Biphasic MM showing homogeneous BAP1 and Ino80 nuclear staining. (Bottom row) Sarcomatoid MM showing focal nuclear staining for BAP1 and for Ino80. Original magnification, ×400. Arrows point at tumor cells showing nuclear staining. HE, hematoxylin and eosin.
      We stained all these MM biopsies for INO80, a protein involved in DNA replication that requires BAP1 for stabilization and localization on the DNA. It has been suggested that BAP1 mutations result in loss of INO80 nuclear staining.
      • Lee H.S.
      • Lee S.A.
      • Hur S.K.
      • Seo J.W.
      • Kwon J.
      Stabilization and targeting of INO80 to replication forks by BAP1 during normal DNA synthesis.
      The concordance between BAP1 and INO80 staining in our sample set was statistically significant (Fig. 3) (chi-square test, p < 0.0001).

      Discussion

      A growing number of MMs are expected in rapidly industrializing countries in which asbestos use is still allowed, such as China and India.
      • Napolitano A.
      • Carbone M.
      Malignant mesothelioma: time to translate?.
      In these countries, information about the incidence and the prevalence of MM is minimal. We conducted an epidemiological study that revealed unique characteristics of MMs in Chinese patients. In eastern China, MMs were significantly more frequent in young individuals, in women, and with a peritoneal localization compared with the U.S., European, and Australian data.

      Mao W, Zhang X, Guo Z, et al. Association of asbestos exposure with malignant mesothelioma incidence in eastern China [e-pub ahead of print]. JAMA Oncol. https://www.ncbi.nlm.nih.gov/pubmed?term=Association+of+Asbestos+Exposure+With+Malignant+Mesothelioma+Incidence+in+Eastern+China&TransSchema=title&cmd=detailssearch, accessed January 12, 2017.

      The mean age of MM diagnosis in the cases presented here was 50.6 years, and the range was 21 to 71 years. The mean age of MM in women was 51.2 and the mean age in men was 47.8. The mean age of MM diagnosis among asbestos-exposed individuals was 55.2, and among non–asbestos-exposed individuals the mean age of diagnosis was 47.3.

      Mao W, Zhang X, Guo Z, et al. Association of asbestos exposure with malignant mesothelioma incidence in eastern China [e-pub ahead of print]. JAMA Oncol. https://www.ncbi.nlm.nih.gov/pubmed?term=Association+of+Asbestos+Exposure+With+Malignant+Mesothelioma+Incidence+in+Eastern+China&TransSchema=title&cmd=detailssearch, accessed January 12, 2017.

      These findings are usually associated with genetic predisposition to MM (such as carriers of germline BRCA1 associated protein 1 gene [BAP1] mutations) or are associated with exposure since birth (such as environmental exposure or growing up in families of asbestos workers).
      • Carbone M.
      • Kanodia S.
      • Chao A.
      • et al.
      Consensus report of the 2015 Weinman International Conference on Mesothelioma.
      The latter hypothesis was ruled out.

      Mao W, Zhang X, Guo Z, et al. Association of asbestos exposure with malignant mesothelioma incidence in eastern China [e-pub ahead of print]. JAMA Oncol. https://www.ncbi.nlm.nih.gov/pubmed?term=Association+of+Asbestos+Exposure+With+Malignant+Mesothelioma+Incidence+in+Eastern+China&TransSchema=title&cmd=detailssearch, accessed January 12, 2017.

      Instead, in the Western world, MM occurs predominantly in asbestos-exposed individuals at a mean age of 72 years, as there is a long latency of about 40 to 50 years from the time of exposure to development of MM.
      • Delgermaa V.
      • Takahashi K.
      • Park E.K.
      • Le G.V.
      • Hara T.
      • Sorahan T.
      Global mesothelioma deaths reported to the World Health Organization between 1994 and 2008.
      • Henley S.J.
      • Larson T.C.
      • Wu M.
      • et al.
      Mesothelioma incidence in 50 states and the District of Columbia, United States, 2003-2008.
      This long latency provides an opportunity to identify and develop biomarkers for early detection.
      • Ostroff R.M.
      • Mehan M.R.
      • Stewart A.
      • et al.
      Early detection of malignant pleural mesothelioma in asbestos-exposed individuals with a noninvasive proteomics-based surveillance tool.
      • Napolitano A.
      • Antoine D.J.
      • Pellegrini L.
      • et al.
      HMGB1 and its hyperacetylated isoform are sensitive and specific serum biomarkers to detect asbestos exposure and to identify mesothelioma patients.
      An additional surprising finding was that among these Chinese patients with MMs, the female-to-male ratio was 4:1 and the peritoneal-to-pleural ratio was 3:1, which is exactly the opposite of what is observed in the Western world.

      Mao W, Zhang X, Guo Z, et al. Association of asbestos exposure with malignant mesothelioma incidence in eastern China [e-pub ahead of print]. JAMA Oncol. https://www.ncbi.nlm.nih.gov/pubmed?term=Association+of+Asbestos+Exposure+With+Malignant+Mesothelioma+Incidence+in+Eastern+China&TransSchema=title&cmd=detailssearch, accessed January 12, 2017.

      • Delgermaa V.
      • Takahashi K.
      • Park E.K.
      • Le G.V.
      • Hara T.
      • Sorahan T.
      Global mesothelioma deaths reported to the World Health Organization between 1994 and 2008.
      • Henley S.J.
      • Larson T.C.
      • Wu M.
      • et al.
      Mesothelioma incidence in 50 states and the District of Columbia, United States, 2003-2008.
      Except for asbestos exposure, the demographics among the patients from the two hospitals were very similar: two of 28 patients with MM from ZJCC (7.1%) had history of asbestos exposure versus 18 of 24 patients with MM from YPH (75%).

      Mao W, Zhang X, Guo Z, et al. Association of asbestos exposure with malignant mesothelioma incidence in eastern China [e-pub ahead of print]. JAMA Oncol. https://www.ncbi.nlm.nih.gov/pubmed?term=Association+of+Asbestos+Exposure+With+Malignant+Mesothelioma+Incidence+in+Eastern+China&TransSchema=title&cmd=detailssearch, accessed January 12, 2017.

      Thus, both hospitals' MM cases comprised mostly young women. Therefore, in eastern China MMs occur predominantly in a different age group and sex than in the United States, and there seems to be no clear association with asbestos exposure. These surprising findings require additional investigation. Ethnicity might be relevant in determining pathogenesis and the molecular characteristics of cancer.
      • Wiencke J.K.
      Impact of race/ethnicity on molecular pathways in human cancer.
      Here we have reported that MM in China is often misdiagnosed—especially in its pleural localization, in which we did not confirm approximately two-thirds of the diagnoses. In addition, to confuse epidemiological and other research studies, cancer misdiagnoses are associated with inappropriate or delayed medical treatment. Moreover, misdiagnoses can lead to unnecessary therapy; for example, a case of chronic pleuritis (a benign condition) is described here.
      Among the cancer types erroneously identified as pleural MM, the most frequent were poorly differentiated metastatic carcinomas from the lung, kidney, and other organs. Metastatic carcinoma of the ovary was the most frequent MM mimicker in the peritoneum. This information should help Chinese pathologists to select more appropriate IHC markers to increase the accuracy of the diagnosis.
      We confirmed that the most sensitive and specific IHC biomarkers to diagnose MMs in Chinese patients are the same as observed in Western countries (i.e., calretinin and WT1). We found that D2-40 was not specific as a MM marker, as it stained not only 88% of MMs but also 80% of carcinosarcomas and lung SCC and 40% of lung adenocarcinomas. CK5/6 stained epithelial type MMs, but it also stained SCC, which in some of the cases we reviewed caused confusion and misdiagnoses. CK5/6 did not stain sarcomatoid MM. Thus, we recommend WT1 and calretinin as MM markers over CK5/6, which however remains a useful additional marker when used in the proper context. Instead, we suggest that CAM5.2 be included in the diagnostic panel because it almost always elicits a positive result, helps document tumor invasion, and helps eliminate outliers such as melanoma and lymphomas, which at times may cause a problem in the differential diagnosis (especially in tumors showing poorly differentiated single small cells among reactive inflammatory cells). Moreover, CAM5.2 is helpful to identify sarcomatoid and biphasic MM (the results of staining for CAM5.2 are almost always positive in both epithelial and spindle cells) and to rule out various types of sarcomas that can show a morphological pattern identical to that of MM but that are not stained by CAM5.2. The results of staining for the epithelial markers TTF-1, Napsin A, CD15, MOC31, PAX8, P63, P40, ER, and PR were consistently (100%) negative in MM; CEA and BerEP4 occasionally stained a small fraction of MM cells.
      On the basis of these data, our experience, and statistical considerations, we would recommend (1) considering invasion of nearby tissues a prerequisite to diagnose MM, which alone would eliminate dubious diagnoses that were made in inadequate specimens (see the 18 cases listed in Table 1), and (2) using supporting IHC with a minimum panel comprising CAM5.2, calretinin, WT1 (except in women with peritoneal tumors), and two or more epithelial markers that need to be chosen on the basis of location (ER, PR, and PAX8 for peritoneal tumors) and histological features (TTF-1 and MOC31 for adenocarcinomas, p63 and p40 when the differential is with SCCs, and PAX8 when the differential includes kidney cancer), as it would significantly increase the correct identification of MM in China and elsewhere.
      Additional recently proposed negative IHC stains for MM diagnoses include BAP1. BAP1 is a deubiquitinase and crucial tumor suppressor in MM, the nuclear expression of which is absent in approximately 65% of MMs.
      • Testa J.R.
      • Cheung M.
      • Pei J.
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      • Carbone M.
      • Ferris L.K.
      • Baumann F.
      • et al.
      BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs.
      • Nasu M.
      • Emi M.
      • Pastorino S.
      • et al.
      High incidence of somatic BAP1 alterations in sporadic malignant mesothelioma.
      Loss of the expression of INO80, which is a protein involved in DNA replication and a BAP1 target, may co-occur with BAP1 loss.
      • Lee H.S.
      • Lee S.A.
      • Hur S.K.
      • Seo J.W.
      • Kwon J.
      Stabilization and targeting of INO80 to replication forks by BAP1 during normal DNA synthesis.
      Our findings, together with those of recent reports,
      • Andrici J.
      • Parkhill T.R.
      • Jung J.
      • et al.
      Loss of expression of BAP1 is very rare in non-small cell lung carcinoma.
      suggest that nuclear BAP1 immunostaining helps differentiate NSCLCs from MM.
      • Carbone M.
      • Shimizu D.
      • Napolitano A.
      • et al.
      Positive nuclear BAP1 immunostaining helps differentiate non-small cell lung carcinomas from malignant mesothelioma.
      Our study has two main limitations: (1) as our analysis started by reviewing putative MM diagnoses by Chinese pathologists, we do not know the percentage of MMs that are misdiagnosed as other cancer types and (2) our analysis was limited to two hospitals in eastern China and the results may therefore not be representative of the whole of China. ZJCC is the largest cancer hospital in the city of Hangzhou (where most patients do not have evidence of asbestos exposure), and YPH is the most important hospital in a nearby area with abundant presence of textile industry that uses chrysotile asbestos.

      Mao W, Zhang X, Guo Z, et al. Association of asbestos exposure with malignant mesothelioma incidence in eastern China [e-pub ahead of print]. JAMA Oncol. https://www.ncbi.nlm.nih.gov/pubmed?term=Association+of+Asbestos+Exposure+With+Malignant+Mesothelioma+Incidence+in+Eastern+China&TransSchema=title&cmd=detailssearch, accessed January 12, 2017.

      These two hospitals should be at the forefront of MM diagnosis in China, yet the percent of misdiagnoses was high.
      The main reason for inaccurate diagnosis of MM in China appeared to be the use of an incomplete set of immunostains and/or the incorrect interpretation of the stain results, as well as an overall tendency to make a definitive diagnosis even when the evidence was inadequate and a repeat biopsy would instead be recommended. Our findings are similar to those of the study reported by Goldberg et al. in 2006, in which a panel of experts reviewed mesotheliomas in France and confirmed the diagnosis in 67% of the cases (we confirmed the diagnosis in 56.5% of cases [52 of 92]), ruled it out in 13% (we ruled it out in 17.4% [16 of 92]), and left it uncertain in 20% (we left it uncertain in 26% [24 of 92]).
      • Goldberg M.
      • Imbernon E.
      • Rolland P.
      • et al.
      The French National Mesothelioma Surveillance Program.
      Thus, the issue of the accuracy of MM diagnosis is not limited to China.
      In our study WT1 proved to be the most specific and reliable marker to identify pleural MM. However, because WT1 can stain ovarian tumors, calretinin is much more specific than WT1 to diagnose peritoneal MM.
      As the role of genetics in MM is increasingly being reevaluated,
      • Testa J.R.
      • Cheung M.
      • Pei J.
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      • Carbone M.
      • Ferris L.K.
      • Baumann F.
      • et al.
      BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs.
      • Carbone M.
      • Flores E.G.
      • Emi M.
      • et al.
      Combined genetic and genealogic studies uncover a large BAP1 cancer syndrome kindred tracing back nine generations to a common ancestor from the 1700s.
      and considering the unique clinicoepidemiological characteristics of MMs in Chinese patients, future studies should address the possible contribution of known and unknown genetic variants to MM in the Chinese population. Genetic studies to investigate whether the same gene alterations found in U.S. and Western MMs
      • Altomare D.A.
      • Menges C.W.
      • Pei J.
      • et al.
      Activated TNF-alpha/NF-kappaB signaling via down-regulation of Fas-associated factor 1 in asbestos-induced mesotheliomas from Arf knockout mice.
      • Bueno R.
      • Stawiski E.W.
      • Goldstein L.D.
      • et al.
      Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations.
      • Lo Iacono M.
      • Monica V.
      • Righi L.
      • et al.
      Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study.
      • 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.
      • Ugurluer G.
      • Chang K.
      • Gamez M.E.
      • et al.
      Genome-based mutational analysis by next generation sequencing in patients with malignant pleural and peritoneal mesothelioma.
      • Yoshikawa Y.
      • Emi M.
      • Hashimoto-Tamaoki T.
      • et al.
      High-density array-CGH with targeted NGS unmask multiple noncontiguous minute deletions on chromosome 3p21 in mesothelioma.
      are found in MMs in Chinese patients would be most informative and might provide clues to the prevalence of peritoneal MM in young Chinese women. Of note, China still uses primary monkey cells to produce polio vaccines.
      • Cutrone R.
      • Lednicky J.
      • Dunn G.
      • et al.
      Some oral poliovirus vaccines were contaminated with infectious SV40 after 1961.
      These cells are often heavily contaminated with simian virus 40 (SV40),
      • Cutrone R.
      • Lednicky J.
      • Dunn G.
      • et al.
      Some oral poliovirus vaccines were contaminated with infectious SV40 after 1961.
      a DNA tumor virus that has been linked to MM.
      • Carbone M.
      Simian virus 40 and human tumors: it is time to study mechanisms.
      • Gazdar A.F.
      • Carbone M.
      Molecular pathogenesis of malignant mesothelioma and its relationship to simian virus 40.
      • Carbone M.
      • Rizzo P.
      • Pass H.
      Simian virus 40: the link with human malignant mesothelioma is well established.
      The process that inactivates polio vaccine does not inactivate SV40; thus, millions were infected with infectious SV40 until 1963 in the United States and until the late 1970s in the USSR.
      • Cutrone R.
      • Lednicky J.
      • Dunn G.
      • et al.
      Some oral poliovirus vaccines were contaminated with infectious SV40 after 1961.
      The possible contribution of SV40 to MM in China should be investigated.
      In conclusion, the accuracy of MM diagnosis should be improved in China. As the need for adequate MM diagnoses will grow in China in the next decades, it is important to identify and address current pitfalls experienced by local pathologists. IHC represents a powerful tool to help correctly diagnose MM. The suggested set of immunostains, if used routinely, would significantly improve the accuracy of diagnosis.

      Acknowledgments

      This research was supported by National Cancer Institute grants 1R01CA198138-01 and by P30CA071789 to Dr. Carbone; by the University of Hawaii Foundation, which received generous donations from Honeywell International Inc. to support mesothelioma research by Dr. Carbone and from Riviera United 4-a Cure to support mesothelioma research by Drs. Yang and Carbone; by grants NCI-R01 CA160715 and DOD CA120355 to Dr. Yang; by donations to support malignant mesothelioma research from Belluck and Fox to Dr. Pass; by the Zhejiang Provincial Natural Science Foundation of China (LY16H160036) to Dr. Guo; by a grant from the Major Science and Technology Project of Medical and Health of Zhejiang Province of China (No. WKJ-ZJ-1403) to Dr. Su; and by a grant as a Key Project of Zhejiang Province Science and Technology Plan (2014C03030) to Dr. Lou. Drs. Carbone, Yang, Su, and Mao were responsible for conceptualization of the research; Drs. Guo and Carbone were responsible for the methodology; Drs. Carbone, Pass, Yu, and Wang were responsible for validation; Drs. Guo, Carbone, Wang, and Yu were responsible for formal analysis; Drs. Guo and Carbone were responsible for investigation; Drs. Guo, Carbone, and Napolitano were responsible for writing; Drs. Lou, Guo, Su, J. Chen, Zhang, Hu, K. Chen, and Mao were responsible for resources; Dr. Gao was responsible for visualization; Drs. Carbone, Su, Yang, Yu, and Mao were responsible for supervision; Drs. Carbone and Mao were responsible for project administration; and Drs. Carbone and Mao were responsible for funding acquisition.

      Supplementary Data

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