Multiplexed (18-Plex) Measurement of Signaling Targets and Cytotoxic T Cells in Trastuzumab-Treated Patients using Imaging Mass Cytometry

Abstract

Purpose: Imaging mass cytometry (IMC) uses metalconjugated antibodies to provide multidimensional, objective measurement of protein targets. We used this highthroughput 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 to discover associations with trastuzumab benefit. Experimental Design: An antibody panel for detection of 18 targets (pan-cytokeratin, HER2 ICD, HER2 ECD, CD8, vimentin, cytokeratin 7, b-catenin, HER3, MET, EGFR, ERK 1-2, MEK 1-2, PTEN, PI3K p110 a, Akt, mTOR, Ki67, and Histone H3) was used with a selection of trastuzumab-treated patients from the Hellenic Cooperative Oncology Group 10/ 05 trial (n ¼ 180), and identified a case-control series. Results: Patients that recurred after adjuvant treatment with trastuzumab trended toward a decreased fraction of HER2 ECD pixels over threshold compared with cases without recurrence (P ¼ 0.057). After exclusion of the lowest HER2 expressers, 5-year recurrence events were associated with reduced total extracellular domain (ECD)/intracellular domain (ICD) ratio intensity in tumor (P ¼ 0.044). These observations are consistent with our previous work using quantitative immunofluorescence, but represent the proof on identical cell content. We also describe the association of the ECD of HER2 with CD8 T-cell infiltration on the same slide. Conclusions: The proximity of CD8 cells as a function of the expression of the ECD of HER2 provides further evidence for the role of the immune system in the mechanism of action of trastuzumab.

Development of a 2-dimensional atlas of the human kidney with imaging mass cytometry

“…In the kidney, endogenous fluorescence, particularly in tubules, can reduce signal-to-noise ratio and compromise image quantitation in fluorescence-based imaging, making a mass spectrometry-based analytic technique particularly appealing (15). IMC has been used to characterize the tumor microenvironment in solid malignancies, yielding information about the identity, number, and spatial relationship between immune and stromal cells (13,16). Additionally, the immune milieu of the human pancreas has recently been characterized using IMC (17,18).…”

A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue

“…Thus, IMC offers a major advantage over classical immunohistochemistry techniques which are limited by the numbers of markers that can be included simultaneously. Together with other studies that have developed antibody panels for FFPE tissue (15,21,22) this sets the stage for detailed studies to determine immune heterogeneity and cell-cell interactions in situ, providing a novel layer of understanding of functioning of the immune system on tissues. We anticipate that our study will guide other researchers that wish to use IMC for analysis of tissue of choice.…”

Multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry

“…Moreover, given that the cells in these lesions had known phenotypes, the supervised approach for thresholding used herein was reasonable. However in the future, unsupervised analysis of data sets generated using the IMC or other multiparametric in situ analysis approaches (Li et al, 2017; Gerner et al, 2012; Moreau et al, 2012; Tsujikawa et al, 2017; Carvajal-Hausdorf et al, 2019; Wang et al, 2019), may identify novel cell types in tissue that are understudied, for example the MS meninges. In addition, discovery of novel cell types using a technique such as IMC can then be recapitulated with standard techniques using multiple well-characterized specimens from established brain banks.…”