Liquid chromatography coupled to multiple reaction monitoring (LC-MRM) for quantification of PD-L1 and PD1-signaling proteins in non-small cell lung carcinoma (NSCLC).

e21040 Background: Improving the predictive biomarkers arena of checkpoint inhibitors (CPIs) beyond PD-L1 immunohistochemistry (IHC) is one of the most important unmet need in NSCLC. Up to 50% of patients that show positive PD-L1 expression by IHC do not respond to anti-PD-L1 treatments, and patients with low/undetectable PD-L1 have significantly improved survival with CPI. Moreover, PD-L1 IHC is heterogeneous, can be affected by tissue fixation time and post-translational modifications like glycosylation. Also, multiple studies indicated that PD-L1 expression alone does not reliably reflect the immune status of the tumor, thus requiring the measurement of other members of the PD1 signalling pathway. Methods: To address these issues, we developed a multiplexed targeted mass spectrometry-based (MS) assay for the quantitation of protein members of the PD-1/PD-L1 axis in formalin fixed paraffin-embedded (FFPE) tissue.The effect of fixation time on protein recovery was determined using differentially fixed H1915 cells. Liquid chromatography (LC) coupled to MRM was used to develop a targeted assay for PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70. Results: After 30 minutes fixation 33.6 µg of protein were extracted per mg of FFPE H1915 cells, while protein recovery after 7 days was 55.8 µg/mg, with greater variability (18% and 28% CV, p-value = ns). The optimized LC-MRM method allows the quantitation of PD-L1 and PD-1 down to 23 amol on-column. We evaluated the utility of our MRM assays using the H1915 FFPE cells (PD-L1 3+ by IHC) and determined an endogenous concentration of PD-L1 and NT5E as being 33.2±0.1 amol/µg of total protein and 4.8±0.5 fmol/µg respectively. Noteworthy, a known glycosylation site of PD-L1 was quantified at 10.9±0.3 amol/µg of total protein (30% of total). As increases sensitivity was required, anti-peptide antibodies were generated against the 15 best peptides. We then evaluated this LC-MRM method using MDA-MB-436 cells with lower levels of PD-L1 protein assessed by IHC. Determination of endogenous PD-L1 concentration (3.0 amol/µg of total protein) was achieved using anti-peptide immuno-enrichment followed by MRM. Conclusions: We developed a fixation time independent extraction technique for FFPE and optimized a highly sensitive LC-MRM method that allows the absolute quantification of our targets. This proteomic workflow allows absolute quantification of the PD-1/PD-L1 axis from FFPE tissue using immuno-enrichment and a multiplexed LC-MRM method.

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