Esheba, Ghada E.
Mesothelioma is a rare, aggressive, and often deadly malignancy. It rarely metastasizes until late in its course; however, it can persistently invade adjacent structures such as the chest wall, diaphragm, mediastinum, and lung parenchyma (Amatya et al., 2009). Malignant mesothelioma often poses a diagnostic problem in surgical pathology. In addition to evaluation by conventional light microscopy, multiple ancillary techniques including electron microscopy and immunohistochemistry have been used to increase the accuracy of diagnosis (Marchevsky, 2008). Although the development of new immunohistochemical markers has improved the diagnostic ability, no single antibody has shown absolute sensitivity or specificity for malignant mesothelioma (Yaziji et al., 2006; Kao et al., 2011). As a result, a panel of immunohistochemical markers known to be either positive or negative for mesothelioma should be used in the differential diagnosis of malignant mesothelioma from its potential histologic mimics, lung adenocarcinoma (LAD) (Suster and Moran, 2006; Ordóñez, 2007; Sandeck et al., 2010; Betta et al., 2012).
D2-40 is a new monoclonal antibody that reacts with a 40-kD antigen in germ cells and germ cell tumors. The antigen is selectively expressed in lymphatic endothelium. This antibody has been proven to be useful in detecting tumors derived from lymphatic tissue as well as lymphatic involvement by a tumor. D2-40 has been recommended recently as a new marker for the diagnosis of malignant mesothelioma (Chu et al., 2005; Chang et al., 2009; Hu et al., 2010; Betta et al., 2012).
Calretinin is a calcium-binding protein of the EF-hand family and is expressed in both the cytoplasm and the nucleus of the mesothelial cells (Suster and Moran, 2006; Yaziji et al., 2006; Ordóñez, 2007; Marchevsky, 2008; Amatya et al., 2009; Sandeck et al., 2010; Kao et al., 2011; Betta et al., 2012).
WT-1 (Wilms’ tumor suppressor gene) is located on chromosome 11p1315 and encodes a transcription factor that contains four zinc-finger motifs at the C-terminus and a proline/glutamine-rich DNA-binding domain at the N-terminus. It plays an essential role in the normal development of the urogenital system. WT-1 protein can be found in the cell nuclei of mesotheliomas and ovarian serous carcinomas, as well as in benign mesothelium and fallopian tube epithelium (Yaziji et al., 2006; Marchevsky, 2008; Amatya et al., 2009; Hartkamp et al, 2010).
Thyroid transcription factor-1 (TTF-1) is a member of the Nkx2 family of homeodomain-containing transcription factors. TTF-1 influences organogenesis and the maintenance of the differentiated phenotypes of various tissues including the thyroid, lung, and brain. TTF-1, which is present in the epithelium of the lung, regulates transcription of the surfactant proteins A, B, and C and is essential for lung morphogenesis. TTF-1 is selectively expressed in tumors of the lung and thyroid (Khoor et al., 1999; Ordóñez, 2000; Srodon and Westra, 2002).
Carcinoembryonic antigen (CEA) is an oncofetal glycoprotein. It is expressed by fetal epithelial cells and in small amounts by normal adult epithelial cells and benign tumors. It is present in large amounts in adenocarcinomas of the gastrointestinal tract (including pancreas), lung, and medullary carcinoma of the thyroid (Suster and Moran, 2006; Ordóñez, 2007; Sandeck et al., 2010; Betta et al., 2012).
Materials and methods
Paraffin-embedded blocks were retrieved from the files of the Department of Pathology, Faculty of Medicine, Tanta University. The study group included 15 cases of pleural malignant mesothelioma including 10 epithelioid, four sarcomatoid, and one case of desmoplastic mesothelioma, and 10 cases of LAD.
The immunohistochemical panel including D2-40, calretinin, WT-1, CEA, and TTF-1 was performed in all cases of mesothelioma and LAD.
For this analysis, formalin-fixed paraffin-embedded tissue was sectioned at 5 mm and air-dried overnight at 37°C, deparaffinized in xylene, and rehydrated in a descending ethanol series. Endogenous peroxidase activity was blocked by immersion for 10 min in 0.3% hydrogen peroxide in methanol, followed by a single wash in PBS, pH 7.4. Sections for D2-40 immunodetection were autoclaved for 7 min at 105°C in 10 mm citrate buffer solution (pH 6.0). Sections were incubated with D2-40 monoclonal antibody (1 : 50 dilution; DakoCytomation, Glostrup, Denmark), calretinin monoclonal antibody (1 : 50 dilution; DakoCytomation, Carpinteria, California, USA), WT-1 (clone 6F-H2, 1 : 400 dilution; DakoCytomation, Carpinteria, California, USA), CEA monoclonal antibody (1 : 50 dilution; DakoCytomation), and TTF-1 monoclonal antibody (1 : 100 dilution; DakoCytomation). The incubation was for 1 h at room temperature for all antibodies. The color was developed using diaminobenzidine (Sigma Chemical, St. Louis, Missouri, USA) as the chromogen. The slides were then counterstained with Mayer’s hematoxylin, dehydrated, and mounted. In order to evaluate the specificity of each antibody, known positive and negative tissues were used as controls. The intensity (on a scale of 0–3), percentage, and pattern of immunostaining were evaluated and recorded semiquantitatively by light microscopy.
The χ2-test was used to determine significant differences in the expression of D2-40, calretinin, WT-1, CEA, and TTF-1 between malignant pleural mesothelioma (MPM) and LAD. The mean, SDs, and P values were reported.
The significance level was 0.05. All analyses were carried out using the SPSS statistical software (SPSS Inc., Chicago, Illinois, USA).
Ten cases (67%) were epithelioid mesotheliomas. The tumors showed a wide range of morphologic patterns including sheet-like (five cases) (Fig. 1a), tubulopapillary (four cases) (Fig. 1b), and adenomatoid (microglandular; one case).
The cells had an eosinophilic cytoplasm with open nuclei and prominent nucleoli, and infrequent mitoses.
Four cases (27%) were sarcomatoid mesotheliomas, with predominant spindle tumor cells with a wavy architecture, and a diffuse infiltrating growth pattern. Only one case (6%) was desmoplastic mesothelioma. The desmoplastic tumor showed cells arranged in between a heavily collagenized stroma. The tumor cells had small, hyperchromatic nuclei.
Six cases of LAD showed a predominantly acinar pattern, whereas four cases showed a papillary pattern. The acinar pattern was characterized by acini and tubules composed of cuboidal or columnar cells with focal mucin production infiltrating the lung parenchyma.
The papillary pattern was characterized by a complex papillary proliferation along fibrovascular cores that replaced the underlying lung architecture.
In Table 1, the immunoreactivity for D2-40, calretinin, WT-1, CEA, and TTF-1 is summarized.
There were 13 out of 15 cases (87%) of mesothelioma that showed positive D2-40 expression in both membranous and cytoplasmic patterns. The 13 cases included 9 cases of epitheloid mesothelioma (Fig. 2c), 3 cases of sarcomatoid mesothelioma (Fig. 3c), and the desmoplastic variant.
The characteristic membranous staining was not typically present in the sarcomatoid mesothelioma.
The desmoplastic variant showed weak positivity with D2-40.
In contrast, all cases (100%) of LAD were negative for D2-40 (Fig. 3d).
There was a statistically significant difference in D2-40 expression between mesothelioma and LAD (P<0.0001).
Thirteen out of 15 (87%) cases showed both cytoplasmic and nuclear staining for calretinin.
All cases of (100%) of epithelioid mesotheliomas were calretinin positive (Fig. 1d). In contrast, only two cases (50%) of sarcomatoid malignant mesotheliomas showed weak-to-moderate calretinin positivity (Fig. 2d). The desmoplastic mesothelioma showed weak positive staining with calretinin.
There was a statistically significant difference in calretinin expression between mesothelioma and LAD (P<0.0001).
However, only two cases (20%) of LAD showed weak cytoplasmic staining with calretinin.
The difference between D2-40 and calretinin expression in mesothelioma was not statistically significant (P=0.8648).
Eight cases (53%) of malignant mesothelioma showed positive nuclear staining for WT-1. The expression was observed in eight out of 10 cases (80%) of epithelioid mesothelioma (Fig. 1e). All cases (100%) of sarcomatoid malignant mesotheliomas were WT-1 negative. The desmoplastic variant was negative for WT-1.
Only one case (10%) of LAD showed patchy weak, positive WT-1 staining.
The difference between WT-1 expression in MPM and LAD was statistically significant (P=0.0151).
There was a statistically significant difference between D2-40 and WT-1 expression in mesothelioma (P=0.0417).
Thyroid transcription factor-1 and carcinoembryonic antigen
The expression pattern, in detail, of TTF-1 and CEA in LAD is shown in Table 1 and Fig. 3.
TTF-1 and CEA staining was present in eight and nine cases (80 and 90%) of LADs, respectively. However, only one case (10%) of epithelioid mesothelioma showed focal weak staining for CEA; however, this case was positive for D2-40, calretinin, and WT-1.
TTF-1 was not detected in any case of malignant mesothelioma.
MPM is a challenging disease in terms of diagnosis and treatment; early and accurate diagnosis can lead to appropriate clinical management.
The histological picture of mesothelioma especially the epithelioid variant, in some cases, has a strong resemblance to that of LAD. This leads to major problems for surgical pathologists in differentiating mesothelioma from LAD, especially when using hematoxylin and eosin-stained sections alone.
Recent studies suggested that D2-40 might be helpful in differentiating MPM from adenocarcinoma because it reacted with MPM but not with other tumors (Takeshima et al., 2009; Sandeck et al., 2010; Kao et al., 2011; Betta et al., 2012).
In the present study, D2-40 expression was found in 87% of MPM.
Ninety percent of epithelioid mesothelioma were positive for D2-40 with characteristic diffuse and strong membranous staining in the majority of epithelioid MPM; however, 75% of sarcomatoid mesothelioma were positive for D2-40, but the staining was less intense than that in the epithelioid variant. In addition, the interpretation of D2-40 staining in sarcomatoid mesothelioma was complicated by the loss of the characteristic membranous staining pattern observed in epithelioid mesotheliomas. These results are consistent with previously published reports (Kushitani et al., 2008; Hu et al., 2010; Kao et al., 2011). In contrast, Ordóñez (2005) was not able to detect D2-40 immunoreactivity in sarcomatoid malignant mesothelioma.
In a previously published study carried out by Hinterberger et al. (2007), on a tissue microarray, the authors found that 66% of the epithelioid and only 30% of the sarcomatoid tumor were positive for D2-40. However, this variation could be explained by the use of full tissue sections in the present study that minimized the rate of false-negative results because of tumor heterogeneity in tissue microarray.
In a recent study carried out by Chirieac et al. (2011) on 24 cases of sarcomatoid mesothelioma, the authors reported that D2-40 is a highly sensitive immunohistochemical marker for sarcomatoid mesothelioma and reported that the sensitivity of D2-40, calretinin, and WT-1 for sarcomatoid mesothelioma is 100, 25, and 33%, respectively.
In this study, all cases of LAD showed negative immunoreactivity for D2-40. However, there have been some reports on its cytoplasmic expression in a few cases of LAD (Mimura et al., 2007).
In the current work, 87% of malignant mesothelioma were positive for calretinin. The latter was expressed in the nucleus and cytoplasm of all cases of epithelioid mesothelioma, and in 50% of sarcomatoid mesothelioma. Cytoplasmic expression was also observed in two cases (20%) of LAD.
Similar results were obtained by Amatya et al. (2009), who also reported that cytoplasmic expression for calretinin was detected in 17.5% cases of LAD.
However, WT-1 was positive in only 53% of malignant mesothelioma, the expression was observed in 80% of epithelioid mesothelioma, and was completely absent in cases of sarcomatoid malignant mesotheliomas. Ten percent of LAD showed focal weak positive WT-1 staining.
The above-mentioned results were in agreement with those of Chu et al. (2005), who reported that 58% of epithelioid mesothelioma showed positive staining for WT-1, whereas none of the sarcomatoid mesothelioma showed such positivity. The latter authors also found that only 7% of LAD was positive for WT-1.
Similarly, in a study carried out by Amatya et al. (2009), the authors found that 60.5 and 7.5% of malignant mesothelioma and LAD were positive for WT-1, respectively.
In this work, there was only one desmoplastic variant of sarcomatoid mesothelioma, which was positive for D2-40 and calretinin, but negative for WT-1. It is noteworthy that there are only limited data on D2-40, calretinin, and WT-1 expression in the desmoplastic variant of mesothelioma in the literature. Nicolini et al. (2011) reported positive D2-40, calretinin, and WT-1 staining in the desmoplastic malignant mesothelioma of the pericardium.
In the present study, it was found that the sensitivity of D2-40 was equal to that of calretinin and markedly better than that of WT-1.
In the differentiation of mesothelioma from LAD, D2-40 showed specificity superior to that of calretinin and WT-1.
These findings suggest that D2-40 is one of the most informative markers for the diagnosis of MPM, because of its high sensitivity and specificity, and is frequently expressed in all histologic types of MPM, in contrast to other mesothelioma markers, which are commonly expressed in the epithelioid component but not in the sarcomatoid component, such as WT-1.
In contrast, the above-mentioned data contradicted those of Hinterberger et al. (2007), who found that the sensitivity of D2-40 was lower than that of calretinin in both epithelioid and sarcomatoid areas; this variation could be attributed to the use of microarray and different antibody clones in their study.
CEA is one of the negative mesothelioma markers available for the differential diagnosis of MPM and LAD.
A systematic review of 51 studies (consisting of 1524 LADs and 1818 epithelioid mesotheliomas) reported that the sensitivities and specificities of CEA for LAD are 83 and 95%, respectively (King et al., 2006).
In the current work, 90% of LADs showed positive staining for CEA; in contrast, only one case of mesothelioma was positive for CEA.
Similar results have been reported previously by Ordóñez (2013).
However, Mimura et al. (2007) and Amatya et al. (2009) could not find any expression for CEA in the mesothelioma cases, whereas CEA expression in LAD, in their studies, was 95.5 and 96.3, respectively.
TTF-1 is expressed in adenocarcinomas originating only in the thyroid or the lungs. Previous studies have shown that TTF-1 expression is present in 100% of thyroid carcinomas and in up to 75% of LADs (Tacha et al., 2012).
In this study, 80% of LAD showed strong staining for TTF-1. In contrast, no TTF-1 expression was found in any of the MPMs examined. These data were in agreement with those of (Mimura et al., 2007).
In this study, a tumor that shows immunoreactivity for D2-40, calretinin, and WT-1 antibodies, but not for CEA and TTF-1 antibodies, is unequivocally MPM, and a tumor negative for D2-40, calretinin, and WT-1, but positive for CEA and TTF-1, is unquestionably not MPM, but LAD.
In summary, the results in this work show that the inclusion of D2-40 in an immunohistochemical panel composed of calretinin, CEA, and TTF-1 will allow a definite diagnosis in the large majority of suspected cases of MPM and can definitely differentiate between MPM and LAD.
Conflicts of interest
There are no conflicts of interest.
Amatya VJ, Takeshima Y, Kohno H, Kushitani K, Yamada T, Morimoto C, Inai K.Caveolin-1 is a novel immunohistochemical marker to differentiate epithelioid mesothelioma from lung adenocarcinoma.Histopathology2009;55:10–19.
Betta P-G, Magnani C, Bensi T, Trincheri NF, Orecchia S.Immunohistochemistry and molecular diagnostics of pleural malignant mesothelioma.Arch Pathol Lab Med2012;136:253–261.
Chang MC, Vargas SO, Hornick JL, Hirsch MS, Crum CP, Nucci MR.Embryonic stem cell transcription factors and D2-40 (podoplanin) as diagnostic immunohistochemical markers in ovarian germ cell tumors.Int J Gynecol Pathol2009;28:347–355.
Chirieac LR, Pinkus GS, Pinkus JL, Godleski J, Sugarbaker DJ, Corson JM.The immunohistochemical characterization of sarcomatoid malignant mesothelioma of the pleura.Am J Cancer Res2011;1:14–24.
Chu AY, Litzky LA, Pasha TL, Acs G, Zhang PJ.Utility of D2-40, a novel mesothelial marker, in the diagnosis of malignant mesothelioma.Mod Pathol2005;18:105–110.
Hartkamp J, Carpenter B, Roberts SGE.The Wilms’ tumor suppressor protein WT1 is processed by the serine protease HtrA2/Omi.Mol Cell2010;37:159–171.
Hinterberger M, Reineke T, Storz M, Weder W, Vogt P, Moch H.D2-40 and calretinin – a tissue microarray analysis of 341 malignant mesotheliomas with emphasis on sarcomatoid differentiation.Mod Pathol2007;20:248–255.
Hu Y, Yang Q, McMahon LA, Wang HL, Xu H.Value of D2-40 in the differential diagnosis of pleural neoplasms with emphasis on its positivity in solitary fibrous tumor.Appl Immunohistochem Mol Morphol2010;18:411–413.
Kao SC-H, Griggs K, Lee K, Armstrong N, Clarke S, Vardy J, et al..Validation of a minimal panel of antibodies for the diagnosis of malignant pleural mesothelioma.Pathology2011;43:313–317.
Khoor A, Whitsett JA, Stahlman MT, Olson SJ, Cagle PT.Utility of surfactant protein B precursor and thyroid transcription factor 1 in differentiating adenocarcinoma of the lung from malignant mesothelioma.Hum Pathol1999;30:695–700.
King JE, Thatcher N, Pickering CAC, Hasleton PS.Sensitivity and specificity of immunohistochemical markers used in the diagnosis of epithelioid mesothelioma: A detailed systematic analysis using published data.Histopathology2006;48:223–232.
Kushitani K, Takeshima Y, Amatya VJ, Furonaka O, Sakatani A, Inai K.Differential diagnosis of sarcomatoid mesothelioma from true sarcoma and sarcomatoid carcinoma using immunohistochemistry.Pathol Int2008;58:75–83.
Marchevsky AM.Application of immunohistochemistry to the diagnosis of malignant mesothelioma.Arch Pathol Lab Med2008;132:397–401.
Mimura T, Ito A, Sakuma T, Ohbayashi C, Yoshimura M, Tsubota N, et al..Novel marker D2-40, combined with calretinin, CEA, and TTF-1: an optimal set of immunodiagnostic markers for pleural mesothelioma.Cancer2007;109:933–938.
Nicolini A, Perazzo A, Lanata S.Desmoplastic malignant mesothelioma of the pericardium: description of a case and review of the literature.Lung India2011;28:219–221.
Ordóñez NG.Thyroid transcription factor-1 is a marker of lung and thyroid carcinomas.Adv Anat Pathol2000;7:123–127.
Ordóñez NG.D2-40 and podoplanin are highly specific and sensitive immunohistochemical markers of epithelioid malignant mesothelioma.Hum Pathol2005;36:372–380.
Ordóñez NG.What are the current best immunohistochemical markers for the diagnosis of epithelioid mesothelioma? A review and update.Hum Pathol2007;38:1–16.
Ordóñez NG.Application of immunohistochemistry in the diagnosis of epithelioid mesothelioma: a review and update.Hum Pathol2013;44:1–19.
Sandeck HP, Røe OD, Kjærheim K, Willén H, Larsson E.Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry.Diagn Pathol2010;5:47.
Srodon M, Westra WH.Immunohistochemical staining for thyroid transcription factor-1: a helpful aid in discerning primary site of tumor origin in patients with brain metastases.Hum Pathol2002;33:642–645.
Suster S, Moran CA.Applications and limitations of immunohistochemistry in the diagnosis of malignant mesothelioma.Adv Anat Pathol2006;13:316–329.
Tacha D, Yu C, Bremer R, Qi W, Haas T.A 6-antibody panel for the classification of lung adenocarcinoma versus squamous cell carcinoma.Appl Immunohistochem Mol Morphol2012;20:201–207.
Takeshima Y, Amatya VJ, Kushitani K, Kaneko M, Inai K.Value of immunohistochemistry in the differential diagnosis of pleural sarcomatoid mesothelioma from lung sarcomatoid carcinoma.Histopathology2009;54:667–676.
Yaziji H, Battifora H, Barry TS, Hwang HC, Bacchi CE, McIntosh MW, et al..Evaluation of 12 antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma: identification of a three-antibody immunohistochemical panel with maximal sensitivity and specificity.Mod Pathol2006;19:514–523.