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Advances in sequencing technologies have progressed to a point where Next Generation Sequencing (NGS)-based diagnostics can be considered for cancer clinical research
John Bean PhD
Emilie Varin PhD
Edit Szepessy MD
Denis Lacombe MD
EORTC Headquarters, Brussels, Belgium
The idea of personalised medicine, tailoring treatments to individual unique disease characteristics so as to administer the right treatment for the right patient, at the right dose, and at the right time, has been around for some time. Yet, despite widespread acceptance of this idea, too many tailored treatment and targeted drugs continue to fail at late stages of their clinical development. Cancer research stakeholders know that there is a problem and there has been a growing recognition that a new integrated model of clinical cancer research is needed to optimise the research and development process.1,2
Simply screening a single mutation for a specific targeted drug is not sustainable. In practice, there would only be a small number of patients who might actually carry the mutation and therefore benefit from the targeted therapy. Moreover, it would not be possible to obtain sufficient amounts of tumour material from patients with certain types of cancer, and the development costs would ultimately be prohibitive.
Cancer biology is complex, so an alternative would be to perform multiple gene assays that could establish the somatic mutational profile of a tumour, and subsequently check if any of the mutations could be addressed therapeutically with agents targeting those specifically defined mutations (for example, BRAF, EGFR, KRAS etc.). The clinician would then be in a position to choose the appropriate agent for the treatment regimen. For this, Next Generation Sequencing (NGS) is one of the most promising technologies for such a high throughput screening approach because it can generate extensive data on a single sample at a reasonable price.
NGS technology is available today, but is it ready for clinical research?
Over the past 10 years, following the disclosure of the Human Genome Project’s results, advances in sequencing technologies have reached a point where NGS-based diagnostics can be considered for cancer clinical research. NGS-based platforms can help clinicians and pharmaceutical companies screen patients for targeted, biomarker driven therapies that are already on the market or in the development phase and they would also prove extremely valuable in terms of biomarker discovery or identification of resistance markers.
Large-scale whole-genome or whole-exome sequencing might not appear to be the first choice for this type of screening strategy, but today high throughput, low cost, targeted genome sequencing is much further developed in the field of cancer research and diagnostics. Numerous NGS-based tests are being developed by both companies and academic labs and these can test for point mutations, short insertions and deletions, copy number alterations and also genomic rearrangements in a wide range of genes known to be somatically mutated in cancer, using paraffin-embedded tissues and in clinically relevant turnaround times in a scalable manner depending on the specific needs.
A large variety of NGS systems are currently available on the market and these demonstrate high performance in terms of throughput and turnaround time alongside a low requirement for tumour sample. There challenges still remain to successfully transition NGS technologies to the clinic, especially in terms of data analysis and storage for which sophisticated bioinformatics pipelines are still needed and also in data interpretation that requires a team of multidisciplinary experts. Whereas data analysis and storage can most likely be overcome with future developments in computer science, the data interpretation is a substantial challenge that must be faced in order to bring NGS into the clinic. It would require reshuffling the order of daily oncology practice to include a panel of clinical and molecular experts who could translate the NGS data into an appropriate treatment decision.
Fortunately, the seeds for the required supporting infrastructure for NGS technologies in cancer clinical research have been sown and hopefully we will soon see the fruits of our labour. The European Organisation for Research and Treatment of Cancer (EORTC), for example, has already launched a novel collaborative molecular screening programme, SPECTA, Screening Patients for Efficient Clinical Trial Access for patients with cancer, an initiative which implements NGS technology to help provide patients with personalised treatment. SPECTA is an integrated and cost-sharing business model for clinical research in oncology and drug development. SPECTA links the effective development of biomarkers in clinical research to secure the safety of patients, stimulate innovative research and new technologies, and ensure high quality, streamlined study operation. It encompasses tumour oriented pan-European and independent platforms for partnership with industry to direct patients to optimal treatment according to the principle of “Screen and Treat”.
SPECTAcolor, the first platform dedicated to colorectal cancer screening, is now enrolling patients with pathologically confirmed metastatic colorectal cancer. Launched in mid-September 2013, over 500 patients have been included in SPECTAcolor as of December 2014, and in the coming years, as new sites continue to open across Europe, it expects to enrol between 600–1000 stage III and IV colorectal cancer patients per year.
Next Generation Sequencing with built-in quality assurance/quality control
Quality assurance for NGS is essential when using this technology for a diagnostic purpose or to make clinical research decisions. Quality controls must be in place along the pipeline, from preanalytical conditions (tumour sample collection from the patient and processing before analysis), through to the sequencing assay itself (including library construction), up until the generation of molecular data via the dedicated bioinformatics pipeline.
In SPECTAcolor, tissue samples are now being collected from all enrolled patients who are then screened for downstream biomarker driven clinical trials. These patients are invited to give their informed consent to test for mutations in biological markers of colorectal cancer. These include KRAS, BRAF, NRAS, PIK3CA and microsatellite instability.
For research purposes, NGS is being used to test additional cancer genes in the hope of allocating these patients into more specific disease subgroups based on their molecular tumour profile. It is hoped that these patients can then be enrolled in downstream therapeutic clinical trials with molecularly described inclusion criteria.
Correlating tumour mutational signatures obtained via NGS with high quality, curated clinical data from long-term patient follow-up for response and resistance is a central element of SPECTAcolor. The incorporated translational research component aims to identify and validate new predictive and/or prognostic biomarkers with companion diagnostics for future biologically driven proof of concept clinical trials.
Participating clinical sites send tissue samples for molecular analysis to a central facility. Formalin fixed paraffin embedded samples, containing primary or metastatic tumour material, are first assessed for their sufficient quality and quantity for downstream molecular testing by a board certified pathologist. After the quality control step, part of the sample is used for DNA extraction and quantification. Centralised sample processing and testing with a set standard operating procedure library from the earliest possible step improves standardisation, reproducibility and implementation of QA/QC measures within all SPECTA disease platforms, in general, and SPECTAcolor in particular. As additional assurance for high compliance with quality assurance criteria, the biobank and central laboratory was assessed by independent reviewers to be followed-up by personal audit of the facilities.
Centralised sample processing and NGS at certified laboratories allows future intertumour and interprogramme studies, since assay outputs are expected to be comparable. In SPECTAcolor, as in all SPECTA platforms, NGS is performed in collaboration with the Wellcome Trust Sanger Institute. The current version of the Sanger screening platform generates DNA sequence for all coding exons of 360 genes recurrently mutated in solid tumours and with roles in cancer development, drug response and progression. The ability to detect nucleotide changes, insertions and deletions, copy number alterations and translocations in a large panel of genes known to be involved in cancer biology and drug response or resistance, using targeted NGS technologies will enable rapid recruitment into biomarker-led clinical trials as well as future studies that correlate mutational signatures with clinical response to novel therapeutic agents. The next NGS panel (version 4) due in early 2015, is adapted to detect clinically actionable mutations at sufficiently high quality levels for making clinical research decisions based on output data.
SPECTA infrastructure includes multidisciplinary experts
This work is assisted by an expert group comprised of SPECTA pathologists, molecular biologists, biobanking and monitoring experts, and clinical investigators participating in any of the SPECTA platforms. Representatives of WTSI are also part of this expert group. This group, called SPECTApath (Figure 1), makes recommendations about human biological sample collection and storage (biobanking), monitoring of molecular data quality, and methods for molecular data interpretation and reporting. They are also involved in the quality assurance of molecular analytical techniques and, in an advisory role, can identify, address and resolve transversal molecular marker and human biological material related issues within SPECTA.
SPECTApath is organised by the EORTC Pathobiology Group, who assign tasks within SPECTApath and monitors its performance. It has an adaptive and flexible design and provides an organisational structure supporting working groups with specified tasks. For these, membership is selected based on expertise.
The availability of high quality tissue samples and their DNA isolates are especially important for reliable and comparable NGS. Consequently, local and central pathology laboratories are one of the most important actors in this type of innovative molecular oriented platform. Assay output variability due to differences in early stage sample preparation, for example, fixative agents, fixation time and DNA extraction, can be high and thus data about these pre-analytical procedures at local hospitals is collected in the EORTC clinical trial database.
PERSOCARE presents model for making personalised medicine decisions
SPECTAcolor platform has shown that the necessary expertise can be functionally assembled within a pan-European infrastructure. Still, these platforms make up just one part of the solution, and there are plans to take this idea a step further. PERSOCARE, (Clinical and Molecular Based Personalised Treatment Decision for Sustainable Healthcare in Europe), is a prime example of a model, in discussion, that could help information garnered from programs such as SPECTA to assist in clinical decision making. PERSOCARE will set out to do this in the realm of targeted therapies, specifically in clinical decisions based on evidence generated by high quality molecular screening programmes. The idea is to pilot this decision making model within the HOPE (European Hospital and Healthcare Federation) network of hospitals, and in this way test societal acceptability and financial viability using standardised Quality Assurance/Quality Control (QA/QC) procedures while taking into account local circumstances and specificities. The goal is to generate the information needed by stakeholders to implement personalised medicine in a real-life setting.
EORTC has a mission to improve treatments for patients with cancer
Patients with cancer are waiting for therapeutic improvement and are constantly asking why is it not possible to deliver the appropriate drugs to treat their particular type of cancer, if we have the appropriate technologies. The truth is, until now we have not yet been able to use these new technologies to their best advantage. The EORTC has made a concerted effort to form new models of partnership together and these include multiple cancer research stakeholders. We veritably are on the verge of changing the drug discovery paradigm.
SPECTAcolor is a step in this direction. With SPECTAcolor, the EORTC has demonstrated that it is indeed possible to gather specialists together across disciplines and across borders, so that the latest technologies can be applied effectively within the cancer drug development process. We just needed to think outside the box, and the various SPECTA platforms will forge the future of cancer therapy for the benefit of patients afflicted with these diseases.