Targeted MS Assay Predicting Tamoxifen Resistance in Estrogen-Receptor-Positive Breast Cancer Tissues and Sera

We
recently reported on the development of a 4-protein-based classifier
(PDCD4, CGN, G3BP2, and OCIAD1) capable of predicting outcome to tamoxifen
treatment in recurrent, estrogen-receptor-positive breast cancer based
on high-resolution MS data. A precise and high-throughput assay to
measure these proteins in a multiplexed, targeted fashion would be
favorable to measure large numbers of patient samples to move these
findings toward a clinical setting. By coupling immunoprecipitation
to multiple reaction monitoring (MRM) MS and stable isotope dilution,
we developed a high-precision assay to measure the 4-protein signature
in 38 primary breast cancer whole tissue lysates (WTLs). Furthermore,
we evaluated the presence and patient stratification capabilities
of our signature in an independent set of 24 matched (pre- and post-therapy)
sera. We compared the performance of immuno-MRM (iMRM) with direct
MRM in the absence of fractionation and shotgun proteomics in combination
with label-free quantification (LFQ) on both WTL and laser capture
microdissected (LCM) tissues. Measurement of the 4-proteins by iMRM
showed not only higher accuracy in measuring proteotypic peptides
(Spearman r: 0.74 to 0.93) when compared with MRM
(Spearman r: 0.0 to 0.76) but also significantly
discriminated patient groups based on treatment outcome (hazard ratio
[HR]: 10.96; 95% confidence interval [CI]: 4.33 to 27.76; Log-rank P < 0.001) when compared with LCM (HR: 2.85; 95% CI:
1.24 to 6.54; Log-rank P = 0.013) and WTL (HR: 1.16;
95% CI: 0.57 to 2.33; Log-rank P = 0.680) LFQ-based
predictors. Serum sample analysis by iMRM confirmed the detection
of the four proteins in these samples. We hereby report that iMRM
outperformed regular MRM, confirmed our previous high-resolution MS
results in tumor tissues, and has shown that the 4-protein signature
is measurable in serum samples.