= Emerging. More than 5 years before clinical availability. (19.79%)
= Expected to be clinically available in 1 to 4 years. (37.97%)
= Clinically available now. (42.25%)
MSACL 2022 : Borchers

MSACL 2022 Abstract

Self-Classified Topic Area(s): Proteomics

Podium Presentation in De Anza 3 on Thursday at 15:55 (Chair: Kamisha Johnson-Davis)

Precise Quantitation of PTEN by Immuno-MRM: A Tool to Resolve the Breast Cancer Biomarker Controversy

Sahar Ibrahim (1, 2, 3), Cathy Lan (4), Catherine Chabot (4), Georgia Mitsa (1, 2), Marguerite Buchanan (4), Adriana Aguilar-Mahecha (4), Mounib Elchebly (4), Oliver Poetz (5, 6), Alan Spatz (1, 4, 7, 8), Mark Basik (1, 4, 9), Gerald Batist (4, 9), Rene P Zahedi (2, 10), Christoph H. Borchers (2, 9, 10)
(1) Division of Experimental Medicine, McGill University, Montreal, QC (2) Segal Cancer Proteomics Centre, McGill University, Montreal, QC (3) Clinical Pathology Department, Menoufia University, Shebeen El Kom, Egypt (4) Segal Cancer Centre, Montreal, QC (5) University of Tübingen, Tübingen, Germany (6) Signatope GmbH, Reutlingen, Germany (7) Department of Pathology, McGill University, Montreal, QC (8) OPTILAB-McGill University Health Centre, Montreal, QC (9) Gerald Bronfman Department of Oncology, McGill University, Montreal, QC (10) Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow, Russia

Christoph Borchers (Presenter)
Jewish General Hospital, McGill University Montreal, QC, Canada

Presenter Bio: Dr. Christoph Borchers is recognized as a pioneer and leading figure in the development of mass spectrometry-based methods for protein quantification using Multiple Reaction Monitoring (MRM). He has also published more than 300 peer-reviewed papers in scientific journals, and is the founder and director of the McGill-Lady Davis Institute Integrated Proteomics Program at the Jewish General Hospital, McGill University, where he is currently a full professor in the Department of Oncology. His research is centred around the improvement, development, and application of proteomics and metabolomics technologies, with a major focus on quantitative targeted proteomics for clinical diagnostics, as well as new mass-spectrometry-based techniques for structural proteomics.
Dr. Borchers received his BS, MSc, and PhD degrees from the University of Konstanz, Germany in 1996. After his post-doctoral training and employment as a staff scientist at NIEHS/NIH/RTP, NC and he became the director of the Duke–UNC Proteomics Facility and held a faculty position at the UNC Medical School in Chapel Hill, NC (2001-2006). From 2006 to 2019, he was a Professor in the Department of Biochemistry and Microbiology, and Director of the University-Genome British Columbia Proteomics Centre at the University of Victoria, British Columbia, Canada, where he held the Rix BC Leadership Chair in Biomedical and Environmental Proteomics.
Dr. Borchers is also involved in promoting proteomics research and education through his involvement with HUPO (International Council Member), the British Columbia Proteomics Network (Executive Committee Member, past Scientific Director) and the Canadian National Proteomics Network (Member, past VP External and Chair of the Board of Directors). He is also a Fellow of the Canadian Academy of Health Sciences.

Abstract

INTRODUCTION:
The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR-signaling and is commonly found to be downregulated in many cancers, including breast cancer (BC), due to transcriptional and post-transcriptional mechanisms. Conflicting immunohistochemistry (IHC) and western blot data have sparked a controversy about PTEN's role as a prognostic and predictive biomarker in these cancers.

OBJECTIVES:
Because the existing data lacks the precision required to correlate minor PTEN-expression changes in tumors with clinical data, our goal was to develop and validate a fully-standardized, highly sensitive, robust anti-peptide immuno-multiple reaction monitoring (iMRM) assay for PTEN quantification.

METHODS:
We selected the PTEN peptide NNIDDVVR as the ideal target after querying databases, enforcing specific sequence and peptide criteria, and analyzing recombinant PTEN by DDA. The LC-MRM method for NNIDDVVR was developed on an Agilent 6495A triple quadrupole mass spectrometer. The LC conditions and collision energies for individual MRM transitions were optimized, and the resulting 11-min LC-MRM method was validated using CPTAC guidelines. Because direct quantitation of PTEN from cell or tissue lysate was not feasible, and because PTEN protein levels are expected to inversely correlate with disease severity, we generated an anti-peptide antibody to enrich NNIDDVVR prior to LC-MRM. The resulting immuno-MRM assay was used to quantify PTEN in cell lines, fresh frozen-, and formalin-fixed paraffin-embedded (FFPE) cancer tissues.

RESULTS:
The average recovery of the anti-NNIDDVVR immuno-enrichment was 90%, and the average accuracy of the complete iMRM assay was 87%. Our iMRM assay enabled precise quantitation of PTEN concentrations in cell lines, fresh frozen- and FFPE tissues, down to 0.1 fmol/10 µg (of extracted protein), with high inter-and intra-day precision (CV 6.3%). iMRM PTEN concentrations in BC-derived patient-derived xenografts (PDX) were consistent (i) across biological replicates, e.g. 5.7±0.1 fmol/10 µg (PTEN-IHC-high) and 0.7±0.0 fmol/10 µg (PTEN-IHC-low); (ii) across technical replicates with an average %CV of 24% for three cores analyzed from the same FFPE block; (iii) generally showed the same trend as the IHC classification. The accuracy of iMRM assays may be affected by the selection of the tissue locus, protein extraction yields, digestion efficiency, and peptide binding capacity, for example, but our results for technical and biological replicates show a low variation in PTEN levels. Although iMRM required substantially less sample-input, minor differences in PTEN-concentrations could be determined even between samples that were deemed to be PTEN-negative based on WB/IHC. Interestingly, the IHC- and iMRM-derived PTEN levels did not agree well with PTEN copy number variations (r 2=0.15), confirming the concept behind proteogenomics, i.e. that genome-only analyses may often be insufficient to capture the phenotype of a tumor and for making optimal treatment decisions. Finally, we applied our PTEN iMRM method to PDXs derived from primary and metastatic triple negative BC samples. The PDXs were treated with paclitaxel and the regression in tumor size was evaluated over 21 days. All the metastatic samples showed a very good correlation (r2=0.86) between PTEN iMRM concentration and treatment-response. More samples will be analyzed to confirm these initial results and to validate PTEN as a clinically relevant biomarker of BC patient's prognosis and response to therapy.

CONCLUSION:
The anti-peptide immuno-multiple reaction monitoring (iMRM) assay which we developed includes an 11-min micro-flow LC-MRM/MS analysis on a triple-quadrupole MS. This assay provides a much-needed tool for the study of PTEN as a potential biomarker in many cancers.


Financial Disclosure

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