Search

The results of EML4-ALK testing are critical to manage ALK tyrosine kinase receptor inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China in 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating three different variants and wild-type samples were collected between 2015 and 2019. Performance was analyzed by evaluating the detection methods, kits, and pre-analytic practices used to further display the landscape of changing conditions of the reliability of EML4-ALK testing. During the 5 years, 3224 results reported from 988 laboratories were evaluated, with an overall error rate of 5.36%. Along with an increasing number of participating laboratories, the error rate within each of the different methods showed a significantly downward trend over the years. No obvious differences in the error rates were found regarding the testing methods or kit manufacturers. Moreover, the individual performance of the laboratories improved when they participated in more PT scheme rounds. The data demonstrated that the performance of individual Chinese laboratories for EML4-ALK testing continuously improved over time by participating PT schemes, regardless of their method. However, care must be taken in standardized operations and validations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to public health. Viral nucleic acid testing is the diagnostic gold standard and can play an important role in the prevention and control of this infection. In this study, bacteriophage MS2 virus-like particles encapsulating specific RNA sequences of SARS-CoV-2 and other coronaviruses were prepared by genetic engineering. The assessment panel, consisting of four positive samples with concentrations of 2.8, 3.5, 4.2, and 4.9 log10 copies/mL and five negative samples with other human coronaviruses, was prepared and distributed to evaluate the accuracy of routine viral RNA detection. Results of 931 panels from 844 laboratories were collected. The overall percentage agreement, positive percentage agreement (PPA), and negative percentage agreement, defined as the percentage of agreement between the correct results and total results submitted for all, positive, and negative samples were 96.8% (8109/8379), 93.9% (3497/3724), and 99.1% (4612/4655), respectively. For samples with concentrations of 4.9 and 4.2 log10 copies/mL, the PPAs were >95%. However, for 3.5 and 2.8 log10 copies/mL, the PPAs were 94.6% (881/931) and 84.9% (790/931), respectively. For all negative samples, the negative percentage agreement values were >95%. Thus, most laboratories can reliably detect SARS-CoV-2. However, further improvement and optimization are required to ensure the accuracy of detection in panel members with lower concentrations of viral RNA.

Genome-edited human cell lines are important resources for producing quality control materials for clinical molecular genetic testing. Generating cell lines with defined mutations through homology-directed repair-based methods are inefficient and can lead to unwanted insertions and deletions in the target loci. Nonhomologous end joining in the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system was harnessed to generate genome-engineered cell lines harboring target mutations. Donor plasmids containing target sites for the single guide RNA (sgRNA) and homologous DNA fragments harboring important cancer gene mutations were cotransfected with the Cas9/sgRNA vector into wild-type human cells. The introduced mutations were validated in-house and in 44 laboratories using various techniques, including next-generation sequencing. Exogenous sequences containing the target mutations were efficiently integrated into the ALK receptor tyrosine kinase (ALK) locus in HEK293T and A549 cells. Successful introduction of artificial mutations was confirmed via both Sanger sequencing and the amplification refractory mutation system. Results of external pilot testing revealed that the DNA samples derived from genome-edited cell lines were widely applicable across multiple platforms and laboratories. This study demonstrates that CRISPR/Cas9-induced nonhomologous end joining is a valuable and novel method for generating artificial mutants for use in quality control applications in clinical molecular genetics.

The echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (EML4-ALK) rearrangement is an important biomarker that plays a pivotal role in therapeutic decision making for non-small-cell lung cancer (NSCLC) patients. Ensuring accuracy and reproducibility of EML4-ALK testing by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing requires reliable reference materials for monitoring assay sensitivity and specificity. Herein, we developed novel reference materials for various kinds of EML4-ALK testing. CRISPR/Cas9 was used to edit various NSCLC cell lines containing EML4-ALK rearrangement variants 1, 2, and 3a/b. After s.c. inoculation, the formalin-fixed, paraffin-embedded (FFPE) samples from xenografts were prepared and tested for suitability as candidate reference materials by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing. Sample validation and commutability assessments showed that all types of FFPE samples derived from xenograft tumors have typical histological structures, and EML4-ALK testing results were similar to the clinical ALK-positive NSCLC specimens. Among the four methods for EML4-ALK detection, the validation test showed 100% concordance. Furthermore, these novel FFPE reference materials showed good stability and homogeneity. Without limitations on variant types and production, our novel FFPE samples based on CRISPR/Cas9 editing and xenografts are suitable as candidate reference materials for the validation, verification, internal quality control, and proficiency testing of EML4-ALK detection.

Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.