Monitoring circulating tumour DNA (ctDNA) is a promising technique that may provide clinicians with a faster, cheaper and less invasive way to evaluate the clinical status and response to therapy of cancer patients.
Dr Hatim Husain, Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA presented findings from a study that used ctDNA in patient urine samples for detection of EGFR T790M resistance mutation in an expanded patient cohort during the ESMO-IASLC Best Abstracts session at the European Lung Cancer Conference, held 15 to 18 April 2015 in Geneva, Switzerland.
Disease progression in patients with metastatic non-small-cell lung cancer (NSCLC) is often heralded by the acquisition of EGFR T790M resistance mutation following treatment with anti-EGFR inhibitors.
Clinical trials of agents that target EGFR T790M are underway but mutation detection remains reliant upon obtaining biopsies from relapsed patients. Dr Husain discussed the need for a non-tissue based approach for T790M detection and the results from a study conducted by his team.
Detection of T790M Mutation by ctDNA Assay Shows 100% Concurrence with Tumour Biopsy Detection
Drug induced apoptosis is indicated by peaks of T790M/Exon 19 deletion mutations in urine ctDNA immediately following therapy, while the subsequent decrease in urine ctDNA mutations is associated with a decrease in tumor burden.
© Hatim Husain
In this study, urine samples were taken from patients with metastatic NSCLC who progressed on erlotinib treatment and ctDNA was extracted by a method that preferentially isolates short, fragmented ctDNA. Droplet digital polymerase chain reaction (ddPCR) was used to quantify the ctDNA and yielded an average of total amplifiable ctDNA per sample of 0.4 µg (range, 0.04 to 2.4 µg). Spiked cell lines were used for analytical characterisation and demonstrated that the EGFR T790M assay had a lower limit of detection of two copies within a background of 60 ng of wild-type DNA, yielding an analytical sensitivity of 0.01%.
EGFR T790M mutation in urine was detected in 15 of 22 (68%) of patients receiving anti-EGFR treatment until progression. Urine EGFR T790M mutation was detected in 10 out of 10 tissue-positive patients, giving 100% concordance. Urine ctDNA testing identified 5 additional patients who may be eligible for treatment with anti-T790M drugs, 3 of whom were tissue negative.
The investigators showed that EGFR T790M mutation can be detected in urinary ctDNA up to 3 months before radiographic progression on first-line anti-EGFR tyrosine kinase inhibitor (TKI).
When T790M positive patients were treated with third generation anti-EGFR TKIs, a decrease in ctDNA T790M load was observed as early as 4 hours after therapy on first day of treatment. The initial decrease in urinary T790M was followed by a spike in T790M during the first week of therapy. Studies to understand if the correlation between size of spikes, tumour burden and/or variation in response are ongoing.
Dr Egbert Smit of the Netherlands Cancer Institute and Vrije Universiteit VU Medical Centre in Amsterdam, The Netherlands, who discussed the study results, said that lung tumours are frequently inaccessable and tumour biopsies have high failure rate in procuring sufficient DNA, while blood and urine samples are easy to obtain, with low complication rate, and repeated sampling is possible.
Advantages of urine over plasma as biosource are its easyness to collect, non-invasivness, potential to avoid out-patient clinics, higher yield of ctDNA, high analytical sensitivity, but it is still uknown if only ctDNA could be detected. The abstract 36O showed that ctDNA EGFR mutation testing in urine is a novel way for non-invasive detection.
Conclusions
This ctDNA assay for multiple genes via next-generation sequencing (NGS) might become a "liquid biopsy" that could serve as an alternative to invasive tissue biopsy, according to Dr Husain, who discussed the team’s development of this platform, which couples extraction of urine ctDNA with an ultra-sensitive NGS mutation enrichment method.
The investigators are the first to demonstrate that this methodology successfully detects EGFR T790M mutation load from ctDNA, which can be harvested in a noninvasive manner from urine samples. This method may enable fast and reliable monitoring of response to anti-EGFR therapy and detect mutations that serve as biomarkers for disease progression without invasive sequential biopsies in relapsed patients with NSCLC.
EGFR T790M mutation in urine was detected months before radiographic detection.
Furthermore, urine ctDNA testing identified tissue negative patients who may be eligible for treatment with third generation anti-EGFR TKIs. Monitoring of urinary T790M dynamics may predict clinical benefit or initial tumour burden.
Reference
The author acknowledged support from the University of California, San Diego Liquid Biopsy Programme and Trovagene.