Persontilpassa kreftbehandling - biomarkørar og kliniske studiar
Highlights from all workstreams illustrating scientific excellence and best practice in process and technique establishment
A project group consisting of clinicians, study nurses and lab technicians has been established to test and create a biobank tracking system, health register and organise physical storage of samples and develop SOPs for use nationally. We are using existing biobanked samples to test new methodologies for detection of circulating tumour DNA.
Next Generation Sequencing (NGS) approaches have been tested using 147 samples from leukemia patients. The results from this analysis have been including a submitted article (Reikvam et al., Genome Medicine, 2016). Data generated from this gene panel has also been used to develop web-based NGS analysis software CoreMine Oncology (PubGene). We have increased the security, and developed the analysis module to include more severity prediction algorithms, higher levels of filtering and in general highly increasing the user friendliness for scientists. To enrich circulating tumour cells (CTC) for downstream analysis, two types of CTC filtration chips have been designed. They have been evaluated and according to the evaluation, new chips have been redesigned and will be tested in 2017. CyTOF for detection of rare cells has shown some success. Methods are being established for quanitification of cells. Enumeration of CTCs using the CellSearch™ platform has generated experience for monitoring treatment of the ongoing Phase I Clinical Trial of cryoimmunotherapy against metastatic castration resistant prostate cancer at HUS. 9 patients have been followed up for more than 6 months. Results indicate CTC counts show good correspondence with aggressiveness of disease and disease progression. The Parsortix™ platform has been compared to the CellSearch™ regarding sensitivity to detect CTCs in peripheral blood and leukapheresis product of patients. The preliminary conclusion is that the CellSearch platform was most sensitive using both peripheral blood and leukapheresis products of patients with metastatic castration resistant prostate cancer. Plans and design are in preparation regarding evaluation of in house microfluidics devices in combination with mass cytometry and other equipment for CTC enumeration and characterization. In operable pancreatic ductal adenocarcinoma different methodologies were evaluated for its ability to detect mutations in KRAS and TP53 in different sample materials from 14 patients (collaboration with Prof A. Molven). Mutations where found in 13/14 samples when using dissected FFPE. Cell free DNA was isolated from pancreatic juice and plasma. ctDNA was present all of the pancreatic juice samples but none in any of plasma sample despite high levels of cell free DNA present. One master student graduated on this project in 2016, and this will be further developed by a recruited PhD candidate in 2017 and close collaboration with SUS and their gene panel. A total 54 patients with advanced pancreatic cancer has been recruited and 314 blood and bone marrow samples have been collected from the patients. A new method for enrichment of circulating tumor cells was reported and validated in a publication in Scientific Reports (Lapin et al, 2016). Analysis of circulating tumor cells and circulating tumor DNA in patient blood samples are ongoing. A new and promising method for circulating tumor DNA detection based on next generation sequencing is near completion. In the study of malignant melanoma treated with Avastin cell free DNA from 39/44 patients was explored guided by results from Sanger and/or NGS gene panel sequencing. By digital droplet PCR ctDNA was detected in 20/24 samples, and a significant change in ctDNA was shown to correlate with clinical data. (manuscript in preparation). A broader mutation profiling of ctDNA will be performed on 10 patients (collaboration with Prof. L. Meza-Zapeda).
Identification and development of methods and procedures for successful delivery
Development in all work areas has begun and essential techniques undergone optimisation, including methods for next-generation sequencing-based ctDNA detection and characterization are being established by adaptation of the Duplex Sequencing Technology. A new method for detection of circulating tumor cells has recently been established.
Sample and tissue collection, processing and storage are very important for the delivery of this project. Time has been taken to develop the correct processes to ensure samples will be taken and processed in a consistent fashion without causing additional discomfort to patients. We are confidence we have procedures that will allow this. For new clinical studies coming on line we have established procedures for bio-banking patient material. We have set up an interim system for labelling, storage and recording of samples for use in this project. SOPs have been prepared to ensure regulatory compliance and consistency across site and teams. This interim system will be superseded by the Haukeland Biobank (being led by the Research and Development department) when it comes online. To ensure a successful transition we have been working closely with the implementation team. For established studies we are transferring them to the new system for example CircSarc, Basket and Mo29518. The REC application for the biobank has been completed and should be approved shortly. We are fortunate to have access to sample material from ongoing or closed studies and therefore have the possibility to address many of the steps independently. Grouped by cancer types we have started different projects on pancreatic cancer, lung cancer, acute myeloid leukemia and endometrial cancer. Throughout 2016 work will begin on melanoma. This work has been completed with our collaborating groups at SUS (Nordgård and Gilja), Bergen (Hovland together with Gjertsen, Molven, Salvesen, Bjørge, Straume dependent on the study) and Trondheim. Establishing procedures for collection and purification of circulating DNA from plasma has been completed in close collaboration with Meza-Zapedas group (OUS) using blood from healthy blood donors. Excellent progress has been made on the identification of cancer-specific circulating endothelial cells. The CyTOF arrived in Bergen in October and has since been set up, staff trained and work has begun on designing panels for future analysis. This will be a focus of 2016 with the appointment of key staff to develop this technology. CHIPs have been designed successfully for the identification and study of circulating tumour cells. Testing will begin in the first half of 2016. The establishment of novel tests for immune-monitoring, testing of procedures to improve immunity of dendritic cells, activity to establish autologous dendritic cell production will continue. The project will need substantial bioinformatics service in the future. So far our Bioinformatician financed by Norwegian Genomics Consortia has spent time collaborating and testing sequence analysis software is being tested and optimized. Discussions with the company PubGene are ongoing for the development and modification of the system to produce a graphical user face for entering and analyzing material. Two applications for affiliated personnel have been successful. Researcher Xiang Wang has received a competitive 3 y researcher grant from the Research Council of Norway. Geneticist Randi Hovland has received a short time research grant at Helse Vest that substantially supports the project. Wang and Gjertsen will submit a FORNY application in collaboration with BTO in April, related to a microfluidics device with huge potential in liquid biopsy. The Project Board first met after a successful kick off meeting in September 2015, Stavanger.
MINDEC-An Enhanced Negative Depletion Strategy for Circulating Tumour Cell Enrichment.
Sci Rep 2016 Jul 19;6():28929. Epub 2016 jul 19
Clinical relevance of circulating KRAS mutated DNA in plasma from patients with advanced pancreatic cancer.
Mol Oncol 2016 Apr;10(4):635-43. Epub 2015 des 15
Identification of Responders by Gene Expression Profiling of Pretreatment Leukemic Cells