The present study demonstrates that GISTs strongly express the carbonic anhydrase II isozyme. Originally this enzyme was not considered an important target in cancer biology, because it is absent or only weakly expressed in most epithelial tumors. By contrast, various studies have included only the membrane-bound isoforms, CA IX and XII, which are overexpressed in several types of cancers (22
). There has been only scattered evidence that CA II is expressed to some extent in malignant cells such as leukemic blast cells (26
) and colorectal (27
), brain (27
) and pancreatic (30
) cancers. A more recent study indicated that CA II expression is induced in neovascular endothelial cells of malignant melanoma and in esophageal, renal and lung cancers (32). It was suggested that CA II associated with the neovascular endothelia could serve as a potential target for cancer therapy. It was also proposed that the presence of CA II in the endothelium could contribute to generation of autoantibody response that would, in turn, be a desired outcome in immune therapy of cancer.
In this study, we demonstrated strong CA II immunoreactivity in GISTs, assessed with two independent antibodies and confirmed CA II expression by Western blotting. Also, CA II transcript expression appears to be stronger in GIST than in many other sarcomas, as demonstrated by gene expression profiles (28
). GISTs have been thought to arise from progenitors of the interstitial cells of Cajal, which are found throughout the gastrointestinal tract from the esophagus to the internal anal sphincter (29
). Cajal cells were the only mesenchymal elements positive for CA II in the normal gastrointestinal tract, indicating shared CA II-positive phenotype with GISTs, as has been previously observed for KIT and DOG1 (31
). Comparison of CA II expression in different tumor categories demonstrated that the CA II immunoreactivity was markedly more common in GISTs than in the other mesenchymal tumors analyzed, suggesting that evaluation of CA II expression could be useful in the differential diagnosis of these entities, especially between GIST and leiomyosarcoma. Even though the specificity of CA II seemed to be quite good, small numbers of unrelated soft tissue tumors such as solitary fibrous tumors and malignant peripheral nerve sheath tumors were also occasionally positive. This result indicated that other markers must also be used, and the clinicopathologic context has to be considered. CA II seems to label GISTs independent of the site of origin or histologic type. Thus, epithelioid gastric GISTs, that are often less strongly KIT- and DOG1-positive, are strongly CA II-positive. Gastric spindle cell GISTs and small intestinal GISTs are nearly uniformly positive also. These findings indicate that CA II has some additional value in GIST diagnostics. Our statistical analysis also suggested that CA II expression may be associated with better prognosis, although this observation needs to be validated with a larger study cohort.
Our results showed that another cytosolic isozyme, CA I, is weakly expressed in GISTs and most cases remained completely negative. CA I was included in the immunohistochemical analysis, because CA1
gene locus is located in close proximity to CA2
gene in the chromosome 8 (8q21.2). This result suggests that high expression of CA II in GISTs is not due to a gene amplification of the region that involves the cluster of genes encoding the cytosolic CAs. Importantly, the high CA II expression in GIST suggests that GIST cell lines may represent a valuable model in the study of the regulation and function of CA II in cancer cells, which has been a poorly understood research area to date. Possibly, CA II could play the same role in GIST as CA IX and XII in other tumors, i.e. modulating intracellular pH balance and promoting extracellular acidification. Lower extracellular pH is generally considered a sign of aggressive phenotype of tumor cells and poorer prognosis (23
, 36), although in our GIST studies, higher CA II expression correlated with better prognosis. Therefore, it seems more likely that CA II expression might be associated with higher differentiation of GIST cells, and therefore, strong immunostaining correlates with favorable prognosis. The Western blotting results showed that the CA II expression levels in GISTs are independent of KIT
In conclusion, our studies show CA II expression in GIST. The prognosis of GISTs with high CA II expression was better than that of GISTs with low or no expression, suggesting that CA II is both a diagnostic and prognostic biomarker for GIST. Further validation studies with other CA antibodies should be undertaken to characterize CA II expression in a larger cohort of GIST and other mesenchymal tumors of the gastrointestinal tract.