Abstract P3-06-04: Investigating clonal dynamics in triple negative breast cancer chemoresistance
Authors: Echeverria GV , Seth S , Moulder S , Symmans W , Chang J , Cai S , Heffernan T , Piwnica-Worms H 
Published: May 2016

Approximately 50% of triple-negative breast cancer (TNBC) patients have extensive residual disease following neoadjuvant chemotherapy (NAC). These patients have a four-fold increase in mortality risk and an increased risk of distant metastases within three years (1). Understanding the molecular basis of resistance to NAC is expected to provide opportunities to better treat patients in the primary setting. Extensive intratumoral subclonal heterogeneity has been well documented in primary, treatment-naïve TNBC (2). Subclonal populations harboring distinct molecular profiles may confound targeted therapy strategies, yet the functional impact of subclonal heterogeneity in TNBC resistance to therapy is unknown. We are implementing DNA barcoding to quantitatively track changes in subclonal architecture pre- and post-treatment in patient-derived xenograft (PDX) models of TNBC in order to design novel combination therapies. Such barcoding strategies have been used to monitor clonal dynamics in breast cancer PDXs with great sensitivity (3).

We have established an orthotopic PDX from a treatment-naïve TNBC patient (PIM1, procured from a patient later found to have chemoresistant disease). In order to model chemoresistance, we treated PIM1 with Adriamycin and cyclophosphamide (AC), standard of care NAC for TNBC patients, which resulted in partial response but left residual disease. To characterize subclonal dynamics in response to NAC, we transduced freshly isolated PIM1 cells with a lentiviral library expressing 25 million unique DNA barcodes (Cellecta) using conditions to ensure each transduced cell contained a single unique barcode. Transduced cells were selected with puromycin, then orthotopically implanted into immuno-compromised mice. High-throughput barcode sequencing revealed reproducible maintenance of greater than 60,000 unique barcodes in PDX tumors. Comparison of barcode distribution in tumors treated with vehicle or NAC will reveal whether NAC selects for a subpopulation of cells during the development of resistance. Future directions will include whole-exome and RNA sequencing to characterize genomic changes associated with alterations in barcode distribution in response to NAC treatment. Our ultimate goal is to identify novel combination therapies to eliminate subclones that contribute to chemoresistance in primary TNBC.

References

1. Cortazar P, et al. (Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis. The Lancet 384(9938):164-172.

2. Shah SP, et al. (2012) The clonal and mutational evolution spectrum of primary triple-negative breast cancers. Nature 486(7403):395-399.

3. Nguyen LV, et al. (2014) DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts. Nat Commun 5.

Citation Format: Echeverria GV, Seth S, Moulder S, Symmans W, Chang J, Cai S, Heffernan T, Piwnica-Worms H. Investigating clonal dynamics in triple negative breast cancer chemoresistance. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-04.