Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections.

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-µm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted ß-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.

Proteomic characterisations of ulcerative colitis endoscopic biopsies associate with clinically relevant histological measurements of disease severity.

AIMS AND METHODS: Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies. RESULTS: Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42-0.72); inverse relationships were detected with cell junction targets (R2=0.49-0.71) and ß-catenin (R2=0.51-0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively. CONCLUSIONS: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

Analysis of Immune Checkpoint Drug Targets and Tumor Proteotypes in Non-Small Cell Lung Cancer.

New therapeutics targeting immune checkpoint proteins have significantly advanced treatment of non-small cell lung cancer (NSCLC), but protein level quantitation of drug targets presents a critical problem. We used multiplexed, targeted mass spectrometry (MS) to quantify immunotherapy target proteins PD-1, PD-L1, PD-L2, IDO1, LAG3, TIM3, ICOSLG, VISTA, GITR, and CD40 in formalin-fixed, paraffin-embedded (FFPE) NSCLC specimens. Immunohistochemistry (IHC) and MS measurements for PD-L1 were weakly correlated, but IHC did not distinguish protein abundance differences detected by MS. PD-L2 abundance exceeded PD-L1 in over half the specimens and the drug target proteins all displayed different abundance patterns. mRNA correlated with protein abundance only for PD-1, PD-L1, and IDO1 and tumor mutation burden did not predict abundance of any protein targets. Global proteome analyses identified distinct proteotypes associated with high PD-L1-expressing and high IDO1-expressing NSCLC. MS quantification of multiple drug targets and tissue proteotypes can improve clinical evaluation of immunotherapies for NSCLC.

Accelerated instability testing reveals quantitative mass spectrometry overcomes specimen storage limitations associated with PD-L1 immunohistochemistry.

Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies (22C3, 28-8, E1L3N, and SP142), raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups (24,737 distinct peptides). PD-L1 quantitation correlated well with IHC expression on unaged sections (R2 = 0.744; P < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal (56%, 58%, and 33% reduction respectively; p < 0.05). Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.

Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this "fit-for-purpose" approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.

Tumor cell expression of vascular endothelial growth factor receptor 2 is an adverse prognostic factor in patients with squamous cell carcinoma of the lung.

A robust immunohistochemical (IHC) assay for VEGFR2 was developed to investigate its utility for patient tailoring in clinical trials. The sensitivity, specificity, and selectivity of the IHC assay were established by siRNA knockdown, immunoblotting, mass spectrometry, and pre-absorption experiments. Characterization of the assay included screening a panel of multiple human cancer tissues and an independent cohort of non-small cell lung carcinoma (NSCLC, n = 118) characterized by TTF-1, p63, CK5/6, and CK7 IHC. VEGFR2 immunoreactivity was interpreted qualitatively (VEGFR2 positive/negative) in blood vessels and by semi-quantitative evaluation using H-scores in tumor cells (0-300). Associations were determined among combinations of VEGFR2 expression in blood vessels and tumor cells, and clinico-pathologic characteristics (age, sex, race, histologic subtype, disease stage) and overall survival using Kaplan-Meier analyses and appropriate statistical models. VEGFR2 expression both in blood vessels and in tumor cells in carcinomas of the lung, cervix, larynx, breast, and others was demonstrated. In the validation cohort, 99/118 (83.9%) NSCLC tissues expressed VEGFR2 in the blood vessels and 46/118 (39.0%) showed high tumor cell positivity (H-score ≥10). Vascular and tumor cell expression were inversely correlated (p = 0.0175). High tumor cell expression of VEGFR2 was associated with a 3.7-fold reduction in median overall survival in lung squamous-cell carcinoma (SCC, n = 25, p = 0.0134). The inverse correlation between vascular and tumor cell expression of VEGFR2 and the adverse prognosis associated with high VEGFR2 expression in immunohistochemically characterized pulmonary SCC are new findings with potential therapeutic implications. The robustness of this novel IHC assay will support further evaluation of its utility for patient tailoring in clinical trials of antiangiogenic agents.

Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation.

There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53±17ng/ml, range=39-102). We also developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC-MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.

A novel, high-sensitivity and drug-tolerant sandwich immunoassay for the quantitative measurement of circulating proteins.

BACKGROUND: Accurate measurement of a total protein target (free plus bound) is essential to optimize dose selection for monoclonal antibody drugs. Herein, we describe a novel sandwich immunoassay format in which the biotherapeutic antibody itself serves as the primary detection antibody. A signal is then generated through the addition of a labeled secondary antibody that recognizes the biotherapeutic antibody. The secondary antibody is conjugated with ruthenium to facilitate electrochemiluminescent analysis. RESULTS: Data are presented from the analysis of two protein biomarkers having disparate size and structure; a 4.5 kDa peptide and a 60 kDa protein. In both cases, validated, highly specific assays were developed and shown to be tolerant to elevated levels of the therapeutic monoclonal antibody in question. CONCLUSION: Our novel format allows drug-tolerant measurement of soluble protein biomarkers targeted by monoclonal antibodies when only two independent epitopes for antibody binding are available and one is recognized by the therapeutic antibody.

Quantification of gemcitabine incorporation into human DNA by LC/MS/MS as a surrogate measure for target engagement.

In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.

The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells.

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B(4) (LTB(4)) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB(4), a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB(4) was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025-25ng/mL for LTB(4) and 0.1-25ng/mL for 5-HETE. The EIA method has a validated range covering 0.025-4ng/mL. When both assays were applied to analyze LTB(4) from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r=0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB(4) and 5-HETE measured by LC/MS/MS after ionophore stimulation (r=0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.