The approach worked on by Dr Taly’s team involved looking for genetic or epigenetic alterations associated with the development of cancer, which serve as acutely specific biomarkers. One of the primary methods for this is liquid biopsy, where fluid samples are investigated for tumour related elements (including nucleic acids or cells) that may be circulating in the blood. Specific DNA sequences are commonly detected using conventional polymerase chain reaction (PCR); improvements in detection methods such as droplet-based digital PCR (ddPCR) for detecting rare events like the early onset of cancers have enabled their use in clinical settings. The prompt detection of cancers through circulating tumour DNA (ctDNA) analysis could have a significant impact on the morbidity and mortality of localised and metastatic colorectal cancer patients.
Complications of Scale Hindering PCR Analysis
However, bloodborne ctDNA concentrations are often fractional relative to concentrations of normal DNA coming from non-tumour cells. “Here comes the need for sensitivity,” Dr Taly explained. “You’re really at the single-molecule level – ctDNA can be detected within blood samples, but since it presides in very low amounts it may only represent a tiny fraction of the total circulating DNA.” Conventional PCR-based procedures presented significant limitations to research when the project began, back in 2008, while the highly sensitive procedures necessary to detect trace ctDNA cannot do so at very low quantities.
“Here comes the need for sensitivity… ctDNA can be detected in blood samples, but since it presides in very low amounts it may only represent a tiny fraction of the total circulating DNA.”
Data collected by the research team on colorectal cancer, lung cancer and pancreatic cancer suggested the typical concentrations of ctDNA that may be anticipated in patients with advanced cases of these cancers. “We looked into how many patients will have less than 2% of ctDNA, and this fraction is quite high,” continued Dr Taly. “It’s 16% in colon cancer, 45% in lung cancer and 26% in pancreatic cancer.” Essentially, a high proportion of patients will demonstrate a low amount of ctDNA even in cases of advanced cancer (as seen in Figure 1), so the challenge for Dr Taly and her team was the development of a reliable method for ctDNA screening that detected as many mutations as possible.
Applications of ddPCR for Localised and Metastatic Colorectal and Cancer Patients
The solution to the TRAM Research Group’s Gordian Knot concerned the use of digital droplet PCR (ddPCR) to enable the high-resolution identification of mutated
Dr Taly’s laboratory assembled what she affectionately described as “homemade microfluidic stations” for development and platform design. Dedicated improvements included a customised optical setup and microfluidic chips; a clinical analysis of samples was carried out in collaboration with Raindance Technologies, using the company’s RainDrop system for assay development and clinical validation. Screening for universal colorectal cancer markers was also assessed in droplets with the use of methylated markers, enabling researchers to discriminate malign tumour tissues from healthy adjacent tissues. An analysis of 56 tumour tissues revealed that all were positive, with 94.3% positive for one marker.
- 📹 Watch: Exclusive Interview with Ed Schuuring of University Medical Center Groningen About the Future of dPCR
- Discover: Potential Applications of Liquid Biopsy for Easy and Difficult Cancers
- Read: NextGen Omics Report on Current Market Trends and Industry Challenges
Dr Taly told the audience that ddPCR allowed for the performance of quantitative and highly sensitive measurements at a resolution of 1/200,000 mutant DNA in wild-type DNA, as opposed to 1/10 in bulk experiments. Even at high dilutions, the detection of target sequences through this procedure is quantitative, making it an efficient means for identifying mutant sequences and a potential tool for addressing challenges in personalised medicine. Highly sensitive methods are required for ctDNA detection and quantification in order to detect cancerous or otherwise malign DNA. Even advanced cancer patients often registered less than 2% ctDNA in their plasma; earlier cancer developmental stages aligned with the lower patient detection through ctDNA monitoring.
Future Applications of Liquid Biopsy for Cancer Detection
The main conclusions from this research were that a ctDNA follow-up could be a pertinent marker of cancer progression, since diagnostics suggest a correlation with conventional markers. Moreover, a ctDNA follow-up could be the only molecular marker of cancer progression for CEA-negative patients. One of the many practical applications of this procedure is that healthcare providers could determine if a particular chemotherapy treatment is effective after one cycle, instead of administering several rounds of unsuccessful therapy. Dr Taly concluded her presentation by asserting that liquid biopsy could be used to track tumour DNA in advanced and early colorectal cancer patients, with optimism for its use in clinical settings in years to come.
Dr Taly will be speaking with Oxford Global again at this year’s NextGen Omics UK event, running from November 09 to November 10. To register your interest or find out more, visit the NextGen Omics portal.
Postel, M., Roosen, A., Laurent-Puig, P., Taly, V. and Wang-Renault, S., 2017. Droplet-based digital PCR and next generation sequencing for monitoring circulating tumor DNA: a cancer diagnostic perspective. Expert Review of Molecular Diagnostics, 18(1), pp.7-17.