The term “liquid biopsy” is defined by the College of American Pathologists (CAP) as assays that analyze proteins, DNA, or cancer cells that circulate in the blood. There is growing interest in the less invasive, less complicated liquid biopsy as an alternative option to traditional biopsies in cases where a biopsy is not obtainable from a patient and/or a reduction in the number of biopsies is required from a patient when monitoring their treatment. This is evidenced by the growing number of patent applications in this field – over a million new inventions can be found on the WIPO PatentScope database.
At Diaceutics, the diagnostics commercialization service provider, we believe tissue is still the gold standard as there is limited standardization in liquid biopsy use, leading to many variables in the pre-analytical workflow. We support harmonization of workflows to ensure high quality liquid biopsy testing and an external assessment program as described in BMC Cancer.i A report about a pilot External Quality Assessment (EQA) program for ctDNA analysis found an error rate of 20% with high error rates observed for RAS testing compared to EGFR analysis. Over-interpretation of wild-type results was found to be an issue with frequent failure to comment on the amount of circulating cell-free DNA (cfDNA) extracted.
The detection, enumeration and the molecular characterization of CTCs (Circulating Tumor Cells), ctDNA (cell-free circulating tumor DNA) and miRNAs (circulating microRNAs) is described in a patent application entitled ‘a method for the quantification of PD-L1 expression’ WO2017072539A1 by Pharmassist as a reliable non-invasive source of blood-based biomarkers useful for molecular profiling before and after treatment. According to the inventors, these blood-based tests are highly important in cases where tumor biopsies are not accessible (NSCLC) and can be used to monitor the efficacy of treatments, identify emerging biomarker-based sources of acquired resistance (i.e. EGFR T790M or ALK kinase domain mutations in ALK fusions) and improve the choice of treatment options.
A combination of liquid biopsy with NGS (Next Generation Sequencing) is reportedly used to improve diagnosis, treatment and screening. The tests are considered to be more accessible for routine monitoring and samples are often combined with a process for the enrichment and extraction of DNA from CTCs. Assaying of CTCs from liquid biopsies is an established procedure and many laboratories offer CTC enrichment for research however the process is expensive and has limitations (sensitivity and specificity). Despite the growing number of publications is this area, there is not enough evidence, at this time, to recommend the routine use of ctDNA tests for early-stage cancer detection, new treatment decisions, monitoring efficacy and screening.
The COBAS liquid biopsy test uses plasma or tumor tissue in the detection of non-small cell lung cancer (NSCLC) and has shown sufficient clinical utility to be FDA approved for mutations in EGFR. ii The FDA approved CellSearchTM system has been used for CTC enumeration with an anti PD-L1 specific antibody and found PD-L1 positive CTCs in 11 out of 16 (68.8%) patients with metastatic breast cancer. This assay could be used for liquid biopsy for monitoring and stratification of cancer patients undergoing immune checkpoint blockade.iii A group led by Oliveira-Costa found a strong cytoplasmatic expression of PD-L1 in CTCs in oral squamous cell carcinoma using immunofluorescence and Nanostring.iv
The detection of PD-L1 expression in CTCs in a quantitative way could be used for monitoring the efficacy of immune checkpoint inhibitors as described in the patent from Pharmassist. There are however concerns that separating CTCs may not yield sufficient cells for an accurate quantitation and this would be an issue for any cell-based assay that required quantifying (issue further compounded by the fact that PD-L1 expression is not a binary assay, but rather a continuum expression requiring an established cut-off for some minimum number of cancer cells present). There is an additional issue of loss of morphological information going from IHC to CTC. Some publications report patterns (as opposed to levels) of PD-L1 immunostaining in relation to surrounding cells which would not be possible from CTC.v The biggest issue with CTC technology is the collection of enough specific cells.
Many hundreds of thousands of new inventions now include liquid biopsy terms in the claims and terms that are related to leukemia or cancer, indicating a growing interest in this area. Liquid biopsy detection of leukemia using closed-loop microfluidics is described in patent WO/2018/093475 from MIT. The inventors claim a new method of detecting blast cells in a blood sample. A patent application WO/2018/132753 from Nantbio INC with the title ‘validation of neoepitope-based treatment’ discusses a method of validating an anticipated neoepitope-based treatment of a patient diagnosed with cancer where a liquid biopsy may be used. The neoepitope-based treatment includes treatment with a transformed dendritic cell that expresses a neoepitope and one or more of a chemokine, a cytokine, and/or checkpoint inhibitor.
Another patent with the title ‘molecular profiling for cancer’ WO/2016/141169 discusses how molecular profiling can be used to identify treatments. The invention provides a system for identifying at least one treatment associated with a cancer in a subject where the sample can be any bodily fluid sample. Molecular profiling of PD-L1 in mutated head and neck squamous cell carcinomas is discussed.
Johns Hopkins University has a patent application WO/2018/005276 of interest – Immune checkpoint inhibitors and resistance in non-small cell lung cancer patients is discussed with use of liquid biopsy from plasma or stool.
Despite the numerous patents and considerable ongoing interest in the area, liquid biopsy currently has limited clinical utility in the primary diagnostic setting. There are opportunities in disease prediction and monitoring and evidence is needed to establish appropriate clinical use.vi There are many reports in the public domain that discuss clinical evidence for the utility of liquid biopsy
The KRAS liquid biopsy in the US is performed in numerous labs and different kits are now available. This is an indicator of how this field is growing in acceptance.