title: Mass-spectrometry-based quantitation of Her2 in gastroesophageal tumor tissue: comparison to IHC and FISH.
abstract: BACKGROUND
  Trastuzumab has shown a survival benefit in cases of Her2-positive gastroesophageal cancer (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, these low-throughput assays often produce discordant or equivocal results.
  
  
  METHODS
  We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/μg) of Her2-SRM protein in cell lines (n = 27) and GEC tissues (n = 139). We compared Her2-SRM protein expression with IHC/FISH, seeking to determine optimal SRM protein expression cutoffs in order to identify HER2 gene amplification.
  
  
  RESULTS
  After demonstrating assay development, precision, and stability, Her2-SRM protein measurement was observed to be highly concordant with the HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM expression of the covariates Met, Egfr, Her3, and HER2 heterogeneity, as well as their interactions (cell lines r (2) = 0.9842; FFPE r (2) = 0.7643). In GEC tissues, Her2-SRM protein was detected at any level in 71.2 % of cases. ROC curves demonstrated that Her2-SRM protein levels have a high specificity (100 %) at an upper-level cutoff of >750 amol/µg and sensitivity of 75 % at a lower-level cutoff of <450 amol/μg for identifying HER2 FISH-amplified tumors. An "equivocal zone" of 450-750 amol/µg of Her2-SRM protein was analogous to IHC2+ but represented fewer cases (9-16 % of cases versus 36-41 %).
  
  
  CONCLUSIONS
  Compared to IHC, targeted SRM-Her2 proteomics provided more objective and quantitative Her2 expression with excellent HER2/CEP17 FISH correlation and fewer equivocal cases. Along with its multiplex capability for other relevant oncoproteins, these results demonstrate a refined HER2 protein expression assay for clinical application.
authors.affiliation: Department of Medicine, Section of Hematology/Oncology, University of Chicago Medical Center, Chicago, IL, 60637, USA. dcatenac@medicine.bsd.uchicago.edu.
authors.forename: Daniel V T
authors.initials: DVT
authors.lastname: Catenacci
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.|NantOmics, LLC, Culver City, CA, USA.
authors.forename: Wei-Li
authors.initials: WL
authors.lastname: Liao
authors.affiliation: Department of Pathology, University of Chicago, Chicago, IL, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Department of Pathology, University of Chicago, Chicago, IL, USA.
authors.forename: Emma
authors.initials: E
authors.lastname: Whitcomb
authors.affiliation: Department of Medicine, Section of Hematology/Oncology, University of Chicago Medical Center, Chicago, IL, 60637, USA.
authors.forename: Les
authors.initials: L
authors.lastname: Henderson
authors.affiliation: Department of Medicine, Section of Hematology/Oncology, University of Chicago Medical Center, Chicago, IL, 60637, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: O'Day
authors.affiliation: Department of Medicine, Section of Hematology/Oncology, University of Chicago Medical Center, Chicago, IL, 60637, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Xu
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.|NantOmics, LLC, Culver City, CA, USA.
authors.forename: Sheeno
authors.initials: S
authors.lastname: Thyparambil
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.|NantOmics, LLC, Culver City, CA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Krizman
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.|NantOmics, LLC, Culver City, CA, USA.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Bengali
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.
authors.forename: Jamar
authors.initials: J
authors.lastname: Uzzell
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.|NantOmics, LLC, Culver City, CA, USA.
authors.forename: Marlene
authors.initials: M
authors.lastname: Darfler
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.|NantOmics, LLC, Culver City, CA, USA.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Cecchi
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.|NantOmics, LLC, Culver City, CA, USA.
authors.forename: Adele
authors.initials: A
authors.lastname: Blackler
authors.affiliation: Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Yung-Jue
authors.initials: YJ
authors.lastname: Bang
authors.affiliation: Department of Pathology, University of Chicago, Chicago, IL, USA.
authors.forename: John
authors.initials: J
authors.lastname: Hart
authors.affiliation: Department of Pathology, University of Chicago, Chicago, IL, USA.
authors.forename: Shu-Yuan
authors.initials: SY
authors.lastname: Xiao
authors.affiliation: Department of Public Health Studies, University of Chicago, Chicago, IL, USA.
authors.forename: Sang Mee
authors.initials: SM
authors.lastname: Lee
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.
authors.forename: Jon
authors.initials: J
authors.lastname: Burrows
authors.affiliation: OncoPlex Diagnostics Inc., Rockville, MD, USA.|NantOmics, LLC, Culver City, CA, USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Hembrough
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human
keywords: Clinical biomarker assay; Companion diagnostic; Esophageal; Gastric; Gastroesophageal adenocarcinoma; HER2 (ERBB2) amplification; Her2 expression; Multiplex protein expression analysis in FFPE tissue; SRM-MS; Selected reaction monitoring mass spectrometry; Stomach cancer