title: Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients.
abstract: Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues.
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Onsum
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Elena
authors.initials: E
authors.lastname: Geretti
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Violette
authors.initials: V
authors.lastname: Paragas
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Kudla
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Sharon P
authors.initials: SP
authors.lastname: Moulis
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Lia
authors.initials: L
authors.lastname: Luus
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wickham
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Charlotte F
authors.initials: CF
authors.lastname: McDonagh
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts.
authors.forename: Gavin
authors.initials: G
authors.lastname: MacBeath
authors.affiliation: Merrimack Pharmaceuticals, Cambridge, Massachusetts. Electronic address: bhendriks@merrimackpharma.com.
authors.forename: Bart S
authors.initials: BS
authors.lastname: Hendriks
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000145:classification / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D005260:Female; D005455:Fluorescent Antibody Technique; D018740:Genetic Heterogeneity; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000145:classification* / Q000378:metabolism* / Q000473:pathology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods*; D013274:Stomach Neoplasms / Q000145:classification / Q000378:metabolism / Q000473:pathology; D046888:Tissue Array Analysis; D001749:Urinary Bladder Neoplasms / Q000145:classification / Q000378:metabolism / Q000473:pathology
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human