Abstract A24: Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in FFPE NSCLC samples.

Background: Binding of PD-L1 expressed on tumor cells to the PD1 on T lymphocytes transduces immuno-inhibitory signals which cripples the T cell9s ability to combat the tumor. Several anti-PD-L1 and anti-PD1 agents are in clinical trials and both regimens have reported promising preliminary results in NSCLC patients. (Brahmer et al., 2012 and Inman, 2013).These studies suggest that tumor expression of PD-L1 is associated with a response to either anti-PD-L1 and anti-PD1 treatment. Immunohistochemistry (IHC) is the current method to assess PD-L1 expression in FFPE tissue; however, PD-L1 IHC has yielded mixed results; some studies showed high false positive by IHC while another study showed that 13% of the PD-L1 negative patients responded to treatment. Moreover, IHC is low throughput and assessing multiple druggable targets by IHC is tissue consuming. As such, there is an urgent need to develop quantitative and highly multiplexed tests to assess biomarker expression. We have developed and clinically validated a quantitative mass spectrometric assay to measure PD-L1 protein expression in FFPE tissue biopsies. Method: We used trypsin digestion mapping of recombinant PD-L1 to identify optimal quantitative peptides. Stable isotope-labeled peptides were synthesized as internal standards, and standard curves were generated in pyrococcus complex matrix to determine LOD, LLOQ, accuracy, precision and linearity of the assay. The PD-L1 assay was pre-clinically validated on 14 cell lines with known expression levels of PD-L1. The assay was then run on archived FFPE sections from in 9 normal tissues, 21 early staged (stage 1 and 2) and 4 advanced staged (stage 3) NSCLC patients. We also used Lung OncoPlex assay to sub-classify NSCLC samples to adenocarcinoma and squamous cell carcinoma. All of the samples were screened in replicates and multiple machines were used to check technical variability. Results: A 10 point calibration curve using five replicates was used to determine the LOD (75 amol) and LOQ (100 amol) for the PD-L1 assay. Fourteen (14) cell lines were assayed for PD-L1 expression by LT-SRM. PD-L1 protein expression was detected in 7 out of 14 cell lines The regression analysis between SRM and mRNA analysis (Broad Institute) demonstrated excellent correlation (R2=0.8894). The NSCLC cell line HCC827 and breast cancer cell line MDA-MB-231 had the highest levels of PD-L1, 374.78 and 298.27 amol/μg protein, respectively. Our initial clinical analysis of NSCLC tissue shows that while no normal lung tissue expresses detectable levels of PD-L1, ~24 % of early stage NSCLC (5/21) and 50 % of advanced stage NSCLC (2/4) express measurable PD-L1 protein. Interestingly, in this initial cohort, all of the PD-L1 positive early staged NSCLC were squamous cell carcinoma while in a small set of advanced staged NSCLC, PD-L1 expression was seen in both squamous cell carcinoma (1/3) and adenocarcinoma (1/1). Characterization of larger cohorts of NSCLC tissue is currently underway and will be presented. Discussion: The need to characterize expression levels of druggable targets in small NSCLC biopsies is becoming ever more critical as new drug targets and biomarkers are identified. Here we describe the development and initial clinical validation of a quantitative proteomic PD-L1 assay which accurately measures PD-L1 expression levels in FFPE tumor tissue. Initial PD-L1 screening using clinical NSCLC samples suggests that more advanced NSCLC patients are more likely to be PD-L1 positive compared to early stage NSCLC patients. Additionally, patients with squamous cell carcinoma are very likely to express PD-L1. Interestingly, Soria et al. (2013) has recently shown a high response rate to PD-L1 therapy in smokers with squamous cell carcinoma. We are currently expanding this initial clinical validation to assess PD-L1 expression levels in larger cohorts, including both adeno and squamous carcinoma. Additional quantitative assays for both lymphocyte (CD3, CD8, CD68) and immunotargets (PD1, B7-H3) are under development. This proteomic assay promises to be a critical component of our multiplexed biomarker analysis, and will allow more accurate identification of potential candidates for PD-L1 or PD1 targeted therapies. Citation Format: Eunkyung An, Wei-Li Liao, Sheeno Thyparambil, Jaime Rodriguez, Ravi Salgia, Ignacio I. Wistuba, Jon Burrows, Todd Hembrough. Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in FFPE NSCLC samples. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A24.