There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2−) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SET ER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors ( ESR1 and PGR ) and robust to preanalytical and analytical influences. We tested SET ER/PR index in biopsies of metastastic HR+/HER2− breast cancer against the treatment outcomes in 140 patients. Then we customized the SET ER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SET ER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955, p = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631, p = 0.001). Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1−98% of ESR1 transcripts (all had high SET ER/PR index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SET ER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2− breast cancer, especially in the absence of mutated ESR1 transcript.

doi: 10.1152/physiolgenomics.00239.2007 You might find this additional info useful... Supplementary material for this article can be found at

Purpose Accurate transcriptional sequencing (RNA-seq) from formalin-fixation and paraffin-embedding (FFPE) tumor samples presents an important challenge for translational research and diagnostic development. In addition, there are now several different protocols to prepare a sequencing library from total RNA. We evaluated the accuracy of RNA-seq data generated from FFPE samples in terms of expression profiling. Methods We designed a biospecimen study to directly compare gene expression results from different protocols to prepare libraries for RNA-seq from human breast cancer tissues, with randomization to fresh-frozen (FF) or FFPE conditions. The protocols were compared using multiple computational methods to assess alignment of reads to reference genome, and the uniformity and continuity of coverage; as well as the variance and correlation, of overall gene expression and patterns of measuring coding sequence, phenotypic patterns of gene expression, and measurements from representative multigene signatures. Results The principal determinant of variance in gene expression was use of exon capture probes, followed by the conditions of preservation (FF versus FFPE), and phenotypic differences between breast cancers. One protocol, with RNase H-based rRNA depletion, exhibited least variability of gene expression measurements, strongest correlation between FF and FFPE samples, and was generally representative of the transcriptome from standard FF RNA-seq protocols. Conclusion Method of RNA-seq library preparation from FFPE samples had marked effect on the accuracy of gene expression measurement compared to matched FF samples. Nevertheless, some protocols produced highly concordant expression data from FFPE RNA-seq data, compared to RNA-seq results from matched frozen samples.