Abstract
Abstract Background: Stratification of molecular subtypes is of outmost importance for clinical decision making according to the 12th St Gallen Consensus conference (1). The molecular subtypes were originally identified by genome-wide mRNA analysis of fresh tissue specimen (2). Conventional immunehistochemistry assessment using ER, PR, HER2 and Ki67 approximate these subtypes with ∼ 80% concordance (3). Here we have tested a predefined Single-Sample-Predictor for molecular subtyping based on mRNA assessment of ESR1, PGR, HER2, Ki67 and RACGAP1 in a prospective-retrospective study design. Materials and Methods: Pretreatment core cut biopsies from n=100 patients with PBC treated within a randomized phase II trial (1) of anthracylin/taxane based NAC were examined. Immunohistochemistry was performed for the Ki67 antigen on an automated IHC platform (Dako Techmate 500). Ki-67 were assessed either by visual scoring (vIHC) or by quantitative image analysis (qIHC). RNA from formalin-fixed, paraffin embedded (FFPE) routine biopsies was extracted using a bead-based extraction method (STRATIFYER XTRAKT kits). Fresh tissue samokes were prepared by using Qiagen kits. ESR1, PGR, HER2, Ki-67 and RACGAP1 as well as CALM2 as a house keeping gene were measured via a multiplex quantitative RT-PCR (qPCR). Stratification into molecular subtypes was done by stepwise analysis of singular genes using predefined cut-off values. Kaplan-Meier survival estimates, patitioning test and correlation analyses were performed using the SAS JMP® 9.0.0 software. Results: Concordance between RNA based and IHC-based subtyping into Luminal A, Luminal B, HER2 positive and Triple Negative breast cancer by using ESR1, PGR, HER2, Ki-67 and RACGAP1 was at 85%. mRNA based analysis of fixed tumor specimen performed equally well as fresh tissue analysis. The rate of pathological complete Remission for the predefined molecular subtypes has been for Luminal A 0%, Luminal B 19%, HER2 positive 21% (without Trastuzumab) und Triple Negative Tumors 22%. qPCR from fresh tissue specimen resulted in almost identical classifications. The 5-year overall survival rate was significantly different between subtypes as expected: Luminal A 100%, Luminal B 85%, HER2 positiv 80% und triple negativ 60%. Luminal tumors predominantly metastasized into the bones. Cancer related deaths within the first three years were restricted tot he Triple negative Tumortype. Conclusion: The Single-Sample-Predictor based on assessment of only five mRNA Markers (ESR1, PGR, HER2, Ki-67 and RACGAP1) reliably stratified into molecular subtypes by using either fixed or fresh pretreatment core needle biopsies. The mRNA based assessment enables objective and highly reproducible subtyping for clinical routine diagnostics. 1) Goldhirsch et al., Annals of Oncology 2011 2) Perou et al., Nature 2000 3) Sotiriou&Pusztai, NEJM 2009 4) Schneeweiss et al, Ann Oncol 2011 Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-19.
Published Version
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