Development of a minimal residual disease assay for anaplastic lymphoma kinase mutations using digital droplet polymerase chain reaction

Julie M. Fischer; Ben Smith; Aubri Waters; Jayasree Krishnamurthy; Dina Parekh; Carol Thiele; Kenneth Lieuw

doi:10.1542/peds.142.1MA7.609

Background: Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. Currently, disease response evaluation requires imaging or bone marrow biopsy. Our objective was to develop a peripheral blood (PB) assay for monitoring minimal residual disease (MRD) in NB utilizing mutations in the anaplastic lymphoma kinase (ALK) gene. Specifically, we targeted mutations in ALKF1174L and ALKR1275Q, which have the highest occurrence. A PB assay with the ability to detect mutation-containing circulating cell-free DNA with adequate sensitivity and specificity would allow clinicians to better identify tumor recurrence and monitor disease response to therapy. As ALK is now being used as a target for NB treatment, a polymerase chain reaction (PCR) based MRD assay would be feasible and clinically useful. Methods: Two NB cell lines containing ALKF1174L and ALKR1275Q mutations were cultured and genomic DNA was isolated. Wild type genomic DNA was spiked with mutant DNA to form the various concentrations of DNA for these experiments. Allele-specific primers were initially designed and reaction conditions were optimized with quantitative real time PCR before runs on digital droplet PCR (ddPCR). To further increase sensitivity, mismatch amplification mutation assay (MAMA) primers were used that add an additional mismatch mutation at the primer’s penultimate 3’ nucleotide resulting in 2 mismatches between the allele to be discriminated against. While regular PCR with this assay is challenged by false positive results due to the rarity of mutant DNA, ddPCR splits a 20ul reaction into 20,000 separate micro droplets allowing for much greater ratio of mutant to wild type, which increases sensitivity and specificity. Results: Real time optimization using MAMA primers showed that we could achieve sensitivity and specificity for both mutations to only about 0.1%, which can detect one mutant copy within 1000 normal copies. However, at levels less than 0.1%, false amplification of the wild type DNA made the assay less useful. When the same conditions were used with ddPCR, the both the ALKF1174L and ALKR1275Q mutant DNAs could be detected reliably below 0.1%, improving both the sensitivity and specificity of the assay. Conclusion: Our assay reveals that ddPCR can improve upon a real time PCR for use in a PB MRD assay. We are currently continuing to improve the sensitivity of our assay by adjusting reaction conditions as well as designing new primers with improved specificity. We believe that the combination of MAMA PCR and ddPCR shows great potential for use as a “liquid biopsy” test with the ability to detect mutation-containing circulating cell-free DNA. Further, such an assay could be applied to surveillance of any cancer where a known mutation is characterized at diagnosis and response to therapy could be followed using this technique.

Development of a minimal residual disease assay for anaplastic lymphoma kinase mutations using digital droplet polymerase chain reaction

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