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European Biopharmaceutical Review

Translating Research into Reality: Part 1

Tissue biopsies, as frozen or formalin-fixed, paraffin-embedded (FFPE) tissue samples, provide a single snapshot of the primary or secondary tumour that has a high likelihood of failing to sample the phenotypic and genomic heterogeneity in both primary and secondary tumours. Metastases may also be inaccessible to a biopsy, and there is significant heterogeneity between different metastatic sites. Add to this the time, cost and patient burden of performing sequential biopsies to monitor disease progression, and it is clear that a more sensitive, specific and non-invasive technology has distinct advantages. The advent of therapies targeted to specific mutations makes such an approach even more imperative.

Advances in DNA sequencing technology have addressed this need in the form of liquid biopsies, broadly defined as non-tissue-based specimens using body fluids – blood, urine and cerebral spinal fluid. However, this article will focus on blood samples.

The term ‘liquid biopsy’ in the context of translational research was first used in conjunction with the analysis of circulating tumour cells (CTCs). Recently, with the development of supporting bioinformatic analysis tools and the increasing focus on the genetics of cancer, liquid biopsy now commonly refers to cell-free DNA or circulating tumour DNA (ctDNA). Although the precise mechanism is unclear, ctDNA enters the circulation as short fragments as a consequence of cell death. Genetic material – that may also include DNA isolated from CTCs, exosomes or RNA – is isolated from diagnostic or on-treatment blood samples. This material is then analysed for the presence of specific mutations or by whole genome analysis for a spectrum of known cancer-associated mutations addressing multiple tumour types.

Building on Research

The information gained from analysis of ctDNA has numerous applications, including: cancer diagnosis; the presence of known cancer risk mutations; success of surgery (absence of ctDNA after treatment); serial sampling to monitor disease recurrence and real time monitoring of treatment; characterisation of acquired resistance and optimal tailoring of subsequent combination therapy; as companion diagnostics; and for early-stage detection/screening and biomarker discovery. As a consequence, the liquid biopsy has advanced from a blood sample primarily concerned with the enumeration of CTCs to a biopsy that is changing the paradigm of drug development and routine treatment in oncology.

Patient selection through screening for specific cancer-associated mutations, that are known to be associated with sensitivity or resistance to a targeted therapy, will help to identify patients that are more likely to respond to that therapy. For example, gefitinib is more likely to provide benefit to patients whose tumours carry an epidermal growth factor receptor (EGFR) mutation, and ovarian or prostate cancers carrying a BRCA1 mutation are more likely to respond to inhibitors of DNA repair. Liquid biopsies may also be used to monitor therapy in real time, as the burden of DNA shed into the circulation is reduced by effective treatment. Monitoring the patient for tumour-specific mutations can identify the emergence of tumour cells with acquired resistance and identify suitable agents for subsequent combination therapy. The burden of ctDNA is generally higher in advanced disease, and at present, the application of ctDNA liquid biopsies is focused on screening for specific mutations and treatment response – which brings us to the technology for DNA testing.

Using Technology


Research into ctDNA technology started approximately 20 years ago. One of the most significant challenges is to distinguish specific signals (cancer-related aberrations) from normal DNA ‘noise’. A 10mL blood sample may yield only 10-30ng DNA containing a few thousand copies of a diploid human genome. Sensitivity then ultimately depends not only on the test itself, but also the yield of ctDNA relative to normal DNA in the blood sample that can vary from 0.01% to more than 90%. Platforms such as droplet digital polymerase chain reaction, BEAMing (beads, emulsions, amplification and magnetics) and next-generation sequencing (NGS) have enabled great leaps in sensitivity whereby the detection of 0.01% mutated DNA is possible. Pharmaceutical companies, working with a diverse collection of technology platform developers and covering multiple sequencing and bioinformatic approaches, have developed mutation-specific ctDNA tests to be used in research or within clinical trial protocols.

Such an approach is exemplified by the first ctDNA test approved by the EMA, the Qiagen therascreen® EGFR RGQ companion diagnostic, to identify non-small cell lung cancer patients who could benefit from treatment with gefitinib. The FDA recently announced the approval of the Cobas EGFR Mutation Test v2 for use with ctDNA to identify patients with specific EGFR mutations suitable for treatment with erlotinib or osimertinib. The broader use of liquid biopsy ctDNA tests for early-stage screening and diagnosis requires further validation due to the low and variable levels of ctDNA associated with different tumour types and the low disease burden of early-stage tumours. Recently, there have been some high-profile warnings from the FDA as a regulatory framework is put in place to deal with the huge expansion of DNA-based tests that are becoming available, from small gene panels to whole genome screening approaches.

Obstacles to Consider

The majority of the FDA-approved diagnostic tests for use in solid tumour disease utilise DNA isolated from FFPE tissue biopsies. There is currently only one FDA-approved NGS platform that allows sequencing of DNA and RNA much quicker and more cheaply than was previously possible (MiSeqDx®, Illumina) and one FDA-approved gene panel test (FoundationOne®, Foundation Medicine). Developing diagnostic tests to support the development of new drugs is a relatively focused activity, generally requiring the analysis of a small number of genes directly linked to the activity of the drug. Validation data for this application are generated alongside a clinical trial that also demonstrates clinical benefit. However, screening for the risk of cancer in otherwise healthy individuals requires time and testing in thousands of people, incurring a not inconsiderable cost. But this has not deterred companies such as Illumina (Grail), Myriad, Genomic Health, Biocartis, Transgenomic Inc, Guardant and others entering the competitive diagnostic space. Although the cost of whole genome sequencing is currently in the region of $1,000 per genome, the cost of a fully analysed genome is in the region of $2,000-5,000, depending on the test.

The gold standard – and currently the only FDA-approved platform for CTCs – is the Veridex CellSearch® immunomagnetic cell separation test based on the expression of the epithelial cell adhesion molecule (EpCAM). First approved for use in metastatic breast cancer and later in colorectal and prostate cancers, a correlate was established between CTC numbers per 7.5mL blood and prognosis. In breast, colorectal and prostate cancers, CTCs above a threshold number of between 3-5 CTCs per 7.5mL, depending on tumour type, was associated with a shorter survival time. As with ctDNA, there are multiple companies developing alternative cell separation/enumeration platforms that are independent of EpCAM expression, utilising dielectrophroesis (such as ApoCell), cell phenotype/morphology (EPIC), or cell density (RareCyte).

All of these platforms have been combined with the analysis of DNA from single CTCs, complementing ctDNA analysis in relation to a more relevant picture of tumour heterogeneity. Although both CTCs and ctDNA are relatively rare with half-lives of 1-2 hours and less than 30 minutes respectively, a number of studies have shown good correlation between ctDNA and CTCs in diverse cancers, including prostate, breast, colon and melanoma; both parameters were associated with more aggressive tumours. Integrating CTC DNA analysis with ctDNA, exosome and epigenetic analysis, is opening up a new window in our understanding of tumour progression.

What Does the Future Hold?

The development of increasingly sophisticated and sensitive ctDNA technology will allow detection of mutations, and deletions of, for example, tumour suppressors, ever-nearer to the start of tumourigenesis, ultimately allowing cost effective screening to become a reality. This will of course need the issue of validation to be resolved to the satisfaction of regulatory authorities.

Even further in the future, a liquid biopsy at the bedside may dictate therapy ‘on the spot’, making personalised medicine just that: identifying the most appropriate treatment and monitoring changes of therapy needed as resistance and/or metastasis occurs.

Becoming a Reality


The applications of liquid biopsies are as exciting as they are numerous. Today, the focus is on later-stage disease, but there is a real potential for early stage diagnosis in asymptomatic individuals. However, current screening is based on known cancer mutations, and there remain many mutations potentially related to cancer with unknown function, so this possibility remains to be demonstrated. Early-stage diagnosis also poses the technical challenge of low ctDNA concentrations compared to that demonstrated in late-stage therapeutic studies. Tissue biopsies for histological and pathological examination will always have a place in the diagnosis and management of cancer, but the interest in liquid biopsy research takes us ever-nearer to making personalised, timely and effective treatments a reality.

Part 2 of this article will explore the application of ctDNA in drug development.

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Paul Elvin, PhD, is Chief Translational Science Officer at Aptus Clinical. He is a specialist in cancer drug discovery with 27 years’ experience in the pharma industry at AstraZeneca, providing scientific and project leadership for small molecule and antibody approaches. In his current role, Paul provides specialist bioscience and translational science expert input into preclinical and clinical drug projects.
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