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Abstract
Background: Mesotheliomas are neoplasms of the serosal lining of the body cavities. Diagnosis requires a multimodal approach of clinical findings, cytology, and histopathology with immunohistochemistry (IHC). The published sensitivity of cytology for diagnosing mesothelioma ranges from 30% to 75%. Aim and Objectives: This study aimed to calculate the incidence of malignant mesothelioma (MM) at our institute and to study the cytological features of MM. Materials and Methods: A retrospective study of pleural, peritoneal, and pericardial fluids submitted at our institute was done. The duration of the study was 8 years (2011–2019). Apart from examining Giemsa smears, a panel of immunocytochemical (ICC) and cell block immunohistochemical (IHC) markers was applied to achieve the diagnosis. These included calretinin, mesothelin, CK5/6, Hector Battifora mesothelial cell antibody (HBME), WT1, MOC31, CK7 and CK20. Histopathological correlation was done wherever possible. Result: In the present study, we compiled four cases of MM over 8 years diagnosed on serous effusion cytology and confirmed by immunocytochemistry (ICC)/cell block immunohistochemistry (IHC)/biopsy. This indicates a rare incidence of MM. The Cytological features of MM were studied. Conclusion: The diagnosis of MM is difficult, especially cytologically. It was found to be a rare entity in the malignant cases diagnosed on effusion cytology.
Keywords: Cytology, fluids, malignant mesothelioma, pericardial, peritoneal, pleural
How to cite this article: Introduction 
Malignant mesotheliomas (MM) are neoplasms arising from the serosal lining of the body cavities.[1] The most common sites of involvement are the pleura (60%–70%) followed by the peritoneum (30%–35%), and pericardium (0.7%).[2] Its incidence is low in India despite it being a major consumer of asbestos, the exposure of which is a major risk factor for the development of the disease. In India, the incidence is 0.05–0.08 per 100,000 men and 0.05–0.1 per 100,000 women.[3]
Diagnosis requires a multi-modal approach. It includes clinical details, radiology, cytology with immunocytochemistry (ICC), and biopsy with immunohistochemistry (IHC). Till now, there are only 60 cases of MM reported in the Indian literature, out of which 15 cases have been detected on serous effusion cytology.[4],[5],[6],[7],[8],[9],[10] The published sensitivity of cytology for the diagnosis of mesothelioma in the worldwide literature ranges from 30% to 75%.[11]
The present study aimed to determine the incidence of malignant mesothelioma at our institute and to study the cytological features.
Material and Methods A retro-prospective review of pleural, peritoneal, and pericardial fluids submitted at our institute over a period of 8 years (January 2011–October 2019) was conducted. The fluid samples were submitted in sterile containers without preservatives, routinely to our department. Complete clinical details along with radiological findings were noted in each case. On the receipt of fluids, cytospin preparations, direct air-dried smears, and alcohol-fixed smears were prepared from each sample. Cytospin preparations were prepared by cyto-centrifugation at 1500 rpm for 10 min and stained with May Grunwald Giemsa (MGG). Alcohol-fixed smears were stained by Papanicolaou stain and air-dried smears with MGG stain. Some slides were left unstained, and cell block was prepared using the plasma thrombin method, subject to the amount of fluid available. The slides were screened and reported by two pathologists. Only the cases that were suspected to be mesotheliomas clinico-radiologically and/or on primary screening were included in the study. A panel of immunocytochemical (ICC) and cell block immunohistochemical (IHC) markers was applied to achieve the diagnosis in these suspicious cases. Histopathological correlation was done wherever possible. The panel of mesothelial cell markers included was calretinin, cytokeratin (CK) 5/6, mesothelin, HBME, Wilms' Tumor 1 (WT-1), and D2-40 (to define mesothelial origin) and epithelial membrane antigen (EMA), desmin, and p53 (to differentiate reactive mesothelial cells from mesothelioma). Other markers like thyroid transcription factor-1 (TTF-1), CK-7 and CK-20, and MOC-31 and carcinoembryonic antigen (CEA) were used to rule out secondary lesions from the lungs, GI tract, and ovaries, respectively. For WT1, nuclear staining; for calretinin, both nuclear and cytoplasmic; for D2-40, membranous; and for CK5/6, cytoplasmic staining were considered positive. A cut-off of staining in at least 10% of the tumor cells was taken as positive. As per the guidelines laid down by the International Mesothelioma Interest Group, at least two mesothelial and two epithelial/carcinoma markers are used to distinguish malignant mesothelioma from other carcinomas.[12] Ethical committee approval on 24th March 2021.
Results A total of 3,372 fluid samples were submitted over a period of 8 years, out of which 2,468 were peritoneal fluids, 732 were pleural, and 172 were pericardial. Of the pleural fluids, 616 were reported as benign, 24 equivocal, and 92 were malignant. Amongst the peritoneal fluids, 2,328 were diagnosed as benign, 32 were equivocal, and 108 were malignant. Out of the total pericardial fluids, 12 were equivocal, 155 benign, and 5 malignant.
A total of 12 cases were suspected to be MM on primary cytospin screening. On further ICC/cell block IHC/biopsy, 4 out of these 12 cases turned out to be MM. The details of these four cases have been summarized in [Table 1] and [Figure 1]. The first three cases: Number 1, 2, and 3 [Table 1] had a similar morphology depicting atypical mesothelial cells, arranged in three-dimensional clusters and balls, showing scalloped edges. The cells had large, single to multiple irregular nuclei with opened up chromatin. The cytoplasm was abundant and basophilic, with vacuolation in some. Window effect and cell engulfment/cannibalism were also observed. Cases included a 42-year-old female with peritoneal involvement and two cases, a 40-year-old male and 47-year-old male with pleural involvement. In these three cases, a provisional diagnosis of mesothelioma was made based on the first cytomorphological impressions under the microscope. This diagnosis was confirmed by ICC/IHC markers on the fluid cell block [Figure 2]a and [Figure 2]b. Case number 4 was a 55-year-old male with pleural effusion, revealing atypical cells arranged in clusters and attempted acini formation with cytoplasmic vacuolations, suggesting adenocarcinoma morphologically on primary screening but turned out to be mesothelioma after IHC/ICC. Positive markers were calretinin, mesothelin, CK 5/6, HBME, and p53. Negative desmin ruled out reactive mesothelial proliferation and negative MOC-31, CK-7, and TTF-1 ruled out adenocarcinomas. In the rest of the eight cases, seven cases were diagnosed as reactive mesothelial proliferations, and one case was diagnosed as adenocarcinoma. The sensitivity and specificity of cytological examination were calculated to be 75% and 96%, respectively. The positive and negative predictive values came out to be 27.2% and 99.4%, respectively.
Figure 1: Case 1 from Table 1: CT scan showing a large intraabdominal mass encasing the adjoining organs. (a) Axial view, (b) Sagittal view
Figure 2: Case 1 from Table 1: (a) 600 × MGG smear showing atypical mesothelial cell cluster with scalloped edges. Cells show large irregular nuclei with moderate to abundant cytoplasm, (b) 400 × cell block showing tumor cells with nuclear and cytoplasmic changes, IHC; (c) calretinin, (d) WT-1, (e) CK5/6, (f) Mesothelin
Figure 3: Case 4 from Table 1: 600 × MGG smear with IHC calretinin (in set)As a total of four cases of malignant mesothelioma were diagnosed, the incidence was calculated to be 0.12%. Out of these cases, three were of pleural origin (total incidence = 0.4% and 3.3% of malignant pleural cases) and one of peritoneal origin (total incidence = 0.04% and 0.9% of malignant peritoneal cases). The age range was 40–55 years with a male: female ratio of 3:1.
Discussion The highest incidence of MM has been reported in some countries in Europe, Australia, and New Zealand.[3] The incidence is much lower in India, accounting for 0.05–0.08 per 100,000 among men and 0.05–0.1 among women.[3] The incidence was found to be 0.12% in the current study. MMs are aggressive and heterogeneous tumors, predominantly seen in males with a peak age incidence in the fifth to sixth decades of life. The most common causative factor in MM is linked to asbestos exposure with a long latency period ranging from 30 to 40 years.[8] The other etiological factors for the occurrence of MM include simian virus 40 and a history of prior irradiation. It has an insidious onset with clinical presentation ranging from pleuritic chest pain, dyspnea, cough, fatigue, and weight loss.[8]
In the present study, we compiled four cases of MM over 8 years, diagnosed on serous effusion cytology and confirmed by ICC/cell block IHC/biopsy. This indicates a rare incidence of MM in this part of the world. Also, none of our cases were linked to asbestos exposure. A total of 60 cases of MM are found in the Indian literature, out of which only two case reports show asbestos exposure.[7],[9] Out of the total cases found, 45 cases were reported from the sSouthern part of India, whereas 15 cases have been reported from the north.[4],[5],[6],[7],[8],[9],[10][Table 2].
The diagnosis of mesotheliomas and differentiating from reactive mesothelial proliferations on cytology samples alone is very challenging due to the lack of evidence of tissue invasion as documented in histopathology. Therefore, multiple investigative methods are utilized to arrive at the diagnosis. Parameters that indicate a malignant growth are the presence of more disorganized clusters, expansile nodules, complex papillae, cellular stratification, the presence of necrosis, and minimal inflammation.[13] IHC/ICC staining with p53, epithelial membrane antigen (EMA), glucose transporter 1 (GLUT 1), and insulin-like growth factor II messenger RNA binding protein (IMP3) favor mesotheliomas, whereas the expression of desmin may support reactive mesothelial proliferations. Molecular markers like homozygous deletion of p16 by fluorescent in situ hybridization (FISH) or the loss of BRCA1 associated protein 1 (BAP1) by IHC may be found only in mesotheliomas. Therefore, a combination of IHC and molecular methods may actually serve the purpose. For the confirmation of morphological diagnosis, the International Mesothelioma Interest Group recommends an IHC panel to include two mesothelioma markers and two markers to rule out other malignancies. In case the results are inconclusive, it is recommended to use a further set of markers.[12] In the present study, EMA, p53, and desmin were utilized for a similar purpose with p53 positive in two, EMA positive in three, and desmin negative in two cases, respectively [Table 1]. Our findings were further supported by classical radiological findings of omental caking, fissure infiltration, and fat invasion besides nodular thickenings and effusions of pleura and peritoneum as described in the literature on imaging of mesotheliomas.[14]
Conclusion To conclude, MM in India is rare. Although a multimodal approach is needed for diagnosis, the significance of our study lies in the fact that cytological techniques can be a good primary and effective method to make a pathological diagnosis of mesothelioma, especially in cases where affected serous fluid is easily accessible as compared to the actual mass. At our institute, serous effusion cytology has 75% sensitivity and 96% specificity for the diagnosis of MM on cytology, which was comparable with the literature [Table 3]. A high degree of suspicion along with ICC/IHC plays a huge role in differentiating adenocarcinoma from MM.
Table 3: Comparison of present studies' sensitivity and specificity with the previous world literatureFinancial support and sponsorship
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Conflicts of interest
There are no conflicts of interest.
References 
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CheckDOI: 10.4103/joc.joc_145_21
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