Propelled by the advent of novel oncogenic pathway-targeting drugs and increasing regulatory expectations to fulfil proof of mechanism endpoints in clinical trials, there is a critical need to reliably evaluate target inhibition by investigational drugs in biologically relevant compartments. However, the ability to monitor modulation of labile oncogenic pathway biomarkers in global hematomalignancy clinical trials is complicated by varying technical expertise at clinical sites, when specimen stability considerations do not permit analysis at a central specialty laboratory. To address this critical gap, we developed a novel preservation system, designated NovaPerm3 (NP3), that enables stabilization of phosphoprotein based biomarker specimens in a single step, requires less than 20 minutes, and can be implemented in routine clinical trial settings. Stabilized specimens can be frozen and analyzed even after 90 days in a specialized laboratory. Using the NP3 preservation system, we systematically developed and validated a flow cytometry assay that not only enables reliable identification of neoplastic plasma cells but also allows for quantitation of phosphoprotein biomarkers in the tumor cells of interest present in both the peripheral blood (PB) and bone marrow (BM). We will describe a) modulation of phosphorylation levels of the S6 ribosomal protein (pS6RP) by a novel anticancer agent in multiple myeloma patients b) qualitative concordance between NP3-flow cytometry and western blot results from multiple drug treated leukemia cell lines c) sensitive detection of neoplastic plasma cells up to 1% of the total white cells and d) specimen (pS6RP) stabilization over a 90 day period. In conclusion, our novel preservation method and clinical trial laboratory validated flow assay enables reliable quantification of signaling biomarkers in hematomalignancy trials with ease in a specialized central laboratory while reducing the procedural burden for collecting biomarker specimens in global clinical trials.

Citation Format: Anil Pahuja, Shyam Sarikonda, Benjamin Lee, Armin Graber, Shabnam Tangri, Naveen Dakappagari. Analytical validation and clinical verification of phosphoprotein biomarker modulation using a novel preservation system-based flow cytometry assay in multiple myeloma clinical trials. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3388. doi:10.1158/1538-7445.AM2015-3388