Reference materials are an essential part of improving the quality of measurements. Reference materials produced by NIST and others are used ensure that the procedures and analytical measurements are working correctly using a well-characterized material with certified values.
Reference materials are made in large batches in with assured homogeneity and stability, and thus have a clear advantage to assessing the quality of measurements over ad hoc laboratory samples.
NIST conducts interlaboratory testing. Another important part of the NIST quality improvements process, using well-characterized reference materials. In some cases, the interlaboratory testing can be an important step in improving reference materials to be better suited for the applications.
SRM 2373® Genomic DNA Standards for HER2 Measurements
The amplification of the gene for HER2 occurs in approximately 20 to 25% of breast cancers. The accurate measurement of this biomarker is important for the proper treatment with anti-HER2 therapeutics. Clinical laboratories are beginning to utilize accurate and sensitive diagnostic tests based on measurements of DNA. We have prepared genomic DNA from 5 human breast cancer cell lines with different amounts of amplification of the HER2 gene. The copy numbers of the HER2 gene and 3 reference genes (not amplified) were measured using quantitative PCR and digital PCR assays. The certified values of the components are the ratios of the HER2 gene copy number to the reference gene copy numbers. The PCR assays were validated and calibrated using NIST SRM®2372 component A (human genomic DNA). The stability of the components was shown by repeated measurements over several years. The DNA concentration determined from absorbance and the PCR assays was provided as informational value.
NIST Reference Materials for Validation of Copy Number Measurements to Support Cancer Precision Medicine
NIST has a collaboration with the Molecular Characterization Laboratory at the Frederick National Laboratory for Cancer Research to work with them to provide reference materials for validation of actionable targets. We used the SRM 2373 genomic DNA for HER2 gene copy numbers to validate their digital PCR and Next Generation Sequeicing (NGS) assays. The details of this are in the article cited below. In addition we are developing additional reference materials for MET and EGFR gene copy number measurements (details below for RM 8366).
Reference Material 8366 (in progress) Copy Number Standards for EGFR and MET
We are in the progress of developing genomic DNA reference materials for copy number measurements of the genes for MET and EGFR (Epidermal Growth Factor). MET and EGFR are both tyrosine kinase proteins found on the surfaces of cells that response to external growth factors. Mutations or amplification of the genes for MET and EGFR can cause increased cellular signaling for cell growth or mobilization resulting in cancer. The accurate measurements of these cancer biomarkers are important to determine the correct diagnostics to determine the treatment, since specific therapeutics are available for both of these targets. We have selected six cancer cell lines with different amounts of gene amplification of MET or EGFR.
The stomach derived cell line Hs 746T also has a MET gene mutation resulting in exon 14 skipping, a mutation found in lung cancers. We are planning on measuring the gene copy numbers of MET, EGFR, and multiple reference genes to obtain reference values. Next generation sequencing will also be used to confirm the copy numbers and mutation status of the samples. An interlaboratory testing of the materials is planned in the second half of 2016, and release soon after the testing is done.
Reference: “Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next Generation Sequencing Methods” ,Chih-Jian Lih, Han Si, Biswajit Das, Robin D. Harrington, Kneshay N. Harper, David J. Sims, Paul M. McGregor, Corinne E. Camalier, Andrew Y. Kayserian, P. Mickey Williams, Hua-Jun He, Jamie Almeida, Steve Lund, Steve Choquette, and Kenneth D. Cole , Journal of Molecular Diagnosis (in press).