Abstract P2-03-02: Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer
Authors: S Gupta, DE Carvajal-Hausdorf, BE Wasserman, K Ho, J Weidler, W Wong, B Rhees, M Bates, DL Rimm
Journal: Cancer Research (2018)

Abstract Background: An on-demand, closed system RT-qPCR (the GeneXpert system, Cepheid, Sunnyvale, CA) has the potential to provide biomarker information in low resourced settings. The system consists of an inexpensive, single-use, disposable, macrofluidic cartridge and an instrument that automates RT-qPCR. Here we use it with a research use only cartridge (STRAT4) that measures the mRNA expression levels of ESR1, PGR, ERBB2, and MKi67 using a single 5uM thick FFPE tissue section from an excisional or core biopsy specimen containing invasive carcinoma of the breast. The assay, results are expressed as a delta cycle threshold (dCt) value, defined as the Ct of a control gene (CYFIP1) minus the Ct of the target gene (ESR1, PGR, ERBB2, or MKi67). We determine whether the dCt result for each marker is equivalent using the entire non-macrodissected section (non m-d) to the dCt results obtained following tumor macro-dissection (m-d) to eliminate non-tumor elements from the assay. Methods: We evaluated the impact of m-d versus non m-d using STRAT4 on a cohort of 62 formalin-fixed paraffin-embedded (FFPE) tumor core needle biopsy specimens with a range of HER2 expression determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). Concordance (sensitivity and specificity) of the STRAT4 ESR1 and HER2 mRNA versus ER and HER2 IHC/FISH measurements were also assessed. Results: We observed excellent agreement of the resulting dCt between the paired samples, m-d versus non m-d, for ESR1 (R2=0.92), PGR (R2=0.90), ERBB2 (R2=0.94) and MKi67 (R2=0.90). No significant difference (P value &amp;gt; 0.99) was observed when we compared the dCt between the paired samples m-d versus non m-d. In addition, using the predefined STRAT4 dCt cutoff for ESR and ERBB2 positivity, we found a significant concordance between RT-qPCR and IHC/FISH for ESR-positivity for the paired samples, m-d (P value &amp;lt; 0.001; sensitivity = 0.98; specificity = 1; PPV = 1; NPV = 0.95) versus non m-d (P value &amp;lt; 0.001; sensitivity = 0.98; specificity = 1; PPV = 1; NPV = 0.95) and HER2-positivity for the paired samples, m-d (P value &amp;lt; 0.001; sensitivity = 0.85; specificity = 0.98; PPV = 0.92; NPV = 0.96) versus non m-d (P value &amp;lt; 0.001; sensitivity = 0.71; specificity = 0.98; PPV = 0.90; NPV = 0.92), respectively. Conclusion: These data suggest that mRNA for ESR and ERBB2 is sufficiently low in surrounding tissues that m-d of whole sections is not required for accurate assessment of key breast cancer mRNA markers in a closed system RT-qPCR assay. The simplicity of the assay workflow may be particularly valuable in low resourced settings where routine access to pathology expertise and to high quality IHC/FISH is challenging. Citation Format: Gupta S, Carvajal-Hausdorf DE, Wasserman BE, Ho K, Weidler J, Wong W, Rhees B, Bates M, Rimm DL. Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-02.