Identification of Bio-Markers for the Bortezomib Resistance in Multiple Myeloma by Mass Spectometry Based Technology.
Johann Micallef, Moyez Dharsee, Jian Chen, Suzanne Ackloo, Hong Chang. University Health Network, Toronto, Canada; Ontario Cancer Biomarker Network, Toronto, Canada
Background: Despite advances in clinical care, multiple myeloma (MM), the second most common blood cancer in adults, remains an almost universally fatal disease. Although bortezomib, a proteasome inhibitor, has improved clinical outcome of MM, only 30-40% of patients responded to the therapy. The mechanisms of drug resistance and prognostic markers have not been completely elucidated
Design: We examined the differentially expressed proteins between 8226-S (bortezomib sensitive) and 8226-R5 (bortezomib resistant) human myeloma cell lines using iTRAQ mass spectrometry (MS). The MS based multiple-reaction-monitoring technique (MRM) was then used to independently verify the quantitative differences of the protein expression levels between the two cell lines. Western blot analysis and immunohistochemistry were used to validate candidate biomarker expression in MM cell lines and primary MM samples.
Results: We identified a list of proteins to be differentially expressed between bortezomib sensitive and resistant cells. Of particular interest was the Myristoylated Alanine Rich C Kinase Substrate (MARCKS) protein which was one of the most highly up-regulated proteins in the 8226/R5 cells (25 times more compared to the 8226/S cells). MARCKS over-expression was verified by MRM. MARCKS up-regulation in the 8226/R5 cells compared to the 8226/S cells was confirmed by Western blot analysis. We also screened an additional 15 MM cell lines, that were not exposed to bortezomib, and identified 7 cell lines that showed a strong expression of MARCKS and 8 cell lines that showed either weak or undetectable levels of MARCKS expression. Similar expression patterns of MARCKS in these cell lines were also confirmed by IHC. . In a pilot study, we prospectively evaluated 10 pre-bortezomib treatment bone marrow biopsies from refractory/relapsed MM patients; 4 achieved objective response (1 complete response, 3 partial response) and 6 failed response following bortezomib treatment. We found all non-responders had moderate to strong MARCKS expression, and 3 of the 4 responders showed undetectable MARCKS by IHC while 1 responder showed only week expression in 10-20% of MM cells.
Conclusions: Our results suggest that MARCKS expression may serve a pre-treatment biomarker indicative of bortezomib resistance and warrant further validation in a larger cohort of MM in a prospective manner.
Tuesday, March 1, 2011 8:45 AM
Platform Session: Section B, Tuesday Morning