Amany Sayed Mawas

Differential microRNA expression profiling of mesothelioma and expression analysis of miR-1 and miR-214 in mesothelioma

Malignant mesothelioma is a highly aggressive cancer with poor prognosis and refractory to currently available therapies. Most of the patients with advanced invasive nature are not amenable to surgical resection and/or available anticancer therapy, thus development of novel effective therapeutic regimes is needed. Aberrant expression of microRNAs (miRNAs) has been proposed to contribute to carcinogenesis and aggressiveness of mesothelioma. We analyzed miRNA expression in mesothelioma cell lines using TaqMan miRNA expression array and found significant number of miRNAs, which showed increased or lost expression. We validated the increased expression of miR-182, and miR-183 in mesothelioma cell lines by individual miRNA assays and SmartFlare miRNA probes. We further investigated the miR-1, and miR-214, which were not expressed in mesothelioma cells by real-time RT-PCR. Transfection of mesothelioma cells, ACC-Meso-1 and CRL5915, with miRNA mimic (hsa-miR-1 mimic and hsa-miR-214 mimic) led to inhibition of cell growth, invasion and migration. We paid attention to PIM1, the target gene of both miR-1 and miR-214 miRNAs and which was found overexpressed in mesothelioma cells, and miR-1 and miR-214 mimic transfection of mesothelioma cell lines showed downregulation of PIM1 by western blot analysis. The miRNAs, miR-1 and miR-214, may play a role in carcinogenesis of mesothelioma thus might be considered as candidate therapeutic targets in mesothelioma.

Identification of Dab2 and Intelectin-1 as Novel Positive Immunohistochemical Markers of Epithelioid Mesothelioma by Transcriptome Microarray Analysis for Its Differentiation From Pulmonary Adenocarcinoma

As there are currently no absolute immunohistochemical positive markers for the definite diagnosis of malignant epithelioid mesothelioma, the identification of additional “positive” markers that may facilitate this diagnosis becomes of clinical importance. Therefore, the aim of this study was to identify novel positive markers of malignant mesothelioma. Whole genome gene expression analysis was performed using RNA extracted from formalin-fixed paraffin-embedded tissue sections of epithelioid mesothelioma and pulmonary adenocarcinoma. Gene expression analysis revealed that disabled homolog 2 (DAB2) and Intelectin-1 had significantly higher expression in epithelioid mesothelioma compared with that in pulmonary adenocarcinoma. The increased mRNA expression of DAB2 and Intelectin-1 was validated by reverse transcriptase polymerase chain reaction of RNA from tumor tissue and protein expression was validated by Western blotting of 5 mesothelioma cell lines. The utility of DAB2 and Intelectin-1 in the differential diagnosis of epithelioid mesothelioma and pulmonary adenocarcinoma was examined by an immunohistochemical study of 75 cases of epithelioid mesothelioma and 67 cases of pulmonary adenocarcinoma. The positive rates of DAB2 and Intelectin-1 expression in epithelioid mesothelioma were 80.0% and 76.0%, respectively, and 3.0% and 0%, respectively, in pulmonary adenocarcinoma. Immunohistochemically, the sensitivity and specificity of DAB2 was 80% and 97% and those of Intelectin-1 were 76% and 100% for differentiation of epithelioid mesothelioma from pulmonary adenocarcinoma. In conclusion, DAB2 and Intelectin-1 are newly identified positive markers of mesothelioma and have potential to be included in future immunohistochemical marker panels for differentiation of epithelioid mesothelioma from pulmonary adenocarcinoma.

MUC4, a novel immunohistochemical marker identified by gene expression profiling, differentiates pleural sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

Sarcomatoid mesothelioma, a histological subtype of malignant pleural mesothelioma, is a very aggressive tumor with a poor prognosis. Histological diagnosis of sarcomatoid mesothelioma largely depends on the histomorphological feature of spindled tumor cells with immunohistochemical reactivity to cytokeratins. Diagnosis also requires clinico-radiological and/or macroscopic evidence of an extrapulmonary location to differentiate it from lung sarcomatoid carcinoma. Although there are promising immunohistochemical antibody panels to differentiate mesothelioma from lung carcinoma, a consensus on the immunohistochemical markers that distinguish sarcomatoid mesothelioma from lung sarcomatoid carcinoma has not been reached and requires further study. We performed whole gene expression analysis of formalin-fixed paraffin-embedded tissue from sarcomatoid mesothelioma and lung sarcomatoid carcinoma and observed significant differences in the expression of MUC4 and other genes between sarcomatoid mesothelioma and lung sarcomatoid carcinoma. Immunohistochemistry demonstrated that MUC4 was expressed in the spindled tumor cells of lung sarcomatoid carcinoma (21/29, 72%) but was not expressed in any sarcomatoid mesothelioma (0/31, 0%). To differentiate sarcomatoid mesothelioma from lung sarcomatoid carcinoma, negative MUC4 expression showed 100% sensitivity and 72% specificity and accuracy rate of 87%, which is higher than immunohistochemical markers such as calretinin, D2-40 and Claudin-4. Therefore, we recommend to include MUC4 as a novel and useful negative immunohistochemical marker for differentiating sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

PIM1 knockdown inhibits cell proliferation and invasion of mesothelioma cells

Malignant mesothelioma is a major asbestos-related cancer with prolonged time lapse from the first exposure of asbestos to the development of mesothelioma. Most of mesothelioma patients show very poor prognosis, thus, an urgent improvement of its treatment is required by development of novel therapeutic strategies. RNA interference (RNAi) is a powerful tool in post-genomic research and cancer therapy through inhibition of gene expression. In the present study, we analyzed the function of PIM1 on mesothelioma cell lines with its knockdown by siRNA transfection. Here, we report that the downregulation of PIM1 led to suppression of cell proliferation by cell cycle arrest at G1 phase and suppression of cell invasion and migration. Considering the mesothelioma as rapidly growing invasive cancer, downregulation of PIM1 may have a potential role for therapeutic management of malignant mesothelioma.

Utility and pitfalls of immunohistochemistry in the differential diagnosis between epithelioid mesothelioma and poorly differentiated lung squamous cell carcinoma.

The aims of this study were to clarify the usefulness of immunohistochemistry in the differential diagnosis of epithelioid mesothelioma with a solid growth pattern [solid epithelioid mesothelioma (SEM)] and poorly differentiated squamous cell carcinoma (PDSCC), and to confirm the validity of a specific type of antibody panel. Additionally, we aimed to clarify the pitfalls of immunohistochemical analyses.

Use of Anti-Noxa Antibody for Differential Diagnosis between Epithelioid Mesothelioma and Reactive Mesothelial Hyperplasia

Objectives: The histological differential diagnosis between epithelioid mesothelioma (EM) and reactive mesothelial hyperplasia (RMH) is not always straightforward. The aim of the present study was to search for new immunohistochemical markers to distinguish EM from RMH. Methods: We evaluated and compared the expression of apoptosis-related genes in EM and RMH by real-time RT-PCR array analysis followed by clustering of significant gene expression. Immunohistochemical staining and statistical analysis of Noxa expression in 81 cases of EM and 55 cases of RMH were performed and compared with the utility of other previously reported antibodies such as Desmin, EMA, GLUT-1, IMP-3 and CD146. Results: Noxa mRNA expression levels were found to be increased in EM when compared to RMH by RT-PCR array analysis. In the immunohistochemical analysis, Noxa showed sensitivity of 69.0%, specificity of 93.6% and positive predictive value of 93.0% as a positive marker of EM in distinguishing it from RMH, and these values were almost similar to IMP-3. Conclusion: Noxa is a marker with relatively high specificity, and can be used to distinguish EM from RMH. It would be a valuable addition to the current antibody panel used for the differential diagnosis of EM and RMH.

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.

Utility of Survivin, BAP1, and Ki‑67 immunohistochemistry in distinguishing epithelioid mesothelioma from reactive mesothelial hyperplasia

Histological distinction between epithelioid mesothelioma (EM) and reactive mesothelial hyperplasia (RMH) can be challenging. The aim of this study was to assess the diagnostic utility of Survivin, Ki-67, and loss of BRCA1-associated protein 1 (BAP1) expressions in distinguishing EM from RMH using immunohistochemistry. Formalin-fixed, paraffin-embedded specimens from 78 cases of EM and 80 cases of RMH were immunohistochemically examined for Survivin, BAP1, and Ki-67. In addition, receiver operating characteristic curve analyses were performed to establish the cut-off values for Survivin and Ki-67 labelling indices. Survivin (cut-off value: 5%) had 67.7% sensitivity and 100% specificity, while Ki-67 (cut-off value: 10%) had 85.1% sensitivity and 87.5% specificity, and BAP1 had 66.2% sensitivity and 100% specificity for the differentiation of EM from RMH. Among the combinations of two markers, the combination of Survivin and BAP1 (Survivin-positive and/or BAP1-loss finding) had the highest diagnostic accuracy (sensitivity: 89.8%; specificity: 100%; accuracy: 95.3%). We recommend using the combination of Survivin and BAP1 to distinguish EM from RMH.