Periodic Reporting for period 1 - FG_WIT (AUTOMATED PROTOTYPE OF CANCER DETECTION FROM CTDNA)
Período documentado: 2023-07-01 hasta 2023-12-31
Early cancer detection appears to be the best strategy to reduce cancer mortality. For instance, when a colorectal cancer is detected in stage 1, chances of survival at 5 years are about 90%, whereas 10% when it is detected in stage 4. The majority of colorectal cancers are detected in stage 3.
Europe has identified that 200, 000 lives could be saved every year with an earlier colorectal cancer detection alone. Early cancer detection faces several challenges:
- Current screening methods show a low acceptability (25% for the FIT in colorectal cancer)
- The sensitivity of current methods is not high enough (45% for the FIT)
Liquid biopsy offer interesting and promising answers to these challenges as the acceptability of blood or urine sampling has been shown to be much higher that the FIT (at least 65%) among targeted populations. The analysis of circulating tumour DNA (ctDNA) allows the detection of cancer from such blood samples. ctDNA can be analysed with different technique trading off sensitivity with scalability. Next Generation Sequencing based technologies are the most scalable ones, but lack sensitivity.
Floating Genes is addressing this challenge by developing new molecular biology tools to detect rare mutations in the liquid biopsy, using next generation sequencing (NGS). Most cancers could be detected earlier, and this technology will also be applied to the monitoring of cancers. Floating Genes is focusing on digestive cancers (colorectal and pancreatic) before expanding to other cancers, through proof of concepts and licensing deals.
The proposed project is key for Floating Genes to reach the market: it will deliver a minimum viable product (MVP), and a market fit by allowing Floating Genes to safely replicate the analysis on human samples (and allow sales in research use only). In other words, it will transform a bench technology in a reliable product usable in clinical practice. Floating Genes has developed a NGS-based technology (patent pending) that can detect rare mutations from circulating tumor DNA with very high sensitivity. The limit of mutation detection in monopolex synthetic experiments is 0.00001%. Enhance-seq will thus be very useful for detecting and tracking mutations in cancer patients from a very early stage.
Europe has identified that 200, 000 lives could be saved every year with an earlier colorectal cancer detection alone. Early cancer detection faces several challenges:
- Current screening methods show a low acceptability (25% for the FIT in colorectal cancer)
- The sensitivity of current methods is not high enough (45% for the FIT)
Liquid biopsy offer interesting and promising answers to these challenges as the acceptability of blood or urine sampling has been shown to be much higher that the FIT (at least 65%) among targeted populations. The analysis of circulating tumour DNA (ctDNA) allows the detection of cancer from such blood samples. ctDNA can be analysed with different technique trading off sensitivity with scalability. Next Generation Sequencing based technologies are the most scalable ones, but lack sensitivity.
Floating Genes is addressing this challenge by developing new molecular biology tools to detect rare mutations in the liquid biopsy, using next generation sequencing (NGS). Most cancers could be detected earlier, and this technology will also be applied to the monitoring of cancers. Floating Genes is focusing on digestive cancers (colorectal and pancreatic) before expanding to other cancers, through proof of concepts and licensing deals.
The proposed project is key for Floating Genes to reach the market: it will deliver a minimum viable product (MVP), and a market fit by allowing Floating Genes to safely replicate the analysis on human samples (and allow sales in research use only). In other words, it will transform a bench technology in a reliable product usable in clinical practice. Floating Genes has developed a NGS-based technology (patent pending) that can detect rare mutations from circulating tumor DNA with very high sensitivity. The limit of mutation detection in monopolex synthetic experiments is 0.00001%. Enhance-seq will thus be very useful for detecting and tracking mutations in cancer patients from a very early stage.
Activities funded with the Women TechEU grant in this program :
• Setup the automated platform :
The Magelia of Inorevia is a microfluidic platform, designed to automate molecular biology protocols, improved their performance, and save up to 40 folds on reagents costs. The closed system it prevents from sample contamination. During the setup of the Magelia, the following activities have been performed:
o Protocol implementation strategy: Floating Genes’ protocol have been broken down, and each step has been prioritized in discussion with the team of Inorevia. It was decided to implement the first two PCR of the protocol, as they are the most standardized steps so far. The ligation and purification that follow these two PCR are currently being implemented.
o Test of microfluidics behaviors of Floating Genes’ reagents (viscosity, merging of droplets…). Samples of reagents were prepared and tested by Inorevia. This step allowed a good fidelity between the
o Installation of the machine and training of the team: the team of Inorevia came to install and calibrate the machine in Floating Genes’ laboratory. This point is crucial, as the machine runs on microfluidics, needs to be calibrated if it is moved. The team of Floating Genes was trained to use the Magelia. Specific attention is required in the sealing of plates, as well as setting up reagents and samples in the machine.
• Test cfDNA reference samples
The objective of this step is to test samples that are closer to clinical samples, than the synthetic samples. For that purpose, we created samples using human genomic DNA purchased from Promega, and digested with an endonuclease to mimic the fragmentation pattern of cell free DNA. We then added synthetic mutant DNA in the mixture in different Variant Allele Frenquencies (VAFs) and tested the protocol as if they were clinical samples.
Here we have shown that the Magelia delivers the expected results in line with the ones obtained until now manually. The Magelia improved the results at low VAF. More data are required to confirm these results.
• Evaluate the performance of the automated protocol vs. the manual implementation
The full protocol has been automated in two steps. The first 2 PCRs were implemented alone, and have shown superior performance. Then the full protocol has been implemented and tested, and improvement are required to tackle false positive resutls.
Comparison between the automated workflow and the manual workflow
The automated workflow has shown the following gains:
• 7 times faster than the manual workflow
• 40% more expensive per sample
• higher sensitivity at 0.06% mutant allele frequency (to be further confirmed)
• comparable erros rates on controls
Business model calibration: the economic comparison revealed that Enhance-seq automated in the Magelia is more expensive than the manual protocol, mostly because of the price of Inorevia’s cartridges. This price is based on a small purchased quantity. By purchasing larger amounts, the price of Inorevia’s cartridges may become competitive. Nevertheless, the gain in reproducibility will make the solution safer and more robust. Indeed the procedures to run the protocol are simpler, and any error can be traced back in the machine logs.
Achievements:
• The critical parts of the protocol of Floating Genes has successfully been translated into the Magelia so far. Further work is in process to make the full protocol automated and reliable on the Magelia.
• Tests on synthetic samples and cfDNA samples have been carried out, and are conclusive on the automated part of the protocol
• The comparison with the manual workflow of the automated part so far shows interesting gains, to be further confirmed.
• Setup the automated platform :
The Magelia of Inorevia is a microfluidic platform, designed to automate molecular biology protocols, improved their performance, and save up to 40 folds on reagents costs. The closed system it prevents from sample contamination. During the setup of the Magelia, the following activities have been performed:
o Protocol implementation strategy: Floating Genes’ protocol have been broken down, and each step has been prioritized in discussion with the team of Inorevia. It was decided to implement the first two PCR of the protocol, as they are the most standardized steps so far. The ligation and purification that follow these two PCR are currently being implemented.
o Test of microfluidics behaviors of Floating Genes’ reagents (viscosity, merging of droplets…). Samples of reagents were prepared and tested by Inorevia. This step allowed a good fidelity between the
o Installation of the machine and training of the team: the team of Inorevia came to install and calibrate the machine in Floating Genes’ laboratory. This point is crucial, as the machine runs on microfluidics, needs to be calibrated if it is moved. The team of Floating Genes was trained to use the Magelia. Specific attention is required in the sealing of plates, as well as setting up reagents and samples in the machine.
• Test cfDNA reference samples
The objective of this step is to test samples that are closer to clinical samples, than the synthetic samples. For that purpose, we created samples using human genomic DNA purchased from Promega, and digested with an endonuclease to mimic the fragmentation pattern of cell free DNA. We then added synthetic mutant DNA in the mixture in different Variant Allele Frenquencies (VAFs) and tested the protocol as if they were clinical samples.
Here we have shown that the Magelia delivers the expected results in line with the ones obtained until now manually. The Magelia improved the results at low VAF. More data are required to confirm these results.
• Evaluate the performance of the automated protocol vs. the manual implementation
The full protocol has been automated in two steps. The first 2 PCRs were implemented alone, and have shown superior performance. Then the full protocol has been implemented and tested, and improvement are required to tackle false positive resutls.
Comparison between the automated workflow and the manual workflow
The automated workflow has shown the following gains:
• 7 times faster than the manual workflow
• 40% more expensive per sample
• higher sensitivity at 0.06% mutant allele frequency (to be further confirmed)
• comparable erros rates on controls
Business model calibration: the economic comparison revealed that Enhance-seq automated in the Magelia is more expensive than the manual protocol, mostly because of the price of Inorevia’s cartridges. This price is based on a small purchased quantity. By purchasing larger amounts, the price of Inorevia’s cartridges may become competitive. Nevertheless, the gain in reproducibility will make the solution safer and more robust. Indeed the procedures to run the protocol are simpler, and any error can be traced back in the machine logs.
Achievements:
• The critical parts of the protocol of Floating Genes has successfully been translated into the Magelia so far. Further work is in process to make the full protocol automated and reliable on the Magelia.
• Tests on synthetic samples and cfDNA samples have been carried out, and are conclusive on the automated part of the protocol
• The comparison with the manual workflow of the automated part so far shows interesting gains, to be further confirmed.
The potential impacts of the automated solutions are the following:
- making the genetic diagnostic test of Floating Genes available as a service for research laboratories in Europe
- allowing highly replicable and robust diagnosis at a small scale, and with a minimal QMS
- early detection of cancer would save many lives
- close and highly sensitive monitoring of cancer patients will increase overall survival to cancer
Further perspectives:
- develop new protocol, especially mutli-omics protocols, which would be too long to be performed manually, but feasible in microfluidics, and would provide a lot of gains in terms of performance (sensitivity, specificity).
- scaling the automated solution to higher throughput would be interesting to reach more patients
- deploy the protocol on the Magelia plateforms already working in European laboratories
Steps needed:
- certification of the solution regarding the IVDR regulation
- further challenge the business model of this solution and scale it to larger populations
- making the genetic diagnostic test of Floating Genes available as a service for research laboratories in Europe
- allowing highly replicable and robust diagnosis at a small scale, and with a minimal QMS
- early detection of cancer would save many lives
- close and highly sensitive monitoring of cancer patients will increase overall survival to cancer
Further perspectives:
- develop new protocol, especially mutli-omics protocols, which would be too long to be performed manually, but feasible in microfluidics, and would provide a lot of gains in terms of performance (sensitivity, specificity).
- scaling the automated solution to higher throughput would be interesting to reach more patients
- deploy the protocol on the Magelia plateforms already working in European laboratories
Steps needed:
- certification of the solution regarding the IVDR regulation
- further challenge the business model of this solution and scale it to larger populations