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Liquid biopsies containing circulating tumor DNA (ctDNA) for diagnosing biomarkers for early detection, metastatic recurrence or targeted therapy: implication for COVID-19 diagnosis

Biomarkers play an indispensable role for cancer management.  Biomarkers include specific molecules or genetic mutations whose presence is indicative of the disease progression.  One major area of its application has been for early detection as late-stage diagnosis portends poor survival for many cancer types.  The early detection of predisposing genotypic biomarkers provides an opportunity to take preemptive measures such as surgery to avoid tumor development.  Another key area has been to detect the onset of recurrent cancer after the initial treatment.  The changes in the expression level of molecular biomarkers could signal the presence of recurrent cancer when it is not visually detectable through imaging.  Further, biomarkers are being used to prognosticate the therapeutic outcome.  Given the adverse side effects associated with various anticancer drugs, there has been a drive to predict the response of specific drugs prior to treatment.

Typically, diagnostic analysis has been performed on solid samples, i.e. tumor specimens resected from cancer patients. Though the data obtained from primary tumor biopsies may accurately reflect the disease status, the approach is being marred by the need for invasive surgery, insufficient specimen for analysis, difficulty of monitoring repeatedly, etc.  Another key finding is that, in the event of recurrence, data obtained from a single biopsy may not reflect the mechanism of drug resistance for the overall cancer.

A solution to the above dilemma may be found in liquid biopsy.  Previous works have shown that primary tumors shed cancer cells into vasculature albeit in a low number (1-10 cells per ml of blood).  For screening, circulating tumor cells (CTCs) can be detected through microscopic imaging using tumor-specific antibodies, which could be indicative of early stage carcinogenesis (Ried et al., 2017).  The changing level of CTC counts could be used to assess therapy efficacy as well as survival prognosis.  The presence of CTCs have been associated with late recurrence (5 year after diagnosis), which occurs in ~50% of hormone receptor-positive breast cancer cases (Sparano et al., 2018). For personalized medicine, CTCs allow gene expression analysis at the single cell level or could be grown into a 3D model to test the efficacy of drugs to tailor the treatment (Potdar et al., 2015). 

To capture, antibody-functionalized carriers that are recovered through magnetic field can be used to separate CTCs from other components using microchips (Hoshino et al., 2011).  With microfluidic channels, unique physical parameters such as greater size, flow velocity, shear force/drag potential have been exploited to distinguish CTCs from normal cells (Nagrath et al., 2007).  Nevertheless, the heterogeneous nature of CTCs and the extreme rarity (1 CTC per 109 hematologic cells in blood) have made them very difficult to isolate.


                       
 

The field of liquid biopsy has advanced further to allow detection of genetic biomarkers without isolating CTCs  Circulating tumor DNA (ctDNA) represent fragments of DNA released into the blood stream by dying tumor cells due to necrosis or apoptosis.  Presumably, ctDNA can be found in other bodily fluids including saliva, urine and spinal fluid.  Like CTCs, the amount of ctDNA detected can be used to gauge disease progression, ex. an increase in ctDNA amount may be indicative of cancer recurrence.

Plasma genotyping performed on ctDNA using next generation sequencing (NGS) was able to detect specific mutations (Iwahashi et al., 2019).  For GWAS (genome wide association study), ctDNA has been used to genotype SNP (single nucleotide polymorphism) using chip-based microarray methodologies, which is based on hybridization.  Using qPCR, mutation could be detected in ctDNA of colorectal cancer patients.  For ctDNA analysis, the presence of normal DNA in plasma could decrease the percentage of mutant allele.  False negatives may require further analysis on tumor biopsies to exclude other mechanisms that give rise to drug resistance.  For positive results obtained, comparison with normal tissue may be necessary to exclude mutations that arose during hematopoiesis.

In a similar vein, the effort to diagnose COVID-19 could be simplified by using liquid biopsy.  Regarding safety, the acquiring of respiratory samples (ex. sputum or other specimens from lower part of lung) could expose clinicians to the virus inadvertently.  Saliva containing COVID-19 coronavirus may be easier to sample and process to detect viral RNA through RT-PCR or RT-LAMP reaction.

The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  For medicinal chemistry, it specializes in peptide synthesis, characterization, modification, purification to generate various peptide-based building blocks as well as pharmaceutical intermediates—in addition to peptide libraries, peptide arrays, peptidomimetics.   Antibody purification, characterization/quantification, modification and labeling are also offered.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA).  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as conjugate to peptidesIt recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides.

 

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References

Hoshino K, Huang YY, Lane N, Huebschman M, Uhr JW, Frenkel EP, et al.  Microchip-based immunomagnetic detection of circulating tumor cells.  Lab Chip. 11:3449-57 (2011).  PMID: 21863182

Iwahashi N, Sakai K, Noguchi T, Yahata T, Matsukawa H, Toujima S, et al.  Liquid biopsy-based comprehensive gene mutation profiling for gynecological cancer using CAncer Personalized Profiling by deep Sequencing.  Sci Rep. 9:10426 (2019).  PMID: 31320709

Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, et al.   Isolation of rare circulating tumour cells in cancer patients by microchip technology.  Nature. 450:1235-9 (2007).   PMID: 18097410

Potdar PD, Lotey NK.  Role of circulating tumor cells in future diagnosis and therapy of cancer.  J. Cancer Metastasis Treat 1, 44-56 (2015).  https://jcmtjournal.com/article/view/1172

Ried K, Eng P, Sali A. Screening for Circulating Tumour Cells Allows Early Detection of Cancer and Monitoring of Treatment Effectiveness: An Observational Study.  Asian Pac J Cancer Prev.  18:2275-2285 (2017).  PMID: 28843267

Sparano J, O'Neill A, Alpaugh K, Wolff AC, Northfelt DW, Dang CT, et al.  Association of Circulating Tumor Cells With Late Recurrence of Estrogen Receptor-Positive Breast Cancer: A Secondary Analysis of a Randomized Clinical Trial.  JAMA Oncol.  4:1700-1706 (2018).  PMID: 30054636