Periodic Reporting for period 2 - diaRNAgnosis (A novel platform for the direct profiling of circulating cell-free ribonucleic acids in biofluids)
Berichtszeitraum: 2023-06-01 bis 2025-05-31
Despite extensive research into circulating cell-free RNAs (ccfRNAs), especially microRNAs (miRNAs), their potential as non-invasive cancer biomarkers has not yet translated into routine clinical use. This gap stems from high costs, complex protocols, and a lack of standardised, scalable detection methods—factors that hinder early and reliable diagnosis of testicular germ cell tumours (TGCTs) and prostate cancer (PCa).
Early detection is critical. TGCTs are the most common cancer in young men, while PCa is a major cause of cancer-related deaths in older men. Current diagnostic delays often lead to advanced disease and costly, aggressive treatments. Using ccfRNA profiles from blood or urine could enable earlier, more accurate diagnoses, reduce reliance on invasive biopsies, and support personalised treatment.
The diaRNAgnosis project addresses these challenges through three key objectives: identifying miRNA signatures for TGCT and PCa, developing a benchtop reader (ODG platform) for rapid, amplification-free ccfRNA detection using dynamic chemical labelling and Silicon Photomultiplier technology, and validating the system with real patient samples.
By combining biomarker discovery with innovative sensing and robust clinical validation, diaRNAgnosis aims to deliver a practical, cost-effective liquid biopsy platform that transforms cancer diagnostics and improves patient outcomes.
Early detection is critical. TGCTs are the most common cancer in young men, while PCa is a major cause of cancer-related deaths in older men. Current diagnostic delays often lead to advanced disease and costly, aggressive treatments. Using ccfRNA profiles from blood or urine could enable earlier, more accurate diagnoses, reduce reliance on invasive biopsies, and support personalised treatment.
The diaRNAgnosis project addresses these challenges through three key objectives: identifying miRNA signatures for TGCT and PCa, developing a benchtop reader (ODG platform) for rapid, amplification-free ccfRNA detection using dynamic chemical labelling and Silicon Photomultiplier technology, and validating the system with real patient samples.
By combining biomarker discovery with innovative sensing and robust clinical validation, diaRNAgnosis aims to deliver a practical, cost-effective liquid biopsy platform that transforms cancer diagnostics and improves patient outcomes.
The diaRNAgnosis project has brought together an international consortium of experts to develop a fast, non‑invasive diagnostic tool for two of the most widespread men’s cancers: testicular germ cell tumours (TGCTs) and prostate cancer (PCa). From day one, the partners organised their work into clearly defined stages, ensuring that biological discovery, device engineering and clinical testing progressed in tandem.
In the first phase, scientists focused on finding reliable molecular fingerprints in blood and urine by studying circulating cell-free RNAs (ccfRNAs). Early results confirmed that a molecule called miR-371a-3p is a highly accurate indicator of TGCT. The team also discovered nine additional microRNAs linked to tumour presence—three of which were rigorously validated using real-time PCR—and identified long non-coding RNAs and microRNAs that appear at elevated levels in the urine of men with prostate cancer. These validated markers formed the foundation for the next development steps.
At the same time, scientists at OPTOI and DESTINA Genomica improved the ODG device. This device was realised by merging dynamic chemical labelling (DCL) technology with a silicon photomultiplier (SiPM) detector, further enhancing signal detection and timing resolution. The first version used time-gated fluorescence to detect RNA molecules labelled with special luminescent tags. To reach lower detection limits, a second version shifted to a chemiluminescent approach enhanced by polystyrene nanoparticles, achieving even greater sensitivity. Key assay reagents—including labelled “SMART” nucleotide (SMART-Base) and magnetic beads functionalized with basic peptide nucleic acid (PNA) to capture target RNAs—were developed in parallel, ensuring that the chosen chemiluminescent workflow worked seamlessly.
Midway through the project, two prototype ODG devices were installed in partner laboratories in Trento (Italy) and Granada (Spain). Automated sample handling minimised user variation, and blinded tests on patient-derived samples confirmed that the ODG platform could detect cancer-derived RNAs without any amplification steps, matching or exceeding the performance of standard PCR-based methods. These successful trials demonstrated that the system is ready for broader clinical evaluation.
To support future impact, the consortium also invested in training and outreach. More than fifty researchers and technicians took part in cross‑sector secondments and workshops, gaining hands-on experience in advanced laboratory methods, data analysis and regulatory processes in the fields covered by the project. The team published peer-reviewed articles detailing their discoveries and platform performance and presented results at international conferences. A public-facing website, social-media updates and participation in events such as the European Researchers’ Night helped share the project’s progress with wider audiences.
By the end of the reporting period, diaRNAgnosis had delivered a fully functioning, amplification-free liquid‑biopsy platform, a robust set of RNA biomarkers for TGCT and PCa, and a clear roadmap for regulatory approval and commercialisation. These achievements mark a major step toward easy-to-use tests that could bring faster, more accurate cancer diagnosis into routine clinical practice, ultimately benefiting patients and healthcare systems worldwide.
In the first phase, scientists focused on finding reliable molecular fingerprints in blood and urine by studying circulating cell-free RNAs (ccfRNAs). Early results confirmed that a molecule called miR-371a-3p is a highly accurate indicator of TGCT. The team also discovered nine additional microRNAs linked to tumour presence—three of which were rigorously validated using real-time PCR—and identified long non-coding RNAs and microRNAs that appear at elevated levels in the urine of men with prostate cancer. These validated markers formed the foundation for the next development steps.
At the same time, scientists at OPTOI and DESTINA Genomica improved the ODG device. This device was realised by merging dynamic chemical labelling (DCL) technology with a silicon photomultiplier (SiPM) detector, further enhancing signal detection and timing resolution. The first version used time-gated fluorescence to detect RNA molecules labelled with special luminescent tags. To reach lower detection limits, a second version shifted to a chemiluminescent approach enhanced by polystyrene nanoparticles, achieving even greater sensitivity. Key assay reagents—including labelled “SMART” nucleotide (SMART-Base) and magnetic beads functionalized with basic peptide nucleic acid (PNA) to capture target RNAs—were developed in parallel, ensuring that the chosen chemiluminescent workflow worked seamlessly.
Midway through the project, two prototype ODG devices were installed in partner laboratories in Trento (Italy) and Granada (Spain). Automated sample handling minimised user variation, and blinded tests on patient-derived samples confirmed that the ODG platform could detect cancer-derived RNAs without any amplification steps, matching or exceeding the performance of standard PCR-based methods. These successful trials demonstrated that the system is ready for broader clinical evaluation.
To support future impact, the consortium also invested in training and outreach. More than fifty researchers and technicians took part in cross‑sector secondments and workshops, gaining hands-on experience in advanced laboratory methods, data analysis and regulatory processes in the fields covered by the project. The team published peer-reviewed articles detailing their discoveries and platform performance and presented results at international conferences. A public-facing website, social-media updates and participation in events such as the European Researchers’ Night helped share the project’s progress with wider audiences.
By the end of the reporting period, diaRNAgnosis had delivered a fully functioning, amplification-free liquid‑biopsy platform, a robust set of RNA biomarkers for TGCT and PCa, and a clear roadmap for regulatory approval and commercialisation. These achievements mark a major step toward easy-to-use tests that could bring faster, more accurate cancer diagnosis into routine clinical practice, ultimately benefiting patients and healthcare systems worldwide.
diaRNAgnosis has pushed well beyond existing diagnostic capabilities by turning circulating microRNA biomarkers into a fully integrated, amplification-free liquid-biopsy platform. Rather than relying on complex extraction or PCR steps, our benchtop ODG device merges dynamic chemical labelling (DCL) reagents with a silicon photomultiplier to interrogate tumour-derived RNAs directly in plasma and urine. In head-to-head comparisons against today’s gold-standard PCR workflows, the ODG device has matched sensitivity and specificity for key markers—miR-371a-3p in testicular germ cell tumours and miR-375 in prostate cancer—while slashing hands-on time and consumable costs. By integrating chemiluminescent detection via biotinylated SMART-Bases, we’ve extended the assay’s dynamic range into sub-femtomolar territory, achieving detection limits that allow PCR-free testing of circulation ccfRNAs.
Building on these breakthroughs, we developed multiplexed assays that measure multiple microRNAs in a single run—rigorously validated against next-generation sequencing and RT-qPCR—paving the way for comprehensive tumor profiling in under an hour. Two ODG prototypes have now completed blinded evaluations on more than 30 patient samples, from benign prostatic hyperplasia to prostate cancer, consistently delivering laboratory-grade results in a fully automated format. Behind the diaRNAgnosis initiative, consortium members are currently securing funding to launch large-scale, multicenter clinical trials that will establish robust performance benchmarks across diverse patient cohorts.
The societal and economic implications could be profound. By delivering rapid, non-invasive, PCR-free absolute quantification of circulating cell-free RNA and interrogating novel molecular signatures, diaRNAgnosis eliminates the biases and variability inherent to amplification-based workflows. This direct-detection approach streamlines sample processing, reduces reliance on invasive biopsies, and accelerates time to diagnosis and treatment. Patients benefit from greater, non-invasive testing and earlier intervention, while hospitals and insurers see smoother logistics and more predictable resource allocation. Thanks to its modular chemistry and adaptable reader, the platform can be quickly reconfigured for other cancers or even infectious diseases, providing a versatile tool for emerging public-health challenges. As we move toward commercialisation, diaRNAgnosis is set not only to redefine personalized oncology diagnostics but also to deliver measurable socio-economic impact and broad societal benefit.
Building on these breakthroughs, we developed multiplexed assays that measure multiple microRNAs in a single run—rigorously validated against next-generation sequencing and RT-qPCR—paving the way for comprehensive tumor profiling in under an hour. Two ODG prototypes have now completed blinded evaluations on more than 30 patient samples, from benign prostatic hyperplasia to prostate cancer, consistently delivering laboratory-grade results in a fully automated format. Behind the diaRNAgnosis initiative, consortium members are currently securing funding to launch large-scale, multicenter clinical trials that will establish robust performance benchmarks across diverse patient cohorts.
The societal and economic implications could be profound. By delivering rapid, non-invasive, PCR-free absolute quantification of circulating cell-free RNA and interrogating novel molecular signatures, diaRNAgnosis eliminates the biases and variability inherent to amplification-based workflows. This direct-detection approach streamlines sample processing, reduces reliance on invasive biopsies, and accelerates time to diagnosis and treatment. Patients benefit from greater, non-invasive testing and earlier intervention, while hospitals and insurers see smoother logistics and more predictable resource allocation. Thanks to its modular chemistry and adaptable reader, the platform can be quickly reconfigured for other cancers or even infectious diseases, providing a versatile tool for emerging public-health challenges. As we move toward commercialisation, diaRNAgnosis is set not only to redefine personalized oncology diagnostics but also to deliver measurable socio-economic impact and broad societal benefit.