Total RET (t-RET) expression in formalin fixed paraffin embedded (FFPE) clinical breast cancer samples and correlation with co-receptors and clinical outcomes


Session type:


Zoe Hudson1,Stacie Marks1,Rob Jones1,Huw Mottram1,Pauline Finlay1,Andrew Green2,Rakha Emad2,Fouad Alchami3,Julia Gee1
1Cardiff University,2Nottingham University,3Weill Cornell Medicine



The tyrosine kinase REarranged in Transfection (RET) is active in breast cancer and associates with endocrine resistance. Accurate assessment of RET expression in formalin-fixed, paraffin-embedded (FFPE) clinical samples is essential in clinical trials investigating the potential benefits of RET targeted therapies


An immunohistochemical assay for t-RET was developed and validated using fresh-cut sections of FFPE cell pellets and clinical breast cancers. The assay was then applied to slides from a historical series of FFPE sections from n=93 primary breast cancers, which had been cut and stored for >10 years. Accompanying clinicopathological information allowed investigation of the relationship of RET expression to co-receptors, kinases, tumour characteristics and clinical outcome.


The final optimised assay involved microwave antigen retrieval in pH9 buffer and overnight primary antibody incubation with RET antibody (Abcam ab134100) at 1:70 to 1:100 dilution in long-term stored or fresh-cut clinical material respectively. The assay showed predominately cytoplasmic staining with scant membrane staining in fresh and stored breast cancer sections, and was validated in cell pellets including staining oestrogen deprivation-resistant cells. In the clinical series, t-RET staining was heterogeneous and positively associated with its co-receptor GFRA1 (p=0.003), tumour size (p=0.03), loss of differentiation (p=0.019) and disease recurrence (p=0.005). Biologically t-RET staining correlated inversely with AKT (p=0.02) while there was some direct association with Shc expression (p=0.085) within the MAPK pathway.


This assay is suitable to assess RET expression in FFPE breast cancer sections even when stored long-term, demonstrating association with poorer prognosis. The assay will now be used to investigate RET expression as a potential biomarker in the FURVA clinical trial, which is evaluating the clinical benefit a RET inhibitor in metastatic ER+/HER2- aromatase inhibitor resistant breast cancer.