In situ characterization of possible cancer stem cells in breast cancer by multiplexing ALDH1, CD44 and cytokeratin on tissue microarrays.
Authors: Neumeister V , Agarwal S , Camp R , Rimm D 
Published: December 2010

Abstract
Abstract #102
Background: Cancer Stem Cells (CSC) have been characterized by CD44 positivity and CD24 negativity in flow cytometric studies. In a recent study ALDH1 was identified as a putative marker for breast cancer stem cells showing overlap with CD44 positivity in a small percentage of the cells (1.6%) and defining cells capable of tumorgenesis in immunodeficient mice. Here we attempt to move this multiplexed, flow-based method of characterization to an in situ method in order to define CSCs in formalin fixed, paraffin embedded breast cancer tissue.&#x2028; Material and Methods: Our breast cancer cohort is a random retrospective collection of 319 node negative and 319 node positive patients with a mean follow up time of 12.6 years. The TMAs were assessed using the AQUA technology, a recently commercialized quantitative microscopy platform that allows reproducible, objective analysis of protein expression levels within molecularly defined architectural compartments. To identify breast CSCs we assessed both ALDH1 in the cytokeratin compartment and also ALDH1 in the CD44 compartment within a cytokeratin mask. Using a CD44 compartment decreases the number of pixels in the denominator of the AQUA score resulting in higher assay sensitivity. Increased sensitivity is achieved by increasing the signal to noise ratio by decreasing the number of pixels in which ALDH1 can be measured.&#x2028; Results: Measurement of ALDH1 expression within a keratin mask was performed on TMAs at 4-fold redundancy and also on a cell line array to define the minimal threshold for ALDH1 expression over background noise. The highest score from each patient's set of 4 spots was used due to tumor heterogeneity in order to identify as many positive patients as possible. This method identified 27 cases (of 546) that were above the AQUA threshold for ALDH1. These cases had significantly worse outcome by Kaplan Meier analysis (log rank p=0.027). Using AQUA to multiplex ALDH1 with CD44 within a cytokeratin mask identified 45 cases (of 397) over the threshold score using only a single fold redundancy. This subset also shows significantly worse outcome (log rank p=0.001). Furthermore after performing multivariate analysis (Cox proportional hazard model) the marker combination remains significant independent of tumor size, histological grade, nodal status, as well as ER-, PR- and HER2-status. The appearance of the cells and their intra-epithelial location was confirmed by examination of positive spots with convolution/deconvolution microscopy.&#x2028; Discussion: These observations suggest a subset of cells can be identified using an in situ technique that is similar to that used to identify CSCs by flow cytometry. The putative CSCs, when present, appear in variable sized clusters. Whether or not they represent true stem cells, they are prognostic for poor outcome independent of the standard prognostic factors used for breast cancer. Identification of these cells may be valuable in identifying high risk patients and potentially lead to a subset of patients that should be targeted for more aggressive therapy.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 102.