MSACL 2018 US Abstract

Topic: Proteomics

Mass Spectrometry Analysis of Immune Checkpoint Proteins in Lung Cancer

Theresa Boyle (Presenter)
Moffitt Cancer Center

Bio: My career has culminated in my position as a Thoracic Molecular Pathologist at the Moffitt Cancer Center with the departments of thoracic oncology, pathology and tumor biology. I am on the clinical investigator track which encompasses clinical work as a molecular genetic pathologist and translational thoracic oncology research. Aspects of this work include biomarker investigation, collaborative involvement as a pathologist for clinical trials, and independent research projects.

Authorship: Theresa Boyle, Kiah Bowers, Victoria Izumi, Bin Fang, Ben Creelan, Eric Haura, John Koomen
H. Lee Moffitt Cancer Center and Research Institute

Short Abstract

Immunotherapy against the programmed death ligand 1 axis (PD-L1/PD-1) has revolutionized the care of lung cancer patients with dramatic response and increase in survival time observed in a subset of these patients. No biomarkers clearly predict which patients will respond, although PD-L1 IHC has been approved as an FDA-approved immunotherapy companion diagnostic. Mass spectrometry is an ideal platform for exploring the role of PD-L1 in relation to other immune checkpoint proteins because it allows for simultaneous quantification of multiple proteins with a small amount of tissue.

Long Abstract


The care of patients with lung cancer has improved significantly with the advent of anti-PDL1/PD1 as an immunotherapy treatment option. Approximately 20% of patients receiving immunotherapy have a dramatic response, and additional patients have stable disease. However, not all patients benefit and nothing clearly predicts who will or will not respond to immunotherapy. Many proteins have been identified which may play a role in how the immune system recognizes and fights against lung cancer cells. However, it is difficult to elucidate their interactive role with current clinical techniques, because most methods assess only one protein at a time. Procedures to obtain lung cancer specimens are associated with risks of pneumothorax, infection, and bleeding, which often limits the amount of tissue that can be collected. There is a critical need to develop methods that can gain more information for clinical guidance and research from limited lung cancer specimens.

Proteomics has the ability to simultaneously quantify multiple proteins in a minute amount of tissue and is ideal for exploring the complex roles and relationships of multiple proteins involved in lung cancer growth and progression. With mass spectrometry, multiple proteins involved in the activation and deactivation of the immune system against lung cancer can be studied.(1) Our immediate goal is to develop a targeted lung cancer biomarker panel with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) to detect and quantify proteins that may influence immunotherapy outcomes. Our ultimate goal is to apply mass spectrometry to guide both the use of currently available therapies and the development of new strategies for patients with lung cancer, enabling selection of the most effective regimen for each individual patient.


Institutional review board (IRB) approval was obtained for a pilot study of 30 patients who consented for the use of their tissue through the Total Cancer Care Program at Moffitt Cancer Center. The first cohort for assay development was composed of 10 cases of non-small small lung cancer (7 adenocarcinoma, 3 squamous cell carcinoma) specimens that had prior immunohistochemistry performed for immune checkpoint proteins (PD-L1, LAG3, TIM3, A2AR, BTLA, and CTLA4) and at least one available formalin-fixed paraffin-embedded block. The second exploratory cohort was composed of 20 cases with available lung cancer specimen blocks from patients treated with immunotherapy within one year of specimen collection. Hematoxylin and eosin (H&E) stained slides were reviewed to assure adequate tumor cellularity (>30%) and quantity (>100 tumor cells) with evaluation of the entire tumor on the slide.

Ten sections were cut and were placed on Arcturus PEN membrane slides to facilitate laser capture microdissection. One slide was stained with H&E, scanned and the images were annotated to guide laser capture microdissection with enrichment of tumor and the tumor microenvironment.

A list of immune checkpoint proteins of potential clinical utility was created based on a review of the literature and clinical trials related to immunotherapy, including PD-1, PD-L1, CTLA4, LAG3, TIM3, BTLA, A2AR, B7-H5 (VISTA), HVEM, B7-H3 (CD276), and MICA. Synthetic proteins were purchased from Origeneā„¢ (Rockville, MD). The proteins were fractionated by SDS-PAGE, digested with trypsin, and the resulting peptides were then analyzed via LC-MS/MS (Q-Exactive Plus, Thermo, San Jose, CA) in a discovery experiment. Data was analyzed with Mascot ( and Skyline (Maccoss Laboratory, UW)(2) to identify the best-performing peptides for assay development. For all selected peptides, unlabeled and stable isotope-labeled standards were synthesized at the 20 micromole scale, evaluated for purity via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF, Sciex) mass spectrometry, and quantified by amino acid analysis (Texas A&M University). The peptides were fragmented into b- and y-ions and the top performing transitions were optimized to determine which voltage created the best collision energy to generate the most sensitive assay. Response curves in both buffer and matrix for each peptide were then measured to determine characteristics including linear range, limit of detection (LOD), and limit of quantification (ULOQ/LLOQ). All peptide measurements were combined into a single scheduled LC-MRM-MS method using selected cell lines as a background matrix to evaluate the sensitivity and effectiveness of the assay panel prior to analysis of FFPE clinical samples.(3-6)

For the first cohort of 10 specimens, bulk protein quantification results will be compared with IHC images and scoring. For the second cohort of 20 specimens, presence and quantity of immune-related proteins will be compared with IHC and patient outcomes.


We have developed a targeted immune checkpoint panel to evaluate the presence and quantity of immune checkpoint and immune-related proteins, including PD-1, PD-L1, CTLA4, LAG3, TIM3, BTLA, A2AR, B7-H5 (VISTA), HVEM, B7-H3 (CD276), and MICA in tumor tissues using LC-MRM-MS.

Conclusions & Discussion

: We are establishing the feasibility for the application of mass spectrometry to measure a large panel of immune-related biomarkers. This panel will be applied to biological and clinical samples in order to identify and quantify the presence of immune checkpoint protein expression to predict the response to immunotherapy and measure the effectiveness of treatment.

References & Acknowledgements:


1. Morales-Betanzos CA, Lee H, Gonzalez Ericsson PI, Balko JM, Johnson DB, Zimmerman LJ, et al. Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma. Mol Cell Proteomics. 2017 Oct;16(10):1705-17. PubMed PMID: 28546465.

2. MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010 Apr 01;26(7):966-8. PubMed PMID: 20147306. Pubmed Central PMCID: PMC2844992.

3. Gerber SA, Kettenbach AN, Rush J, Gygi SP. The absolute quantification strategy: application to phosphorylation profiling of human separase serine 1126. Methods Mol Biol. 2007;359:71-86. PubMed PMID: 17484111.

4. Kirkpatrick DS, Gerber SA, Gygi SP. The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods. 2005 Mar;35(3):265-73. PubMed PMID: 15722223.

5. Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):6940-5. PubMed PMID: 12771378. Pubmed Central PMCID: PMC165809.

6. Barnidge DR, Dratz EA, Martin T, Bonilla LE, Moran LB, Lindall A. Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards. Anal Chem. 2003 Feb 01;75(3):445-51. PubMed PMID: 12585469.

Acknowledgements: We acknowledge the support of the following at the H. Lee Moffitt Cancer Center and Research Institute: Lung Cancer Center of Excellence (director:Eric Haura; honest broker: Katie Fellows); Proteomics Core (Director, John Koomen; scientist, Kiah Bowers); Digital Microscopy Core (Director, Joseph Johnson); and Tissue Core (Manager, Michelle Fournier; staff scientist, Jeanette Rheinhardt; pathologist, Dr. Alex Lopez). We also thanks Dr. Benjamin Creelan for providing us with lung cancer slides stained by immunohistochemistry for immune checkpoints by Mosaic Laboratories.

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