Abstract P5-05-07: Elucidating molecular resistance to trastuzumab using next generation sequencing in isogenic cell models
Authors: Abukhdeir Abde M , Najor Matthew , Turturro Sanja , Pergande Melissa R , Borgia Jeffrey A , Khalak Hanif G , Cobleigh Melody 
Published: May 2015

A minority of all breast cancers will express increased levels of the ERBB2 protein. They are eligible for trastuzumab-based therapy. Some will respond, but all will progress. Thus, the problem of resistance to trastuzumab has generated an urgent need to determine the underlying mechanisms of that resistance.

Cancer is a genetic disease and the mechanism of trastuzumab resistance is likely also genetic in nature. However, the significant genomic heterogeneity between and within patient tumors greatly complicates the identification of a genetic mechanism of resistance for trastuzumab. In order to overcome some of these challenges, we looked to an isogenic model of trastuzumab resistance. We acquired the trastuzumab-sensitive breast cancer cell line, BT474 and two clones of this cell line that were conditioned to exhibit trastuzumab resistance.

To investigate a possible genetic mechanism of trastuzumab-resistance, we performed whole exome sequencing using Ampliseq chemistry on the Ion Torrent platform from Life Technologies and paired-end RNA-sequencing on the Illumina HiSeq platform. Next-generation sequencing data was bioinformatically analyzed using tools that allowed us to filter relevant variants based on statistical and functional significance. Variants of interest were those that that arose during drug treatment, which were identified as those in each of the resistant clones, which were novel compared to the parent clone. Proteins from whole cell lysates were resolved in two dimensions using 3-10 nonlinear strips for isoelectric focusing followed by resolution via 4-20% SDS-PAGE. Proteins were visualized via Gelcode blue and cored with a biopsy punch, trypsinized, and submitted for protein ID on an LTQ XL mass spectrometer. We performed functional validation of genetic alterations through the use of somatic cell gene targeting of an ERBB2-expressing clone of the MCF-10A cell lines, a non-tumorigenic model of breast cancer, which is sensitive to trastuzumab.

Exome sequencing initially yielded more than 10,000 unique DNA variants across the three clones, which after bioinformatic analysis resulted in ∼1000 variants of interest. Two-dimensional gel electrophoresis revealed 25-30 differentially expressed proteins per sample. Correlation between the sequencing and proteomics data provided us with a candidate gene list of less than 100 genes and several cellular pathways related to growth signaling and immunity. Genetic alterations were tested for their ability to cause trastuzumab resistance in MCF-10A clones.

We describe herein a detailed molecular analysis for a model of trastuzumab resistance. Validated genetic alterations will be investigated in a unique collection of archival specimens, which we hope will open the path towards the development of novel agents to augment the effects of trastuzumab.

Citation Format: Abde M Abukhdeir, Matthew Najor, Sanja Turturro, Melissa R Pergande, Jeffrey A Borgia, Hanif G Khalak, Melody Cobleigh. Elucidating molecular resistance to trastuzumab using next generation sequencing in isogenic cell models [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 P5-05-07.