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Liquid biopsies in clinical practice

Angharad Williams PhD
9 August, 2017  
Over the past few years, there has been much interest in the molecular characterisation of circulating cell-free tumour DNA (ctDNA) in patients with cancer1 where tumour biopsy analysis is complicated by a lack of sufficient sample or good quality DNA. For some patients with cancer, tumour tissue biopsy is not possible altogether.2 However, despite promising research into the application of ctDNA analysis in specific clinical scenarios, work continues to establish clearly defined clinical validity and utility of ctDNA testing, as well as assessing cost and logistical issues.
 
Stratified medicine services 
The All Wales Medical Genetic Service (AWMGS) has been providing stratified medicine services for cancer patients in Wales and the surrounding regions since 2009. These services have historically focussed on the genetic analysis of formalin-fixed paraffin embedded tissue (FFPE) to determine treatment options for non-small cell lung cancer (NSCLC) patients, metastatic colorectal cancer patients, melanoma and gastrointestinal stromal tumour patients. The number of stratified medicine-based genetic analyses performed at AWMGS has grown year on year, and in 2016, approximately 1700 patient samples were analysed within the laboratory.
 
Until recently, the standard protocol for a patient with potential stratified medicine treatment options would be that the biopsy/resection taken from the tumour would be formalin-fixed; this FFPE would then be histologically assessed and the area of highest neoplastic cell content macrodissected prior to automated DNA extraction using a Maxwell® 16 FFPE Plus LEV DNA Purification kit. The presence or absence of specific mutations in the patient tumour DNA sample, detected using in-house designed pyrosequencing or Sanger sequencing assays, would then inform clinical treatment decisions; for example, a NSCLC patient with an epidermal growth factor receptor (EGFR)-sensitising mutation would be likely to benefit from EGFR oral tyrosine kinase inhibitors.3,4
 
Unfortunately, there are a number of issues with performing genetic analysis on FFPE tissue:
  • FFPE tissue generally yields poor quality DNA owing to DNA damage (including deamination, oxidation and double strand breaks) caused by the formalin fixative. FFPE-based genetic testing therefore fails in a small percentage (~5%) of patients.
  • Some cancer patients are not well enough to have a biopsy taken, therefore no FFPE is available. This issue is particularly of note for non-small cell lung 
  • cancer patients.
  • FFPE tissue blocks are exhausted, in some cases owing to extensive histological assessments that can be required in cancer diagnosis.
These factors mean that a number of cancer patients cannot access stratified medicine treatment options because genetic analysis of the tumour is not possible. Therefore there was a clinical need to identify a means of expanding the current service in order to reach more cancer patients. Local lung oncologists were particularly keen for such a service to be available for NSCLC patients because a high proportion of these patients fall into the second category above, where there is no tumour sample available for analysis. 
 
The benefits and uses of liquid biopsies in clinical practice
Apoptotic or necrotic tumour cells discharge DNA fragments into the circulating blood system; these DNA fragments are called cell-free ctDNA. Somatic genetic alterations specific to the tumour can be detected in ctDNA; ctDNA is thus a highly specific biomarker.5 Cell-free DNA (cfDNA) is also shed from normal cells into the circulation; in general the volume of normal cfDNA is considerably higher than circulating tumour DNA (ctDNA). CtDNA is obtained by means of a minimally invasive blood test, termed ‘liquid biopsy’. Blood samples for ctDNA analysis can be processed with a quick turnaround time, saving valuable time for patients who need to promptly commence anticancer treatments. Because the blood test can be taken in most clinical settings, serial liquid biopsies can be taken and processed in ‘real-time’, as a patient undergoes anti-cancer therapy. This offers several advantages to conventional tests, such as radiological monitoring and repeat tumour biopsies.
 
There is the potential to assess ctDNA in different settings, such as disease screening and disease diagnosis.6 It can act as a predictive biomarker for the response to various targeted anticancer therapies, and it also offers the potential to monitor disease response to therapy, monitor periods of minimal residual disease,7 and to help to detect relapsed disease.8 It is also possible to glean molecular mechanisms of therapeutic resistance.9
 
One example of a clinical application for ctDNA testing is detecting sensitising EGFR mutations in metastatic NSCLC patients, which is a positive predictive biomarker for response to oral tyrosine kinase inhibitors.3,4 Gefitinib (Iressa®, AstraZeneca) is a National Institute for Health and Care Excellence (NICE)-approved oral tyrosine kinase inhibitor for patients in the UK with EGFR mut+ve ctDNA lung cancer, specifically where tumour tissue biopsy testing is unavailable.10
 
Another application for ctDNA testing is in the detection of the frequently acquired EGFR c.2369C>T p.(Thr790Met) resistance mutation (commonly known as T790M) in ctDNA, in patients with EGFRmut+ve lung cancer treated with first-line EGFR tyrosine kinase inhibitors.9 In the UK, the third-generation EGFR tyrosine kinase inhibitor osimertinib (Tagrisso®, AstraZeneca) has been given NICE approval for availability on the Cancer Drugs Fund.11 Clinicians can prescribe this therapy to patients, on the detection of T790M in ctDNA, without the necessity for re-biopsy of tumour tissue.12
 
Establishing the ctDNA-based NSCLC stratified medicine service in the AWMGS
The AWMGS already had experience of working with ctDNA on a research basis owing to the laboratory’s involvement in clinical trials that utilised ctDNA extracted from blood in order to monitor the tumour genotypes of advanced cancer patients. A number of feasibility studies on ctDNA had also been performed within the laboratory by students and staff in preparation for the use of ctDNA in trials. Therefore, we had already internally validated cfDNA extraction protocols and assays, and were aware of the issues surrounding the rapid transportation of blood for downstream ctDNA analysis, as discussed below.
 
Cell-free circulating DNA is rapidly cleared from the blood; therefore careful consideration was taken to decide on the most appropriate route for diagnostic testing using such a labile biomarker. An overview of the final service procedure is given in Figure 1. Important points to consider were:
  • What is the best type of blood sampling tube to preserve ctDNA?
  • What was the optimum pathway to get the sample to the laboratory within the strict time frame?
  • Education of the requesting clinicians and nurses on the importance of timely sample receipt and limitations of the 
  • testing.
  • What is the most sensitive, cost effective and time efficient testing method to use in a clinical setting?
 
Strict precautions were implemented to maintain the integrity of the ctDNA during blood collection, dispatch and processing. Investigations into the stability of ctDNA in blood in EDTA collection tubes compared with specialist preservative tubes and communication with other research groups13 led to our decision to only use ctDNA preservative tubes such as CellSave Preservative Tubes (Janssen Diagnostics) or Cell-Free DNA BCT® (Streck). These tubes preserve DNA for up to 96 hours,13 which provides adequate time for samples to reach the laboratory with confidence that the ctDNA quality has been maintained. Our experience with clinical trials, already collecting plasma for ctDNA, demonstrated that using a guaranteed next-day delivery postal service was the best way of ensuring rapid delivery of samples. 
 
The fact that the ctDNA service needs specialised tubes and has precise delivery requirements means that this system is more complex than other sample submission processes within the clinical laboratory. To prevent any confusion by potential service users, we prepared a simple guidance leaflet to accompany the testing request forms. This leaflet provided information on what tubes to use, how to use them, and details on how to send the sample to the laboratory. In addition, the Service Lead visited a number of centres around Wales and the surrounding regions that would benefit from access to the new ctDNA service, to speak to lung oncology teams about the logistics of requesting testing, and to raise awareness of the new service. CtDNA in peripheral blood is usually at low levels.1
 
Its presence is masked by the background levels of wild-type cfDNA and a highly sensitive technology was needed to ensure detection of low level ctDNA mutations could be achieved. A number of methods were considered, including next generation sequencing and quantitative polymerase chain reaction, before it was decided that a new fluorescence based technology, droplet digital polymerase chain reaction (ddPCR), would be used. DdPCR offered the sensitivity required and there were researchers already experienced with the process and the equipment within the laboratory. 
 
The Bio-Rad QX200 droplet digital PCR system separates the PCR reaction into thousands of nanolitre-sized droplets.14 With mutation-specific fluorescence probes, each of the thousands of droplets can be individually read to give an extremely low sensitivity of approximately 0.0001% mutant DNA in a wild-type background.14 Using this method, we have been able to detect EGFR mutations in patient ctDNA as low as 0.7% in the blood sample; this would have been unachievable using the conventional tumour tissue testing methods such as pyrosequencing, which is validated within our laboratory to detect mutant alleles down to 2% in a background of wild-type DNA.
 
One of the strengths of the validation process was the collaboration with the clinical oncology community in Wales. Effective communication during validation stages allowed us to track NSCLC patients, who had already had FFPE-based genetic testing within AWMGS, and request a follow-up blood sample for ctDNA analysis in real-time. This resulted in assurance that the pathways for blood sample collection and dispatch were in place within Wales, as well as adding confidence to our validation process via the successful detection of EGFR mutations in ctDNA from real patient samples.
 
The success of the ctDNA clinical service
The EGFR ctDNA service was launched in April 2016 with immediate participation from clinicians across Wales. The service continues to grow and we are now involved with an AstraZeneca service evaluation involving successive FFPE and ctDNA analysis of patient samples for both first-line and resistant EGFR mutations for treatment across Wales and South West England. 
 
As of February 2017, the ctDNA clinical service has tested more than 100 patient samples, 62% of which were for T790M resistance testing, avoiding the need for unpleasant repeat biopsies for progressing patients (Table 1). Six patients, who would not otherwise have accessed molecular testing due to inability to biopsy or failed FFPE-based testing, are now receiving first-line EGFR tyrosine kinase inhibitor therapy due to positive results from the ctDNA clinical service. 
 
 
The response from the lung oncology community has been extremely positive and we will soon be issuing an official feedback questionnaire to collate users’ thoughts and improvement suggestions regarding our current ctDNA service. In December 2016, the laboratory team was awarded the MediWales Innovation NHS Judges’ prize for the ctDNA service and its positive impact on patient care. In the near future, we will be expanding the circulating biomarkers for stratified medicine sections with increased interest from metastatic colorectal cancer and melanoma clinicians. The research team at the AWMGS are continuing to investigate the use of ctDNA in other tumour types, and the feasibility of other circulating biomarkers such as exosomes and circulating tumour cells. 
 
References
1 Heitzer E, Ulz P, Geigl JB. Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem 2015;61(1):112–23. 
2 Vanderlaan PA et al. Success and failure rates of tumor genotyping techniques in routine pathological samples with non-small-cell lung cancer. Lung Cancer 2014;84(1):39–44. 
3 Douillard JY et al. Gefitinib treatment in EGFR mutated caucasian NSCLC: circulating-free tumor DNA as a surrogate for determination of EGFR status. J Thorac Oncol 2014;9(9):1345–53. 
4 Goto K et al. Epidermal growth factor receptor mutation status in circulating free DNA in serum: from IPASS, a phase III study of gefitinib or carboplatin/paclitaxel in non-small cell lung cancer. J Thorac Oncol 2012;7(1):115–21. 
5 Bettegowda C et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 2014;6(224):224ra24.
6 Chen KZ et al. Circulating tumor DNA detection in early-stage non-small cell lung cancer patients by targeted sequencing. Sci Rep 2016;6:31985. 
7 Dawson S-J et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 2013;368(13):1199–209.
8 Forshew T et al. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med 2012;4(136):136ra68. 
9 Murtaza M et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 2013;497(7447):108–12.
10 National Institute for Health and Care Excellence. Gefitinib for the first-line treatment of locally advanced or metastatic non-small-cell lung cancer. Technology appraisal guidance (TA192), 2010. www.nice.org.uk/guidance/ta192 (accessed March 2017).
11 National Institute for Health and Care Excellence. Osimertinib for treating locally advanced or metastatic EGFR T790M mutation-positive non-small-cell lung cancer. Final appraisal determination (TA10022). 2016. 
12 Sundaresan TK et al. Detection of T790M, the acquired resistance EGFR mutation, by tumor biopsy versus noninvasive blood-based analyses. Clin Cancer Res 2016;22(5):1103–10. 
13 Rothwell DG et al. Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample. Mol Oncol 2016;10:566–74. 
14 Hindson BJ et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 2011;83(22):
8604–10.