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Reliable Novel Liquid Biopsy technology for early detection of colorectal cancer

Periodic Reporting for period 2 - Liqbiopsens (Reliable Novel Liquid Biopsy technology for early detection of colorectal cancer)

Reporting period: 2017-07-01 to 2018-12-31

According to the World Health Organization (WHO), cancer is one of the leading causes of death worldwide, with 12.7 million cancer cases and 8.2 million deaths in 2012. The most frequently occurring forms of the disease in the EU are colorectal, breast, prostate and lung cancers. In men, lung cancer is the most frequent cause of cancer death, while in women, it is breast cancer. In both men and women, colorectal cancer is the second most common cause of cancer death. Although significant advances are being made in the fight against the disease, cancer remains a key public health concern and a tremendous burden on European societies.
In the past, EU-level cooperation has proven to be of added value in the fight against cancer. The Commission is therefore continuing its efforts to support Member States in their efforts to protect the health of European citizens by taking concrete action. The ambitious goal set by EC is to reduce cancer incidence by 15% by 2020.
The overall aim ofLIQBIOPSENSproject is the further development and validation in real settingsof a novel diagnostic platform for the early and fast detection of ctDNA and their KRAS and BRAF mutations associated to colorectal cancer through blood samples. The main features of LIQBIOPSENS are:
• Reliability
• Low-cost
• Multiplexing Capacities
• Sensitivity
• Short analysis time
• User-friendly
• Flexibility
The ultimate goal of LIQBIOPSENS is the introduction of our novel diagnostic system, which have significant advantages with respect to the current methods into the market place. One of the key points in LIQBIOPSENS approach is that our system will be evaluated by professional end-users in order to assure commercial success.
Work performed in LIQBIOPSENS project can be divided in eight main activities:

1) Development of a disposable test cartridge that acts as the basic sensing unit of the whole system. During the project, a disposable test cartridge prototype has been developed and characterized. A complete production procedure, including quality control and sensor surface functionalization methods, has been implemented.
2) Development of a kit of chemical reagents to be used with the disposable test cartridge for the determination of up to 27 different mutations in the KRAS and BRAF genes. During the project, DestiNA has designed DGL probes and the chemistries needed for the preparation of SMART Nucleobases containing different tags, the “so called” BOT-SMART Bases. These reagents have been characterized and tested.
3) Optimal integration of both the sensing technology of AWSENSORS and the chemical DNA testing technology of DESTINA. FORTH has developed a complete acoustic detection protocol that has been applied to the final LIQBIOPSENS platform.
4) LIQBIOPSENS project deals with the market-oriented evolution of the LIQBIOPSENS platform. A prototype platform has been developed considering the very useful feedback from the end-users and liquid biopsy market trends. Developments carried out include mechanical and thermal design, electronic hardware, firmware and software implementations, and the integration of an automatic liquid handling systems capable of running different assay protocols autonomously.
5) Validation of the prototype in real application scenarios. During the project, the clinical validation study has been planned. A cohort of CRC patients with different mutations in KRAS and BRAF genes have been recruited for the validation study. Samples have been obtained and stored and patient data kept in a sample data base. Quality of the extracted DNA has been checked. A training program has been followed by the validation team to learn how to use the Liqbiopsens platform. Finally, real samples have been evaluated by Liqbiopsens platform and the results compared with reference analytic methods.
6) Innovation management allowed us to define a methodology to select and prioritize the implementation of new features. A very comprehensive report has been generated on companies developing or commercializing their own liquid biopsy tests or platforms. Following EC recommendations, Liqbiopsens market positioning study has been included, as well as a preliminary FtO (Freedom to Operate) analysis.
7) Exploitation activities consisted on the development of a comprehensive business plan to transfer the results of the project to the society. Liquid biopsy market study carried out within innovation management activities have been used as starting point to develop our exploitation strategy. A Joint Venture company has been selected as the optimal vehicle to implement the necessary steps to commercialize the valuable results of the project.
8) Last but not least, efforts in dissemination have also been made during these 36 months. A dissemination, communication and exploitation strategy has been planned. A project website has been developed based on this strategy. Two LIQBIOPSENS brochures have been designed and released. Press notes and articles for the general public have been launched. Consortium has attended workshops and conferences and scientific papers have been published in international open source peer reviewed journals. Other important achievement encompassed in the dissemination activities is the Foundation of “International Society of Liquid Biopsy”.
LIQBIOPSENS proposal addresses a relevant problem for the health sector: finding a reliable tool for early diagnostic and evolution monitoring of colorectal cancer. Traditionally, invasive and costly outpatient procedures (colonoscopy, flexible sigmoidoscopy) and/or non-specific specimen testing (FOB, FIT) have been used as screening methods for CRC. If tumours are located, tissue biopsy is employed to determine their nature.
While this procedure can provide important information about the patient disease, tissue biopsy can be painful, represents a single snap-shot in time, is subjected to significant selection bias and if the tumour tested was first detected by palpation or imaging, it may already be so large that the disease is well advanced. Furthermore, when the tumour tissue is removed or it is inaccessible, further analysis is impossible. Therefore, cancer remains a ghost disease when primary tumours are removed through surgery, meaning that there are not tools to assess the efficiency of treatments or prevent metastasis
LIQBIOPSENS approach is based on the ‘liquid biopsy’ concept. A blood sample form the patient is used to study KRAS and BRAF mutations in ctDNA related to CRC. LIQBIOPSENS removes significant barriers for the widespread implantation of screening methods for CRC by:
· Avoiding invasive and painful outpatient procedures.
· Reducing costs of the current screening procedures. (between 40 and 50 €)
· Being specific and reliable. (detect ctDNA in 100% of patients with stage II–IV CRC and in 75% of patients with stage I, with 100% specificity).
· Reducing required time analysis (30-60 minutes)

LIQBIOSENS will provide another tool in this field, facilitating some of the issues that other technologies have:
· Multiplexing capabilities of LIQBIOPSENS system allow the user to evaluate 27 different KRAS and BRAF mutations in one single measurement (30-60 minutes).
· LIQBIOPSENS platform cost is reduced if compared with alternatives due to its inexpensive acoustic wave transduction method.
· Reduction of typical “false positives” of PCR-based technologies
Cartridge inserted to the platform
X-ray image of the array
Disposable Test Cartridge
Liqbiopsens Platform prototype
Array wafer
Detail of the cartridge connection interface
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