Soluble mesothelin related peptides (SMRP) and osteopontin as protein biomarkers for malignant mesothelioma: analytical validation of ELISA based assays and characterization at mRNA and protein levels.
BACKGROUND
There is a need to identify reliable markers for malignant mesothelioma. Soluble mesothelin related peptides (SMRP) and osteopontin (OPN) have gained interest in recent years for this purpose.


METHODS
SMRP (Fujirebio Diagnostics Inc.) and OPN (R&D Inc.) ELISA methods were evaluated for multiple parameters. Concentrations were measured in blood from patients with mesothelioma, normal healthy volunteers, and patients with other (non-mesothelioma) cancers. In silico analysis was performed using the GeoProfiles database. At the protein level, SMRP and OPN were measured in cell culture supernatants, and values were compared in patients pre- and post-extrapleural pneumonectomy.


RESULTS
The SMRP assay demonstrates intra-assay CVs of 2.3% and 3% (at 4.6 nM and 13.7 nM, respectively), and inter-assay CVs of 3.5% and 3.7% at the same concentrations. The limit of detection (LOD) is 0.182 nM. The OPN assay demonstrates intra-assay CVs of 5.8%, 4.1%, and 5.2% (at 1.9, 5.1, and 11.1 ng/mL, respectively), and inter-assay CVs of 8.5%, 8.4%, and 12.1% at the same concentrations. The LOD is 0.032 ng/mL. Both SMRP and OPN in mesothelioma patients were significantly higher than in patients with other (non-mesothelioma) cancer and in healthy volunteers. The two markers do not appear to correlate with each other and exhibit different tissue expression patterns. Protein concentrations of these markers are highest in different sets of cell lines. Finally, SMRP but not OPN concentrations were decreased in five of seven consecutive patients after extrapleural pneumonectomy, compared to their respective pre-operative values.


CONCLUSIONS
These assays provide reliable and reproducible quantitation of SMRP and OPN proteins. Both are increased in mesothelioma patients compared to non-mesothelioma controls. However, the two analytes do not correlate with each other and show distinct expression profiles and protein expression. Concentrations of SMRP but not OPN are decreased in post-surgical samples. Our results further characterize these markers, establish assay performance characteristics, and lay the groundwork for further studies to measure these markers in blood.
Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY 10032, USA. alex.rai@columbia.edu
Alex J
AJ
Rai
Raja M
RM
Flores
Anu
A
Mathew
Rita
R
Gonzalez-Espinoza
Matthew
M
Bott
Marc
M
Ladanyi
Valerie
V
Rusch
Martin
M
Fleisher
D000368:Aged; D014408:Biomarkers, Tumor / Q000032:analysis / Q000097:blood / Q000235:genetics; D016022:Case-Control Studies; D018929:Cell Culture Techniques; D016208:Databases, Factual; D004797:Enzyme-Linked Immunosorbent Assay; D058851:GPI-Linked Proteins; D020869:Gene Expression Profiling; D006801:Humans; D057230:Limit of Detection; D008562:Membrane Glycoproteins / Q000737:chemistry*; D000090204:Mesothelin; D008654:Mesothelioma / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D053495:Osteopontin / Q000032:analysis* / Q000097:blood / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000097:blood / Q000235:genetics / Q000378:metabolism; D011013:Pneumonectomy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D058851:GPI-Linked Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D053495:Osteopontin; D000090204:Mesothelin