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Level of mesothelin expression can indicate the prognosis of malignant pleural mesothelioma

Background Malignant pleural mesothelioma (MPM) is a fatal, treatment-resistant tumor. The median survival of MPM is 9–12 months and its early prognostic markers remains uncertain. The objective of this study was to determine that the level of mesothelin expression can be as a predictor of prognosis in MPM patients. Methods Level of mesothelin expression was detected in 38 MPM tissue specimens by immunohistochemistry analysis. The relationship of MPM prognosis and mesothelin expression was evaluated by univariate and multivariate Cox regression, Kaplan-Meier survival curves. Results High level of mesothelin expression was significantly associated with non-epithelioid type of MPM and smoking. Meanwhile, higher level of mesothelin expression indicated a shorter total survival. Conclusions The present study suggested that mesothelin is a dependent prognostic factor in MPM patients and might be a novel potential target for immunotherapy in MPM.


Introduction
Malignant pleural mesothelioma (MPM) is an aggressive form of cancer that is caused by exposure to asbestos, which incidence is increasing worldwide (1). The global mesothelioma burden is estimated to be in the range of 36,300 to 38,400 annual deaths, according to the database of The World Health Organization (WHO) (1994 to 2014) (2). MPM has very poor prognosis due to its chemoresistance and radioresistance, with the median survival time (MST) of 9-12 months (3,4). MPM is difficult to early diagnosis resulting in few surgery opportunities for most patients with MPM. Therefore, early detection and diagnosis of MPM may help to increase its surgery opportunity and improve its prognosis.
In nowadays, several molecules, including estrogen receptors (5), matrix metalloproteinase 2 and 9 (6,7) have been reported as biomarkers for MPM. However, these factors were not available for early diagnosis due to lack of specificity. Therefore, it is very valuable to find a new prognostic factor for early diagnosis of MPM. Mesothelin is a glycosylphosphatidylinositol (GPI)-anchored cellsurface protein, which level of expression is low in normal tissues and high in a variety of malignant tumors, such as ovarian cancer, pancreatic cancer (8), and mesotheliomas (9). It has been reported that mesothelin can be as a marker of neoplastic progression and immunotherapy target in gastroesophageal cancer (10). However, the prognostic value of soluble mesothelin in malignant pleural mesothelioma remains unclear.
In the present study, the mesothelin expression profile in MPM tissues was analyzed using immunohistochemistry (IHC) staining method, and its prognostic value was evaluated using statistic methods. Counter-stained with hematoxylin, the sections were rinsed, dehydrated and covered.

Immunohistochemical scoring
The randomly selected slides were evaluated by two independent pathologists who were blinded to the clinical status of patients. The intensity of mesothelin expression was scored from 0 to 3+ according to the percentage of positive cells: 0 (trace or 1-25% positive expression), 1+ (26-50% positive expression), 2+ (51-75% positive expression), 3+ (76-100% positive expression). Scores of 0 and 1+ were classified as low expression and scores of 2+ and 3+ were classified as high expression.

Statistical analysis
SPSS 17.0software (SPSS Inc., Chicago, IL) was used to perform statistical analysis. The correlation between mesothelin expression and pathologic and staging features was analyzed by Fisher's exact test. Survival information was plotted using the Kaplan-Meier curves and the log rank test.
Univariate and multivariate analysis of prognostic factors was performed using Cox's proportional hazards regression model. P<0.05 was used as the cut-off value and considered statistically significant.

Mesothelin expression and patient characteristics in MPM
A total of 38 patients were enrolled in the present study, including 30 males and 8 females. Median patient age was 66.11±9.45 years. Twenty-six cases (68%) had a smoking history. 29 (76%) patients were epithelioid type and the others were non-epithelioid type, which included sarcomatoid and biphasic. More details were shown in Table 1. Non-epithelioid 2 7 *indicates statistical significant difference.

A B C D
The high-level expression of mesothelin were significantly associated with higher in smokers (P=0.030) and non-epithelioid type (P=0.002). There is no significant difference of mesothelin expression in other clinic characteristics. Representative pictures for mesothelin staining are shown in Figure 1.

Relationship between overall survival of MPM and patient characteristics or mesothelin expression
To determine the prognostic factors of MPM, the overall survival (OS) of MPM patients was analyzed based on IHC between patient characteristics and mesothelin expression respectively ( Figure 2).
It is worth noting that the patients with high expression (7.47±1.48 months) of mesothelin had shorter OS than those with low expression (14.72±1.30 months) (P=0.005 Figure 2F). However, no significant difference of OS was observed in subgroup of gender, age and stage ( Figure 2B,C,D).
To determine which factors had the influence on the MPM patients' overall survival, various possible predictive factors were engaged in univariate analysis ( Table 2). The results indicated that smoking status (P=0.023), pathological type (P<0.001), and mesothelin expression (P=0.009) had significant effects on OS, which confirmed to the results of Kaplan-Meier overall survival curve. By multivariate Cox analysis, it was confirmed that non-epithelioid type (P=0.037) and high mesothelin expression (P=0.038) were the independent negative prognostic factors of OS for patients with MPM ( Table 3).

Discussion and conclusion
High-level expression of mesothelin has been reported     in several type of malignant tumors including MPM, lung, ovarian, pancreatic and esophageal cancer (8)(9)(10). However, its prognostic value in patients with MPM was still unknown. In the present study, the relationship between mesothelin expression and prognosis of MPM was investigated.
Mesothelin is a 40-kDa glycosyl-phosphatidylinositollinked cell-surface glycoprotein (11). It has been reported that it might play an important role in cell adhesion and metastasis in ovarian cancers (8) and be a potential marker of neoplastic progression (12,13). In human lung cancer and mesothelioma cells, overexpression of mesothelin can promote epithelialto-mesenchymal transition and tumorigenicity, then leads to tumor growth and metastasis (9).
In MPM, it has been known that male gender, age and asbestos exposure are considered as poor prognostic factors (14,15). Perspective from pathology in MPM, nonepithelioid type may have a worse prognosis and a longer history of asbestos exposure of higher intensity (16,17). In the present study, it has been showed that the high-level expression of mesothelin was prevalent in the smokers and non-epithelioid type. Meanwhile, the patients with high mesothelin expression showed a significantly lower OS than those with low expression. Multivariate analysis determined that mesothelin was a independent negative prognostic factor in OS of patients. As the mesothelin expression could be easily detected by immunohistochemistry, it can be served as a promising tumor biomarker for prognosis in MPM patients.
Though the previous study presented the survival advantage in female MPM patients compared to male (17). There is no significant difference of OS between different genders in the present results. Moreover, patients in early stage have not showed longer survival duration than those in late stage. The reason might be the small number of enrolled cases. Therefore, a multi-institutional study which should enroll more MPM cases will be performed.
Several studies showed that mesothelin might be a novel target for immunotherapy among several cancers (18). The treatment based on mesothelin antibody has been studied in ovarian, pancreatic cancer (8), gastroesophageal cancer (10), glioblastoma (11) and malignant mesothelioma (19). The present study confirmed that high-level expression of mesothelin in MPM predicted the poor prognosis and act as a n independent factor. Therefore, the down-regulation expression of mesothelin in MPM would be considered as a novel target therapy for MPM.