BackgroundImmune check point proteins play a pivotal role in immune evasion by the tumor. Recent trials involving inhibitors of the immune checkpoint protein pairs, PD-1 and PD-L1 have demonstrated anti-tumor activity. Measuring the levels of immune check point proteins and other members of the immunological synapse will help clinicians personalize therapy. Currently, immunohistochemistry (IHC) is the preferred diagnostic to assess PD-L1 status; however, PD-L1 positivity varies based on the antibody that is used. Additionally, PD-L1 negative patients by IHC have responded to anti-PD-L1 therapy implicating disconnect between PD-L1 diagnostics and response. We have developed and clinically validated a quantitative mass spectrometric technique that not only quantitates PD-L1 in formalin fixed paraffin embedded (FFPE) tissue but can concurrently quantitate other members (B7H3, B7.1, B7.2, OX40L) of the immunological synapse using the same tissue section.

MethodRecombinant PD-L1 protein was used to identify optimal quantitative peptides for PD-L1 assay. Standard curves were generated using labeled and unlabeled peptides. 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. In addition PD-L1 was also assayed in bladder, breast and gastric cancer.


PD-L1 protein expression was detected in 7 out of 14 cell lines The regression analysis between SRM and mRNA analysis demonstrated moderate correlation (R2 = 0.8894). Normal lung tissue did not express PD-L1; ∼24% of early stage (5/21) and 50% of advanced stage NSCLC (2/4) expressed measurable PD-L1 protein. PD-L1 was detected more frequently in squamous cell carcinoma than adenocarcinoma. We are currently assessing the levels of PD-L1 and other targets of the immunological synapse using multiplex mass spectrometry and comparing it with IHC in 100 cholangiocarcinoma and possible inclusion of PD-L1diagnostics in clinical trials.

DiscussionThe need to characterize expression levels of druggable targets in small biopsies is becoming ever more critical as new drug targets and biomarkers are identified. 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. Laser microdissection (LMD) can be used to specifically microdissect the immunological synapse. Additional quantitative assays for both lymphocyte (CD8, CD68) and immunotargets (B7-H3,B.1, B7.2 etc) have been developed for assessing the ‘immune profile’ in tumor associated stroma via LMD. This immuno-proteomic assay of the key immunological synapse members within tumor and/or stroma may lead to improved personalized immunotherapy.

Citation Format: Sheeno P. Thyparambil, Fabiola Cecchi, Eunkyung An, Wei-Li Liao, Jon Burrows, Todd Hembrough, Daniel Catenacci. Development and clinical validation of a quantitative mass spectrometric assay for immuno-oncology targets in FFPE samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4255. doi:10.1158/1538-7445.AM2015-4255