Immuno-multiple reaction monitoring (iMRM) for quantitation of PD-L1 and PD-1-signaling proteins in non-small cell lung carcinoma (NSCLC).

2627 Background: More accurate predictive biomarkers of response to checkpoint inhibitors (CPIs) still is a major unmet need in oncology. PD-L1 immunohistochemistry (IHC) limitations include its analytical variability and the post-translational modifications of PD-1 signaling-associated proteins like glycosylation. Moreover, PD-L1 IHC is an imperfect surrogate of the tumor immune microenvironment, and immunoscoring is important but difficult to assess in a clinical setting. Proteomic based technologies can overcome these challenges, but the low concentration of these proteins and the presence of high background noise in formalin-fixed paraffin embedded (FFPE) tumors were limiting obstacles. In this study, we evaluate the benefit of a new approach we used with anti-peptide antibodies to purify surrogate peptides, while liquid chromatography (LC) was coupled to multiple reaction monitoring mass spectrometry (iMRM) to improve specificity and precision of protein quantitation. Methods: To determine the concentration of PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70, we used unique and well detectable proteolytic peptides as surrogates. In a refined protocol, we optimized protein extraction and digestion, peptide immuno-enrichment, LC and MRM parameters to maximize recovery, increase target-specific signal and reduce noise. Plus, we assessed the glycosylation status of PD-L1, PD-L2, and PD-1. The entire workflow was fully validated using 31 NSCLC FFPE tumors. PD-L1 quantitation by iMRM was compared to PD-L1 IHC clone 22C3. Results: On average, 71±29 µg (n = 52) of protein could be extracted from each 1–3 mm 3 NSCLC tumor FFPE core. The optimized iMRM method allowed the quantitation of PD-L1 and PD-1 down to 21 amol on-column. Inter- and intra-day repeatability were well below FDA guidelines (coefficients of variation [CV] < 20%) with average CVs of 5.2±4.0% (intra-day) and 4.5±2.6% (inter-day). Sample storage had no significant effect on peptide quantitation. The final multiplexed iMRM assay enables quantitation all targets and glycosylation states for > 40 samples in only 3 days (including external calibration and quality controls) and was used to quantify the PD-1/PD-L1 axis proteins successfully in all 31 NSCLC FFPE tumors. PD-L1 expression ranged from 2 amol/μg to 61 amol/μg of total protein. As expected, iMRM results correlated moderately (R = 0.56, ρ < 0.001) with PD-L1 IHC. PD-L1 glycosylation status ranged from 99.9±0.2%, and therefore did not explain the discrepancies between IHC and iMRM for these samples. Conclusions: Herein a robust iMRM workflow was developed for the quantitation of the PD-1/PD-L1 axis in FFPE. This proof-of-concept supports that MS-based assay can provide otherwise unavailable data (e.g., PD-L1 concentration, glycosylation status). CPI treated patient tumors are being currently processed to validate the predictive value of the assay.

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