MUC4 immunohistochemistry is useful in distinguishing epithelioid mesothelioma from adenocarcinoma and squamous cell carcinoma of the lung

The differential diagnosis of epithelioid mesothelioma from lung adenocarcinoma and squamous cell carcinoma requires the positive and negative immunohistochemical markers of mesothelioma. The IMIG guideline has suggested the use of Calretinin, D2–40, WT1, and CK5/6 as mesothelial markers, TTF-1, Napsin-A, Claudin 4, CEA as lung adenocarcinoma markers p40, p63, CK5/6, MOC-31 as squamous cell markers. However, use of other immunohistochemical markers is still necessary. We evaluated 65 epithelioid mesotheliomas, 60 adenocarcinomas, and 57 squamous cell carcinomas of the lung for MUC4 expression by immunohistochemistry and compared with the previously known immunohistochemical markers. MUC4 expression was not found in any of 65 cases of epithelioid mesothelioma. In contrast, MUC4 expression was observed in 50/60(83.3%) cases of lung adenocarcinoma and 50/56(89.3%) cases of lung squamous cell carcinoma. The negative MUC4 expression showed 100% sensitivity, 86.2% specificity and accuracy rate of 91.2% to differentiate epithelioid mesothelioma from lung carcinoma. The sensitivity, specificity, and accuracy of MUC4 are comparable to that of previously known markers of lung adenocarcinoma and squamous cell carcinoma, namely CEA, Claudin 4 and better than that of MOC-31. In conclusion, MUC4 immunohistochemistry is useful for differentiation of epithelioid mesothelioma from lung carcinoma, either adenocarcinoma or squamous cell carcinoma.

We recently proposed the addition of two immunohistochemical markers, Intelectin-1 and DAB2, as the positive immunohistochemical markers of epithelioid mesothelioma 5 . In this study, we also found the down regulation of many other genes including MUC4 in epithelioid mesothelioma as mentioned in its supplementary data.
Therefore, we evaluated the diagnostic applicability of MUC4 immunohistochemistry for differentiation of epithelioid mesothelioma to lung adenocarcinoma and squamous cell carcinoma.

Materials and Methods
Patients and Histologic Samples. Pathological specimens (formalin-fixed paraffin-embedded tissue blocks) of 65 epithelioid mesotheliomas and 60 lung adenocarcinoma and 57 squamous cell carcinoma were obtained from the tissue archives of the Department of Pathology, Hiroshima University. We also reviewed the patient's clinical details and chest computed tomography findings for confirmation of the tumor localization. The mean age of lung adenocarcinoma patients was 69 with range from 43 to 84 (male 38, female 22), that of lung squamous cell carcinoma was 69 with range from 39 to 86 (male 49, female 7), and that of epithelioid mesothelioma 69 with range from 33-92 (male 61, female 4).
All histological sections were examined and reclassified by three pathologists (VJA, KK, and YT) according to recent WHO classification 6 . Pathologic diagnosis was confirmed by histologic findings and the immunohistochemical marker panel recommended by 2012 International Mesothelioma Interest Group (IMIG) guideline 4 . The collection of tissue specimens for this study was carried out in accordance with the "Ethics Guidelines for Human Genome/Gene Research" enacted by the Japanese Government. Ethical approval was obtained from the institutional ethics review committee (Hiroshima University E-974). All experimental procedures were in accordance with the with ethical guidelines. Samples used were linked-anonymised archival specimens, and individual consent was not required for this research.
Immunohistochemical Procedures and Evaluation of MUC4 Expression. Immunohistoc hemistry was performed using 3 μm tissue sections prepared from the best representative formalin-fixed paraffin-embedded blocks of epithelioid mesothelioma, lung adenocarcinoma, and squamous cell carcinoma cases. Immunohistochemical staining was performed using the Ventana Benchmark GX automated immunohistochemical station (Roche Diagnostics, Tokyo, Japan). The antigen retrieval methods and antibodies used in this study are summarized in Table 1. Incubation with the secondary antibody and detection was performed with Ventana ultraView Universal DAB Detection Kit (Roche Diagnostics). Immunoreactivity was evaluated as either negative or positive. Nuclear staining of calretinin, WT1, p40, p63, and TTF-1, cytoplasmic staining of MUC4 and Napsin-A, CK5/6, CEA, membranous staining of podoplanin (clone: D2-40), Epithelial Related Antigen (clone: MOC-31) and claudin 4 were considered as 'positive' .
Statistical analyses were performed using the Fisher's exact test for calculation of p-value of positivity of individual markers, Mann Whitney U test for calculation Z-score and the p-value of immunohistochemical score of individual markers. Sensitivity, specificity, and accuracy rate are calculated using a 2 × 2 contingency table model.

Results
Immunohistochemical Result. The percentages and immunohistochemical scores of MUC4 expression in epithelioid mesothelioma, lung adenocarcinoma, and squamous cell carcinoma and other immunohistochemical markers are shown in Table 2.   Calretinin, D2-40, WT1, and CK5/6. Calretinin expression was present in 64 cases (98.5%) of epithelioid mesothelioma with the immunohistochemical score of 3+ in 57. Calretinin expression was also present in 17 of 60 (28.3%) cases of lung adenocarcinoma, and 28 of 56 (50%) cases of squamous cell carcinoma. The immunohistochemical of 3+ was not present in adenocarcinoma but was present in 5 cases of squamous cell carcinoma. D2-40 expression was present in 63 cases (96.9%) of epithelioid mesothelioma with the immunohistochemical score of 3+ in 54 cases. D2-40 expression was also present in 7 of 60 (11.7%) cases of lung adenocarcinoma and 34 of 56 (60.7%) cases of squamous cell carcinoma. The immunohistochemical score of 3+ was not present TTF-1, Napsin-A, p63, p40. Nuclear expression of TTF-1 and cytoplasmic expression of Napsin-A are the positive immunohistochemical markers of lung adenocarcinoma. TTF-1 and Napsin-A expression were not present in any of 65 (0%) cases of epithelioid mesothelioma and only 54 (90%) cases and 48 (80%) cases of lung adenocarcinoma. The immunohistochemical score of 3+ for TTF-1 and Napsin-A was observed in 46 and 36 cases respectively. TTF-1 expression was also present in 5 (8.9%) cases, and Napsin-A expression in 2 (3.6%) cases of lung squamous cell carcinoma but with the immunohistochemical score of 1+.
Nuclear p63 expression and recently nuclear p40 expression are the positive immunohistochemical markers of lung squamous cell carcinoma. However, we found p63 and p40 expression in 15 (23%) and 3 (4.6%) cases of epithelioid mesothelioma although the immunohistochemical score was 1+. The p63 and p40 expression were present in 56 (100%), 55(98.2%) cases of squamous cell carcinoma and most of them with the immunohistochemical score of 3+. However, 32 (53.3%) and 6 (10%) cases of adenocarcinoma also showed immunoreactivity for p63 and p40 respectively but with the immunohistochemical score of 1+. Table 3 shows the sensitivities, specificities, and accuracy rates of the immunohistochemical markers to differentiate epithelioid Mesothelioma from lung adenocarcinoma and squamous cell carcinoma. Among the negative mesothelioma markers, MUC4, CEA, Claudin 4, TTF-1, and Napsin-A showed 100% sensitivity, p40 showed 95.4%, P63 showed 76.9%, and CK5/6 showed 69.2%. When the histological type was known as lung adenocarcinoma or squamous cell carcinoma, the specificity of TTF-1, Napsin-A and p40 were high, but with combined lung adenocarcinoma and squamous cell carcinoma, it remains around or less than 50%. Among the positive mesothelioma markers, WT1 showed the highest specificity of 98.6%, but sensitivity was limited to 86.2%. The sensitivities of calretinin and D2-40 were high with 98.5% and 96.9% respectively, but the specificities were low with 61.2%and 64.7% specificity. Among the negative mesothelial markers, CEA has the highest accuracy rate of 98.9% followed by Claudin 4 of 97.8%. The accuracy of MUC4 is 91.2% which is better than that of MOC-31.

Discussion
MUC4 is a transmembrane mucin expressed in normal epithelial cells including epithelial mucosa of the digestive tract, ductal epithelium of salivary gland and lacrimal gland, larynx and trachea, lung, stomach, intestine, uterus, cervix, mammary gland, ovary, and kidney 7 . Many human malignancies like carcinomas of the pancreas 8 , ovary 9 , salivary gland 10 , lung 11 , stomach 12 , breast 13 show MUC4 expression. MUC4 expression has been reported in the non-small cell lung cancer more in adenocarcinoma than do squamous cell carcinomas and large cell carcinomas 14 . MUC4 expression was also present significantly in solid adenocarcinoma of the lung 15 . In addition to poor prognosis correlated with MUC4 expression in the biliary tract, pancreas, ovary, and colorectal junction 16 , the MUC4 mRNA expression is related to the tumor histological type and its differentiation 17 . The tubular and papillary patterns of adenocarcinomas and the nests and in squamous pearls of squamous cell carcinomas show strong membranous and less cytoplasmic expression of MUC4 18 . MUC4 plays a role in cell differentiation rather than cell proliferation of the normal goblet cells, the stratified squamous epithelial cells, and malignant epithelial tumor cells 19 . We previously reported the MUC4 expression in sarcomatoid carcinoma of the lung and its utility to differentiate from sarcomatoid mesothelioma 20 .
In this study, We found MUC4 expression in 50/60 (83.3%) cases of lung adenocarcinoma, 50/56 (89.3%) cases of squamous cell carcinoma, and none (0%) of epithelioid mesothelioma. Kwon et al. 14 and Llinares et al. 21 previously reported the diagnostic value of MUC4 expression in distinguishing epithelioid mesothelioma and lung adenocarcinoma. They reported the diagnostic value of MUC4 immunostaining in distinguishing mesothelioma and lung adenocarcinoma. They raised the polyclonal antibody against a KLH conjugate of a synthetic peptide corresponding to the tandem repeat sequence of MUC4 for their study. They found that MUC4 was expressed in 0 of the 41 epithelioid mesotheliomas and 32 of the 35 (91%) lung adenocarcinoma. Our study utilized the commercially available antibody and is the validation of the previous study. Other things different from this study is the inclusion of squamous cell carcinoma which also showed similar frequencies of positive cases like that of lung adenocarcinoma. Our data of MUC4 expression in adenocarcinoma and squamous cell carcinoma of lung is also similar to another study by Kwon et al. who studied MUC4 expression in lung adenocarcinoma and squamous cell carcinoma of the lung but without mesothelioma.
In this study, we analyzed the utility of MUC4 to distinguish epithelioid mesothelioma from lung adenocarcinoma and squamous cell carcinoma. We have previously reported the MUC4 expression in sarcomatoid carcinoma of the lung and its utility to differentiate from sarcomatoid mesothelioma 20 . In our previous report, we emphasized the MUC4 expression in spindled cells of sarcomatoid carcinoma of lung. None of sarcomatoid mesothelioma had the expression of MUC4 expression. MUC4 was expressed in 72% of the sarcomatoid carcinoma of lung.
Ordonez et al. 22 reported that there is no absolute specific and sensitive marker of epithelioid mesothelioma, although the various immunohistochemical markers are currently available for the diagnosis of epithelioid mesotheliomas. He also added the location and histologic features of the tumor, the sex of the patient, and the clinical findings need to be considered when selecting the markers for accurate diagnosis of epithelioid mesothelioma from the various other carcinomas 22 .  The sensitivity and specificity of MUC4 to differentiate epithelioid mesothelioma from lung adenocarcinoma were 100% and 83.3% respectively with the accuracy rate of 92% and those of MUC4 to differentiate epithelioid mesothelioma from lung squamous cell carcinoma 100% and 89.3% respectively with the accuracy rate of 95%. We found CEA is the best marker with the sensitivity of 100%, specificity of 98.3%, and accuracy rate of 98.9% followed by Claudin 4 which had the sensitivity of 100%, specificity of 96.6%, and accuracy rate of 97.8%. MUC4 expression showed a similar sensitivity of 100%, but the lower specificity of 86.2% and accuracy rate of 91.2%. However, it showed better sensitivity, specificity and accuracy rate than that of MOC-31. Also, we found positive immunoreactivity for MUC4 in some cases of adenocarcinoma and squamous cell carcinoma with no CEA and/or Claudin 4 expression. Two cases of CEA-negative adenocarcinoma, one of which was negative for Claudin 4 too, showed MUC4 expression (immunohistochemical score of 2+ or 3+). Moreover, 3 cases of Claudin 4-negative adenocarcinoma, two of which were negative for CEA too, showed MUC4 expression (immunohistochemical score of 1+ or 2+). There was one adenocarcinoma case which was negative for both CEA and Claudin-4 but positive MUC4 expression. So, MUC4 has the potential for the use as an additional negative marker of epithelioid mesothelioma for differentiation from lung cancer including adenocarcinoma or squamous cell carcinoma.
International Mesothelioma Interest Group Guideline for pathologic diagnosis of malignant mesothelioma has recommended various immunohistochemical negative markers, CEA, MOC-31, TTF-1, Napsin-A to differentiate it from adenocarcinoma and p40, p63, MOC-31 to differentiate it from squamous cell carcinoma 4 . Ordonez et al. later added Claudin 4 as one of the best broad-spectrum carcinoma markers to discriminate epithelioid mesotheliomas from both lung adenocarcinomas and squamous cell carcinomas 23 . In our previous publication too, we found CEA and Claudin 4 were the best positive carcinoma markers for discriminating epithelioid mesotheliomas from squamous cell carcinomas 24 . Although p63 is the marker of squamous cell carcinoma, it was at least focally positive in many of adenocarcinoma (32/60, 53.3%) and epithelioid mesothelioma (15/65, 23.1%) in the present study. So, limiting the value of the p63 expression for differentiation of epithelioid mesothelioma from squamous cell carcinoma.
In conclusion, MUC4 can be added as additional positive marker of non-small cell carcinoma (both lung adenocarcinoma and squamous cell carcinoma) and negative immunohistochemical marker to differentiate epithelioid mesothelioma from lung adenocarcinoma and squamous cell carcinoma. This study includes the limited cases (65 epithelioid mesothelioma and 116 cases of lung adenocarcinoma and needs follow up study consisting of large numbers of cases.