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Abstract
Patients with Malignant Mesothelioma (MM) develop unidentified auto-antibodies to MM tumour antigens. This study was conducted to identify the targets of MM patient auto-antibodies in order to try to understand more of the anti-tumour response and to determine if these antibodies might be helpful for diagnosis or prognostication. Using MM patient sera in a Western immunoblott screening strategy, no common immunoreactive proteins were identified. The sera from one long-term survivor recognised a protein band of 50–60 kDa present in cell lysates from four of five MM cell lines tested. The immunoreactive proteins in this band were identified by 2D electrophoretic separation of a MM cell line protein lysate, followed by analysis of excised immunoreactive proteins on a MALDI TOF mass spectrometer and peptide mass fingerprinting. The immunoreactive proteins identified were vimentin (accession gi55977767) and the ATP synthase (F1-ATPase) beta chain (accession gi114549 and gi47606749). ELISA assays were developed for antibodies to these proteins. Neither vimentin (median and 95% CI 0.346; 0.32–0.468 for MM patients, 0.327; 0.308–0.428 for controls) nor ß-F1-ATPase (0.257; 0.221–0.453 for MM patients, 0.263; 0.22–0.35 for controls) showed significant differences in autoantibody levels between a group of MM patients and controls. Using a dichotomized antibody level (high, low) for these targets we demonstrated that vimentin antibody levels were not associated with survival. In contrast, high ß-F1-ATPase antibody levels were significantly associated with increased median survival (18 months) compared to low ß F1 ATPase antibody levels (9 months; p = 0.049). Immunohistochemical analysis on a MM tissue microarray showed cytoplasmic staining in 28 of 33 samples for vimentin and strong cytoplasmic staining in14 and weak in 16 samples for ß-F1-ATPase. Therefore antibodies to neither vimentin nor ß-F1-ATPase are useful for differential diagnosis of MM, however high antibody levels to ß-F1-ATPase may be associated with increased survival and this warrants further investigation.
Highlights
New clinical biomarkers are needed for malignant mesothelioma (MM), an aggressive, asbestos-induced incurable tumour
Using the serological identification by recombinant expression cloning (SEREX) approach [14] we identified tumour associated antigens recognised by MM patient sera, the majority of specificities were uniquely associated with individual patients though some common reactivities were observed including against topoisomerase IIb, U2AF(65) [15] and ß-F1-ATPase
Using an one dimensional Western immunoblotting screening strategy we have previously demonstrated that some MM patients exhibit high titre antibodies to MM proteins expressed on cultured MM cell lines [16]
Summary
New clinical biomarkers are needed for malignant mesothelioma (MM), an aggressive, asbestos-induced incurable tumour. Using the serological identification by recombinant expression cloning (SEREX) approach [14] we identified tumour associated antigens recognised by MM patient sera, the majority of specificities were uniquely associated with individual patients though some common reactivities were observed including against topoisomerase IIb, U2AF(65) [15] and ß-F1-ATPase (unpublished data). SS-F1-ATPase was recognised as an antigen by two other patients in a SEREX screening strategy (unpublished observations), the clone/recombinant protein which contained an open reading frame corresponding to the C-terminal portion of the protein (nt 595–1736) was not recognised by the Patient #145’s sera by Western immunoblotting These results are likely to be due to the effect of the denaturing conditions on the auto-antibody epitopes [17].
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