Abstract 468: Feasibility of digital PCR for the detection of ALK rearrangement in the plasma of non-small cell lung cancer patients
Authors: Cho William C. , Fong Alvin H. , Fung King Y. , Chik Jeannie Y. , Li Jacky Y. 
Published: July 2021

Abstract
Introduction: Currently, the three primary methods of detecting ALK rearrangement are fluorescent in situ hybridization, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR). The former two methods use tissue as the laboratory materials, but some patients do not have sufficient tissue to perform the tests. The RT-PCR method can be conducted by both tissue and blood, but its detection of ALK rearrangement seems not sensitive enough. Digital PCR (dPCR) is a biotechnology refinement of conventional PCR methods that may be a more precise method than RT-PCR.
Materials and Methods: An EML4-ALK variant 1 (v1) synthetic sequence, a variant 2 (v2) synthetic sequence, and a variant 3 (v3) non-small cell lung cancer (NSCLC) cell line (NCI-H2228) were used to evaluate the performance of our dPCR assay. To examine the performance PCR on clinical samples, we used both dPCR and RT-PCR assays to quantify the expression levels of EML4-ALK variants (v1, v2, and v3) in the circulating tumor RNAs derived from 414 plasma samples of 30 NSCLC patients during their ALK inhibitor treatment.
Results: We have designed a multiplex dPCR assay for the detection of EML4-ALK in a v1 synthetic sequence (with 8,000 copies), a v2 synthetic sequence (with 10,000 copies), and a v3 cell line (with 100 copies). Our results showed high specificities and precisions (8,757 ± 592 copies, 11,070 ± 7,791 copies, and 93 ± 14 copies, respectively) for detecting all three variants simultaneously. On the other hand, our results observed good linearities (R2 = 0.9996, 0.9985, and 0.9991, respectively) in all three variants. To determine our dPCR assay's limit of detection for v1 and v2 synthetic sequences (each with four copies), our assay successfully measured the copy numbers of both v1 and v2 (4.1 ± 1.6 and 3.9 ± 1.8 copies, respectively). Comparing the sensitivities of our dPCR assay with RT-PCR assay, dPCR was able to detect down to 2 copies (for v1 and v2) and 20 pg of cDNA (for v3) while RT-PCR could only detect 200 copies (for v1 and v2) and 200 pg of cDNA (for v3). After confirming the promising performance of our dPCR assay, we tried to detect EML4-ALK variants by both dPCR and RT-PCR assays in the plasma samples of our recruited patients but mostly negative. Only one sample detected 39.2 copies of EML4-ALK v3 using dPCR assay.
Conclusion: dPCR assay is a precise method with good performance at detecting EML4-ALK variants in synthetic sequences and cell line, which is more sensitive than RT-PCR. However, EML4-ALK variants could not be detected in the majority of our plasma samples. This may be due to insufficient circulating RNAs for detection. In addition, RNA may be degraded by RNase in plasma and that platelet may be a better alternative than plasma for the detection of RNAs in patient samples. Additional testing is warranted to detect EML4-ALK variants in the platelet samples of another NSCLC patient cohort.
Citation Format: William C. Cho, Alvin H. Fong, King Y. Fung, Jeannie Y. Chik, Jacky Y. Li. Feasibility of digital PCR for the detection of ALK rearrangement in the plasma of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 468.