Challenges in using liquid biopsies for gene expression profiling.

Area of Science:

• Biomolecular analysis
• Cancer diagnostics
• Molecular pathology

Background:

• Circulating tumor cells (CTCs) are valuable biomarkers for understanding tumor biology through liquid biopsies.
• Accurate gene expression profiling of CTCs is crucial for potential clinical applications in treatment selection.
• The nCounter NanoString assay with the PAM50 CodeSet is a potential tool for CTC gene expression analysis.

Purpose of the Study:

• To evaluate the accuracy of the nCounter NanoString assay for gene expression profiling of CTCs.
• To identify potential biases and confounding factors in CTC analysis using this assay.
• To establish best practices for CTC molecular profiling in liquid biopsy studies.

Main Methods:

• CTCs were isolated from spiked blood samples using the ANGLE Parsortix system.
• Breast cancer cell lines (Hs578T, SkBr3, MDA-MB-231, MCF7) were used as positive controls.
• Unspiked blood processed by Parsortix served as negative controls for differential gene expression analysis.
• NanoStringDiff statistical method was employed for validation.

Main Results:

• An average of 12 significantly differentially expressed genes were identified between spiked and unspiked samples.
• Targeted pre-amplification in the NanoString assay introduced false positive results due to amplification bias.
• Amplification of non-cancer genes from leukocytes confounded CTC gene expression profiling.

Conclusions:

• Pre-amplification bias is a significant concern for gene expression profiling assays used in CTC analysis.
• The use of unspiked negative controls is recommended when evaluating CTC technologies for gene expression.
• Careful consideration of pre-analytical variables, like amplification bias, is essential for reliable liquid biopsy studies.