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Molecular Urine Test Identifies Lung Carcinoma Mutations

By LabMedica International staff writers
Posted on 20 May 2015
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Image: Droplet digital polymerase chain reaction system (Photo courtesy of Bio-Rad).
Image: Droplet digital polymerase chain reaction system (Photo courtesy of Bio-Rad).
Image: The MiSeq table top sequencer (Photo courtesy of Konrad Förstner).
Image: The MiSeq table top sequencer (Photo courtesy of Konrad Förstner).
The monitoring of circulating tumor 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.

Disease progression in patients with metastatic non-small-cell lung cancer (NSCLC) is often heralded by the acquisition of epidermal growth factor receptor (EGFR) T790M resistance mutation following treatment with anti-EGFR inhibitors.

Scientists at Moores Cancer Center (La Jolla, CA, USA) took urine samples 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 system (Bio-Rad; Hercules, CA, USA) was used to quantify the ctDNA and yielded an average of total amplifiable ctDNA per sample of 0.4 µg (range, 0.04 µg to 2.4 µg). Spiked cell lines were used for analytical characterization 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 status was analyzed using a PCR method that amplifies short target DNA fragments using kinetically-favorable binding conditions for a wild type blocking oligonucleotide, followed by massively parallel deep sequencing using a MiSeq desktop sequencer (Illumina; San Diego CA, USA).

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 five additional patients who may be eligible for treatment with anti-T790M drugs, three of whom were tissue negative. The investigators showed that EGFR T790M mutation can be detected in urinary ctDNA up to three 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 four 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.

Hatim Husain, MD, the lead author of the study and his colleagues concluded that 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. EGFR T790M mutation in urine was detected months before radiographic detection, and furthermore, urine ctDNA testing identified tissue negative patients who may be eligible for treatment with third generation anti-EGFR TKIs and monitoring of urinary T790M dynamics may predict clinical benefit or initial tumour burden. The study was presented at the EUROPEAN Lung Cancer Conference held April 15–18, 2015, in Geneva (Switzerland).

Related Links:

Moores Cancer Center
Bio-Rad
Illumina


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