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Development and Interlaboratory Evaluation of a NIST Reference Clin Chem Lab Med 2019; 57(8): 1142–1152 Hua-Jun He*, Biswajit Das, Megan H. Cleveland, Li Chen, Corinne E. Camalier, Liang-Chun Liu, Kara L. Norman, Andrew P. Fellowes, Christopher R. McEvoy, Steve P. Lund, Jamie Almeida, Carolyn R. Steffen, Chris Karlovich, P. Mickey Williams and Kenneth D. Cole* Development and interlaboratory evaluation of a NIST Reference Material RM 8366 for EGFR and MET gene copy number measurements https://doi.org/10.1515/cclm-2018-1306 (approximately 1 ×, 5 × and greater than 30 ×), whole Received December 7, 2018; accepted February 21, 2019; previously exome sequencing (WES), and two different pan-cancer published online May 21, 2019 gene panels. The WES data were analyzed using three dif- Abstract ferent bioinformatic algorithms. Results: The certified values (digital PCR) for EGFR and Background: The National Institute of Standards and MET were in good agreement (within 20%) with the values Technology (NIST) Reference Material RM 8366 was devel- obtained from the different NGS methods and algorithms oped to improve the quality of gene copy measurements of for five of the six components; one component had lower EGFR (epidermal growth factor receptor) and MET (proto- NGS values. oncogene, receptor tyrosine kinase), important targets for Conclusions: This study shows that NIST RM 8366 is a cancer diagnostics and treatment. The reference material valuable reference material to evaluate the performance is composed of genomic DNA prepared from six human of assays that assess EGFR and MET gene copy number cancer cell lines with different levels of amplification of measurements. the target genes. Keywords: cancer; digital PCR; EGFR; MET; next genera- Methods: The reference values for the ratios of the EGFR tion sequencing; reference material. and MET gene copy numbers to the copy numbers of refer- ence genes were measured using digital PCR. The digital PCR measurements were confirmed by two additional lab- oratories. The samples were also characterized using Next Introduction Generation Sequencing (NGS) methods including whole Reference materials (RMs) are intended to provide a genome sequencing (WGS) at three levels of coverage uniform source of stable samples that can be used to ensure reliable measurement results. RMs can be used to *Corresponding authors: Hua-Jun He and Kenneth D. Cole: track and compare the performance over time of different Biosystems and Biomaterials Division, National Institute methods, instruments, laboratories and operators. NIST of Standards and Technology, 100 Bureau Drive, MS 8312, has developed reference material RM 8366 to improve the Gaithersburg, MD 20899, USA, Phone: +301-975-2169, gene copy number measurements of EGFR (epidermal Fax: +301-330-3447, E-mail: [email protected] (H.-J. He); [email protected] (K.D. Cole) growth factor receptor) and MET (proto-oncogene, recep- Biswajit Das, Li Chen, Corinne E. Camalier, Chris Karlovich and tor tyrosine kinase). The amplification (increased copies) P. Mickey Williams: Molecular Characterization and Clinical Assay of the EGFR gene and its protein overexpression are useful Development Laboratory, Frederick National Laboratory for Cancer biomarkers for determining the therapeutic treatments Research, Frederick, MD, USA and predictive clinical outcomes of cancer patients in Megan H. Cleveland and Carolyn R. Steffen: Biomolecular response to anti-EGFR targeted therapy [1, 2]. Abnormal Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, USA MET activation in cancer, which may be triggered by MET Liang-Chun Liu and Kara L. Norman: Thermo Fisher Scientific, overexpression, correlates with poor prognosis, tumor Fremont, CA, USA growth and metastasis and tumor angiogenesis [3]. Clini- Andrew P. Fellowes and Christopher R. McEvoy: Peter MacCallum cal trials are ongoing to evaluate the safety and efficacy of Cancer Centre, Melbourne, Victoria, Australia selective MET inhibitors in cancer patients [4]. Steve P. Lund: Statistical Engineering Division, National Institute of Standards and Technology, Gaithersburg, MD, USA Rapid and specific quantitative PCR (qPCR) and digital Jamie Almeida: Biosystems and Biomaterials Division, National PCR (dPCR) assays are used to measure gene copy number Institute of Standards and Technology, Gaithersburg, MD, USA measurements of cancer biomarkers in patient samples. Open Access. © 2019 Hua-Jun He and Kenneth D. Cole et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. Brought to you by | NIST Library-National Institute of Standards and Technology Authenticated Download Date | 9/25/19 3:14 PM He et al.: EGFR and MET reference materials 1143 The results from qPCR analysis for ERBB2 (HER2) testing Materials and methods positively correlated with the results from immunohisto- chemistry and fluorescence in situ hybridization methods Cell lines and cell culture [5]. Next-generation sequencing (NGS) assays are being used more frequently in clinical laboratories and provide The NIST RM 8366 consists of genomic DNA samples prepared from a powerful tool to detect multiple genetic alterations in six human cancer cell lines. The cell lines were obtained from ATCC a quantitative manner. However, the assessment of copy (Manassas, VA, USA) as frozen stocks and cultured in the NIST labo- ratory using standard tissue culture methods. The identities of the number variation (CNV) poses challenges because differ- cell lines were authenticated when received from the repository and ent NGS assay platforms may use different chemistries after production of the genomic DNA using short tandem repeat (hybrid capture versus amplification-based target enrich- (STR) DNA genotyping (Supplementary material). This project was ment), different bioinformatic approaches for the calcula- approved by the NIST Human Subjects Protection Office for human tion of copy number alteration, and different algorithms subject research and ethical principles. to adjust for tumor cellularity in the specimen tested. While several NGS platforms have demonstrated strong DNA extraction and purification correlations with fluorescence in situ hybridization in the assessment of CNV [6–8], not all laboratories have access Large batches of cells were prepared from each cell line and used to to FISH for CNV validation. The availability of CNV refer- prepare the genomic DNA. The cells were sub-cultured for four or five ence materials evaluate the performance of NGS assays. passages and were harvested when they reached 85% to 95% conflu- NIST developed Standard Reference Material (SRM) ence from 10 T-175 flask cultures. The culture medium was removed, 2373 for the measurement of HER2 (ERBB2) gene ampli- and the cells were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS). The cells were detached from the flask surface using fication and showed that the reference material was 0.25% (w/v) Trypsin-0.53 mM EDTA solution (Life Technologies, useful for evaluating NGS assay performance and increas- Carlsbad, CA, USA, Cat# 25200-056). Large scale DNA extraction was ing confidence in CNV measurements [9, 10]. Digital and accomplished using the modified Zymo Quick-gDNA™ midiPrep kit quantitative PCR measurements were used for the deter- (Cat# D3100) procedure. After the initial extraction, the samples were mination of ERBB2 (HER2) copy number levels in the five pre-treated with bovine pancreatic ribonuclease A before re-extrac- tion. All purified genomic DNA samples were dissolved or eluted in components (genomic DNA from breast cancer cell lines) TE-4 buffer (10 mmol/L Tris, 0.1 mmol/L EDTA, pH 8.0) and stored at of SRM 2373. 4 °C (range 4–6 °C). Digital PCR (dPCR) is a sensitive and mature tool for the measurements of DNA target concentrations. Efforts are underway at NIST to make dPCR a traceable measure- Digital PCR assays and control gene selection ment method [11–13]. Guidelines on the quality manage- ment of NGS in clinical applications have been proposed, We used the guidelines for the minimal information of quantitative including test validation, quality-control procedures, PCR experiments to guide the development and reporting of the digi- proficiency testing and the use of reference materials [14]. tal PCR assays [17]. NGS assays intended for clinical oncology applications Four sets of PCR assays were developed for both EGFR and MET, have been performance evaluated using pooled cancer and the details and characterization of the assays are contained in the cell lines and clinical samples [15, 16]. Supplementary materials section. The dPCR assays were done using a Bio-Rad QX200 digital PCR system using TaqMan™ fluorescent probe- In this report, a new NIST reference material (RM) based methods. All the assays worked well according to the minimal 8366 was shown to be useful to evaluate and monitor information for publication of quantitative digital PCR experiments the performance of assays for EGFR and MET gene copy guidelines [17]. The PCR products obtained from the four EGFR primer number measurements. We developed, and performance pairs and the four MET primer pairs were analyzed by agarose gel evaluated new dPCR assays for the target genes, EGFR and electrophoresis (details and results in the Supplementary materials, Figure S1). For each assay, one PCR product band was detected at the MET. Digital PCR assays for the reference genes have been expected position. These results indicate the success of using such performance evaluated
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