Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods

Lih, Chih-Jian; Si, Han; Das, Biswajit; Harrington, Robin; Harper, Kneshay N.; Sims, David; McGregor, Paul M.; Camalier, Corinne E.; Kayserian, Andrew Y.; Williams, P. Mickey; He, Hua‐Jun; Almeida, Jamie L.; Lund, Steven P.; Choquette, Steve; Cole, Kenneth D.

doi:10.1016/j.jmoldx.2016.05.008

Cited by 20 publications

(17 citation statements)

References 44 publications

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“…These assays were developed for the certification of NIST cancer biomarker standards (23,24). The calculation of copy number measurements from droplet digital PCR requires an accurate measurement of the droplet size.…”

Section: Resultsmentioning

confidence: 99%

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples

Stein

DeRose

et al. 2018

BioTechniques

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We compared different methods (absorbance, fluorescent dye-binding, and digital PCR) for measuring the concentrations of human genomic DNA from cultured cells and absorbance measurements of a synthetic DNA oligonucleotide. NIST Standard Reference Material (SRM) 2082, a pathlength absorbance standard, was used to benchmark the absorbance measurements done with microvolume spectrophotometers and a microvolume plate reader. Control absorbance values were measured on a high accuracy spectrophotometer and a NIST calibrated pathlength cuvette. Measurements of the human genomic DNA sample were done with several types of fluorescent dye binding assays using different DNA calibrators. The fluorescent dye binding methods gave different results for genomic DNA depending upon the type of DNA calibrator and the fluorescent dye that was used. The human genomic DNA sample was also characterized by using six different droplet digital PCR assays (amplicons located on different chromosomes) to measure the average copy number. Conversion of the digital PCR data to copy numbers was sensitive to the droplet size used for calculations and conversion to mass concentration was dependent upon the molecular weight of the human genome used for the calculations. The results from the different methods were compared and the caveats for each measurement method were discussed.

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Section: Resultsmentioning

confidence: 99%

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples

Stein

DeRose

et al. 2018

BioTechniques

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“…These sequencing platforms are being used for screening of genomic loci copy number alterations. In application to HER2, the NGS determination would still require confirmatory testing with validated assays 52 .…”

Section: Her2 Targeting In Gastroesophageal Cancermentioning

confidence: 99%

Perspectives of HER2-targeting in gastric and esophageal cancer

Gerson

Skariah

Denlinger

et al. 2017

Expert Opinion on Investigational Drugs

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Introduction The blockade of HER2 signaling has significantly improved the outlook for esophagogastric cancer patients. However, targeting HER2 still remains challenging due to complex biology of this receptor in gastric and esophageal cancers. Areas covered Here, we review complex HER2 biology, current methods of HER2 testing and tumor heterogeneity of gastroesophageal cancer. Ongoing and completed clinical research data are discussed. Expert Opinion HER2 overexpression is a validated target in gastroesophageal cancer, with therapeutic implications resulting in prolonged survival when inhibited in the front-line setting. With standardized HER2 testing in gastro-esophageal cancer, the ongoing trials are testing newer agents and combinations including combination of anti-HER2 antibodies with immunotherapy. Clonal heterogeneity and emergence of resistance will challenge our approach to treating these patients beyond the frontline settings.

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“…A call for the development of a cell line HER-2 measurement standard in 2003 19 led to a HER-2 genomic DNA standard this year (2016) 20,21 but, at least so far, no HER-2 protein standard. Other descriptions regarding the use of cell lines in IHC testing, sometimes in the context of proficiency testing, often do not characterize cell lines in terms of the number of HER-2 molecules per cell.…”

Section: Discussionmentioning

confidence: 99%

Analytic Response Curves of Clinical Breast Cancer IHC Tests

Vani

Sompuram

Schaedle

et al. 2017

J Histochem Cytochem.

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An important limitation in the field of immunohistochemistry (IHC) is the inability to correlate stain intensity with specific analyte concentrations. Clinical immunohistochemical tests are not described in terms of analytic response curves, namely, the analyte concentrations in a tissue sample at which an immunohistochemical stain (1) is first visible, (2) increases in proportion to the analyte concentration, and (3) ultimately approaches a maximum color intensity. Using a new immunostaining tool ( IHControls), we measured the analytic response curves of the major clinical immunohistochemical tests for human epidermal growth factor receptor type II (HER-2), estrogen receptor (ER), and progesterone receptor (PR). The IHControls comprise the analytes HER-2, ER, and PR at approximately log concentration intervals across the range of biological expression, from 100 to 1,000,000 molecules per test microbead. We stained IHControls of various concentrations using instruments, reagents, and protocols from three major IHC vendors. Stain intensity at each analyte concentration was measured, thereby generating an analytic response curve. We learned that for HER-2 and PR, there is significant variability in test results between clinical kits for samples with analyte concentrations of approximately 10 molecules/microbead. We propose that the characterization of immunostains is an important step toward standardization.

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Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods

Cited by 20 publications

References 44 publications

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples

Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples

Perspectives of HER2-targeting in gastric and esophageal cancer

Analytic Response Curves of Clinical Breast Cancer IHC Tests

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