Title: Point-of-care Analysis by Non-invasive Dielectrophoresis for ORAl cancer diagnosis (the PANDORA project)
Funder: Department of Health (England)

Abstract: Research Plan As part of due diligence to license the technology to DEPtech, IP searches were performed by the University of Surrey and DEPtech prior to licensing the patent. The International Search Report listed 5 X's and 4 A's.The EP examination report referred to this citing WO99/60392. This disclosed only 3 conductive layers whereas the DEP-Well invention requires ten to fifty conductive layers. This increase in the number of layers was not considered to be inventive by the Examiner. It was argued that it was not obvious and the required number of layers was not disclosed in the prior art. This response was filed Feb 09 but no reply has been received yet.USA - In addition to the same objection raised by the EP examination the claims were said to be anticipated by US6149789 (Benecke et al).The UK patent agent believes we have a strong case for overcoming these objections and it has gone to appeal in the US. Aims RESEARCH QUESTIONS1) Is it possible to develop a non-invasive and fast-responding automated dielectrophoresis prototype system to identify oral cancer cells at the point of care? 2) Will an initial pilot clinical study demonstrate statistically significant results regarding accuracy of the technology prototype in detecting cancer cells (via sensitivity, specificity, positive and negative predictive value analysis)? PROJECT PLANPANDORA project is part of a long-term collaboration between the Research Team members to study the potential application of dielectrophoresis (DEP) to oral cancer diagnosis. DEP is the motion of a cell in a non-uniform electric field; this motion is frequency-dependent, following a frequency spectrum which uniquely relates to the biophysical characteristics of cell membrane and cytoplasm. Genetically or geometrically similar cells have more similar, but still distinct, responses at given frequencies, allowing subtle differences between the cells to be detected. We have shown in our early in vitro and ex-vivo study using an early prototype of DEP Well Electrode System that oral cancer cells have a significantly higher cytoplasmatic conductivity, and lower specific membrance capacitance than normal oral epithelial cells. The overall bench-to-bed vision of our research into DEP and oral cancer has progressed and will continue via these 4 milestones:1) In vitro DEP study (2004-2007 completed and published): This was an investigation of electrostatic characteristics of in vitro oral cancer cell versus normal oral epithelial cell lines. The very early prototype of DEP technology used primitive, labour-intensive needle electrodes to show a basic difference in the behaviour of cell lines. We demonstrated that in-vitro cultured oral cancer cells have a highly different DEP phenotype to normal cultured oral cells, hence paving the way for pilot testing in humans. This work was published in Oral Oncology in 2007.2) Preliminary first-in-human proof-of-concept study (2008-2010 just completed - not published yet): This ex-vivo study was supported by a NIHR RISC grant and followed the encouraging results of the in vitro study. Oral cells were collected via a painless brush from oral cancer patients and healthy controls and tested in the DEPwell system prototype. With respect to the in vitro study, this prototype provided highly accurate data using a computer-based analysis system and novel electrode that allowed a thousand cells to be measured simultaneously, though data were still collected serially. We observed that cancer cells non-invasively collected from oral cancer patients have a different DEP spectrum than normal oral cells.3) Further development of technology, including pilot clinical validation (current stage; 2011-2013.): This is the study outlined here. The first-in-human proof-of-concept study has demonstrated that DEP prototype system can differentiate between oral cancer and normal cells. The technology needs to be developed into a fully-automated portable DEP prototype system that will be validated in a pilot clinical study to test its diagnostic specificity, sensitivity, positive and negative predictive value. This study will deliver: - A fully-functional, fully-automated portable DEP prototype - Preliminary clinical data, to form the basis for future clinical validation.- A plan for intellectual property and commercial implementation of the technology, to inform future commercial and intellectual property exploitation. 4) Final development and clinical validation of the technology/credible IP and commercial plan (future stage). This study will follow the i4i Early Stage project (if successful) and will be based on collaboration between industry, academic and clinical researchers (i4i Late Stage). The technology will be finalised (from prototype to final device) and clinically validated so to obtain robust evidence of efficacy. A business and management plan will be defined. RESEARCH METHODOLOGYThe present study will address the research questions via work divided into 6 distinct packages, each containing several deliverables. Package 1: Following our first indicative studies, we need to refine the unique DEP fingerprint that identifies oral cancer from non-malignant cells etc and define the optimum conditions of field and suspension medium to accentuate that difference from other cells. This will be achieved via intense testing of oral cancer versus normal brush samples. This extends the work performed during the NIHR RISC proof-of-concept study, in particular by characterising other oral lesions which may act as confounding factors. Package 2: Engineering the current prototype system into a fully-automated software-integrated prototype. At present, the system is operator-dependant and is based around several electronic sub-components and a microscope. This will need to be re-engineered and miniaturized into an easy-to-use, portable system. Particular attention will need to be taken in integrating the electronics, control/analysis software and sample handling. Package 3: Study and development of handheld device (PandoraBrush) for repeatable and painless cell sampling of oral mucosa - in order to standardize and reduce inter-operator variability.Package 4: Pilot/feasibility clinical study of newly developed technology with initial preliminary analysis of sensitivity, specificity, and positive and negative predictive value.Package 5: Study of EU regulatory requirements and plan to apply for CE marking.Package 6: Development of a plan for intellectual property and commercial implementation of the technology Detailed description of workpackages, milestones and deliverables is provided in Annex 2, with a Gantt chart in Annex 3. In order to perform this work, the project will be undertaken by two teams, working on separate frameworks (Engineering and Clinical) in an integrated manner. These are: Engineering Framework: The work carried out to date by the applicants has examined the principle of cellular analysis by DEP to discriminate between healthy oral keratinocytes and cancerous cells. This is performed using DEPchips developed (and patented) at Surrey, as well as a range of ancillary signal generators, image acquisition apparatus and computer analysis software constructed around a laboratory microscope. Over the course of the present project, the Engineering Team will convert this lab-based apparatus into a portable automated system with low-cost components and a simple user interface (Pandora's Box), into which an untrained practitioner could repeatably insert cells for accurate analysis. The key deliverables of the engineering framework are:- Development of the optical path required to analyse the system in the final prototype (current equipment uses a microscope and camera system);- Associated electronics (currently several instruments controlled by a MatLab program) will be re-engineered to provide suitably controlled signal generation, data acquisition and analysis;- Development of dedicated software to increase accuracy and specificity by using signal processing techniques to identify cancer cells in samples with low cell counts and debris;- Identification of DEP fingerprint that signify differences between cell types, in order to maximise selectivity and minimise false positive diagnoses;- Development of a handheld device (PandoraBrush) for repeatable and painlessly cell sampling of oral mucosa in order to standardize and reduce inter-operator variability. To this end we aim at developing a small, handheld, battery-powered device with a motorised brush that the user places over the lesion, and a cowl to both retain the sample and allow the user to accurately place the device. Activation of the device will engage the brush to collect cells which would then be easily recovered for analysis. The design, construction and testing of this handheld device will run alongside the construction of the main unit.- Business plan and regulatory issuesClinical Framework: Our initial clinical work demonstrated that DEP analysis can be performed using cancer cells collected from patients' mouth using a relatively painless brush. The clinical team has also provided a major contribution to the study through identification of necessity for technology development within this clinical field and potential benefit for patients and the NHS. Clinical researchers have also contributed to clinical testing of the technology via collection of samples from cases and controls. The key deliverables of the clinical framework are:- Collection of samples from cancer patients and controls to finalize set up of the system. The main clinical centre (UCLH EDH) will also coordinate sample collection at the other 2 collaborating centres;- Collection of samples from cancer patients, controls and also patients with non-malignant oral disease to test diagnostic accuracy of the newly developed portable full-automated prototype. The main clinical centre will also coordinate sample collection at the other 2 collaborating centres;- Training of new clinical investigators re use of manual brush- Initial pilot statistical analysis such as sensitivity, specificity, positive and negative predictive value (via dedicated statistician at UCL Clinical Trial Unit); - Clinical contribution to design and develop the PandoraBrush, including comparative clinical testing vs traditional manual brush.- Business plan It is expected that at least 35 participants per centre (105 samples in total: 53 cases+52 controls) will be needed to finalize the set up of DEP system (Package 1). A minimum of further 102 patients will be enrolled (34 cases+34 controls+34 benign oral diseases) for the subsequent pilot/feasibility clinical study of newly developed system (see Annex 2 for sample calculation) Benefit - Current standards of service in NHS primary care (based on oral visual examination) often fail to identify oral cancer at an early stage. Most individuals are referred to Cancer Unit when their oral cancers are advanced and treatment is burdened by hig


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS