Abstract P3-03-06: Characterization of HER2-positive breast cancer (BC) cells selected for tolerance to trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC)
Authors: Biswas T , Fritzemeier R , Mark A , Meißner T , Young B , Jones BL , Pegram M 
Published: March 2017

Abstract
Cellular mechanisms of trastuzumab resistance include alteration(s) in cell signaling pathways (PTEN loss, activation of PI3K/Akt signaling), steric hindrance of antibody binding (by Muc-1/Muc-3), over-activation of alternate receptor kinases (HER3/c-Met/IGF-1R), and proteolysis of HER2 extracellular domain harboring target epitopes for antibody-based therapeutics. Prior studies of trastuzumab resistance have focused largely on cells selected ex vivo with the antibody in absence of human immune effector cells. We developed a selection model, wherein human HER2 positive BC cells (BT474, SKBR3) were subjected to acute ADCC (&gt;90% cell death), trastuzumab concentration 100ug/mL, effector-target ratio 100:1, using human peripheral blood mononuclear cells (PBMCs) as effectors. Surviving cells were allowed to recover to confluence over 8-10 weeks, for 10 total rounds of ADCC selection ex vivo. Mock-treated parent, IgG1 isotype control, trastuzumab, and PBMCs alone were used as controls. ADCC assays based on calcein fluorescent labeling of live target cells, revealed significant reduction (maximum 20%, p&lt;0.005) in cell lysis in immune-selected BT474 cell lines compared to parental controls (immune-selected SKBR3 cells exhibited a non-significant trend towards reduced ADCC). Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 x 75 bp paired-end, median of 46 million paired-end reads/sample), coupled with pathway enrichment analysis (Reactome), followed by q-PCR validation, confirmed significant changes in expression in immune-selected cells (compared to parent control) for genes including: ALDH1, ANK1, TMPRSS3, HINT1, DNM2, TNNC1, COL4A4 in BT474; and ALDH1, ANK1, CAMP1, CPE, IDO1 in SKBR3 cells. Whole-genome sequencing (Illumina HiSeq X, 150 bp paired end, 30x coverage) elucidated 180 genes with single nucleotide variations (SNVs) in immune-selected cells compared to parent in BT474 cells, and 215 genes in SKBR3 cells. Thirty-four SNVs were shared in both cell lines. Further screening and validation confirm genes with SNVs demonstrating significant transcript up-regulation. These include: COL4A3, LEP, SOX-9 in BT474; and HLA-B, TNFRSF10B, HLA-B, PSMA6 in SKBR3. In further phenotypic analysis, ADCC-conditioned BT474 cells exhibit an elongated fibroblast-like morphology with multiple processes, in contrast to control. Immune-selected SKBR3 cells (and not BT474 cells) demonstrate significantly increased motility compared to control in transwell migration assays (p&lt;0.001), and demonstrated increased cell proliferation (MTT assay, 10-15%, 48h; p=0.0242) as compared to parent controls. Our data indicate immune-selection by effector cells contributes to ADCC tolerance in vitro, and is associated with distinct genotypic and phenotypic alterations. Future investigation will determine whether Fc-engineered MAbs (afucosylated), antibody drug conjugates (T-DM1), or potentiation of ADCC by co-stimulatory agonist CD137 antibodies will re-sensitize ADCC-tolerance. This investigation will help to elucidate potentially targetable pathways that emerge from immune-selection with trastuzumab.
Citation Format: Biswas T, Fritzemeier R, Mark A, Meißner T, Young B, Jones BL, Pegram M. Characterization of HER2-positive breast cancer (BC) cells selected for tolerance to trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-06.