Achieving high accuracy in diagnosing tumours involving the serosal cavities is made difficult by several factors, including the large number of origins for metastatic carcinomas involving this anatomic site, the antigens shared by MM and carcinomas, and the morphological changes undergone by reactive mesothelial cells in various benign conditions. The paucity of consistently reliable markers for the differentiation between MM and adenocarcinoma is exemplified in the case of serous OC/PPC, due to overlapping clinical presentation (especially within the peritoneal cavity), morphological resemblance, and co-expression or lack of expression of multiple markers. Markers that are expressed by both tumours in surgical specimens include podoplanin, calretinin, CK5/6, WT1 and mesothelin, while both tumours are CEA negative [reviewed in 4-5
], and similar difficulties are encountered in effusion cytology.14,33
In order to expand the diagnostic panel available to date, we applied gene expression array technology to the differential diagnosis between OC/PPC and DMPM.8
In follow-up studies based on the array findings, we have identified several new potential markers that are significantly more highly expressed in OC/PPC compared with MM and/or reactive mesothelial cells, including the gap junction protein claudin-3,34
and folate receptors,17
as well as a new MM marker, tenascin-X.37
In the present study, we investigated the diagnostic role of PINCH-2 as a potential MM marker.
Significantly higher levels of PINCH-2
mRNA were found in MM effusions compared with OC/PPC, in agreement with the Affymetrix array data,8
with comparable differences between MM and breast carcinoma effusions. While no high cut-off levels that were diagnostic for MM were found, low cut-off expression levels below which the diagnosis of MM was unlikely were observed. While applying this cut-off will not result in correct classification of the majority of OC/PPC using the PINCH-2
assay alone, our recent observations with respect to TNXB37
and the folate receptors FOLR1
suggest that the combination of several genes in the qPCR assay may be highly informative. We are currently studying several additional genes that may differentiate OC/PPC from MM, with the aim of designing such a qPCR panel in the near future.
The diagnostic role of PINCH-2 was subsequently studied at the protein level. Flow cytometry analysis of PINCH-2 protein expression showed higher expression in MM compared with OC/PPC and breast carcinoma, in agreement with the mRNA expression data. However, this difference was of a moderate degree, as reflected by the overlapping expression extent among the three cancer types, suggesting that this assay has little diagnostic value in the differential diagnosis between these three malignancies. Nevertheless, the high expression of PINCH-2 in effusions suggests a biological role for this protein in metastatic cancer cells, a possibility that we attempted to explore by analysing the relationship between this molecule and previously studied molecules with a role in adhesion, metastasis and regulation of apoptosis. This analysis was applied to the OC/PPC specimens analysed using qPCR, as this tumour category was the largest and best characterized one in our material.
The expression of ILK, one of the central molecular partners of PINCH proteins9,10,18-20,23,24
has not been investigated in our material to date. However, we have previously studied the expression of the α6, αv and β1 integrin subunits, which are molecular partners of ILK, in OC/PPC.26,27
Analysis of the association between PINCH-2
mRNA levels and expression of these integrin subunits did not reveal any significant association. Similarly, no relationship was found with the presence and levels of E-cadherin, Snail, Slug, SIP-1, claudins, IAP members or NFjB p65 in our material. Further research is necessary in order to characterize the molecular partners of PINCH-2 in serosal cancers, primarily through a study of ILK and PINCH-2 expression in a large series of MM and OC/PPC, as well as in vitro
MM is an aggressive tumour with extremely poor outcome in the majority of cases. As we observed large variation in PINCH-2 mRNA levels in OC/PPC effusions, we hypothesized that patients with effusions characterized by a high level of this gene may have poor outcome. However, no significant association between PINCH-2 mRNA levels and clinicopathological parameters, including survival, were found, suggesting that PINCH-2 is not a strong prognosticator in this cancer. While the number of MM specimens analysed in this study does not allow evaluation of the clinical role of this molecule in MM, future investigation of this issue may be of interest.
In conclusion, qPCR analysis confirmed the higher levels of PINCH-2 mRNA in MM compared with OC/PPC and breast carcinoma effusions, suggesting a diagnostic value for this molecule as part of a larger gene panel. PINCH-2 protein is frequently expressed in cancer cells in effusions, but further research is required in order to identify its potential molecular partners and clinical role in this setting.