{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T21:39:50Z","timestamp":1782509990634,"version":"3.54.5"},"reference-count":36,"publisher":"Oxford University Press (OUP)","issue":"7","license":[{"start":{"date-parts":[[2021,6,17]],"date-time":"2021-06-17T00:00:00Z","timestamp":1623888000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"funder":[{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R33CA173300"],"award-info":[{"award-number":["R33CA173300"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["U24CA160034"],"award-info":[{"award-number":["U24CA160034"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["U01CA214114"],"award-info":[{"award-number":["U01CA214114"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01CA235575"],"award-info":[{"award-number":["R01CA235575"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R50CA211499"],"award-info":[{"award-number":["R50CA211499"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Fred Hutchinson Experimental Histopathology Shared Resource"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,7,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by immunohistochemistry and\/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>HER2 immuno-MRM-MS assay linearity was \u2265103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1093\/clinchem\/hvab047","type":"journal-article","created":{"date-parts":[[2021,3,10]],"date-time":"2021-03-10T20:12:02Z","timestamp":1615407122000},"page":"1008-1018","source":"Crossref","is-referenced-by-count":47,"title":["Quantification of Human Epidermal Growth Factor Receptor 2 by Immunopeptide Enrichment and Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded and Frozen Breast Cancer Tissues"],"prefix":"10.1093","volume":"67","author":[{"given":"Jacob J","family":"Kennedy","sequence":"first","affiliation":[{"name":"Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey R","family":"Whiteaker","sequence":"additional","affiliation":[{"name":"Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura C","family":"Kennedy","sequence":"additional","affiliation":[{"name":"Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dustin E","family":"Bosch","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa L","family":"Lerch","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Regine M","family":"Schoenherr","sequence":"additional","affiliation":[{"name":"Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Zhao","sequence":"additional","affiliation":[{"name":"Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"ChenWei","family":"Lin","sequence":"additional","affiliation":[{"name":"Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shrabanti","family":"Chowdhury","sequence":"additional","affiliation":[{"name":"Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark R","family":"Kilgore","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kimberly H","family":"Allison","sequence":"additional","affiliation":[{"name":"Department of Pathology, Stanford University Medical Center, Palo Alto, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pei","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew N","family":"Hoofnagle","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA"},{"name":"Department of Medicine, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Geoffrey Stuart","family":"Baird","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA"},{"name":"Department of Medicine, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amanda G","family":"Paulovich","sequence":"additional","affiliation":[{"name":"Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2021,6,17]]},"reference":[{"key":"2021070811033680000_hvab047-B1","doi-asserted-by":"crossref","first-page":"1951","DOI":"10.1200\/JCO.19.02488","article-title":"HER2-low breast cancer: pathological and clinical landscape","volume":"38","author":"Tarantino","year":"2020","journal-title":"J Clin Oncol"},{"key":"2021070811033680000_hvab047-B2","doi-asserted-by":"crossref","first-page":"1364","DOI":"10.5858\/arpa.2018-0902-SA","article-title":"Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology\/College of American Pathologists Clinical Practice Guideline focused update","volume":"142","author":"Wolff","year":"2018","journal-title":"Arch Pathol Lab Med"},{"key":"2021070811033680000_hvab047-B3","doi-asserted-by":"crossref","first-page":"1887","DOI":"10.1200\/JCO.19.02318","article-title":"Antitumor activity and safety of trastuzumab deruxtecan in patients with HER2-Low-Expressing Advanced Breast Cancer: Results From a Phase Ib Study","volume":"38","author":"Modi","year":"2020","journal-title":"J Clin Oncol"},{"key":"2021070811033680000_hvab047-B4","doi-asserted-by":"crossref","first-page":"2786","DOI":"10.1074\/mcp.O115.049049","article-title":"Quantification of HER2 by targeted mass spectrometry in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues","volume":"14","author":"Steiner","year":"2015","journal-title":"Mol Cell Proteomics"},{"key":"2021070811033680000_hvab047-B5","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1016\/j.jmoldx.2013.03.002","article-title":"Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue","volume":"15","author":"Hembrough","year":"2013","journal-title":"J Mol Diagn"},{"key":"2021070811033680000_hvab047-B6","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.molonc.2015.09.002","article-title":"High HER2 protein levels correlate with increased survival in breast cancer patients treated with anti-HER2 therapy","volume":"10","author":"Nuciforo","year":"2016","journal-title":"Mol Oncol"},{"key":"2021070811033680000_hvab047-B7","doi-asserted-by":"crossref","first-page":"1339","DOI":"10.1093\/clinchem\/hvaa178","article-title":"Clinical application of multiple reaction monitoring-mass spectrometry to human epidermal growth factor receptor 2 measurements as a potential diagnostic tool for breast cancer therapy","volume":"66","author":"Do","year":"2020","journal-title":"Clin Chem"},{"key":"2021070811033680000_hvab047-B8","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.jim.2009.11.017","article-title":"Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring-mass spectrometry","volume":"353","author":"Schoenherr","year":"2010","journal-title":"J Immunol Methods"},{"key":"2021070811033680000_hvab047-B9","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1074\/mcp.O114.043133","article-title":"Anti-peptide monoclonal antibodies generated for immuno-multiple reaction monitoring-mass spectrometry assays have a high probability of supporting Western blot and ELISA","volume":"14","author":"Schoenherr","year":"2015","journal-title":"Mol Cell Proteomics"},{"key":"2021070811033680000_hvab047-B10","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1373\/clinchem.2015.250563","article-title":"Recommendations for the generation, quantification, storage, and handling of peptides used for mass spectrometry-based assays","volume":"62","author":"Hoofnagle","year":"2016","journal-title":"Clin Chem"},{"key":"2021070811033680000_hvab047-B11","year":"2017"},{"key":"2021070811033680000_hvab047-B12","doi-asserted-by":"crossref","first-page":"1436","DOI":"10.1016\/j.cell.2020.10.036","article-title":"Proteogenomic landscape of breast cancer tumorigenesis and targeted therapy [English]","volume":"183","author":"Krug","year":"2020","journal-title":"Cell"},{"key":"2021070811033680000_hvab047-B13","doi-asserted-by":"crossref","first-page":"3997","DOI":"10.1200\/JCO.2013.50.9984","article-title":"Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology\/College of American Pathologists clinical practice guideline update","volume":"31","author":"Wolff","year":"2013","journal-title":"J Clin Oncol"},{"key":"2021070811033680000_hvab047-B14","doi-asserted-by":"crossref","first-page":"2717","DOI":"10.1021\/acs.jproteome.6b00245","article-title":"Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of Formalin-Fixed, Paraffin-embedded tissues","volume":"15","author":"Kennedy","year":"2016","journal-title":"J Proteome Res"},{"key":"2021070811033680000_hvab047-B15","doi-asserted-by":"crossref","first-page":"1253","DOI":"10.1002\/pmic.201100587","article-title":"Multiplexed quantification of estrogen receptor and HER2\/Neu in tissue and cell lysates by peptide immunoaffinity enrichment mass spectrometry","volume":"12","author":"Schoenherr","year":"2012","journal-title":"Proteomics"},{"key":"2021070811033680000_hvab047-B16","doi-asserted-by":"crossref","first-page":"907","DOI":"10.1074\/mcp.M113.036095","article-title":"Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach","volume":"13","author":"Carr","year":"2014","journal-title":"Mol Cell Proteomics"},{"key":"2021070811033680000_hvab047-B17","doi-asserted-by":"crossref","first-page":"941","DOI":"10.1373\/clinchem.2014.224840","article-title":"From lost in translation to paradise found: enabling protein biomarker method transfer by mass spectrometry","volume":"60","author":"Grant","year":"2014","journal-title":"Clin Chem"},{"key":"2021070811033680000_hvab047-B18","doi-asserted-by":"crossref","first-page":"966","DOI":"10.1093\/bioinformatics\/btq054","article-title":"Skyline: an open source document editor for creating and analyzing targeted proteomics experiments","volume":"26","author":"MacLean","year":"2010","journal-title":"Bioinformatics"},{"key":"2021070811033680000_hvab047-B19","year":"2021"},{"key":"2021070811033680000_hvab047-B20","year":"2020"},{"key":"2021070811033680000_hvab047-B21","year":"2020"},{"key":"2021070811033680000_hvab047-B22","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1038\/modpathol.2013.134","article-title":"The estimation of tumor cell percentage for molecular testing by pathologists is not accurate","volume":"27","author":"Smits","year":"2014","journal-title":"Mod Pathol"},{"key":"2021070811033680000_hvab047-B23","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1016\/j.jaad.2005.05.015","article-title":"Subcutaneous fat in normal and diseased states: 2. Anatomy and physiology of white and brown adipose tissue","volume":"53","author":"Avram","year":"2005","journal-title":"J Am Acad Dermatol"},{"key":"2021070811033680000_hvab047-B24","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/j.ab.2008.05.024","article-title":"The normalization of gene expression data in melanoma: investigating the use of glyceraldehyde 3-phosphate dehydrogenase and 18S ribosomal RNA as internal reference genes for quantitative real-time PCR","volume":"380","author":"Giricz","year":"2008","journal-title":"Anal Biochem"},{"key":"2021070811033680000_hvab047-B25","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1006\/gyno.1998.5195","article-title":"Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in human cervical cancers","volume":"71","author":"Kim","year":"1998","journal-title":"Gynecol Oncol"},{"key":"2021070811033680000_hvab047-B26","doi-asserted-by":"crossref","first-page":"2357","DOI":"10.1074\/mcp.M114.047050","article-title":"Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma","volume":"14","author":"Abbatiello","year":"2015","journal-title":"Mol Cell Proteomics"},{"key":"2021070811033680000_hvab047-B27","doi-asserted-by":"crossref","first-page":"7381","DOI":"10.1158\/1078-0432.CCR-09-1735","article-title":"Loss of HER2 amplification following trastuzumab-based neoadjuvant systemic therapy and survival outcomes","volume":"15","author":"Mittendorf","year":"2009","journal-title":"Clin Cancer Res"},{"key":"2021070811033680000_hvab047-B28","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1038\/modpathol.2017.171","article-title":"Clinical relevance and concordance of HER2 status in local and central testing\u2014an analysis of 1581 HER2-positive breast carcinomas over 12 years","volume":"31","author":"Pfitzner","year":"2018","journal-title":"Mod Pathol"},{"key":"2021070811033680000_hvab047-B29","doi-asserted-by":"crossref","first-page":"1660","DOI":"10.5858\/arpa.2012-0670-OA","article-title":"Review of estrogen receptor, progesterone receptor, and HER-2\/neu immunohistochemistry impacts on treatment for a small subset of breast cancer patients transferring care to another institution","volume":"137","author":"Jorns","year":"2013","journal-title":"Arch Pathol Lab Med"},{"key":"2021070811033680000_hvab047-B30","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1007\/s10549-019-05148-5","article-title":"Impact of the updated 2018 ASCO\/CAP guidelines on HER2 FISH testing in invasive breast cancer: a retrospective study of HER2 fish results of 2233 cases","volume":"175","author":"Liu","year":"2019","journal-title":"Breast Cancer Res Treat"},{"key":"2021070811033680000_hvab047-B31","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1016\/j.molonc.2013.02.018","article-title":"Influence of DNA copy number and mRNA levels on the expression of breast cancer related proteins","volume":"7","author":"Myhre","year":"2013","journal-title":"Mol Oncol"},{"key":"2021070811033680000_hvab047-B32","doi-asserted-by":"crossref","first-page":"688","DOI":"10.1158\/2159-8290.CD-19-1014","article-title":"Targeting HER2 with trastuzumab deruxtecan: a dose-expansion, phase I study in multiple advanced solid tumors","volume":"10","author":"Tsurutani","year":"2020","journal-title":"Cancer Discov"},{"key":"2021070811033680000_hvab047-B33","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1309\/AJCP4A7KTAYHZSOE","article-title":"Distinguishing score 0 from score 1+ in HER2 immunohistochemistry-negative breast cancer: clinical and pathobiological relevance","volume":"140","author":"Lambein","year":"2013","journal-title":"Am J Clin Pathol"},{"key":"2021070811033680000_hvab047-B34","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1373\/clinchem.2015.245266","article-title":"First steps toward harmonization of LC-MS\/MS thyroglobulin assays","volume":"62","author":"Netzel","year":"2016","journal-title":"Clin Chem"},{"key":"2021070811033680000_hvab047-B35","doi-asserted-by":"crossref","first-page":"2817","DOI":"10.1056\/NEJMoa041588","article-title":"A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer","volume":"351","author":"Paik","year":"2004","journal-title":"N Engl J Med"},{"key":"2021070811033680000_hvab047-B36","doi-asserted-by":"crossref","first-page":"4434","DOI":"10.3390\/ijms21124434","article-title":"Analysis of Spatial Distribution and Prognostic Value of Different Pan Cytokeratin Immunostaining Intensities in Breast Tumor Tissue Sections","volume":"21","author":"Vranes","year":"2020","journal-title":"Int J Mol Sci"}],"container-title":["Clinical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/academic.oup.com\/clinchem\/article-pdf\/67\/7\/1008\/38875308\/hvab047.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/academic.oup.com\/clinchem\/article-pdf\/67\/7\/1008\/38875308\/hvab047.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,7,8]],"date-time":"2021-07-08T19:45:08Z","timestamp":1625773508000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/clinchem\/article\/67\/7\/1008\/6301170"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,17]]},"references-count":36,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,6,17]]},"published-print":{"date-parts":[[2021,7,6]]}},"URL":"https:\/\/doi.org\/10.1093\/clinchem\/hvab047","relation":{},"ISSN":["0009-9147","1530-8561"],"issn-type":[{"value":"0009-9147","type":"print"},{"value":"1530-8561","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2021,7,1]]},"published":{"date-parts":[[2021,6,17]]}}}