A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

Abstract

Background A major perspective for the use of circulating tumor DNA (ctDNA) in the clinical setting of non-small cell lung cancer (NSCLC) is expected as predictive factor for resistance and response to EGFR TKI therapy and, especially, as a non-invasive alternative to tissue biopsy. However, ctDNA is both highly fragmented and mostly low concentrated in plasma and serum. On this basis, it is important to use a platform characterized by high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the newly developed and commercially available SensiScreen® EGFR Liquid assay platform (PentaBase) with regard to sensitivity, linearity, repeatability and accuracy and finally to compare it to our already implemented methods. The validation was made in three independent European laboratories using two cohorts on a total of 68 unique liquid biopsies. Results Using artificial samples containing 1600 copies of WT DNA spiked with 50% - 0.1% of mutant copies across a seven—log dilution scale, we assessed the sensitivity, linearity, repeatability and accuracy for the p.T790M, p.L858R and exon 19 deletion assays of the SensiScreen® EGFR Liquid assay platform. The lowest value detectable ranged from 0.5% to 0.1% with R2≥0,97 indicating good linearity. High PCR efficiency was shown for all three assays. In 102 single PCRs each containing theoretical one copy of the mutant at initiating, assays showed repeatable positivity in 75.5% - 80.4% of reactions. At low ctDNA levels, as in plasma, the SensiScreen® EGFR Liquid assay platform showed better sensitivity than the Therascreen® EGFR platform (Qiagen) and equal performance to the ctEGFR Mutation Detection Kit (EntroGen) and the IOT® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific) with 100% concordance at the sequence level. Conclusion For profiling clinical plasma samples, characterized by low ctDNA abundance, the SensiScreen® EGFR Liquid assay is able to identify down to 1 copy of mutant alleles and with its high sensitivity, linearity and accuracy it may be a competitive platform of choice.

Роль Мутації Т790М У Першій Та Другій Лініях Терапії Пацієнтів З EGFR-позитивним Недрібноклітинним Раком Легені

“…Саме тому малоінвазивні методи діагностики є пріоритетнішими. При цьому на останніх стадіях захворювання концентрація пухлинної ДНК, що циркулює в крові, є досить високою, та сучасні набори молекулярногенетичної діагностики з використанням плазми крові в якості біологічного матеріалу мають досить високу чутливість -виявляють концентрацію у крові <0,1% мутантних копій за семилогарифмічною шкалою розведення, а ефективність становить близько 80% (тобто тільки 20% результатів можуть бути хибно-негативними) [27]. Варто пам'ятати, що у разі отримання негативного результату з використанням плазми крові, згідно з рекомендаціями Європейського товариства медичної онкології (European Society for Medical Oncology -ESMO) та Національної загальної онкологічної мережі (National Comprehensive Cancer Network -NCCN), на наступному етапі необхідно провести дослідження вже з використанням пухлинної тканини.…”

Integration of liquid biopsy and pharmacogenomics for precision therapy of EGFR mutant and resistant lung cancers

“…A diagnostic tool comprising of a panel of miR-21, miR-126, miR-210, miR-486-5p detected NSCLC with a sensitivity and specificity of 86.2 and 96.5% respectively [110]. The SensiScreen ® EGFR Liquid kit that was commercially launched recently detects EGFR mutations (T790M, L858R, exon 19 deletions) at a higher sensitivity and specificity, which outperforms the established assay platforms with a robust ability to detect single copy mutations [117].…”

Circulating Tumor DNA in Pediatric Cancer

“…The differences in genetic mutations are an important issue to consider, as the mutation to be detected will depend on the purpose of ctDNA testing, e.g., detecting treatment resistance to tyrosine kinase inhibitors for Philadelphia chromosome-like ALL, a high-risk subtype of pediatric ALL ( Zhang et al, 2018 ). Current ctDNA assays have been used for the detection of somatic mutations such as EGFR ( Jensen et al, 2021 ), however these somatic mutations have relatively lower frequencies in childhood cancer due to less exposure to mutagens including smoking and ionizing radiation. Also, liquid biopsy sampling for adult cancers is generally 5–20 ml of blood and in children, this amount is considerably lower, depending on age and weight ( Kahana-Edwin et al, 2021a ).…”