Developing Novel Liquid Biopsy Oncology Tests

In the quest to improve the detection and monitoring of cancer and other diseases, interest in developing innovative liquid biopsy technologies continues to grow. By detecting and analyzing novel biomarkers from bodily fluids such as blood, liquid biopsies can provide clinicians with deep insights into a patient’s cancer, while avoiding some of the limitations associated with traditional diagnostic methods. However, despite the advantages liquid biopsies can offer, wider use of these tests is still limited by several factors.

To learn more about the potential that liquid biopsies present, as well as challenges that are limiting their wider clinical adoption, Technology Networks spoke to Aiguo (Adam) Zhang, CEO, DiaCarta. In this interview, Zhang also discusses some of the technologies that DiaCarta has developed within the liquid biopsy space and shares his thoughts on what the future may hold for the field.

AM: Can you tell us about the origins of DiaCarta and its technology?

AZ: DiaCarta is a molecular diagnostics company borne out of decades of research in next-generation sequencing (NGS), circulating cell-free DNA (cfDNA) monitoring, and cancer diagnostic assays. Our team of scientists have dedicated several years to investigating alternative methods for early cancer detection such as liquid biopsy and novel methods of diagnosing infectious and oncologic diseases. By combining our founding members’ expertise within the diagnostics space with their entrepreneurial spirit, we founded DiaCarta in 2011 to advance diagnostics using novel sequencing methods and technologies for a variety of indications, including gene mutation detection, pre-cancerous and cancer diagnoses, and cancer therapy toxicity monitoring. DiaCarta’s diagnostic test kits are powered by one of its two proprietary platforms, XNA technology or SuperbDNA™.

AM: How can circulating tumor DNA (ctDNA) be used to detect and manage diseases such as cancer? What benefits does it offer as a biomarker?

AZ: Personalized medicine – specifically precision oncology – nowadays provides molecular characterization of a patient’s tumor via tissue biopsy to help guide treatment decisions. However, to fully implement personalization in the field of oncology, it is necessary to have an easily accessible and less invasive way to determine and follow the molecular makeup of a tumor from the moment of its detection and over the course of treatment of the disease. One such approach is through a liquid biopsy, where the genetic characterization of the tumor may be assessed through a biofluid sample. The term “liquid biopsy” refers to tumor-derived analytes sampled from various biological fluids, usually blood, as well as other clinical specimens such as urine, saliva, ascites and cerebrospinal fluid.

cfDNA or cell-free DNA are degraded DNA fragments that are released into the blood from cells. Several different types of cfDNA have been described in the circulation of blood and other biological fluids. ctDNA or circulating tumor DNA is a small portion of cfDNA that indicates the presence of a tumor in the body. The vast majority of cfDNA is non-cancerous and is often referred to as wild-type DNA. Compared to traditional tissue biopsy, liquid biopsy has the advantage of easy accessibility and is less invasive to the patient. Doctors do not need to perform a procedure to retrieve a tissue sample from a tumor and may obtain biomarkers from a simple blood draw. Liquid biopsy is a growing resource of genomic and genetic molecular biomarkers present in ctDNA and cfDNA.

The ongoing development of technologies to detect cfDNA with high sensitivity has facilitated the employment of liquid biopsies in diverse clinical applications, including in oncology. Circulating tumor DNA obtained from the circulating tumor cells (CTC) of human plasma may be analyzed at multiple time points throughout the course of the disease. Liquid biopsies act as methods of patient stratification for treatment (known as “companion diagnostics”), screening, monitoring response to the selected treatment, and detection of minimal residual disease (MRD) after surgery.

AM: What challenges currently limit greater use of ctDNA in diagnostics?

AZ: Despite the prevalence of liquid biopsy tests, many physicians still use older diagnostic methods to identify cancerous or precancerous conditions and genetic mutations that serve as signals to the condition. Traditionally, to diagnose colorectal cancer (CRC), doctors prescribe a stool-based testing kit (FIT) that can be used either at home or in a clinical setting. Stool samples are then shipped to the laboratory that provided the kit for analysis, with physicians and patients receiving the results in several weeks, if not months. During a traditional tissue biopsy, doctors perform a surgical procedure to obtain a sample of tissue from a tumor that is then analyzed to identify if the sample is cancerous.

New, innovative methods of blood-based liquid biopsy tests also come with a variety of limitations. Despite higher specificity in detecting various forms of cancer and gene mutations, the detection sensitivity of the biomarkers in liquid biopsy also poses a challenge to doctors and is a bottleneck for wider use of these tests. Many liquid biopsy tests rely on target amplification, meaning mutant-type DNA sequences are amplified when a sample is being analyzed. Target amplification methods often cause signal interference as the vast majority of amplified sequences are wild-type DNA.

Patients and physicians are required to be in a clinical care setting to draw blood instead of the patient using a self-administered testing kit at home. Some blood-based tests are not compatible with standard laboratory equipment and require specialized instruments that are not widely available at most laboratories. Liquid biopsy testing similarly experiences the problem of conventional diagnostic methods in which samples are sent to an external laboratory for analysis. In spite of delivering precise results with multiple datapoints, currently available liquid biopsy methods are inefficient tools for healthcare providers and high-risk patients who must wait weeks to receive results.

AM: DiaCarta has two main technology platforms, SuperbDNA Signal Amplification Technology and the XNA Molecular Clamp Technology. Can you describe each technology and its diagnostic applications?

AZ: DiaCarta’s diagnostic tests and blood-based assays are based on two proprietary technology platforms of XNA technology and SuperbDNA Signal Amplification Technology. XNA, xenonucleic acids, are synthetic nucleic acid molecular oligomers that hybridize with target wild-type DNA sequences and can be employed as molecular clamps in quantitative real-time polymerase chain reactions (qPCR). The XNA are complementary to wild-type DNA sequences and are designed to loosely bind to mutant sequences, allowing for the suppression of wild-type sequences and amplification of mutant-type sequences in a PCR reaction. This allows DiaCarta’s XNA-mediated assays to have a limit of detection as low as 0.1% VAF for ctDNA using blood from a patient.

DiaCarta is developing several XNA-mediated assays that have demonstrated high specificity and sensitivity in detecting small amounts of target DNA sequences in oncology and beyond. DiaCarta’s QClamp® Gene Mutation Detection Tests use XNA technology to detect stage 0 through IV gene mutations that signal precancerous and cancerous conditions through a simple blood draw. The ColoScape™ colorectal cancer mutation detection kit identifies noncancerous growths known as advanced adenoma or polyp that signal when CRC is forming, or a patient is at risk of developing CRC.  

DiaCarta has also developed SuperbDNA technology to qualitatively or quantitatively measure the presence of target DNA or RNA, aiding physicians in determining the course of treatment for cancer patients. Other molecular diagnostic methods amplify the DNA or RNA targets in samples, sometimes leading to cross-contamination problems or difficulty in analysis of an assay. SuperbDNA technology instead amplifies the signals used in detection of target DNA or RNA directly. Through a series of hybridization reactions between capture probes and target probes, SuperbDNA technology amplifies the signal from target probes in a way that is proportional to the sample and without any DNA or RNA extraction step.

The QuantiDNA™ cfDNA test, powered by SuperbDNA technology, is used to quantify the total amount of cfDNA directly from a plasma sample while the patient is undergoing cancer chemotherapy or radiation therapy. These therapies not only kill tumor cells, but also normal cells. When these therapies cause damage to normal cells, clinicians refer to this as “toxicity”.  DiaCarta’s RadTox™ cfDNA liquid biopsy test may be used specifically for measuring radiation therapy toxicity in cancer patients, enabling doctors to directly monitor side effect severity and tumor response within days of radiation treatment initiation. Most cancer patients have a similar baseline level of cfDNA in the blood, with radiation increasing cfDNA to different levels for different individuals. By determining the cfDNA level in cancer patients’ blood after radiation, doctors can identify patients’ personalized radiation sensitivity and estimate the highest risk for radiation toxicity. RadTox may allow dose escalation for tumors in less sensitive patients and reduction of overtreatment risks in highly sensitive patients, giving doctors the ability to identify which patients are at risk of adverse effects or varying sensitivities prior to treatment. RadTox may give doctors the opportunity to set a baseline for care prior to treatment and to adjust dosing levels accordingly during treatment.

AM: How do DiaCarta’s technologies compare to alternative technologies such as droplet digital PCR (ddPCR) and NGS?

AZ: The first advantage that DiaCarta’s technologies have over ddPCR or NGS is cost. DiaCarta’s XNA platform and the SuperbDNA signal amplification technology are compatible with commonly available tabletop thermal cyclers or PCR machines and do not require specialized and expensive equipment like the other two. This allows DiaCarta’s platforms to analyze samples in a clinical setting or in a standard laboratory setting, reducing the need for a large transportation infrastructure as part of the supply chain. Moreover, DiaCarta’s proprietary scientific platforms deliver accurate and sensitive results in significantly less time than NGS and ddPCR – both NGS and ddPCR take 3–5 days to deliver results whereas DiaCarta’s technologies deliver in the order of hours. Our technologies offer an inexpensive and time efficient alternative to current standard procedures of point mutation detection and have much higher throughput capability than ddPCR or NGS. Additionally, they are effective even at extremely low sample size: XNA-mediated QClamp® Gene Mutation Detection Tests detect 0.1% VAF to 0.5% VAF mutant DNA out of wild-type DNA for targeted mutations. This low limit of detection allows DiaCarta’s assays to be used in the early detection of pre-cancerous and cancerous tumors, whereas conventional technologies require a larger sample size and are less sensitive to targeted gene mutations.  

AM: DiaCarta has a growing body of peer-reviewed publications supporting its diagnostic technologies. Can you provide more details on these recent studies?

AZ: DiaCarta continues to research the clinical applicability, sensitivity and specificity of its diagnostic tests, and several peer-reviewed publications have been published about its platforms and products. A recent publication in PLOS ONE found that the ColoScape gene mutation detection kit had a clinical specificity of 100% and clinical sensitivity of 92.2% for the detection of CRC, and a clinical specificity of 95% and clinical sensitivity of 62.5% for the detection of precancerous lesions. This study validates the diagnostic application of ColoScape to detect precancerous lesions and colorectal cancer and aid in early cancer detection.

A study published in Nature Scientific Reports demonstrates that DiaCarta's cfDNA biomarker algorithm may function as a prognostic biomarker and efficacy predictor for responses to chemotherapy in non-small cell lung cancer (NSCLC) patients. Using samples measured by DiaCarta’s QuantiDNA™ cfDNA test, researchers found that the kinetics of plasma cfDNA correlated with clinical response and progression free survival in NSCLC patients undergoing chemotherapy based on an algorithm.

AM: DiaCarta is also developing diagnostic tests outside of oncology. Can you tell us more about the other disease areas that DiaCarta is targeting?

AZ: DiaCarta’s QuantiVirus™ SARS-CoV-2 Tests rely on XNA-mediated technology to identify known variants of the virus, including the Omicron variant. XNA’s ability to act as complementary to wild-type sequences allows our SARS-CoV-2 Tests to be adapted to target and differentiate between emerging COVID-19 variants. 

Both the first generation QuantiVirus™ SARS-CoV-2 Test and second generation QuantiVirus SARS-CoV-2 Multiplex Test Kit received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA) among several other regulatory approvals across the globe. In September 2020, the U.S. FDA published performance data on 58 different molecular coronavirus tests and recognized both DiaCarta’s QuantiVirus SARS-CoV-2 Test Kit and QuantiVirus SARS-CoV-2 Multiplex Test Kit as two of the most sensitive test kits authorized under EUA in the United States. Outside of the United States, our QuantiVirus SARS-CoV-2 Test kits have been CE/IVD marked and approved in the European Union, North America, and Asia.

We are also developing and commercializing our CE/IVD-certified QuantiVirus HPV (human papillomavirus) E6/E7 mRNA test to help clinicians identify individuals who are most at risk of persistent infection. HPV has shown a major risk factor in the development of pre-invasive or invasive carcinoma, and other detection methods do not distinguish between persistent and transient infections. Our research concludes that HPV E6/E7 mRNA detection in cervical exfoliated cells can be a potential triage option for HPV-positive women who are most likely at risk for cervical cancer.

Another new development at DiaCarta is the interest in pursuing companion diagnostics for rare diseases that have been previously overlooked.

AM: What can we expect for the future of liquid biopsy and for DiaCarta?

AZ: Liquid biopsy will continue to grow as healthcare providers address the need for accurate early cancer detection and treatment toxicity monitoring. Liquid biopsy developers must also commercialize liquid biopsy tests, so they are widely available and easily attainable for healthcare professionals. DiaCarta continues to develop its diagnostic technologies by initiating more in vivo studies and gathering additional evidence that its tests are not only clinically applicable, but also highly sensitive and specific for each condition. We also envision our technologies expanding into other indications besides cancer and COVID-19 diagnostics. Because XNA are designed to be 100% complementary to wild-type DNA sequences, this technique may be adapted across a variety of indications and diagnostics. DiaCarta recognizes the potential of XNA and SuperbDNA technology to detect and monitor other disease states through targeting specific RNA and DNA sequences, and we look forward to advancing our liquid biopsy technologies in other diagnostic applications.

Aiguo Zhang was speaking to Anna MacDonald, Science Writer for Technology Networks.