With the advent of cancer immunotherapy (CIT), the number of clinical targets and opportunities for oncology treatment has grown rapidly. This situation has presented a challenge to clinicians about how to select and optimize combinations for chemotherapy, particularly given the genetic variation between patients and known biochemical differences between target organs. Clearly, the best place to search for the answers to these and other important questions lies in analysis of the tumor microenvironment (TME) through direct analysis of biopsy tissue. Because tumor specimens are routinely formalin-fixed and paraffin-embedded (FFPE), analysis of FFPE is a pre-requisite for TME investigation. Although this limits common biomarker methods, such as flow cytometry and ligand binding assays, the number of methods capable of FFPE analysis is expanding. FFPE-related methods can be divided into those which permit imaging and those that do not. While image analysis provides essential contextual information about cell type and location, imaging methods are only semi-quantitative. Thus, co-application with non-imaging methods capable of analyzing more analytes with greater quantitative ability is a commonly adopted strategy. The default method for FFPE image analysis is immunohistochemistry (IHC). In recent years, several methods have been introduced to extend IHC by permitting multiplexed protein analysis. Despite successful delivery of increased information content, these methods are costly and cumbersome to execute. Non-imaging analysis is currently dominated by molecular methods that use next generation sequencing to allow facile profiling of either genes or transcripts from FFPE. Mass Spectrometry (MS) is a versatile, highly specific alternative for quantitative analysis of proteins in FFPE and offers both imaging and non-imaging formats. The most viable imaging option uses antibodies tagged with lanthanide metals directed towards target tissue proteins. MS detection of specific metal ions occurs following sample ablation using a laser or other means. The focus of the present talk will be on the role and advantages of non-imaging analysis of FFPE by high-resolution LC/MS/MS for multiplexed protein quantification in the TME. In the method described, single FFPE sections (5 um) were extracted and digested with trypsin allowing either global or targeted analysis using < 20 ug of total protein as determined spectrophotometrically prior to digestion. Both methods employed orbitrap high resolution mass analysis following peptide separation by basic reverse phase fractionation and on-line nano LC (C18). Targeted analysis occurred using surrogate peptides with detection by parallel reaction monitoring (PRM). A targeted panel was prepared for 10 immuno-oncology drug targets: CD40, GITR, ICOSLG, IDO-1, LAG3, PD-1, PDL-1, PDL-2, VISTA and TIM-3. Quantification was based on comparison to stable isotope labelled peptide standards spiked into each sample. Data will be shared from the analysis of 46 FFPE specimens obtained from NSCLC patients. In addition to targeted and global MS analysis, each specimen was also analyzed by next generation sequencing for DNA and mRNA along with IHC analysis for a limited set of targets. Insights from this comparative analysis will be provided along with discussion on the emerging role of MS for protein quantification in the TME.
Upon completion, the participant will understand the main tools used to examine the tumor microenvionment and the emerging role of quantitative mass spectrometry for protein analysis.
Upon completion, the participant will understand the role and difference between imaging and non-imaging tools for the analysis of FFPE and, importantly, the need to use both in strategies to study the tumor microenvironment.
A key learning point from the talk is how nimble current generation MS tools are for rapid implementation and quantitative aseessment of unique protein panels. This advantage is underpined by the fact that antibody enrichment is not needed, even for low abundance markers, when tissue is analyzed.
The PD-1/PD-2/PDL1 axis has been widely studied in cancer immunotherapy. Data presented from MS analysis shows that addditional key learning is obtained by having access to MS data. In addition, the importance of direct measurement of proteins by MS, as opposed to relying on mRNA will become readily apparent to the participants.
Upon completion, the participant will understand how vital it is to measure proteins in the tumor microenvironment and that LC/MS/MS is far and away the preferred option. Further, the possible options for clinical use will be disclosed to the participants.