Establishment of potential reference measurement procedure and reference materials for EML 4-ALK fusion variants measurement

Yi Yang ^{1 2}, Yu Zhang ¹, Shujun Zhou ², Xia Wang ¹, Chunyan Niu ¹, Yongzhuo Zhang ¹, Huafang Gao ^{3 4}, Xiaohua Jin ^{3 4}, Shangjun Wang ⁵, Meihong Du ⁶, Xiaoyan Cheng ⁶, Lingxiang Zhu ⁷, Lianhua Dong ⁸

Abstract

Echinoderm microtubule-associated protein 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene detection is of great significance for personalized tumor treatment. With the continuous improvement of detection technology for EML4-ALK fusion variants, it is urgent to establish a metrological traceability system to ensure the consistency and comparability of clinical detection results. Traceability establishment relies on SI-traceable reference materials (RMs) and qualified reference measurement procedures (RMPs). In this study, a potential RMP based on reverse transcription digital PCR (RT-dPCR) was developed for the quantitative detection of EML4-ALK V1, V3 fusion variants and reference ALK gene (ALK-ref, covering wild-type, V1 and V3 genotypes), and corresponding EML4-ALK fusion gene RMs were prepared. The established RMP exhibited high specificity, favorable inter-laboratory reproducibility (CV < 7.3 %) and excellent linearity (0.92 < slope < 1.06, R² ≧ 0.99). The limits of detection (LoD) for V1, V3 and ALK-ref were 2 copies/reaction, 2 copies/reaction and 1 copy/reaction, respectively. Inter-laboratory verification using the prepared RMs and established RMP demonstrated consistent detection results in terms of the copy concentrations of EML4-ALK fusion variants, ALK-ref, and the EML4-ALK/ALK-ref ratio across participating laboratories. The validated RMP with high specificity and accuracy is applicable for the value assignment of EML4-ALK RMs. The established RMP and RMs can effectively construct the traceability system for EML4-ALK fusion gene quantification and improve the consistency and comparability of clinical detection results.