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Point-of-care blood device for fast and reliable prediction of drug response in non- small-cell lung carcinoma patients from blood samples

Final Report Summary - LUNGCARD (Point-of-care blood device for fast and reliable prediction of drug response in non- small-cell lung carcinoma patients from blood samples)

Executive Summary:
The partners
The LungCARD Consortium is composed by SMEs with a significant experience in their respective fields.
◦ STAB Vida: extensive expertise on DNA-based diagnostics.
◦ MicroLIQUID: SME manufacturer of micro fluidic chip.
◦ Moltech: manufacturer of biomedical devices, including optical, robotic and software systems.
Considering the complex nature of the project, SMEs sub-contracted RTD and demonstration activities to RTD partners:
• Faculty of Science and Technology, Portugal (FFCT): Research on genetics, nanodiagnostics and nanovectors for therapy
• University of Hull, United Kingdom (UHULL): Research in microreactor and micro fluidic technology over the last 15 years.
• Institute of Photonic Technology, Germany (IPHT): Research of novel optical (plasmonic – optics on the nanoscale) effects on metal and hybrid nanostructures.
Furthermore, Castle Hill Hospital (partner Hey) from United Kingdom offers world class care for patients with cancer and blood disorders and helps RTDs to design the diagnostic tool in accordance with clinicians/patients needs. Also they provided the patients samples for demonstration activities.

The product
Despite EGFR testing is of vital importance for NSCLC patients, to date, there's no product on the market with the following features:
• Results within ≤6 hours
• Test price below 200 Euro
• 100% of Specificity and 90% of Sensitivity
• Usability at the point-of-care
• No complex equipment to use

The market
-Lung Cancer cases: 1.8 million new cases in 2012, 12.9% of the total (2008: 1.6 million, 12,7% of all new cancer patients )
-NSCLC cases: between 75 and 85% of the total
-EGFR mutations occur in 10 to 20 percent of NSCLC tumors
-Market potential using a conservative scenario: at least 216000 patients per year (considering NSCLC as 80% of total and EGFR mutation positive as 15%)
-Considering that EGFR tests will follow the evolution of personalised medicine, a brief analysis will clarify the market potential:
.Global NSCLC market is expecting to rise at a compound annual growth rate (CAGR) of 3.3%, from $5.7 billion in 2012 to $7.9 billion in 2022, and is currently dominated by Roche’s Avastin (bevacizumab) and Tarceva (erlotinib), according to a recent Global Data report
Project Context and Objectives:
According with Globocan 2012 (http://globocan.iarc.fr/) lung cancer has been the most common cancer in the world for several decades, with an estimated 1.8 million new cases in 2012 worldwide. The main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Together, NSCLCs make up about 75% of all lung cancers (www.emedicinehealth.com). Today most of european and american hospitals follow the same treatment guidelines for NSCLC patients: all NSCLC patients should be tested for EGFR mutations by taking a biopsy. If patients have a mutation in EGFR gene should receive an epidermal growth factor receptor-tyrosine kinase inhibitor such as gefitinib (Iressa® from Astrazeneca) and afitinib (Gilotrif® from Boehringer Ingelheim). However if the patients don´t have mutations they would be treated with chemotherapy. Some patients (e.g. 30% in UK) may never get histological confirmation (biopsy is not possible) because they are too sick. The clinical laboratories use PCR-based and Sanger Sequencing technologies to perform the EGFR analysis from tumour biopsies (Fixed Paraffin Embedded samples), however low quality of results are observed. According with EMQN scheme 2014 (where STAB VIDA also participated) there were around 3% of genotyping errors mainly due to poor quality/low yield of DNA extracted from FFPE samples and highly variability of laboratory methods used for DNA extraction and quantification. The limitations associated with the poor condition of patients to collect biopsy and the current laboratory methods (as described below), brings an excellent opportunity to develop a novel tool with clear advantages for clinicians and lung cancer patients. The proposed research addresses SMEs need:
i) a point-of-care tool easy to use, allowing easy interpretation of results, accurate and relatively cheap that integrates “all laboratory-based process steps” in one single step, improve the treatment effectiveness in non-small-cell lung carcinoma patients. For this propose, micro fluidics is the adequate technical answer. Microliquid is a SME producing micro fluidics chips but needs to subcontract research for designing a suitable chip for DNA testing.
ii) replace the tumour-based assay with a blood based test, which is a minimally invasive approach for sample collection, avoiding all the problems associated with poor DNA extraction from FFPE samples. A further benefit of the blood-based assay is that the test can be done prospectively, prior to surgery and also used repeatedly as a prognostic tool to measure efficacy of cancer therapy and/or early recurrence.

Therefore, the Lung CARD proposal aims the development of a cheaper, rapid and reliable fully integrated assay that combines automatic blood sample processing and EGFR genotyping in one single step. To date no such product is available on market. The development of this tool, requires technological knowledge in several fields: genetics, nanotechnology, micro fluidics, photonic, electronics and software. The SMEs (STAB VIDA, Moltech and MicroLiquid) together will subcontract research activities to RTD performers (FFCT-UNL, UHULL and IPHT) in order to develop a biochip and bioanalyser that combines blood sample processing, detection of EGFR somatic mutations (exon 19 and 21 mutations, which together comprise 90% of EGFR mutations) and software interface for easy interpretation of results. In order to achieve the main objective, the following objectives were set forward:

Scientific and technical objectives:
• Development of a micro fluidic card pre packed with reagents for sample pre-treatment (DNA extraction from circulating lung cancer cells in the blood, purification and amplification of the specific region between exon 19-21) and genetic analysis (hybridisation of gold silver nanoprobes to the target DNA and mutation detection by micro capillary electrophoresis with an estimated cost below 50 euros per chip ready-to-use (link to D2.2 and MS4; Project result nº1 and 4, month 18)
• Development of user-friendly software interface for simple and unambiguous interpretation of EGFR genotyping results (link to D4.3 and MS5, month 20)
• Development of a bioanalyser to analyse the micro fluidic chip with an estimated cost below 15000 euros (link to D4.4 and MS5; Project result nº2, month 21)
• Analytical validation of blood test in terms of limit of detection (LOD), sensitivity, specificity, and reproducibility using 50 human blood samples. A limit of detection between 1-10% should be achieved (link to D5.4 and MS6, month 24)
• Development of a blood test suitable for EGFR mutations detection in less than 2 hours (link to D5.4 and MS6; Project result nº 3, month 24)

Legal, societal and economic objectives:
• Fulfilment of In Vitro Diagnostic Medical Device directives (93/42/EEC, 90/385/EEC and 98/79/EEC) (link to D7.5 and MS10, month 24)
• Fulfilment the clinicians and lung cancer patients needs (link to D5.4 and MS6, month 24)
• Filing for CE mark approval (link to D7.5 and MS10, month 24)
• Dissemination of diagnostic device to end-user clinicians, clinical laboratories, hospitals (link to D6.2 and MS7, month 24)
• Transfer the project results to the consortium through training activities planned to promote the uptake of the results by SME partners and (link to D7.1 and MS9, month 24)
• Ensure the protection of intellectual property rights and the exploitation of the project results (link to D7.4 and MS10, month 24)
• Increase the competitiveness of SME participants across the European Union (link to D7.1 D7.4 and MS9, month 24)
• Improvement of treatment effectiveness in advanced non-small cell lung cancer (NSCLC) patients (link to D5.4 and MS6, month 24)

Project Results:
Project result nº1: The Lung CARD lab-on-a-chip containing all reagents

The development of LungCARD chip generates two versions: LC4iv2, which includes all the reactions on chip (from circulating tumour cells capture to mutation detection) and LC4i v3, which includes the integration of circulating tumour cells capture from blood and PCR functions. The last version (LC4i v3) was made as a contingency plan due to difficulties with the electrophoretic separation of AuNP as explained below in 3).

The integrated Lungcard chip (LC4i v2) consists of a 4x4 inch square device made of three layers of glass. The largest feature is a sample chamber with a volume of approximately 12 ml, more than large enough to hold the 9 -10 ml of blood required by existing test standards for circulating tumour cell (CTC) analysis. One corner of the sample chamber leads to a valve chamber and magnetic bead (MB) wash area. This then leads to a PCR chamber, a hybridisation chamber and a separation channel. Also included a storage chamber for AuNP probes and associated channels for the electrophoretic transfer of DNA PCR products and nanoprobes. There are five inter-related chip modules that are required for chip function. A photo of LungCARD chip and represented in schematic form is shown in figure 1 (see Technical annex attached).
a) LungCARD chip metrics:
.Device dimensions: 4x4 inches, 101.6 mm x 101.6 mm.
.Bottom etched layer, approximately 100 x 100 mm.
.Top sealing layer, approximately 75 x 60 mm.
.Overall chip thickness: 4.3 mm (3 layers: 1.15 + 3 + 1.15 mm).
.Electrode wells 1 to 4 are 4 mm diameter. Electrode well 5 is 5 mm diameter.
.All five wells are 3.1 mm deep.
.PCR/hybridisation chamber fill inlets 2 mm diameter.
.Sample (Blood) chamber: 67.5 mm x 55 mm x 3.1 mm, volume: approx 12 ml.
.PCR chamber: 5.2 mm diameter, 500 micron deep.
.AuNP storage well: 3 mm diameter, 3.1 mm deep.
.Hybridisation chamber: 3.2 mm diameter, 500 micron deep.
.Electrophoresis separation channel: 250 microns wide (at widest part), 100 microns deep.
.Interconnecting channels of fluidic network (other than separation channel) 400 um wide, 100 um deep.
PPM = Porous polymer monolith; AuNP = Gold nanoprobes.
b) Chip reagents:
.Sample (Blood) chamber (capacity = 12mL): 10 EpCAM coated magnetic beads/uL of blood
.PCR Chamber (capacity=10uL): High Fidelity buffer (0.75x) MgCl2 (5 mM), dNTPs (1mM), Target DNA primer forwards (0.5 uM), Target DNA primer backwards (0.5uM) BSA/PVP/Tween (0.1%), Taq enzyme (1ul/10 ul reaction), Deionised water (make up to 10 ul)
.Wash chamber and Channel from electrode 1 to PCR chamber: PCR chamber solution
.Channel from electrode 2 to Hybridisation chamber: ion bridge (5:1:2 mixture of 60% Diallyldimethylammomium chloride solution with polyethylene glycol diacrylate and deionised water, sensitised with 1% by weight of Irgacure 651)
.Channel from electrode 3 to AuNP chamber: 3% (w/v) Linear Polydimethyl Acrylamide (LPDMA) in 1x Tris Borate EDTA pH 8.3)
. Channel from electrode 4 to Separation channel: 3% (w/v) Linear Polydimethyl Acrylamide (LPDMA) in 1x Tris Borate EDTA pH 8.3)
. AuNP storage well: 15 ul of AuNP probe mixture
. Hybridisation channel: 3% (w/v) Linear Polydimethyl Acrylamide (LPDMA) in 1x Tris Borate EDTA pH 8.3)
. Separation channel: 3% (w/v) Linear Polydimethyl Acrylamide (LPDMA) in 1x Tris Borate EDTA pH 8.3)

A simplified Lungcard chip was produced (contingency plan), LC4i v3, which comprised just two integrated functions. These are tumour cell capture and on chip PCR. The footprint of the chip was the same, although the lower PCR chamber filling inlet was moved to the bottom of the PCR chamber and all downstream features were deleted. A single inlet/outlet channel connected to the PCR chamber was retained (figure 2 - see Technical annex attached). This simplified LungCARD chip (LC4i v3) is operated in the same way as the fully integrated chip (LC4i v2), except that after the PCR step has completed, the chip is removed from the integrated system box and the contents of the PCR chamber are removed. To do this, the seals over the PCR chamber inlet and outlet are removed. A syringe with a Luer fitting is placed into the lower PCR chamber inlet and this is used to extract the PCR products. Then the PCR products retrieved from the chip are analysed using conventional, commercially available capillary electrophoresis systems. The process of chip fabrication is described in detail in D2.2.

Project result nº2: The biochip analyser

The LungCARD biochip analyser first prototype is physically slip into two units and a computer. A photo of the prototype is in figure 3 (see Technical annex attached). The first unit contents all the analysing systems, where the bio-chip is loaded to and the second unit is the controlling electronics. Such design offered better flexibility during the development of the system. Based on the several different methods used during the evaluation of the biochip, the system can be split into four different sub-units: (1) Magnetic separation unit (including chip carriage between units), (2) Thermal management units (3) Electrophoretic unit and (4) Optical detection unit. Detailed documentation of each sub-unit as well system performance are described in D4.2 and D4.4 and controlling software to operate the units is described in D4.3. The 3D technical drawing of the mechanical parts in the analyzing system is presented in figure 4 (see Technical annex attached).

a) Magnetic separation unit

The magnetic separation unit is used for the magnetic capture of circulating tumour cells (CTC) in the blood reservoir in the LungCARD chip. In order collect the cells required for analysis, the magnetic stirring unit moves two magnets across the Lung card chip. Due to the size of the sample chamber X = 67.5 Y = 55, and Z = 3.1 mm, with a volume approximately 11.4 ml, it is necessary to manipulate the magnetic particles within the sample chamber to allow the most effective capture possible through all three axis (X, Y and Z). In addition the external permanent magnet also moves the magnetic beads attached to the cells from blood reservoir to PCR unit.


b) Thermal management units

This unit is used of for a lysis of the captured CTC cells, PCR cycling for the specific DNA sequence generation and for finally for a hybridization of the specific DNA sequences with metal (gold) nanoparticle probes. The initial proposal was that the all the thermal management in the microfluidic chip can be achieved by a single Peltier system attached to one side of the chip. The Peltier size is 15x15 mm^2. The Peltier unit has a built in thermometer to measure the temperature on the active side of the Peltier. On the other side is the cooling element for quicker removal of excess heat. The temperature is controlled through the developed electronics. In total three examples were produced, for each R&D partner one. Software controlling the temperature with Graphical User Interface was made for Windows based platforms.


c) Electrophoretic unit

The electrophoretic unit is used of for an electrophoretic movement of the gold nanoprobes in a microfluidic channel on the chip. The electrophoretic unit consists of a 5 channel HVPSU capable of supplying 6kV on one channel, -6kV on one channel and +/- 6 kV on 3 channels, each at up to 500 uA and be controlled by software (Channel 1 = -6KV, Channels 2, 3 and 4 = ±6KV and Channel 5 = -6KV.


d) Optical detection unit

The optical detection unit is used of for a detection of gold nanoprobes in a microfluidic channel on the chip. The detection system (D4.1) consists of light broad band source, guiding optics, collection optics and spectral analyser. The high sensitivity of this system is achieved by two aspects. First, the collection optical fiber is design to collect only light from narrow channel in the microfluidic channel. This substantially suppresses the background signal and increase sensitivity. Second, the analysis of the whole spectral range from 400nm to 900nm obtained from spectral analyser. This enables separation of the signal from the NP-probes and the varying background signal due to the thermal drift of the light source and mechanical instability of the system. The method of the signal decomposition into the signal and background was content software packages in delivery report D4.3.

e) Integration of the systems into the whole system

All the core system units were integrated into a first prototype of LungCARD biochip analyzer (figure 5 -see Technical annex attached). The magnetic separation unit is composed by a fork with attached permanent magnet carries out the movement of the magnetic beads in the blood reservoir in the microfluidic chip and at the end of this process (CTC capture) the magnetic beads linked to CTC are moved via linear bearings on two rails to the area with thermal management unit, electrophoretic unit and optical detection unit. A clamping unit housing the Peltier thermal cycler and the top heater is used to secure the chip in place, ensure efficient thermal contact (during PCR reaction) and connect the electrodes to the chip. The bottom Peltier unit with the cooler is attached to the bottom movable manifold. The heater unit is attached to the top movable manifold. The electrical leads are attached to the top movable manifold and they get into a contact with the electric pad on the microfluidic chip, when the manifold is pressed down. After PCR and hybridization reactions, it is necessary to align the electrophoresis channel in the chip with the optical detector (for detection) by measure of the total transmitted signal. This adjustment is performed by micrometres (located on the X). The carriage is pulled into position by springs (located on the X) during this operation to ensure it is firmly held in position. The light source and the spectrometer were placed next to the chip holder and only the fibers were connected to the top and bottom manifold. Electrical interface for the electronics is placed in a separate box. This box contains all the necessary control electronics, data grabbers, and power supply for the electrophoretic unit, magnetic unit, thermal management unit and optical detection unit. Further, this box also contains electronics for a USB based communication with a computer system. This system was developed in collaboration with the partner University of Hull.

f) Safety design and manufacturer of LungCARD analyzer

The LungCARD biochip analyzer was designed and manufactured in order to reduce the risks to the operator, such as risks of accidental electric shocks, minimize risks from incorrect connections, minimize the risks of fire or explosion are presented. In the design five major safety measures very implemented. The first safety measure is the construction of cover lid which ensures that the operator will not interfere with the instrument during the measurement. This will avoid accidental injury of the operator by the moveable magnetic arm, getting the electrical shock from the high voltage leads, getting burn from the heating element on the PCR unit. The second safety measure is power switch connected with the lid. By opening the cover lid on the instrument the switch disconnect the high-voltage power leads, movement of the magnetic unit and switch off the power supply for the thermo element unit. This significantly reduces the risk of the injury of the operator. Next safety measure is the use of certified shortcut resistant high voltage power leads connecting the biochip reader with the electronic box. This cables are save to level of the voltage used in the HVPS separation unit. Regarding the thermal element unit, the heaters were placed in a location, which minimize the accidental contact with the operator. The heaters are secured below a thick plastic cover. The last measure was to direct the illumination source in optical detection unit towards the bottom of the unit. This avoids any accidental illumination of the operator’s eyes.

Project result nº3: The process to extract and analyse tumour DNA from blood samples

The complete process from blood to tumour DNA analysis is described in D2.1 D3.2 and D4.3 however here it will be described the main steps:

1.Add 10 magnetic beads (functionalized with EpCAM antibodies) per uL of blood (magnetic beads working solution of 1.3x10^4 beads/uL) to 13mL of whole blood. Mix well (up and down)

2. Load the blood sample into the chip and place the chip on the carriage in the LungCARD analyzer. Run Magnetic actuator software (around 2 hours for 13mL of blood) which allows to move the functionalised magnetic beads sequentially in x,y,z coordinates through the blood in a series of motions, each step with a 20 second hold time.

3. After magnetic capture, the beads linked to the cells are transferred to the PCR chamber by moving the external magnet in one continuous motion. The upper and lower manifold actuator arms are activated to bring the Peltier into contact with the bottom of the chip and the heater into contact with the top of the chip. The electrodes in the upper manifold also make spring-loaded contact with the conductive polymer pads on the top of the chip. Run PCR software. Thermal cycling parameters were programmed using the optimized conditions achieved in D3.2.

4. Gold-Nanoprobes (Au-NP) hybridisation on chip is achieved by electrophoretically driving AuNP from their storage well to the hybridisation chamber and by pulling the products from PCR chamber through the PPM to hybridisation chamber, using the HVPSU software. Voltage and time settings for moving the DNA PCR product and AuNP for hybridisation on chip were optimized as described in D2.1. Once the DNA and AuNP are in the hybridisation chamber, run the Hybridization program using the optimized conditions achieved in D3.2.

5. Align the optical system with the channel and switch on the lamp on the front of the main control box. Run the optical system and HPSVU software simultaneously using the selected HVPSU settings to form a plug of particles for injection into the separation channel. The injected plug of AuNP travels around the separation channel to the detector and results in a change in the output signal level as they pass the detector. The result is displayed as an instantaneous electropherogram at the bottom part of the display. When the HVPSU sequence has finished, the optical system software stop.

6. The data obtained can be analysed using the developed data analysis program. The software will then display the electropherogram and the corresponding voltage and current values for that run. The software has a range of post processing options including the report with input data (patient data, chip ID) and genotyping results (including internal control and the mutations detected).

7. The chip can then be removed from the instrument by moving the manifold levers to release the manifolds and then the chip carriage can be slid along the carriage to where it can be accessed and removed. The optical detection software must be reset, then the instrument is then ready for another chip for analysis.

The full process is controlled by a software package developed (see D4.3) which includes the graphical user interface (GUI). The GUI indicates the state of the experimental sequences in a visible, intuitive way to the operator. The program fulfills all software steps automatically and no user-interaction will be necessary after the optical alignment anymore. The report with the genotyping information (presence/absence of RGFR mutations) for the clinician will be also generated automatically (figure 6 - see Technical annex attached).

At this stage the controls experiments have shown that the LungCARD units are working, except the electrophoretic separation of AuNP (see D2.1 - Individual chip modules optimized_supporting info). More than 100 experiments were carried out, and the main electrophoresis separation results are the following:
a) If gold nanoprobes (AuNP) are re-suspended in Phosphate buffer, focussing of the AuNP was observed and this would lead to a long plug of AuNP, of 10 mm or more in length becoming confined to a band one or two mm in length. It did not matter what type of AuNP was injected, or whether they were hybridised, or even whether they were a mixture of hybridised probes, the results were always similar: two closely spaced intense peaks, or an intense peak followed by a shoulder,
b) When probes were re-suspended in 0.5x TBE, the AuNP moved far more slowly and gradually dispersed. Weak signals could be picked up at the detector, but these were on the limit of detection and were very broad. These bands varied from one run to the next and were not repeatable as they did not follow any coherent pattern.
c) Increasing the TBE concentration to 1x TBE, AuNP began to interact with the walls of the channel and an injected plug of AuNP probes gradually dispersed as it travelled along the channel. The AuNP in the centre of the channel moved more quickly than the AuNP at the Wall. This created a long and dispersed plug moving slowly along the channel wall
d) When the TBE concentration was increased to 2xTBE, the wall interaction became so strong that the AuNP probes became stuck on the channel walls after injection and then they did not move at all (similar to what was observed when using POP6 and 7 commercial separation gels (Life Technologies) and is probably due to the relatively high conductivities of these gels.
e) Passivation of the channel walls with perfluoro-trichloro-silanol eliminated these wall effects and it was possible to move the AuNP probes through the chip even with 3xTBE. However, the injection of a plug of AuNP probes would disperse and increase in length by a factor of 10 after travelling just a couple of cm from the injection point and by the time the plug reached the halfway point around the separation channel, it became so diffuse it was no longer possible to observe it.
f) Increasing the conductivity of the buffer by using more concentrated TBE was hoped to reduce electrodispersion effects; however, this led to increased Joule heating. The separation voltage was reduced in an attempt to reduce Joule heating, but even at a total separation voltage of just 2000 V, any injected plugs of AuNP probes dispersed rapidly and they could not be observed after travelling along the first half of the separation channel.
For the reasons described above, the separation of AuNP was not successful and a simplified Lungcard chip was produced as a contingency plan, LC4i v3, which comprised just two integrated functions (tumour cell capture and PCR on chip). The generated PCR products on-chip were analysed off chip by capillary electrophoresis. All demonstration activities were carried out using the LungCARD biochip LC4i v3.


Project result nº4: The application of gold nanoprobes to lung cancer pharmacogenomics

Gold nanoprobes were designed and synthesized for the detection of EGFR mutations (figure 7 and 8 -see Technical annex attached). Here it is summarized the main steps during their development and the results achieved. Detailed information is described in D3.1 and D3.2:
1. Gold-Nanoparticles were synthesized by the citrate reduction method and and characterised by TEM and UV/Vis.
2. Gold-Nanoprobes were produced with the functionalization of gold nanoparticles with Thiol-modified oligonucleotides by Salt-aging method. The Thiol-modified oligonucleotides were designed to be specific for each EGFR mutation. In total it was synthesized seven oligonucleotides to detect six EGFR mutations (Exon 19 – WT, Exon 19 – Del E746-A750, Exon 19 – Del E746-T751, Exon 19 – Del L747-P753, Exon 19 – Del S752-I759 and Exon 21) and one internal control (ACTB).
3. The synthesized Au-nanoprobes were used in hybridization assays using synthetic oligos and/or PCR amplicons for the existing DNA/cell samples. Hybridization and target sequence identification was assessed via the noncross-linking colorimetric method. In addition, the hybridisation efficiency was also assessed through Ferguson plot analysis by measuring the distance migrated at a given field strength. The gold nanoprobes can successfully detect the respective DNA targets harbouring the relevant EGFR alterations and the internal control.
Potential Impact:
Impact on LungCARD partners:

The development of a blood test to guide the therapy in non-small cell lung cancer patients will have impact for all LungCARD partners. The LungCARD team has been working during these two years (2013-2015) in order to solve the problems faced by the oncologists in the selection the best therapy for NSCLC patients and at the same time to increase the competitiveness of the SMEs involved. The current EGFR testing approaches requires a tissue sample (usually stored in FFPE) which represents critical limitations not only for clinicians/patients but also for the laboratories that performed the EGFR analysis. The impact of LungCARD project for each partner is described as follows:

Castle Hill Hospital (partner HEY):
In the UK, 30% of patients may never get histological confirmation because they are too sick or is too dangerous. Therefore the clinicians do not know how to treat them. If patients have a mutation they should receive a tyrosine kinase inhibitor (e.g gefitinib), if not they would do better with chemotherapy. With the LungCARD blood test, all patients can be tested and receive the most efficient treatment. In addition, the EGFR testing requires biopsy analysis and can take 3-4 weeks to get a result. By then a significant proportion of patients will be dead. With the LungCARD blood test, the result could be available after 3 days and more lives could be saved.

SMEs (STAB VIDA, MicroLiquid and Moltech):
The developed new technological approach is highly innovative when compared to the existing offer on the market and will automatically provide a market advantage, thus improving the competitiveness of the 3 SME partners. All together, the SMEs will start to compete in a market where, right now, none of them has any kind of penetration – personalized medicine a fast-developing market. Markets and Markets reports that the market for personalized medicine is estimated to reach close to $150 billion by 2015, and cross $900 million by 2020. The profitability analysis for LungCARD carried out by partner Moltech suggested that net revenue in 2021 will be around 7 million € with a market penetration of 20%. Considering that the world market for NSCLC analysis is huge, even in the most conservative scenario of reaching 1% only of market penetration, in the first year of production the SMEs will start to generate value. Incomes will be enough to cover the costs and generate profits. Therefore ROI and ROS will be positive too, which means that the invested capital is starting to generate profits and the sales are beginning to produce an income margin. Despite the favorable financial indicators, it was the first time that SMEs lead a project which involved the participation of patients with lung cancer. SMEs have the opportunity to work with a reference hospital in United Kingdom with high background in lung cancer (Castle Hill hospital) and with an interdisciplinary and expert team which contributed to increase their know-how and give them an advantage to gradually positioning as innovative players in this market. STAB VIDA expert on DNA-based diagnostics, gained know how to further develop novel services and products for diagnostics based on micro fluidics and nanotechnology. Moltech a manufacturer of biological devices acquired knowledge in optical detection and bioanalysers functioning and MicroLiquid, a manufacturer of micro fluidic chip acquired experience in more complex fabrication processes, increasing their competitiveness in point-of-care market. Furthermore, the developed technology could be used for other biomarkers in a wide variety of tumours (e.g. KRAS in colorectal cancer). LungCARD project has significantly contributed in creating new partnerships with Universities and relevant research centres, together with strengthening existing relations with SMEs already participating in other EU funded projects. All SMEs are strongly committed to put the LungCARD product in the market and have decided not to publish the project foreground, submit a provisional patent (see D7.3) and continue the work post-project according with the plan established in the last Steering Committee report and in deliverable D7.4.

Faculty of Sciences and Technology (RTD FFCT partner):
The LungCARD project allowed to strengthen the research group in areas of biosensing and microfluidics and to create a network of excellence with relevant European partners within an Industry framework, which impacted strongly in the awareness of solutions that meet society and of relevance to effective translation. However, due to the SME oriented nature of LungCARD, publication record was low and, hence, academically it impacted negatively considering the dedication to the project (two full years, no publications thus far). Globally, LungCARD impacted positively in networking, internacionalisation, bridging the gap to Industry, and specific expertise in microfluidics.

University of Hull (RTD UHULL partner):
The LungCARD project is the first microfluidic device from the University of Hull (UoH) that has been tested on patients with cancer. This study has allowed the “gold standard” analysis to be tested against the microfluidic device’s performance. Groups within the Departments of Chemistry, Biological Sciences and the Hull York Medical School from across UoH have worked successfully to deliver the project. The combination of skills from the different SMEs and academic groups has provided UoH with an extensive network for future research and commercialization activities. The project has provided excellent training opportunities in interdisciplinary research for five postdoctoral research assistants. In addition, more than 8 undergraduate students (Biological Sciences and Chemistry) have worked alongside the research assistants undertaking their final year research dissertations; the skills of interdisciplinary working in the bioclinical area are being widely disseminated. The integrated prototype device has been used as an exemplar of how a microfluidic device can alter clinical practice and has been used to leverage more funds from the University of Hull. In addition the lessons and principles used in the LungCARD device are being applied in other tumour systems including glioblastoma, mesothelioma and colorectal cancer. It s anticipated that the microfluidic device produced during the LungCARD project will be used as an impact case study for the applied health submission from UoH in the United Kingdom’s research excellence framework (REF) exercise in 2020.

Institut fuer Photonische Technologien E.V (RTD IPHT partner):
The LungCARD project had a very positive impact on our research teams.
It was very useful experience to be involved in this international project. We experienced the profit and obstacles associated with having partners across Europe and witnessed the importance of good project management. We learned a lot about the current issues with the diagnosis of lung cancer and have now better insight into the industrial needs into bringing their products on the market.
Through the co-operation with other research groups, our team got familiar with new techniques of for DNA extraction, multiplex PCR and detection.
All the experience we gained will be used in our next projects. We can now employ, either by ourselves or possible by the partners in this project, these techniques in other detection schemes. Finally, we enjoyed to work in the international team of research groups and industry.

Socio-economic impact:
Lung cancer is one of the most common cancers in the EU. EU Member States, regions and populations differ significantly in lung cancer incidence and mortality. These variations reflect differences in the development of the tobacco epidemic, as described in the EUphact Smoking. Southern (Italy, Greece) and Eastern and Central European (Hungary, Czech Republic) men currently have the highest incidence rates of lung cancer within the EU. As smoking is currently very prevalent among younger women, an increase of the female lung cancer rates can be expected. In many EU countries the incidence of smoking and lung cancer is significantly higher in lower socio-economic groups. Exposure to smoking at an earlier age, as well as lower rates of successful cessation contribute to the higher risk of lung cancer in this segment of the population. At present time, patients suspected to have cancer diseases are subjected to a precarious quality of life, since they would need the close and real time control of their health conditions, which can only be assured with the patient hospitalization to perform the necessary tests, with the evident negative consequences on the quality of life of the patient, as well as of his family scene. Thus, enhancing the level of service offered to European patients and ensures that healthcare providers become more effective in achieving their ultimate goal: saving lives. The possibility to perform a blood test (no need for a biopsy), using a portable device compatible with their daily life, puts in evidence the strong impact of the Lung CARD system on the European citizens quality of life and the security of the patients. Furthermore, the expected improvement of the whole biomedical sector, derived from the higher quality of the final product to the service of the entire Community, will open the way for making new investments in the sector, thus creating new job opportunities.

Dissemination activities and exploitation of results:
The main focus point of this proposal is to use and disseminate the project results by further developing and marketing the Lung CARD system. A Dissemination Plan (DP) and Exploitation Agreement (EA) were developed under work packages 6 and 7, respectively. The EA define how the partners, both as a consortium and as individual organizations, intend to exploit the results of the work carried out. During LungCARD project the following activities were taken to assure the optimum uptake and use of the main project results:

.Demonstration - During the last 3 months, demonstration activities (WP5) were performed by Castle Hill hospital (partner HEY) and University of Hull (partner UHULL) in order to validate both the LungCARD analyser and biochip using 10 blood samples from lung cancer patients and healthy volunteers. These activities were lead by SME MicroLiquid and supported by the other SME partners (STAB VIDA and Moltech). Although the system is working (D5.4) still lacks accuracy, robustness and reproducibility and additional experiments post-project are running at STAB VIDA laboratories as planned in deliverable D7.4.

.Dissemination - All dissemination activities (WP6) were lead by SME Moltech. After launching the project´s website in month 2, all partners took an active role in disseminate the project results through participation in international conferences (e.g uropean Society of Pharmacogenomics and Theranostics and 7èmes Assises de Génétiques Humaine et Médicale) and info days for students, industry and investors (e.g participation in Biopharm 2014 in Boston). STAB VIDA also gave an interview for a national newspaper where was mention the purpose of LungCARD project. All dissemination activities are described in detail in deliverable D6.5. In order to ensure that no confidential information is disclosed, the approval of content to be published was carried out by Steering Committee managed by Orfeu Flores (STAB VIDA´s CEO).

.IPR protection and exploitation agreement– During the last 3 months, the SMEs carried out a patentability and a freedom-to-operate analysis. Both studies suggested that considering the described state of the art and the given invention, it derives that the microfluidic apparatus for extracting CTCs (Circulating Tumour Cells) from small volumes of blood samples (p.ex. 10 mL) comprising a biochip, magnetic actuator, optical reader and other elements, for detecting gene mutations affecting the expression of EGFR (Epidermal Growth Factor Receptor) in tumour cells present in blood samples from NSCLC (Non-Small Cell Lung Cancer) patients, is new and inventive. The method and the biochip are new, although they are to be inventive only when used with the microfluidic apparatus of the invention – also maintaining unity of the invention. Therefore a patent application for the bioanalyzer comprising the biochip and method implemented therein has a better chance of being granted than a patent application claiming independently the biochip, bioanalyzer and method, which could be objected by lack of unity of invention (multiple inventions) and lack of inventive step for the biochip. Considering the Patentability analysis and the results of LungCARD, a provisional patent has been written and submitted to the Portuguese Patent Office – INPI, on January 15th, 2015, granting this priority date for other countries. In addition, a Business Plan was established for the potential exploitation of LungCARD’s IPR and also an exploitation agreement to formalise the modalities and the conditions that will govern the commercial exploitation of the project results. Detailed information on IPR protection, business plan and exploitation agreement is described in deliverable D7.3.

.Training – the training activities for SMEs (WP7) took place in month 24 to ensure that the SME participants will be able to assimilate the results of the project. A training session took place in FFCT laboratories where STAB VIDA were able to learn about the reagents manufacturing for the Biochip. At University of Hull both SMEs MicroLiquid and STAB VIDA received training in biochip manufacturing and procedures for the analysis of blood samples in LungCARD analyzer. SME Moltech had a training session at IPHT to learn how to construct the LungCARD bioanalyzer and also the controlling software package. All these training sessions allowed to guarantee the complete transfer of knowledge and results to the SMEs. Details are defined in D7.1.
According to LungCARD Dow it is estimated that the LungCARD prototype is 2 to 3 years far to the market. Following the plan described in last Steering Committee report and deliverable D7.4 additional laboratory experiments are running at STAB VIDA laboratories and at the same time we are writing the LungCARD II proposal to submit in a H2020 call. The LungCARD II project aims to build improved versions of LungCARD chip and analyser and test them in clinical environment. Moreover it will be implemented the manufacturing process according with ISO 13485 in order to fulfill the regulatory requirements for CE marking.
List of Websites:
1) Website: www.lungcard.eu

2) Coordinator contacts:
Carla Clemente
Head of Clinical Sequencing and Quality Manager
clemente@stabvida.com
Tel: +351 210438607
Fax: +351 210438608
Skype: Carla from STAB VIDA
Adress: Madan Parque, Rua dos Inventores, Sala 2.18
2825-182 Caparica - Portugal
www.stabvida.com