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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - ULTRAPLACAD (ULTRAsensitive PLAsmonic devices for early CAncer Diagnosis)

Teaser

Summary

Colorectal cancer is a lethal disease responsible for more than 200,000 deaths per year in Europe, with similar absolute numbers for men and women. It is the second most common cancer in Europe in terms of incidence rates (after breast cancer) and also the second cancer in terms of mortality, with only lung cancer contributing more cancer deaths.
Early detection through population screening has been shown to reduce the mortality from colorectal cancer. Unfortunately, colorectal cancer screening campaigns suffer from low compliant rates linked to the invasive currently available diagnostic methods. Standard clinical protocols for people at high risk of developing colorectal cancer detection are based on colonoscopy and tissue biopsy, which involve sampling cells with the use of small-gauge needle. Tissue biopsy is also applied for the surveillance and follow-up of patients with colorectal cancer, and in these cases the procedure is even more invasive, and sometimes technically challenging or even impossible.
Cancer biomarkers circulating in body fluids are widely used, but they lack sensitivity and specificity, reducing their applicative value. This problem has been tackled by attempting to develop cost-effective, highly reliable and minimally-invasive biomarkers and integrated diagnostic approaches. However, barriers remain that prevent a widespread use of these approaches.
ULTRAPLACAD aims at the development of a novel robust in vitro diagnostic system for cancer diagnosis, prognosis, patient follow-up and therapy efficacy assessment based on molecular analysis of peripheral blood (liquid biopsy).
ULTRAPLACAD is a plasmonic-based device with integrated nanostructures for the detection of mutated DNAs, microRNAs and tumor autoantibodies (a-TAAs). The detection of all these molecular cancer biomarkers freely circulating in blood of colorectal cancer patients are combined in a single device that is expected to overcome hurdles and limitations of the available approaches. The ULTRAPLACAD project has developed assays for the detection of circulating DNA and microRNA with no need for preliminary amplification of the nucleic acid sequences. In addition, a-TAAs are detected with an improved sensitivity with respect to conventional fluorescence detection platforms.
ULTRAPLACAD has developed a bimodal industrial prototype integrating novel surface plasmon resonance imaging (NESPRI) and plasmon-enhanced fluorescence (PEFSI) sensing technologies, thus providing an unique and up to now unprecedented platform for the comprehensive detection of nucleic acids and protein biomarkers in blood plasma. Automated fabrication processes suitable for low cost mass production have been also developed and applied to produce disposable integrated chips.

Work performed

The project has started with the identification of end users requirements and technical specifications of ULTRAPLACAD final industrial prototype. Efficient synthetic probes for the detection of nucleic acid sequences have been designed and synthesized and procedures for the needed chemical modification of nanostructures have been selected and tested. Nanoparticle-enhanced assays that operate with attomolar sensitivity in detecting DNA and femtomolar sensitivity in detecting microRNAs have been developed. PEFSI detection of a-TAAs is performed by attaching specifically designed and synthesized thermoresponsive polymers on PEFSI nanostructures.
The industrial prototype of a bimodal reader for NESPRI and PEFSI detection has been developed and tested thanks to the knowledge acquired by designing and fabricating NESPRI and PEFSI laboratory prototypes. A leaflet describing the industrial prototype was prepared to attract potential customers.
Animal models of liquid biopsy have been developed and retrospective cohorts of colorectal carcinoma patient plasma samples have been recruited. An internal mini-bank comprising tumor tissue, plasma, and purified analytes was organized and samples shared among partners.
The process for the high-volume fabrication of nanostructured disposable chips has been developed and demonstrated. The microfluidics part of the disposable chip has been produced by demonstrating the high-volume manufacturing capability. The integration of the nanostructured optical surface and microfluidics part in large-volume production has also been demonstrated. Protocols for the PCR-free detection of circulating DNAs and microRNAs from human plasma have been defined.
The ULTRAPLACAd industrial prototype has been installed at the Laboratory of Clinical Pathology of the Italian National Cancer Institute (Rome) and a public demonstration was given during the ULTRAPLACAD end-user workshop.
The project results were made available to the scientific community through 19 Open Access peer review publications and to broad public through the project website and YouTube video (https://www.youtube.com/watch?v=88n3IRsWTm8). Exploitation strategies were discussed and defined. They include participation to new EU funded projects (short term), commercialize the platform (long term) and agreement with companies outside the consortium (long term).
To summarize, the main results achieved are:
- NESPRI and PEFSI nanostructure geometries have been identified;
- Probes for the selected all-RAS mutations and microRNAs have been synthesized;
- Candidate antigenic peptides for a-TAA detection have been defined;
- Attomolar SPRI detection of all-RAS mutations and femtomolar detection of microRNAs have been demonstrated;
- The high-volume fabrication of nanostructured disposable chips has been demonstrated;
- The ULTRAPLACAD industrial prototype, based on the NESPRI and PEFSI readers lab prototypes, has been fabricated, tested and public demonstration given.

Final results

ULTRAPLACAD has developed a new plasmonic system for minimal-invasive colorectal cancer diagnosis based on the ultrasensitive analysis of nucleic acids and protein biomarkers circulating in human blood. The detection of mutated DNA sequences and microRNAs in human plasma samples, without PCR amplification, and a-TAAs, with an improved sensitivity with respect to conventional fluorescence detection platforms, has been demonstrated. These characteristics put ULTRAPLACAD at the forefront of molecular diagnosis technologies as a unique and up to now not existing platform for the comprehensive detection of nucleic acids and protein biomarkers in blood plasma. Moreover, it offers the advantage of reduced sample contaminations, analysis time, assay costs due to reduced complexity of assays and respective readers.
Very few FDA approved non-invasive methods for colorectal cancer screening have been available only recently. Based on cost analysis and benchmarking of the existing commercial systems (digital PCR) ULTRAPLACAD system seems to be very competitive, with estimated manufacturing cost for aTAA (200 analytes), ctDNA (50 spots) and miRNA (8 spots) reasonable, if compared to dPCR cost.
ULTRAPLACAD platform will improve diagnosis and also enable a more specific selection of patients for therapy, as well as allows therapy monitoring from liquid biopsies, thus reducing invasive procedures and improving patient management. The development of ULTRAPLACAD platform enables a broad range of clinical applications and is, therefore, a step to saving thousands of lives and, at the same time, avoiding additional strain on the healthcare systems in developed countries.