Reference materials (RMs) are an essential part of improving the quality of measurements. RMs 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.
RMs are made in large batches with assured homogeneity and stability, and thus have a clear advantage to assessing the quality of measurements over ad hoc laboratory samples.
Another important part of the NIST quality improvements process is using well-characterized RMs to conduct interlaboratory testing. In some cases, the interlaboratory testing can be an important step in improving RMs to be better suited for the applications.
The amplification of the gene for Human Epidermal Growth Factor Receptor 2 (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. Diagnostic tests like HER2 assays make it possible to select the best personalized treatment for individual patients based on the genetic makeup of their tumors.
SRM 2373 consists of purified genomic DNA from five breast cancer cell lines with different amounts of HER2 gene (Fig. 2).
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 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. This SRM is available from NIST as SRM 2373®.
NIST RMs for Validation of Copy Number Measurements to Support Cancer Precision Medicine
NIST has an ongoing collaboration with the Molecular Characterization Laboratory (MCL) at the Frederick National Laboratory for Cancer Research to provide them with RMs for validation of actionable targets. SRM 2373 genomic DNA for HER2 gene copy numbers was used by MCL scientists to validate the performance of digital PCR and Next Generation Sequencing (NGS) assays for evaluating gene variations associated with an increased risk of breast cancer. The details of this work are described in the article cited below.
We have developed genomic DNA RM for copy number measurements of the genes for MET and EGFR (Epidermal Growth Factor Receptor). 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 for the correct diagnostics to determine the treatment, since specific therapeutics are available for both of these targets.
RM 8366 consists of purified genomic DNA isolated from six different cancer cell lines with different amounts of amplification of the EGFR and MET genes (Fig.5). It is intended to harmonize the measurements of ratios of the human EGFR and MET genes to unamplified reference genes. This RM is available from NIST as RM 8366.
The reference values for the ratio of the EGFR and MET gene copy number to the copy numbers of reference genes were determined by digital PCR. Interestingly, the component E (produced from a stomach derived cell line Hs 746T) also has a MET gene mutation resulting in exon 14 skipping, a mutation commonly found in lung cancers. NGS was used to confirm the copy numbers and mutation status of the samples. The comparison of different NGS methods used is shown in Fig.6 and Fig. 7 for EGFR and MET copy number measurements, respectively. An interlaboratory testing of the RMs has been completed and a manuscript is in preparation describing the use of the RM 8366 for the validation of NGS and digital PCR methods.