title: Quantifying insulin receptor isoform expression in FFPE breast tumors.
abstract: BACKGROUND
  The development of predictive biomarkers for IGF targeted anti-cancer therapeutics remains a critical unmet need. The insulin receptor A isoform (InsR-A) has been identified as a possible biomarker candidate but quantification of InsR-A in widely available formalin fixed paraffin embedded (FFPE) tissues is complicated by its similarities with the metabolic signaling insulin receptor isoform B (InsR-B). In the present study, qPCR based assays specific for InsR-A, InsR-B and IGF-1R were developed for use in FFPE tissues and tested for feasible use in clinical archived FFPE estrogen receptor (ER)+and ER- breast cancer tumors.
  
  
  METHODS
  FFPE compatible primer sets were designed with amplicon sizes of less than 60 base pairs and validated for target specificity, assay repeatability and amplification efficiency. FFPE tumors from ER+ (n=83) and ER-(n=64) primary untreated breast cancers, and ER+ hormone refractory (HR ER+) (n=61) breast cancers were identified for feasibility testing. The feasible use of InsR-A and InsR-B qPCRs were tested using all tumor groups and the feasibility of IGF-1R qPCR was determined using HR ER+ tumors.
  
  
  RESULTS
  All qPCR assays were highly reproducible with amplification efficiencies between 96-104% over a 6 log range with limits of detection of 4 or 5 copies per reaction. Greater than 90% of samples were successfully amplified using InsR-A, InsR-B or IGF-1R qPCR primer sets and greater than 88% of samples tested amplified both InsR isoforms or both isoforms and IGF-1R. InsR-A was the predominant isoform in 82% ER+, 68% ER- and 100% HR ER+ breast cancer. Exploratory analyses demonstrated significantly more InsR-A expression in ER+ and HR ER+ groups compared to InsR-B (ER+ p<0.05, HR ER+ p<0.0005) and both groups had greater InsR-A expression when compared to ER- tumors (ER+ p<0.0005, HR ER+ p<0.05). IGF-1R expression of HR ER+ tumors was lower than InsR-A (p<0.0005) but higher than InsR-B (p<0.0005). The InsR-B expression of HR ER+ tumors was significantly reduced compared other tumor subgroups (ER+ and ER-, p<0.0005) and lead to a significant elevation of HR ER+ InsR-A: InsR-B ratios (ER+ and ER-, p<0.0005).
  
  
  CONCLUSIONS
  The validated, highly sensitive InsR-A and InsR-B qPCR based assays presented here are the first to demonstrate the feasible amplification of InsR isoforms in FFPE tissues. Quantification data generated from this feasibility study indicating InsR-A is more predominant than InsR-B in breast cancer support the use of these assays for further investigation of InsR-A and InsR-B as predictive biomarkers for IGF targeted therapeutics.
authors.affiliation: Department of Oncology, Mayo Clinic, Rochester, MN 55905, United States.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Harrington
authors.forename: S John
authors.initials: SJ
authors.lastname: Weroha
authors.forename: Carol
authors.initials: C
authors.lastname: Reynolds
authors.forename: Vera J
authors.initials: VJ
authors.lastname: Suman
authors.forename: Wilma L
authors.initials: WL
authors.lastname: Lingle
authors.forename: Paul
authors.initials: P
authors.lastname: Haluska
mesh_terms: D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D005240:Feasibility Studies; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D020033:Protein Isoforms; D011960:Receptors, Estrogen; C435941:INSR protein, human; D011972:Receptor, Insulin