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Lung cancEr fusiOn geNes: a new dIagnostic Device

Periodic Reporting for period 2 - LEONID (Lung cancEr fusiOn geNes: a new dIagnostic Device)

Reporting period: 2016-04-01 to 2016-12-31

Lung cancer is the leading cause of cancer-related mortality worldwide. Approximately 5% of Non Small Cell Lung Carcinoma (NSCLC) cases show a fusion between EML4 and ALK genes, that generates a constitutively active chimerical protein, targeted by the antitumor drug crizotinib. Other chromosomal translocations conferring sensitivity to crizotinib or other tyrosin-kinase inhibitors involve ROS1 and RET genes.
Identification of the appropriate subset of NSCLC patients likely to benefit from molecular targeted therapy is critical to the success of personalized medicine. The success of crizotinib in ALK-driven tumors and emerging clinical evidence of response in ROS1- and RET-rearranged tumors led to the need of simple, reliable and inexpensive methods for fusion genes detection in order to allow the screening of a large population of lung cancer patients and the fast identification of those suitable for treatment with molecular targeted agents as crizotinib.
The LEONID project aims to develop and validate a new diagnostic device able to detect and quantify with the Nanostring platform transcripts of fusion genes involving ALK, ROS1 and RET in lung cancer as reliable as the methods currently employed in clinical practice, but easier, cheaper and faster and possibly more accurate.
In WP1 (Diagnostic device development) DPGx tested both NanoString chemistries, Standard and Fusion Elements, in order to select the best one in terms of functionality, specificity and sensitivity. After several tests the Standard chemistry was chosen for the device development because Fusion Elements showed cross-reactivity, high background and lower sensitivity.
In the same WP the NanoString Standard Chemistry codeset (probes panel) has been upgraded and a commercially available human RNA has been identified as a possible positive reaction control for the device and tested in all runs.
In WP3 (Design & development of the analysis software) BM studied the regulations about software certification as a medical device, NanoString technology and output and requirements provided by DPGx. Then BM together with DPGx defined the first draft of calculation algorithms that have to “translate” the NanoString raw data into information suitable for a medical report.
Other activities performed by BM in this WP for the development of the software prototype are: database design, technology selection and definition of user interfaces and reporting.
In WP4 (External validation of the diagnostic device and the associated analysis software) GL staff was trained on NanoString pre-analytic and analytic procedure.
The WP5 (Collection of clinical samples) has been carried out by DPGx with the support of two Subcontractors: Laboratory of Biosciences, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, IRCCS, Meldola (IRST) and Laboratory of Molecular Pathology, Azienda Ospedaliera Universitaria Pisana, Pisa (AOUP).
During the first 6 months the procedure for the submission of the clinical trial application data for an Ethics Committee opinion and the preparation of supporting documents have been performed.
The clinical study has been approved by CEIIAV and CEAVNO Ethics Committees on December 2015.
Afterwards 51 NSCLC patients have been selected: 6 from IRST, 45 from AOUP, both positive and negative for ALK/ROS1/RET rearrangements. IHC-ALK slides, FISH-ALK/ROS1/RET digital images and sets of FFPE tissue sections for RNA extraction have been collected for 12 samples, FISH-ALK digital images have been collected for most of them
In WP6 (Data mining and correlation) for the quantification of tissue features and morphology DPGx provided IHC-ALK slides and FISH-ALK/ROS1/RET digital images to the subcontractor Definiens, that was in charge of developing a software solution. So far the development of three FISH translocation analysis algorithms (ALK, ROS1 and RET) have been completed. Development of ALK-IHC analysis algorithm is work in progress.
For WP8 (Project Management) a Consortium agreement has been signed and a Steering Committee, a Technical Board and a Project Management Team have been settled.
Three general meetings took place for activities alignment, problems troubleshooting and update about the project progress.
A Project Plan for monitoring the progression of all WPs and tasks and a Quality Assurance Plan defining quality assurance & risk assessment topics have been prepared and shared among partners.
Regarding the administrative management a cost breakdown structure has been created in order to support the correlation of costs per WP and all the guidelines and documents for costs and time management have been prepared.
Communication with EU and Project officer has been implement and maintained as well.
The LEONID project will create the first CE-IVD diagnostic device able to detect all fusion genes involving ALK, ROS1 and RET in the same well and quantify each new transcript, based on the Nanostring platform.
The software associated to the diagnostic device will guarantee the standardization of the results analysis and, being part of an oncological electronic health record as module, could encourage the adoption of this EHR in the hospitals where the device will be used.
A unique correlation between patient outcomes, molecular results coming from Nanostring, and tissue morphology signatures will be searched in order to better stratify patients and improve treatment decision and sample selection.
The new Nanostring-based diagnostic device and its associated software should bring relevant benefits.
The reduction of costs deriving from multiplexing and from a lower error rate would positively contribute to the sustainability of health care systems.
The lower complexity of procedure and analysis would allow to reinvest trained technicians and expert pathologists in other tests/techniques requiring highly skilled staff.
Higher throughput and faster workflow make the Nanostring assay suitable as a prescreening tool in both research and clinical practice for the identification of a subset of patients with NSCLC most likely to benefit from specific targeted therapies.
The internal quality controls, the associated analysis software and the CE-IVD validation of the device, together with the high sensitivity and the semiautomated feature of the technique would allow the detection of the main lung cancer fusion genes in a more reliable, standardized and less operator dependent way, all to the good for NSCLC patients and their management by the oncologists.
The results of LEONID would have important impacts on all components of its consortium. For DPGx, the new device will be complementary to the existing products for pharmacogenetics analysis in oncology and will boost the company’s approach of potential European distributors. GL can offer the test to low throughput labs and can speed up the entrance into the oncology business. BM can extend the panel of features of its proprietary oncology EHR dedicated to the analysis of laboratory tests’ results and can provide its new software through DPGx or directly.
As further LEONID’s impact at the social level, the employment by DPGx of two people should be considered. These new employees are in charge of most of WP5 activities.
Diatech Pharmacogenetics' researcher at work on LEONID project