Reactive Mesothelial Hyperplasia Research Articles

Investigation of MTAP and BAP1 staining loss and P16/CDKN2A deletion in pleural cytology specimens and its role in the diagnosis of mesothelioma.

Mesothelioma is a malignant neoplasm with a poor survival rate. We aimed to investigate the importance of BAP1, MTAP (IHC), and p16/CDKN2A homozygous deletion (FISH) in cytologic material obtained from pleural effusion sampling, which is a less invasive procedure in the diagnosis of mesothelioma. Our study discussed pleural cytology samples of cases with histopathologically proven mesothelioma diagnoses between 2017 and 2022. As the control group, materials that had pleural effusion sampling for other reasons and reactive mesothelial hyperplasia were included in the study. Cell blocks prepared from these materials were subjected to fluorescent in situ hybridization for p16/CDKN2A homozygous deletion and immunohistochemistry for BAP1 and MTAP. The specificity of the P16/CDKN2A homozygous deletion in diagnosing mesothelioma is 100%. Its sensitivity is 68.75%. The specificity of BAP1 immunohistochemical nuclear expression loss is 95%, while the sensitivity is 60%. Loss of nuclear expression of MTAP alone has the lowest specificity and sensitivity, with a specificity of 86% and a sensitivity of 43%. The highest sensitivity is reached when BAP1 loss and p16/CDKN2A homozygous deletion are evaluated together, increasing to 81%. The specificity is 95%. It has been determined that any marker alone cannot be used for a definitive mesothelioma diagnosis in pleural effusion cytological specimens; however, sensitivity increases in some combinations. The combination of BAP1 immunohistochemistry and p16/CDKN2A homozygous deletion detected by FISH, which has a higher specificity and sensitivity, can be routinely used in the diagnosis of mesothelioma under the guidance of clinical and radiologic information.

Cytological Diagnosis of Malignant Pleural Effusion Caused by Metastatic Squamous Cell Carcinoma: A Series of Four Cases

The majority of malignant pleural effusions are caused by adenocarcinomas, with the most common primary sites being the lung and breast in men and women, respectively. Metastatic squamous cell carcinoma in serous effusions is rare, accounting for less than 3% of all malignant effusions. The most common primary site of origin for metastatic squamous cell carcinoma involving serous effusions is the lung, followed by the head and neck, oesophagus, and cervix. Well-differentiated squamous cell carcinoma in pleural effusion, characterised by keratinised cells with hyperchromatic nuclei, tadpole cells, and fiber cells, is exceedingly rare. Poorly differentiated squamous cell carcinomas often present a diagnostic challenge and can be mistaken for poorly differentiated adenocarcinoma, malignant mesothelioma, or reactive mesothelial hyperplasia. Immunohistochemistry is often required for a definitive diagnosis. Making an accurate diagnosis is crucial for providing optimal treatment to the patient. In this series, four cases (56 years old male,69 years old male, 60 years old female and 81 years old male) of malignant pleural effusion caused by metastatic squamous cell carcinoma arising from the lung, oropharynx, cervix, and oesophagus were examined. While one of the cases involved a well-differentiated squamous cell carcinoma with characteristic cellular morphology that allowed for a straightforward diagnosis, the other three cases were poorly differentiated squamous cell carcinomas that required cell block preparation and immunohistochemistry for confirmation.

UTILITY OF HBME1 IMMUNOSTAIN IN DIFFERENTIATING REACTIVE MESOTHELIAL CELLS LESIONS FROM MALIGNANT EPITHELIAL CELLS LESIONS IN SEROUS EFFUSIONS

Objective: Parietal and the visceral layers of the mesothelium are separated by a thin layer of lubricating fluid and are made up of a single layer of flat cells. The three body cavities pleura, peritoneum, and pericardium are lined by mesothelium. The collection of fluid inside these cavities is called an effusion and may be due to a variety of inflammatory, infective, and neoplastic causes and often causes reactive mesothelial cells hyperplasia that sometimes is very hard to differentiate from malignant epithelial cells in cytopathological examinations of these fluids. Methods: We studied the utility of the Human Battifora Mesothelial Epitope-1 (HBME1) immunostain to differentiate these conditions. All the fluids from various effusions collected at the department of pathology, at a tertiary care institute in Lucknow were included in the study. Detailed history, examination findings, blood investigations, imaging findings, and histopathology reports were also noted. Fluids that showed reactive mesothelial hyperplasia and/or malignant epithelial cells on microscopic examination were further analyzed by the application of HBME1 on the cell block. Results: A total of 50 fluids were studied finally including 30 cases from the positive malignant cells group and 20 cases from the reactive mesothelial cells group. Out of 30 cases included in the malignant cells group, 16 cases (53.33%) were immunoreactive for HBME1, and out of the 20 cases included in the reactive mesothelial cell group, 18 cases (90%) showed immunoreactivity for HBME1. Conclusion: Observing this we can conclude that HBME1 immunoreactivity was significantly associated with the presence of reactive mesothelial cells compared to the malignant cells group.

The cytological features of effusions with mesothelioma in situ: A report of 9 cases.

The diagnosis of mesothelioma in situ (MIS) is now accepted by the WHO as a pre-invasive neoplastic mesothelial proliferation and considered a diagnosis based on histologic evaluation only. Although the definition of MIS includes recurrent effusions, little is known about the cytologic features of such effusions. Since mesothelioma is usually diagnosed at an advanced stage and has a poor prognosis, early detection of a neoplastic mesothelial population in such effusions can potentially have a positive impact on the management of such a dire disease. We reviewed a total of 18 pleural effusions from nine patients with recurrent effusions. Of these, five patients had follow-up biopsies diagnosed as MIS and the remaining four cases had negative radiology and malignant cytology proven by molecular markers (BAP1, MTAP or CDKN2A deletion) and at least 1 year follow-up with no overt mass identified by radiology. Initial effusions may mimic reactive mesothelial hyperplasia or exhibit atypia. As effusions recur, the cellularity and atypia increase and the mesothelial proliferation becomes morphologically indistinguishable from mesothelioma. Molecular alterations diagnostic of mesothelioma can be detected in these effusions, even in the initial-benign/reactive appearing ones. The cellularity and atypia detected in such effusions surpassed those noted on the biopsies, raising questions regarding the cause of such discrepancy. The diagnosis of MIS can be suspected based on malignant effusion cytology supported by molecular alterations. We propose that the proliferation of neoplastic mesothelial clones represent a clinically silent "liquid phase MIS stage" corresponding to in situ stage in other organs.

Immunohistochemical Study of 5-Methylthioadenosine Phosphorylase (MTAP) and Epithelial Membrane Antigen (EMA) in Differentiating Epithelioid Pleural Mesothelioma from Reactive Mesothelial Hyperplasia

Immunohistochemical Study of 5-Methylthioadenosine Phosphorylase (MTAP) and Epithelial Membrane Antigen (EMA) in Differentiating Epithelioid Pleural Mesothelioma from Reactive Mesothelial Hyperplasia

New candidates in the differential diagnosis of malignant mesothelioma from benign mesothelial hyperplasia and adenocarcinoma; DARS2 and suprabasin

ObjectivesMalignant mesothelioma (MM) is a primary malignant tumour with a very bad prognosis, which develops from the mesothelial cells lining the serosal surfaces. Because MM can show a wide variety of histological patterns and its cytomorphological features are quite extensive, it is often confused with lung adenocarcinomas (LAC) and reactive mesothelial hyperplasia (RMH). The immunohistochemical examination method is the most useful method for discrimination. In this study, we aimed to determine the value of suprabasin and DARS2 markers in the differential diagnosis of RMH, MM and LAC. MethodsThirty MM, 30 LAC and 30 RMH samples selected from the archive of Fırat University Hospital Pathology Department Laboratory were included in this study. Suprabasin and DARS2 markers were applied to the samples immunohistochemically and their place in the differential diagnosis was examined. ResultsAlthough DARS2 expression was observed in RMH, MM and adenocarcinoma samples, Suprabasin expression was only observed in adenocarcinoma. There was a significant difference between the groups in terms of DARS2 and Suprabasin expression. No suprabasin expression was detected in MM and RMH. ConclusionSuprabasin and DARS2 may be proposed as new biomarkers to differentiate MM from LAC.

Sneaky Diagnosis of Pleural Malignant Mesothelioma in Thoracic Surgery: All That Glitters Is Not Gold.

Malignant Pleural Mesothelioma (MPM) is a highly aggressive disease whose diagnosis could be challenging and confusing. It could occur with atypical presentations on every examined level. Here, we present three unconventional cases of the complex diagnostic process of MPM that we have experienced during routine practice: a patient with reactive mesothelial hyperplasia mimicking MPM, an unexpected presentation of MPM with persistent unilateral hydropneumothorax, a rare case of MPM in situ. Then, we review the relevant literature on each of these topics. Definitive biomarkers to confidently distinguish MPM from other pleural affections are still demanded. Patients presenting with persistent hydropneumothorax must always be investigated for MPM. MPM in situ is now a reality, and this raises questions about its management.

5-hmC loss is another useful tool in addition to BAP1 and MTAP immunostains to distinguish diffuse malignant peritoneal mesothelioma from reactive mesothelial hyperplasia in peritoneal cytology cell-blocks and biopsies.

The differentiation between reactive mesothelial hyperplasia (RMH) and diffuse malignant peritoneal mesothelioma (DMPM) is challenging especially when applied on peritoneal small samples. The use of BRCA-associated protein 1 (BAP1) and methylthioadenosine phosphorylase (MTAP) immunostains is familiar to identify malignant mesothelial proliferation. Recently, nuclear 5-hydroxymethylcytosine (5-hmC) was reported to be a new recognition tool of pleural mesothelial malignancy on surgical specimens. However, application of 5-hmC immunostaining has not yet studied in peritoneal specimens from small biopsies or cytology cell-blocks. The aim was to assess the diagnostic accuracy of this new marker combination to distinguish DMPM from RMH in biopsies and cell-blocks. Seventy-five cases were analyzed; among which, 38 were of cytological specimens including 6 RMH and 32 DMPM, and 37 tissue biopsies with 7 RMH and 30 DMPM. BAP1, MTAP, and 5-hmC immunostains were performed on all cases. RMH cases exhibited a retained staining with all immunostains. Among DMPM, BAP1 was lost in 71.8% of cytology cell-blocks and 66.7% of biopsies. MTAP was lost in 40.6% of cytology cell-blocks and 33.3% of biopsies. 5-hmC was lost in 40.6% of cytology cell-blocks and 30% of biopsies. The combination of BAP1, MTAP, and 5-hmC showed the best accuracy in differential diagnosis between RMH and DMPM (sensitivity = 0.84, specificity = 1 in cytology cell-blocks; sensitivity = 0.90, specificity = 1 in biopsy). The best diagnostic combination in peritoneal cytology effusion fluids and biopsies samples provided by BAP1, MTAP, and 5-hmC should be applied on a diagnostic step-wise algorithm by pathologists involved into the management of DMPM, because of their therapeutic implications.

Nivolumab-induced peritonitis with peritoneal mesothelial hyperplasia mimicking metastatic mesothelioma.

A 57-year-old man developed a mesothelial proliferation in the peritoneum, several months after he was diagnosed with biopsy-proven epithelioid mesothelioma of the pleura and having undergone several treatments with checkpoint inhibitor immunotherapy. The differential diagnosis was metastatic mesothelioma from the lung primary, versus a reactive process. A diagnosis of atypical mesothelial proliferation was made. Follow-up CT showed no evidence of abdominal disease 5 months later. The complication of serositis following checkpoint inhibitor therapy is reviewed, as well as the differential diagnosis between reactive mesothelial hyperplasia and epithelioid mesothelioma.

Insulin-Like Growth Factor 2 mRNA Binding Protein 3 Promotes Cell Proliferation of Malignant Mesothelioma Cells by Downregulating p27Kip1.

Malignant mesothelioma is a tumor with a poor prognosis, mainly caused by asbestos exposure and with no adequate treatment yet. To develop future therapeutic targets, we analyzed the microarray dataset GSE 29370 of malignant mesothelioma and reactive mesothelial hyperplasia, downloaded from the Gene Expression Omnibus (GEO) database. We identified insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) as one of the significantly upregulated genes in malignant mesothelioma. IGF2BP3 functions as an oncoprotein in many human cancers; however, to our knowledge, this is the first study on the biological function of IGF2BP3 in malignant mesothelioma cells. The knockdown of IGF2BP3 in malignant mesothelioma cells resulted in the suppression of cell proliferation with an increase in the proportion of cells in the G1 phase of the cell cycle. Furthermore, knockdown of IGF2BP3 inhibited cell migration and invasion. We focused on the cell cycle assay to investigate the role of IGF2BP3 in cell proliferation in malignant mesothelioma. Among the various proteins involved in cell cycle regulation, the expression of p27 Kip1 (p27) increased significantly upon IGF2BP3 knockdown. Next, p27 siRNA was added to suppress the increased expression of p27. The results showed that p27 knockdown attenuated the effects of IGF2BP3 knockdown on cell proliferation and G1 phase arrest. In conclusion, we found that IGF2BP3 promotes cell proliferation, a critical step in tumorigenesis, by suppressing the expression of p27 in malignant mesothelioma.

The panorama of different faces of mesothelial cells.

All effusions in serous cavities represent a pathologic processes secondary to inflammatory, neoplastic, hemodynamic, or mechanical/traumatic etiologies. This elicits reactive changes in the extremely sensitive mesothelial cells lining the serosal surfaces. The result is hypertrophy and hyperplasia which lead to broad changes with a wide range of morphological appearances. These reversible alterations may resolve entirely after the recovery of underlying pathology.Under the tertiary care situations, neoplastic effusion specimens are encountered more frequently. Although some non-neoplastic pathologic process may demonstrate a few diagnostic features, cytologic evaluation of malignant effusions usually show diagnostic malignant cells. However, the most versatile mesothelial cells demonstrate a very wide cytomorphological spectrum secondary to reactive challenges. These mesothelial cells are usually referred to as ‘reactive mesothelial cells’. In addition other terms such as reactive mesothelial proliferation, reactive mesothelial hyperplasia, irritated mesothelial cells, activated mesothelial cells, hyperplastic mesothelial cells, hypertrophic mesothelial cells, and proliferative mesothelial cells. Rarely atypical mesothelial cells, although not recommended, is used inadvertently. Although there is a lack of general agreement defining these terms, some of these including atypical mesothelial cells, should not be preferred. With reference to this CMAS series, usually favored term ‘reactive mesothelial cells’ is preferred.The size of reactive mesothelial cells range from 15 to 30 µm (but may be up to 50 µm). These polyhedral cells with variable amount of cytoplasm and enlarged nuclei may show variation in sizes and shapes with conspicuous nucleoli. Bi- and multi-nucleation is frequent. Cohesive groups of mesothelial cells as sheets and three dimensional groups may be present. Some floridly reactive mesothelial cells with hyperchromatic enlarged nuclei with prominent nucleoli and scant cytoplasm may resemble malignant cells. This astonishingly wide morphological spectrum of reactive mesothelial cells is a significant interpretation challenge in effusion fluid cytology. Methodical interpretation approach with appropriate knowledge about this wide spectrum is important aspect in diagnostic cytopathology of effusion fluids.

SOX6 Expression Is Sensitive for Peritoneal Epithelioid Malignant Mesothelioma, But Not Specific in the Differential Diagnosis With Tubo-ovarian Serous Neoplasia.

Primary peritoneal malignant mesothelioma (MM) can demonstrate morphologic overlap with low-grade and high-grade tubo-ovarian serous neoplasms; it is also biologically and prognostically distinct from benign mesothelial proliferations. Currently, there is no single biomarker that can definitively distinguish these neoplasms. Sex-determining region Y box 6 (SOX6) immunohistochemistry has been recently described to differentiate pleural epithelioid MM from lung adenocarcinoma, but it has not been evaluated in the peritoneum. SOX6 immunohistochemistry was performed on 43 peritoneal epithelioid MM, 7 peritoneal biphasic MM, 5 well-differentiated papillary mesotheliomas, 5 serous borderline tumors, 29 low-grade serous carcinomas (LGSCs), 20 high-grade serous carcinomas (HGSCs), and 25 cases of peritoneal reactive mesothelial hyperplasia. Quantitative SOX6 expression in epithelioid MM (median, 100% of tumor cells) was significantly greater than in LGSC/serous borderline tumor (median, 90%; P=0.004) and HGSC (median, 45%; P=0.0001). However, when SOX6 is expression is defined as ≥10% of tumor cells, there was no significant difference in the rate of SOX6 positivity between epithelioid MM (41/43, 95%), LGSC (28/29, 97%; P=1.0), and HGSC (17/20, 85%; P=0.16). Quantitative extent of SOX6 expression in epithelioid MM was significantly greater than in biphasic MM (median, 0%; P=0.0001), well-differentiated papillary mesothelioma (median, 20%; P=0.001), and reactive mesothelial hyperplasia (median, 20%; P=0.0001), but not significantly different from flat quiescent mesothelium (median, 90%; P=0.82). SOX6 immunohistochemistry is 95% sensitive for peritoneal epithelioid MM, but is also consistently expressed in LGSC and HGSC, negating its usefulness in this common differential diagnosis. SOX6 also shows variable expression across the spectrum of reactive, benign neoplastic, and malignant mesothelial lesions of the peritoneum, and does not appear to be diagnostically useful in distinguishing benign from malignant mesothelial proliferations.

DNA Methylation Profiling Discriminates between Malignant Pleural Mesothelioma and Neoplastic or Reactive Histologic Mimics

The diagnosis of malignant pleural mesothelioma (MPM) is challenging because of its potential overlap with other neoplasms or even with reactive conditions. DNA methylation analysis is effective in diagnosing tumors. In the present study, this approach was tested for use in MPM diagnosis. The DNA methylation patterns of a discovery cohort and an independent-validation cohort of MPMs were compared to those of 202 cases representing malignant and benign diagnostic mimics (angiosarcoma, desmoid-type fibromatosis, epithelioid sarcoma, leiomyosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, nodular fasciitis, reactive mesothelial hyperplasia, sclerosing fibrous pleuritis, solitary fibrous tumor, and synovial sarcoma). By both unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analysis, MPM samples in the discovery cohort exhibited a DNA methylation profile different from those of other neoplastic and reactive mimics. These results were confirmed in the independent validation cohort and by in silico analysis of the MPM-The Cancer Genome Atlas data set. Copy number variation profiles were also inferred to identify molecular hallmarks of MPM, including CDKN2A and NF2 deletions. Methylation profiling was effective in the diagnosis of MPM, although caution is advised in samples with low tumor cell content.

Diagnostic Utility of BAP1, EZH2 and Survivin in Differentiating Pleural Epithelioid Mesothelioma and Reactive Mesothelial Hyperplasia: Immunohistochemical Study.

Background: Epithelioid mesothelioma (EM) is the commonest subtype of malignant pleural mesothelioma. Its histopathological discrimination from reactive mesothelial hyperplasia (RMH) could be challenging. Thus, an immunohistochemical panel is mandatory for better discrimination. BAP1 is a newly identified diagnostic marker whose loss is specific to malignant mesothelioma. EZH2 overexpression is reported in different cancers, but its relation to BAP1 in malignant mesothelioma has not been fully understood. Survivin expression is said to be significantly higher in EM than in non-neoplastic pleural tissue, but its diagnostic utility as an immunohistochemical marker has not been thoroughly investigated in this field. To the best of our knowledge, no previous studies have been conducted to assess the diagnostic accuracy of the combined use of these three nuclear markers (BAP1, EZH2 and Survivin) in discriminating pleural EM from RMH. Methods: This retrospective study includes two groups: 81 cases of pleural EM and 67 cases of RMH, retrieved from the archives of Pathology Department of Ain Shams University Hospitals and Ain-Shams University Specialized Hospital during the period from January 2016 to December 2019. An immunohistochemical study was performed using BAP1, EZH2 and Survivin antibodies. Results: There were highly statistically significant relations between study groups as regards the studied markers (p = 0.001 for each). The specificity was 100% for all combinations of immunohistochemical markers. Sensitivity of any combination of the immunohistochemical markers used in this study was found to be higher than the sensitivity of any of these markers used individually. The combination of all three markers showed the highest diagnostic accuracy (95.9%) and the highest sensitivity (92.6%). However, the combination of Survivin and EZH2 yielded the same diagnostic accuracy and sensitivity. Conclusion: Adding EZH2, Survivin and BAP1 to the diagnostic IHC panel for differentiating pleural EM and RMH could enhance diagnostic sensitivity. Moreover, Survivin is a potentially promising marker in this context, especially when combined with EZH2.

The International System for Reporting Serous Fluid Cytopathology: How to Incorporate Molecular Data in Cytopathology Reports

Serous effusion cytology is widely employed in the initial evaluation of the etiology of effusions with a high diagnostic sensitivity. To standardize practices, The International System for Reporting Serous Fluid Cytology (TIS) was developed following best international practices, the most up-to-date literature, and expert consensus. In the context of this system, ancillary techniques play an important role. Besides defining basic principles in laboratory specimen handling, adequacy criteria, and a standardized reporting terminology with five diagnostic categories, TIS provides an actionable framework for using immunohistochemical and molecular testing in effusion samples, namely, in atypical, suspicious of malignant samples. For diagnostic purposes, these tests may be employed to distinguish between a primary and secondary neoplasm, to confirm a diagnosis of malignant mesothelioma vs. reactive mesothelial hyperplasia, and to correctly classify and determine the primary location of a metastasis. Theranostic molecular tests may also be used for these samples to evaluate potential therapeutic targets. Pathologists play a central role in guiding this process by determining adequacy and selecting appropriate ancillary tests. The activity in this area of research should increase in the near future as new therapeutic targets are discovered and new drugs enter the clinical practice.

Diagnostic utility of BRCA1-associated protein 1 (BAP1), Programmed Cell Death4 (PCD4) and Epithelial membrane antigen (EMA) expression in differentiation of malignant mesothelioma from reactive mesothelial cell hyperplasia in both cytology and cell block

Background: Malignant pleural mesothelioma (MPM) is the most lethal tumor arising from the mesothelial cells. Sometimes, it is difficult to be diagnosed based on morphology alone as reactive mesothelial hyperplasia (RMH) and some metastatic carcinomas may be confused with mesothelioma. Aim of the study: Our study aims to evaluate diagnostic utility of BRCA1 “Breast Cancer gene” associated protein-1 (BAP1), Programmed Cell Death (PCD4), and Epithelial membrane antigen (EMA) in differentiation of malignant mesothelioma (MM) from reactive mesothelial hyperplasia (RMH) by immunohistochemistry (IHC).Material and methods: Retrospective study, include 60 patients, was carried out in Chest and Pathology Departments of Zagazig University from October 2016 till August 2020. The expression levels of BAP1, PCD4and EMA were investigated using cytological analysis compared with cell block method in all cases of MPMs and RMHResults: BAP1loss was detects malignant mesothelioma confirmed by cytology in 19 out of 20 patients with sensitivity of 95%, specificity 92.5%, PCD4 was positive in 39 out of 40 patients of RMH and can diagnose reactive mesothelioma with a sensitivity of sensitivity of 85.7%, specificity 100%. EMA was positive 95% in MM confirmed by cytology and can diagnose malignant mesothelioma with a sensitivity of 95%, specificity 97.5%. Conclusion: Increased sensitivity of diagnosis by cell block method was observed in cases that were reported as reactive mesothelial hyperplasia by conventional smears. BAP1 loss, negative PCD4 immunostaining and positive EMA favor mesothelioma.Keywords: BAP1, PCD4, EMA, malignant mesothelioma.

CDKN2A and MTAP Are Useful Biomarkers Detectable by Droplet Digital PCR in Malignant Pleural Mesothelioma: A Potential Alternative Method in Diagnosis Compared to Fluorescence In Situ Hybridisation.

BackgroundThe diagnosis of malignant pleural mesothelioma (MPM) can be difficult, in part due to the difficulty in distinguishing between MPM and reactive mesothelial hyperplasia (RMH). The tumor suppressor gene, CDKN2A, is frequently silenced by epigenetic mechanisms in many cancers; in the case of MPM it is mostly silenced via genomic deletion. Co-deletion of the CDKN2A and methylthioadenosine phosphorylase (MTAP) genes has been researched extensively and discovered to be a highly specific characteristic of MPM. Most studies have used FISH to detect the deletion of CDKN2A and IHC for MTAP as a surrogate for this. In this study, we aim to investigate and validate droplet digital PCR (ddPCR) as an emerging alternative and efficient testing method in diagnosing MPM, by particularly emphasizing on the loss of MTAP and CDKN2A.MethodsThis study included 75 formalin fixed paraffin embedded (FFPE) MPM tissue, and 12 normal pleural tissue and 10 RMH as control. Additionally, primary MPM cell lines and normal pleural samples were used as biomarker detection controls, as established in our previous publication. All FFPE specimens were processed to isolate the DNA, that was subsequently used for ddPCR detection of CDKN2A and MTAP. FFPE samples were also analyzed by fluorescence in situ hybridization (FISH) for CDKN2A and MTAP deletion, and for MTAP IHC expression. Concordance of IHC and ddPCR with FISH were studied in these samples.Results95% and 82% of cases showed co-deletion of both MTAP and CDKN2A when determined by FISH and ddPCR respectively. ddPCR has a sensitivity of 72% and specificity of 100% in detecting CDKN2A homozygous loss in MPM. ddPCR also has a concordance rate of 92% with FISH in detecting homozygous loss of CDKN2A. MTAP IHC was 68% sensitive and 100% specific for detecting CDKN2A homozygous loss in MPM when these losses were determined by ddPCR.ConclusionOur study confirms that MTAP is often co-deleted with CDKN2A in MPM. Our in-house designed ddPCR assays for MTAP and CDKN2A are useful in differentiating MPM from RMH, and is highly concordant with FISH that is currently used in diagnosing MPM. ddPCR detection of these genetic losses can potentially be utilized as an alternative method in the diagnosis of MPM and for the future development of a less-invasive MPM-specific detection technique on MPM tumor tissue DNA.

Can BAP1 expression loss in mesothelial cells be an indicator of malignancy?

BackgroundMalignant mesothelioma is a highly aggressive tumor that can be confused with a benign mesothelial lesion, especially cytomorphologic lesions. BRCA1-associated protein 1 (BAP1) acts as a tumor suppressor. In this study, we aim to investigate the value of BAP1 staining of malignant mesothelioma cases with expression loss and diagnosis in cell block and biopsy tissue. MethodsBetween January 2009 and March 2017, 64 mesotheliomas, 117 reactive mesothelial hyperplasias, and 20 fibrinous pleuritis/pericarditis were diagnosed with morphologic and immunohistochemical findings in our pathology clinic and were included in the study. Formalin-fixed, paraffin-embedded tissues were immunohistochemically examined for BAP1. Inflammatory and stromal cells were used as positive internal controls. BAP1 was assessed for nuclear staining in mesothelial cells.ResultsExaminations of the relationship between patient diagnosis and BAP1 biopsy status showed that the BAP1 loss rate (76.6%) was significantly higher in malignant mesothelioma cases than in other benign diseases (0%) (p<.001). Sensitivity and specificity were 76.56% and 100%, respectively, for biopsy tissue from malignant mesothelioma. Sensitivity and specificity were both 100% for BAP1 test on cell block tissue. Furthermore, the consistency between BAP1 cell block and biopsy results was excellent (ĸ=0.90) and the correlation was significant (p<.001).ConclusionsThis study shows that BAP1 expression loss in both cytology and biopsy tissue in biopsy-confirmed malignant mesothelioma cases is an essential parameter for malignant mesothelioma diagnosis.

The "Brescia panel" (Claudin-4 and BRCA-associated protein 1) in the differential diagnosis of mesotheliomas with epithelioid features versus metastatic carcinomas.

The distinction between mesothelioma with epithelioid features and metastatic carcinoma may be challenging, particularly on cytology. A novel 2-hit Claudin-4 and BRCA-associated protein 1 (BAP1) panel was investigated. The objective of this study was to determine the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the panel on cytology from pleural effusions and matched biopsies, including 49 malignant mesotheliomas on cytology with 43 matched biopsies, 49 normal/reactive mesothelial proliferations, and 49 pleural metastatic carcinomas from different primaries with 21 matched pleural biopsies. The diagnostic role of the 4 categories obtained by crossing the immunostaining results was analyzed. Claudin-4 strongly stained all metastatic carcinomas and tested completely negative in normal mesothelium, benign reactive mesothelial hyperplasia, and malignant mesothelioma. All normal and benign mesothelial proliferations and all carcinomas except 1 were immunoreactive for BAP1, whereas BAP1 loss was observed in 88% of malignant mesotheliomas. The expression of Claudin-4 alone excluded all benign and malignant mesothelial growth, consistently characterizing all metastatic carcinomas. Double negativity was evident in all malignant mesotheliomas, and double positivity was observed in all metastatic carcinomas. BAP1-positive/Claudin-4-negative status was observed only in malignant mesotheliomas and benign mesothelial proliferations. A single metastatic anal squamous cell carcinoma had BAP1-negative/Claudin-4-positive staining. Claudin-4 expression was completely specific and sensitive for metastatic carcinoma, excluding mesothelial proliferations. BAP1 staining characterized 98% of metastatic carcinomas and 100% of benign mesothelial proliferations, whereas negativity was observed almost exclusively in mesotheliomas. This 2-hit panel is probably the best compromise for differentiating malignant mesothelioma and metastatic carcinoma on either cytology or biopsy specimens.

Comparative study of BAP1 and CD147 expression in the diagnosis of malignant mesothelioma

Background: Malignant mesothelioma (MM) is a lethal tumor of serosal surfaces. Its differentiation from reactive mesothelial hyperplasia is mandatory, and may be problematic in many situations. Thus, the application of a targeted panel of specific markers permits proper diagnosis in the majority of cases. Aim: This study aimed to determine the potential use of BRCA associated protein-1 (BAP1) and CD147 antibodies to differentiate between MM and reactive mesothelial hyperplasia. Materials and Methods: The current work was carried out on 120 cases (56 reactive mesothelial hyperplasia and 64 malignant mesothelioma cases), retrieved as 70 cell blocks and 50 tissue samples. Immunohistochemical staining, using calretinin (to confirm the mesothelial lineage), BAP1, and CD147 antibodies, was performed for each case. Results: Lost nuclear BAP1 expression was detected in 45.3% of the mesotheliomas versus 19.6% of reactive mesothelial hyperplasia cases. Positive membranous CD147 expression was found in 84.4% of mesothelioma cases versus 8.9% of reactive mesothelial hyperplasia cases. BAP1 showed 44.6% sensitivity and 80% specificity, while CD147 showed 84.3% sensitivity and 91% specificity in the diagnosis of malignant mesothelioma. Conclusions: The loss of BAP1 expression and positive CD147 expression could be of diagnostic value for malignant mesothelioma and can exclude the diagnosis of reactive mesothelial proliferations in both biopsy and cytology specimens. CD147 exhibits higher sensitivity and specificity than BAP1.