Cell-free DNA analysis allows noninvasive tumour genotype monitoring

Mar 24 2014

By Lynda Williams, Senior medwireNews Reporter

Noninvasive tumour genotyping using cell-free plasma (cf) DNA analysis is feasible, say US researchers who believe the technique will aid patient diagnosis and monitoring.

In a proof-of-concept study, the team developed droplet digital polymerase chain reaction (ddPCR) assays to detect endothelial growth factor receptor (EGFR) L858R and exon 19 deletions in patients with advanced lung cancer or melanoma.

ddPCR was able to detect EGFR mutations at a sensitivity of between five and 50 copies in a background of 10,000 wild-type copies, report Geoffrey Oxnard (Dana-Farber Cancer Institute, Boston, Massachusetts, USA) and co-authors.

They used plasma samples from two sets of 23 patients with KRAS-mutant lung cancer – known to have no overlapping EGFR mutations – to set the threshold for a positive test for EGFR L858R and exon 19 deletions at 1 copy/mL and 5 copy/mL, respectively.

The team also successfully created ddPCR assays to detect KRAS mutations and BRAF V600E mutations.

Measuring human long interspersed element (LINE)1 allowed the researchers to quantify and qualify the quality of cfDNA in samples from 32 patients with EGFR- and KRAS-mutant lung cancer. Detection of mutant alleles improved with increasing levels of LINE-1, possibly suggesting that “LINE-1 levels may assist in identifying which plasma specimens are vulnerable to [a false] negative genotyping result”, note Oxnard et al.

Repeat assays in a patient with EGFR-mutant non-small-cell lung cancer (NSCLC) being treated with chemotherapy after erlotinib failure showed that EGFR L858R increased with the development of brain metastases, whereas levels fell after beginning a new treatment. A second patient's EGFR L858R levels fell when pleural drainage was resolved, despite imaging indicating stable disease.

Changes in KRAS-mutant NSCLC and BRAF-mutant levels were also reported in NSCLC and melanoma patients, respectively, as they underwent treatment or had a change in pain status, even when imaging failed to show any measurable disease.

Finally, analysis of plasma from 13 patients treated for advanced EGFR-mutant NSCLC receiving first-line erlotinib showed that eight of the nine patients who responded to treatment also had a complete plasma response.

Moreover, plasma levels increased in six patients with progression between 4 and 24 weeks before progression was confirmed by the Response Evaluation Criteria in Solid Tumors.

T790M – thought to be a biomarker for patients with EGFR-mutant lung cancer and acquired resistance – was also present in the plasma of these patients, which was confirmed by rebiopsy.

“ddPCR is an attractive technology as its speed, cost, and ease of use are similar to other PCR-based assays, yet the sensitivity and quantitative nature of this assay offers broader clinical application,” Oxnard et al conclude in Clinical Cancer Research.

“Prospective validation based upon this initial experience is needed, and is under way.”

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