Overcome the Endogenous Levels in Biomarker Quantitation Using LC-MS

The role of PD-L1 as a prognostic and predictive biomarker is an area of great interest. However, there is a lack of consensus on how to deliver PD-L1 as a clinical biomarker. At the heart of this conundrum is the subjective scoring of PD-L1 IHC in most studies to date. Current standard scoring systems involve separation of epithelial and inflammatory cells and find clinical significance in different percentages of expression, e.g., above or below 1%. Clearly, an objective, reproducible and accurate approach to PD-L1 scoring would bring a degree of necessary consistency to this landscape. Using a systematic comparison of technologies and the application of QuPath, a digital pathology platform, we show that high PD-L1 expression is associated with improved clinical outcome in Triple Negative breast cancer in the context of standard of care (SoC) chemotherapy, consistent with previous findings. In addition, we demonstrate for the first time that high PD-L1 expression is also associated with better outcome in ER- disease as a whole including HER2+ breast cancer. We demonstrate the influence of antibody choice on quantification and clinical impact with the Ventana antibody (SP142) providing the most robust assay in our hands. Through sampling different regions of the tumour, we show that tumour rich regions display the greatest range of PD-L1 expression and this has the most clinical significance compared to stroma and lymphoid rich areas. Furthermore, we observe that both inflammatory and epithelial PD-L1 expression are associated with improved survival in the context of chemotherapy. Moreover, as seen with PD-L1 inhibitor studies, a low threshold of PD-L1 expression stratifies patient outcome. This emphasises the importance of using digital pathology and precise biomarker quantitation to achieve accurate and reproducible scores that can discriminate low PD-L1 expression.

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Strategies for H-score Normalization of Preanalytical Technical Variables with Potential Utility to Immunohistochemical-Based Biomarker Quantitation in Therapeutic Reponse Diagnostics

Analytical Cellular Pathology ◽

10.1155/2011/583182 ◽

2011 ◽

Vol 34 (3) ◽

pp. 159-168 ◽

Cited By ~ 12

Author(s):

William E. Pierceall ◽

Michele Wolfe ◽

Jessica Suschak ◽

Hua Chang ◽

Yan Chen ◽

...

Keyword(s):

Dynamic Range ◽

Immunohistochemical Analysis ◽

Average Intensity ◽

Positive Staining ◽

Breast Cancers ◽

Potential Utility ◽

Quantitative Expression ◽

Broad Dynamic Range ◽

Score Normalization ◽

Biomarker Quantitation

Digital quantitative immunohistochemical analysis of protein biomarker expression offers a broad dynamic range against which clinical outcomes may be measured. Semi-quantitative expression data represented as an H-score is produced by computer generated average intensity of positive staining given weight by the percentage of cells showing positive staining. While patient H-scores vary for biological reasons, variation may also arise from preanalytic technical issues, such as differences in fixation protocols. In this study, we present data on two candidate calibrator nuclear-localized proteins, SNRPA and SnRNP70, with robust and consistent expression levels across breast cancers. Quantitative expression measurement of these two candidate biomarkers may potentially be used to eliminate the effect of differences in preanalytic processing of specimens by normalizing H-scores derived from test biomarkers of interest. To examine the effects of preanalytical fixation variation on biomarker quantitation and potential utility of candidate calibrators to address such issues, 6 surgically-resected human breast cancer patient specimens were divided into 6 portions and fixed under distinct conditions (fixation following resection in formalin for 2 hr, 8 hr or 48 hr, or held overnight at 4°C in buffered saline prior to formalin fixation for 2 hr, 8 hr, or 48 hr). We find H-score variation between fixation conditions within individual patient's tumors that were stained for XPF, ATM, BRCA1, pMK2 and PARP1. Most interestingly, detectable expression of SNRPA and SnRNP70 is covariant to test biomarkers under distinct fixation conditions and so these hold the potential for serving as calibration standards for general antigen preservation and reactivity.

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Multiplexed MRM with Internal Standards for Cerebrospinal Fluid Candidate Protein Biomarker Quantitation

Journal of Proteome Research ◽

10.1021/pr500317d ◽

2014 ◽

Vol 13 (8) ◽

pp. 3733-3747 ◽

Cited By ~ 34

Author(s):

Andrew J. Percy ◽

Juncong Yang ◽

Andrew G. Chambers ◽

Romain Simon ◽

Darryl B. Hardie ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Internal Standards ◽

Protein Biomarker ◽

Candidate Protein ◽

Biomarker Quantitation

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Mass spectrometry assay as an alternative to the enzyme-linked immunosorbent assay test for biomarker quantitation in ecotoxicology: Application to vitellogenin in Crustacea (Gammarus fossarum)

Journal of Chromatography A ◽

10.1016/j.chroma.2010.06.015 ◽

2010 ◽

Vol 1217 (31) ◽

pp. 5109-5115 ◽

Cited By ~ 22

Author(s):

Romain Simon ◽

Guillaume Jubeaux ◽

Arnaud Chaumot ◽

Jérôme Lemoine ◽

Olivier Geffard ◽

...

Keyword(s):

Mass Spectrometry ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Gammarus Fossarum ◽

Biomarker Quantitation ◽

Mass Spectrometry Assay

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Targeted Biomarker Quantitation by LC-MS

10.1002/9781119413073 ◽

2017 ◽

Cited By ~ 1

Keyword(s):

Biomarker Quantitation

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Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1) and Data-Independent (MS2) Acquisitions

International Journal of Proteomics ◽

10.1155/2013/791985 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 17

Author(s):

Jason M. Held ◽

Birgit Schilling ◽

Alexandria K. D'Souza ◽

Tara Srinivasan ◽

Jessica B. Behring ◽

...

Keyword(s):

Mass Spectrometry ◽

High Resolution ◽

Posttranslational Modifications ◽

Development Time ◽

Assay Development ◽

Selected Reaction Monitoring ◽

Label Free ◽

Data Independent Acquisition ◽

Key Indicator ◽

Biomarker Quantitation

The receptor tyrosine kinase ErbB2 is a breast cancer biomarker whose posttranslational modifications (PTMs) are a key indicator of its activation. Quantifying the expression and PTMs of biomarkers such as ErbB2 by selected reaction monitoring (SRM) mass spectrometry has several limitations, including minimal coverage and extensive assay development time. Therefore, we assessed the utility of two high resolution, full scan mass spectrometry approaches, MS1 Filtering and SWATH MS2, for targeted ErbB2 proteomics. Endogenous ErbB2 immunoprecipitated from SK-BR-3 cells was in-gel digested with trypsin, chymotrypsin, Asp-N, or trypsin plus Asp-N in triplicate. Data-dependent acquisition with an AB SCIEX TripleTOF 5600 and MS1 Filtering data processing was used to assess peptide and PTM coverage as well as the reproducibility of enzyme digestion. Data-independent acquisition (SWATH) was also performed for MS2 quantitation. MS1 Filtering and SWATH MS2 allow quantitation of all detected analytes after acquisition, enabling the use of multiple proteases for quantitative assessment of target proteins. Combining high resolution proteomics with multiprotease digestion enabled quantitative mapping of ErbB2 with excellent reproducibility, improved amino acid sequence and PTM coverage, and decreased assay development time compared to typical SRM assays. These results demonstrate that high resolution quantitative proteomic approaches are an effective tool for targeted biomarker quantitation.

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