Table 2.

Summary of Workshop Findings: Implications to the Field of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) Biomarkers.

Recent Technological & Methodological Advances

No enrichment approaches: - EPIC Sciences unique “no cell left behind” approach allows for detection of CTCs without an initial enrichment step typical for other methods. Identification and isolation of CTC subpopulations: - Both the technology developed by EPIC Sciences and the microfluidic approach described by Shana Kelley described the ability to identify and isolate CTCs from distinct CTC subpopulations (e.g., EpCAM expression). Analysis of these distinct populations holds promise for understanding disease biology. Assessment of CTCs at the single-cell level: - Recent improvements in technology have begun to allow for the isolation and interrogation of individual CTCs. These advances provide opportunities to assess overall disease heterogeneity, a commonly discussed theme at this workshop. Development of multi-marker gene panels for assessment of cfDNA: - A commonly discussed theme throughout this workshop was shift in focus from single-gene assessment to multi-marker panels capable of extensive genomic mutation and copy number change assessment, thus providing a more comprehensive overview of disease.

Considerations for Incorporation of CTCs and cfDNA into Clinical Trials:

Trials should be clinical decision-driven not biomarker-driven, with appropriate biomarkers significantly adding to the prognostic and/or predictive information already obtained via validated methods. In addition, physicians need to have the appropriate tools to address this elevated risk. Trials need to be performed in disease settings with tools and treatments that are known to be effective in order to appropriately assess the value of CTC/cfDNA to treatment efficacy and disease outcome. When designing trials, investigations must consider the impact of treatment itself on the biomarker of interest. It is likely that the biomarker load will significantly decrease following treatment, making assessment of its true value difficult or even impossible. Special consideration must be placed on designing, implementing and validating standard operating procedures (SOPs) for the collection and analysis of samples. Appropriate selection of the timing of sample collection is critical, and should be based on the specific biology of each disease (e.g., baseline, throughout treatment, following treatment completion, and follow-up samples). Must determine if characterization is necessary or if enumeration will suffice. If characterization is deemed necessary, one must then decide at what level the collected sample will need to be assessed (DNA, RNA, protein, functional assays) to properly answer the posed question(s). Technology selection is important, especially with regards to previous trial data, and widespread feasibility based on overall cost must be considered. Appropriate statistical evaluation of the number of patients required to answer posed questions. Typically, larger number of patients will be required than standard clinical trials due to the rare nature of CTCs/cfDNA and overall disease heterogeneity. Most importantly, how results will be analyzed and interpreted, and if the obtained data can be compared head-to-head with previously performed or ongoing clinical trials.

Moving Forward: General Considerations for the Future Use of CTCs and cfDNA

CTC and cfDNA analysis should be incorporated into ongoing clinical trials where blood collection is mandatory, thus allowing for greater generalizability and more impactful results. Need to develop SOPs for cfDNA and CTC sample archiving, and make this routine practice for ongoing clinical trials, thus allowing reassessment or further assessment of archived samples following technological advances. Need to design trials that incorporate both CTCs and cfDNA to allow for direct comparison and determination of each biomarker’s role and value in various disease settings. Need rationally designed prospective trials from which to draw meaningful conclusions.