(683g) Quantitative, Multiparameter Analysis of Negatively Enriched CTC

CTCs have been isolated in the peripheral blood samples of patients with various kinds of solid tumor malignancies, and elevated CTC numbers correlate with adverse clinical outcomes. While there is an accumulating body of evidence supporting CTCs as prognostic biomarkers, the most important clinically relevant questions including how to best utilize CTCs in clinical decision making remain unanswered, and are actively being investigated. Even more fundamentally, the question of whether CTC numbers alone are sufficient to begin to address these clinical situations or whether additional markers for CTC characterization need to be taken into account remains unanswered.

To help address these questions, recent research investigations have shifted from purely detection to phenotypic characterization of CTCs since there may be subpopulations of CTCs that have more aggressive and metastatic potential. We have assembled a collaborative team, comprised of engineers, chemists, microscopists, medical oncologists and statisticians, to develop comprehensive multiparameter characterizations of CTCs that are isolated in the peripheral blood of patients with solid tumor malignancies. Based on significant research, discussions, and experimental results, our team reached the conclusion that quantitative multiparameter expression testing of CTCs was essential for furthering our understanding of how to best utilize CTC information clinically. By multiparameter analysis, we are referring to more than four markers per individual cell. Once multiparameter technology is developed, then clinical validation studies will be performed to determine the clinical significance of CTC subtypes in addition to CTC numbers.

However, typical current epifluorescent microscopy of more than three markers has significant challenges including: 1) a judgment call on the part of the investigator as to whether the cell is “positive” or “negative” for the marker,  2) potential for bleed through or cross-talk between different “channels” presenting the possibility that a “positive” signal is actually from another probe/marker, 3) bleach out of the dye, such as in the case of FITC, 4) current limit to approximately four different colors due to dye and technological limitations, and 5) most confocal microscopes do not have the capability to quantitatively measure the intensity of the fluorescence from specific cells, and 6) in the case of the confocal system, challenges of getting enough “confocal time”.

To address these challenges, we are developing multispectral imaging technology which will allow, potentially, up to 8 different markers in a quantitative manner. Our approach utilizes computer controlled microscope stage, modified commercial Nunance Multispectral Imaging technology, and highly optimized and customized antibodies and fluorescent probes. This presentation will present our progress on actual breast and head and neck cancer patients circulating tumor cells.