PMID- 24983965 OWN - NLM STAT- MEDLINE DCOM- 20151106 LR - 20211021 IS - 1932-6203 (Electronic) IS - 1932-6203 (Linking) VI - 9 IP - 7 DP - 2014 TI - Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue. PG - e100586 LID - 10.1371/journal.pone.0100586 [doi] LID - e100586 AB - BACKGROUND: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for 'high Met' expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. METHODS: After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). RESULTS: Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/microg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/microg to 4669.5 amol/microg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of >/=1500 amol/microg was 100% sensitive (95% CI 0.69-1) and 100% specific (95% CI 0.92-1) for MET amplification. CONCLUSIONS: The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC. FAU - Catenacci, Daniel V T AU - Catenacci DV AD - Department of Medicine, Section of Hematology & Oncology, University of Chicago, Chicago, Illinois, United States of America. FAU - Liao, Wei-Li AU - Liao WL AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. FAU - Thyparambil, Sheeno AU - Thyparambil S AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. FAU - Henderson, Les AU - Henderson L AD - Department of Medicine, Section of Hematology & Oncology, University of Chicago, Chicago, Illinois, United States of America. FAU - Xu, Peng AU - Xu P AD - Department of Medicine, Section of Hematology & Oncology, University of Chicago, Chicago, Illinois, United States of America. FAU - Zhao, Lei AU - Zhao L AD - Department of Pathology, University of Chicago, Chicago, Illinois, United States of America. FAU - Rambo, Brittany AU - Rambo B AD - Department of Medicine, Section of Hematology & Oncology, University of Chicago, Chicago, Illinois, United States of America. FAU - Hart, John AU - Hart J AD - Department of Pathology, University of Chicago, Chicago, Illinois, United States of America. FAU - Xiao, Shu-Yuan AU - Xiao SY AD - Department of Pathology, University of Chicago, Chicago, Illinois, United States of America. FAU - Bengali, Kathleen AU - Bengali K AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. FAU - Uzzell, Jamar AU - Uzzell J AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. FAU - Darfler, Marlene AU - Darfler M AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. FAU - Krizman, David B AU - Krizman DB AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. FAU - Cecchi, Fabiola AU - Cecchi F AD - Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. FAU - Bottaro, Donald P AU - Bottaro DP AD - Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. FAU - Karrison, Theodore AU - Karrison T AD - Department of Health Studies, University of Chicago, Chicago, Illinois, United States of America. FAU - Veenstra, Timothy D AU - Veenstra TD AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. FAU - Hembrough, Todd AU - Hembrough T AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. FAU - Burrows, Jon AU - Burrows J AD - OncoPlex Diagnostics Inc., Rockville, Maryland, United States of America. LA - eng GR - K12 CA139160/CA/NCI NIH HHS/United States GR - UL1 TR000430/TR/NCATS NIH HHS/United States GR - ImNIH/Intramural NIH HHS/United States GR - K12 CA139160-01A/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, N.I.H., Intramural PT - Research Support, Non-U.S. Gov't DEP - 20140701 PL - United States TA - PLoS One JT - PloS one JID - 101285081 RN - EC 2.7.10.1 (MET protein, human) RN - EC 2.7.10.1 (Proto-Oncogene Proteins c-met) SB - IM MH - *Esophageal Neoplasms/genetics/metabolism/pathology MH - Female MH - *Gene Amplification MH - Humans MH - Immunohistochemistry/methods MH - Male MH - Mass Spectrometry/*methods MH - *Proto-Oncogene Proteins c-met/genetics/metabolism MH - *Stomach Neoplasms/genetics/metabolism/pathology PMC - PMC4077664 COIS- Competing Interests: WLL, ST, KB, JU, MD, DBK, TDV, TH, and JB are/were paid employees and stock owners at Oncoplex Dx. DVTC received collaborative research funding from Oncoplex Dx. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. EDAT- 2014/07/02 06:00 MHDA- 2015/11/07 06:00 PMCR- 2014/07/01 CRDT- 2014/07/02 06:00 PHST- 2014/04/14 00:00 [received] PHST- 2014/05/25 00:00 [accepted] PHST- 2014/07/02 06:00 [entrez] PHST- 2014/07/02 06:00 [pubmed] PHST- 2015/11/07 06:00 [medline] PHST- 2014/07/01 00:00 [pmc-release] AID - PONE-D-14-16057 [pii] AID - 10.1371/journal.pone.0100586 [doi] PST - epublish SO - PLoS One. 2014 Jul 1;9(7):e100586. doi: 10.1371/journal.pone.0100586. eCollection 2014.