The Liquid Biopsy: The Promise and Challenge behind CTCs and cfDNA

The Liquid Biopsy: The Promise and Challenge behind CTCs and cfDNA

Kalorama Information offers comprehensive analysis of the cancer diagnostics market, including cancer testing services, withThe World Market for Cancer Diagnostics, 6th Edition. Liquid biopsy technology is also reviewed inThe World Market for Molecular Diagnostics, 6th Edition. A free Kalorama Information white paper regarding the market for laboratory-developed oncology tests is availablehere.

The localization of disease can make diagnostic insights inaccessible by routine IVD testing formats. The problem has been encountered in several lab testing disciplines: the characterization of sexually transmitted infections (STIs) without a site sample; lack of definitive neurodegenerative disease diagnosis without a central nervous system (CNS) sample; inherited diseases and chromosomal abnormalities without in utero sampling; and the characterization of cancer without a tissue or tumor sample. In these cases, the invasivity of traditional sample collection impeded screening or entailed risks that precluded screening. Many IVD success stories have consequently featured the introduction of less-invasive sampling procedures, whether STI self-testing, prenatal testing using maternal blood draws, and now complex cancer diagnostics using blood sampling. While previous non-invasive sampling technologies represented IVD market boons, liquid biopsies for cancer testing may prove to be the grail of the clinical diagnostics market.

While previous non-invasive sampling technologies represented IVD market boons, liquid biopsies for cancer testing may prove to be the grail of the clinical diagnostics market.

Liquid biopsy is an alternative to traditional biopsies or invasively collected tissue samples, instead a venous blood draw is used. Combined with next-generation sequencing (NGS), liquid biopsy-based tests may be able to address the entire spectrum of cancer treatment from primary diagnosis through remission and recurrence monitoring. The diagnosis of cancer in asymptomatic patients is a proposed role for liquid biopsies and a new frontier for IVD, but does not represent the comprehensive value of the technology. Genetic screening of liquid biopsy samples could more effectively detect heterogeneities in tumor cells, including shifting drug susceptibility and resistance. The accessibility of liquid biopsies also could also enable routine monitoring of diagnosed cancer patients through remission and for recurrence.

Liquid biopsies or blood draws become suitable oncology samples through the isolation of cell-free DNA (cfDNA) or the enrichment and extraction of DNA from circulating tumor cells (CTCs). Assaying of CTCs from liquid biopsies is the more established procedure with a FDA-cleared CTC enrichment and analysis platform, CELLSEARCH, from Janssen Diagnostics (Veridex). The CELLSEARCH workflow includes magnetic nanoparticles, epithelial cell-specific antibodies and leukocyte-specific antibodies to separate tumor cells from the centrifuged plasma and contaminant white blood cells. However, subsequent CTC-based assays beyond enumeration remain at a developmental stage due to analytical limitations.

Circulating Tumor Cells (CTCs)

A high number of additional vendors offer solutions for CTC enrichment and extraction in addition to Veridex, but often only for research use only (RUO) or, in some cases, CE-marked. The enrichment of samples for CTCs is crucial to subsequent analyses and live cell assays. The process is also highly cost-intensive and subject to technical limitations - restricting the size of the CTC IVD market. While obstacles remain in terms of CTC enrichment sensitivity and specificity, CTCs are an unrivaled resource for the study, detection and monitoring of metastasis and recurrence.

A number of techniques are used for CT enrichment including “positive” capture (antibodies against CTC cell surface markers); “negative” capture (commonly antibodies against leukocyte cell surface markers); cell size separation or exclusion filtration (smaller blood cells filtered out using membranes or post nanostructures); density gradient separation; dielectrophoresis (separation via an electric field); and spiral microchannels (separation via drag forces). These methods often suffer from a lack of sensitivity - loss of tumor cells from the enriched sample - or lack of specificity. Antibody-based separation in particular can prove problematic due to non-specific binding to non-tumoral, non-epithelial cells or binding to naturally circulating epithelial cells unrelated to cancer. Notably, commercialized enrichment methods such as CELLSEARCH use a combination of methods to produce a purified sample. A major limitation to antibody-based enrichment methods, protein-based tumor markers are not available for many tumor types and are subject to variable expression through the progression of disease.

A major limitation to antibody-based enrichment methods, protein-based tumor markers are not available for many tumor types and are subject to variable expression through the progression of disease.

The above enrichment methods are useful for cell counting and cell assays as they avoid lysis. Following capture and enrichment, a diverse set of analytical methods are available for the characterization of CTCs, including immunocytochemistry (ICC) or use of antibodies to probe for CTC surface markers; fluorescent in situ hybridization (FISH) or direct molecular probing (no amplification) of nucleic acid sequences related to aberrant or mutated genes; and reverse transcriptase PCR (RT-PCR) for the detection of mRNA relating to oncogene expression. Cell-based assays such as ICC and FISH are high-complexity tests not as amenable to routine performance; amplification-based tests have become increasingly preferred through their high sensitivity, automated nature and applicability to the study of different nucleic acid populations (mRNA, miRNA, DNA).

CTCs can be analyzed on multiple omic levels (genome, transcriptome, proteome) with clear analytical signals in terms of protein expression and cellular pathways otherwise obscured by the noise of the complex matrices of blood samples.

The preservation of CTCs and continued viability of the cells through enrichment make them a uniquely power resource in both research and clinical testing. The cells can be analyzed on multiple omic levels (genome, transcriptome, proteome) with clear analytical signals in terms of protein expression and cellular pathways otherwise obscured by the noise of the complex matrices of blood samples. Enriched CTCs represent a basis for the comprehensive study of ‘seed cells’ responsible for metastasis. However, current CTC enrichment methods can sometimes fail to discriminate between active seed cells and other present CTCs unable to initiate metastasis or tumor growth in other tissues. Further development of ‘label-free’ enrichment methods or methods not reliant on cell surface markers is desired to isolate active seed cells for effective post-enrichment analysis.

CTC tests could develop into a lead diagnostic for cancer monitoring and detection of cancer recurrence. The detection and counting of CTCs against baselines could be used to determine recurrence risk. Characterization of viable tumor cells in the bloodstream after initial remission could also reveal emergent resistance to the implemented therapy. However, CTC enrichment methods remain highly complex and in need of refinement. While able to be more frequently performed than tissue biopsies, CTC enrichment cannot be as routinely performed as more direct testing methods requiring only lysis or nucleic acid extraction. CTCs thus remain impractical for asymptomatic cancer screening and may not be the most cost-effective resource for tumor profiling.

Cell-free DNA (cfDNA)

Genetic markers can offer a potentially higher level of specificity for tumor profiling than CTCs. With the ongoing difficulty of capturing and differentiating heterogeneous CTCs, researchers and labs have begun development of cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) found in the bloodstream as an analyte. Combined with sequencing, liquid biopsied cfDNA coulde prove a rich analytical resource for cancer diagnostics. Most currently available cfDNA assays are lab developed tests (LDTs) used to sequence and detect known cancer mutations in order to characterize tumors in diagnosed patients.

Circulating tumor DNA or oncology-relevant cfDNA is now recognized as a promising biomarker with wide application from asymptomatic detection through recurrence detection.

The basis of cfDNA testing was discovered by researchers in the 1990s: dying cells, including tumor cells, shed DNA into the bloodstream. Advances in genetics technology since have laid the foundation for cfDNA testing, including the development of next-generation sequencing (NGS) and maturation of non-invasive prenatal testing (NIPT). Circulating tumor DNA or oncology-relevant cfDNA is now recognized as a promising biomarker with wide application from asymptomatic detection through recurrence detection.

For now, virtually all clinical ctDNA testing is performed on diagnosed cancer patients. An important early application has been in testing of non-small cell lung cancer (NSCLC). An estimated one-third of NSCLC patients cannot provide an adequate tissue biopsy needed for testing prior to targeted therapy. The cfDNA assay for NSCLC patients targets EGFR mutation as a biomarker for EGFR-TKI therapy candidates.

The envisioned role of cfDNA expands beyond the detection of targeted mutations. The relatively short turnaround time for cfDNA testing compared to CTCs and the accessibility of liquid biopsies would allow for routine monitoring during therapy or after intervention. Circulating tumor DNA is being considered as a relative indicator of tumor volume, particularly during remission. Higher rates of recurrence have also been demonstrated in patients with detectable ctDNA after operation.

Tumor heterogeneity or inconsistent presence of mutations among tumor cells can potentially also be better captured by cfDNA than highly localized tissue biopsies. Cancer therapy can give rise to heterogeneous tumor cells as resistant cells proliferate. Routine liquid biopsies and cfDNA testing would then reveal the relative quantities of cfDNA related to resistant and non-resistant tumor volume, informing changes in therapy.

Researchers have yet to define the threshold for ctDNA quantities or sequences indicative of early onset cancer requiring intervention, and non-lethal cancers or malignant cells that can be handled by the patient’s immune system.

Cell-free DNA has also been proposed as a means of primary diagnosis in asymptomatic patients, though the accuracy of diagnosis remains problematic due to the residual presence of ctDNA in healthy patients. Researchers have yet to define the threshold for ctDNA quantities or sequences indicative of early onset cancer requiring intervention, and non-lethal cancers or malignant cells that can be handled by the patient’s immune system. Sensitivity is also an obstacle for NGS-based cfDNA assays; developed NIPT assays detect fetal cfDNA present at levels of 10% of all cfDNA in maternal blood while ctDNA assays must search for a veritable needle in the haystack or 0.1% of present cfDNA.

Researchers anticipate collaborative roles for CTC and cfDNA-based analyses in future cancer diagnostics. Asymptomatic detection and initial profiling of tumors could be accomplished by more cost-effective ctDNA detection, potentially followed by CTC testing for better determination of the cancer stage. Either cfDNA or CTCs could be used for tumor profiling, with CTCs representing a more comprehensive analytical resource for the determination of intracellular cancer mechanisms. Circulating tumor DNA could be used for routine patient monitoring with its relative level monitored to determine disease progression or remission. Halted remission or cancer recurrence could prompt CTC analyses to inform the selection of new therapies. Liquid biopsies may ultimately become the standard sample for custom panels incorporating both CTCs and ctDNA.