Procedures
ΔCT
BLOOD
Whole-blood samples were collected in PAXgene Blood RNA tubes and processed to total RNA with the Qiagen PAXgene Blood RNA Kit (Qiagen, Valencia, CA, USA). Target-gene amplification was done in a quantitative PCR reaction with TaqMan Universal PCR Master Mix (Applied Biosystem, Division of Life Technologies Corporation, Carlsbad, CA, USA) and Precision Profiles (Source MDx, Boulder, CO, USA). Individual target-gene amplification was multiplexed with the eukaryotic 18S rRNA endogenous control and run-in triplicate in a 384-well format on the 7900HT fast real–time PCR system. Normalized CT values (ΔCT) for each amplified target gene replicate were calculated. Resulting in triplicate ΔCT values for individual target genes were averaged yielding a final ΔCT value. The 6–gene score was calculated based on the model: 2×ABL2+SEMA4D+ITGAL–C1QA–TIMP1–CDKN1A. Maximum change to each patient’s 6–gene score while on treatment was compared to baseline values and recorded as a percentage change.Peripheral blood samples were collected, and serum was separated out for testing using the TIMP-1 and TIMP-2 Duoset ELISA kit (R&D Systems) according to manufacturer instructions. Serum samples were diluted 400-fold (1 μL diluted in 399 μL of reagent diluent), and a standard curve was run along with the serum samples. Optical density is measured on a plate reader at 450 nm.