Abstract PD6-3: Recurrent ESR1 fusion transcripts are associated with endocrine resistance in estrogen receptor positive, HER2 negative breast cancer

Breast cancer proliferation measured by Ki67 immunohistochemistry after short-term antiestrogen therapy has been shown to correlate with disease-free survival. This suggests the use of biomarkers of the early effects of endocrine therapy on ER+ tumors will identify resistant cancers. Thus, we hypothesized that profiling operable ER+ tumors after short term treatment with an aromatase inhibitor would discover actionable molecular alterations causally associated with resistance to estrogen deprivation. We performed whole exome sequencing, RNA-Seq and quantitative immunofluorescence (QIF) of ER, PR, HER2, and Ki67 in biopsies from 130 patients with an operable ER+/HER2– breast cancer that had received letrozole for 10-21 days prior to surgery. Tumors were categorized by the natural log of 2-week post-letrozole Ki67 as sensitive, intermediate, or resistant. We sequenced RNA from 50 frozen tumors and performed fusion transcript analysis using 4 programmatic algorithms (dRanger, TopHat, DeFuse, Chimera Scan), resulting in 304 candidate gene fusions in 44 tumors. Primers with universal sequencing tags were designed against 3’ and 5’ sites of breakpoints mapping to RefSeq exon coding regions (n=187); fusion sequences were amplified by qRT-PCR from tumor and breast cancer cell line RNA. Single or multiple distinct product bands were visualized by gel electrophoresis in 96 tumor samples and Sanger-sequenced. Results were mapped to the human RNA reference transcriptome using BLAST. Overall, 9% of putative fusion transcripts (n=27 from 16 unique tumors) were validated by mapping to the open reading frames of predicted 3’ and 5’ genes. Fusion transcripts called by more than one program were more likely to validate (13 of 24 redundant versus 14 of 269 unique; p Using the 2-week Ki67 to stratify for response to treatment, the validated ESR1 fusions were present only in tumors that maintained high (≥7.4%) to intermediate (>2.7%) Ki67 labeling indices upon estrogen deprivation with letrozole (p=0.01). PR expression was lower (p=0.003) and ER expression higher (p=0.05) in ESR1 fusion+ tumors compared to fusion negative tumors. RNA extracted from 14 additional tumors were screened for ESR1 fusions by qRT-PCR and the ESR1:CCDC170 fusion was validated in 1 of 8 resistant/intermediate and 0 of 6 sensitive tumors. In summary, biomarkers of early response to antiestrogens are needed in order to identify ER+ cancers that are treatment resistant. In a prospective trial of operable ER+/HER2− breast tumors, we discovered recurrent intrachromosomal ESR1 fusion transcripts associated with intrinsic resistance to estrogen deprivation with letrozole. Additional work investigating the genomic basis and function of the fusion transcripts is underway. Citation Format: Jennifer M Giltnane, Justin M Balko, Thomas L Stricker, Christian Young, M Valeria Estrada, Nikhil Wagle, Eliezer van Allen, X Jasmine Mu, Violeta Sanchez, Jaime Farley, Kerry Fitzgerald, Armin Graber, Joseph A Pinto, Franco Doimi, Henry Gomez, Monica Rizzo, Thomas B Julian, Vandana Abramson, Ingrid Mayer, Mark Kelley, Ashwini Yenamandra, Ferrin C Wheeler, Melinda Sanders, Levi Garraway, Ingrid Meszoely, Carlos L Arteaga. Recurrent ESR1 fusion transcripts are associated with endocrine resistance in estrogen receptor positive, HER2 negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-3.