Abstract P2-09-18: Multiplexed (18-Plex) measurement of protein targets in trastuzumab-treated patients using imaging mass cytometry
Authors: Carvajal-Hausdorf DE , Stanton KP , Patsenker J , Villarroel-Espindola F , Esch A , Montgomery RR , Psyrri A , Kalogeras KT , Kotoula V , Fountzilas G , Schalper KA , Kluger Y , Rimm DL 
Published: February 2018

Abstract
Introduction: Recent studies have shown that the molecular heterogeneity of HER2 intracellular (ICD) and extracellular (ECD) domains, as well as overall immune infiltration, are associated with response to adjuvant trastuzumab. Traditional strategies for in situ measurement in the tumor microenvironment allow the combination of up to 6 targets, limiting our capability for in-depth interrogation of tissues. Imaging Mass Cytometry (IMC) uses metal-conjugated antibodies to provide multidimensional, objective measurement of protein targets. We used this high-throughput multiplexing platform to perform an 18-plex assessment of HER2 ICD/ECD, cytotoxic T cell infiltration and other structural and signaling proteins in a cohort of patients treated with trastuzumab.
Methods: An antibody panel for detection of 18 targets (Pancytokeratin, HER2 ICD, HER2 ECD, CD8, vimentin, cytokeratin 7, beta-catenin, HER3, MET, EGFR, ERK 1-2, MEK 1-2, PTEN, PI3K p110 alpha, Akt, mTOR, Ki67 and Histone H3) was conjugated to unique metals for detection in an IMC instrument (Fluidigm). All assays were objectively standardized and validated using quantitative immunofluoresce (QIF). Finally, the IMC technique was validated against HER2 single marker assays by QIF. We used a collection of trastuzumab-treated patients from the HeCOG 10/05 trial (n=180), and identified a case:control series using 5-year recurrence events (n=19), which were matched to controls (n=41) by age and TNM stage. Formalin-fixed, paraffin embedded tissues in tissue microarray format were ablated in the IMC attachment to the CyTOF flow cytometer for simultaneous detection of markers. Image visualization was conducted using MCD Viewer (Fluidigm). Statistical analyses were performed using a range of platforms.
Results: Patients that recurred after adjuvant treatment with trastuzumab showed a decreased fraction of HER2 ECD pixels over threshold in a compartment determined by CK and HER2 ICD compared to cases without recurrence (p=0.057). After exclusion of the lowest HER2 expressers (that would have fallen below the threshold for positive by current HER2 assays), 5-year recurrence events where associated with reduced total ECD/ ICD ratio intensity in tumor (p=0.044). Patients below the median for total ECD/ICD ratio showed a trend for decreased benefit from trastuzumab (p=0.066). Levels of cytotoxic T cell infiltration, depicted by total CD8 intensity, were lower in patients with recurrences (p=0.05).
Conclusion: Objective measurement of highly multiplexed protein targets in routine, fixed breast cancer tissues shows that a decreased ratio of HER2 ECD/ ICD is associated with 5-year recurrence after trastuzumab treatment. This observation is consistent with our previous work using QIF but represents the first time this has been done on identical cell content (on a single tissue section). Additionally, on the same section we found that lower levels of overall cytotoxic T cell infiltration were associated with worse outcome. Further analysis of the multiplexed data, including both correlative and distance-based analyses are underway.
Citation Format: Carvajal-Hausdorf DE, Stanton KP, Patsenker J, Villarroel-Espindola F, Esch A, Montgomery RR, Psyrri A, Kalogeras KT, Kotoula V, Fountzilas G, Schalper KA, Kluger Y, Rimm DL. Multiplexed (18-Plex) measurement of protein targets in trastuzumab-treated patients using imaging mass cytometry [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-18.