CCQM-K176 breast cancer biomarker HER2 copy number variation (CNV) measurement
Authors: Dong Lianhua , Wang Xia , Devonshire Alison , Huggett Jim , Ellison Steve , Gonzalez Ana Fernandez , French David , Morris Phattaraporn , Temisak Sasithon , Bae Young , Vonsky Maxim , Runov Andrey , Baoutina Anna , McLaughlin Jacob L H , Burke Daniel , Flatschart Roberto Becht , Medeiros Marcelo N , Saraiva Antonio Marcos , Leguizamon John Emerson , Torres Andres Leon , Divieto Carla , Pegoraro Mattia , Revel Laura , Yalcinkaya Burhanettin , Taslak Hava , Akgoz Muslum 
Published: October 2024

Main text
Breast cancer is the most common malignant tumor in women all over the world. Breast cancers with amplification of the human epidermal growth factor receptor 2 (HER2) gene (referred to as HER2-positive breast cancer) accounts for about 20% ~ 30% of invasive breast cancer, and this type of tumor is characterized by high invasiveness, high risk of recurrence, rapid progression and poor prognosis. The determination of HER2 gene expression or copy number variant (CNV) (HER2 copy number normalized to cell number or a reference gene) in tumor cells of breast cancer patients is beneficial to the choice of treatment, through targeted therapies such as trastuzumab, and prognosis. Testing of the core competencies of laboratories to deliver services of CNV measurements for different genetic analytes has not been covered previously. Agreement was received in the October 2019 meeting of the NAWG in Torino to conduct the HER2 CNV measurement as a key comparison. Evidence of successful participation in formal, relevant international comparisons is needed to support calibration and measurement capability claims (CMCs) made by national metrology institutes (NMIs) and designated institutes (DIs).
Ten National Metrology Institutions participated in the Track A Key Comparison: CCQM-K176 "Breast cancer biomarker HER2 copy number variation (CNV) measurement". Participants were requested to evaluate the copy number concentration (expressed in [μL-1]) and copy number ratio of defined genomic sequences in eukaryotic intact genomic DNA at an indicative concentration of 90~130 ng/μL. Participation in K176 required assay selection/design and quantitative detection of the analyte in the buffer. The objectives of the key comparison were to measure 1) HER2 copy number concentration; 2) the copy number concentration of the single copy reference gene ribonuclease P RNA component H1 (RPPH1); 3) the HER2/ RPPH1 copy number ratio, using two defined sequences representative of the two genes (HER2 and RPPH1) in mixed genomic DNA samples, and 4) provide evidence for CMC claims by participating laboratories when measuring target sequence content in purified genomic DNA. All participants performed measurements by digital PCR (dPCR), with alternative assays and pre-treatment of samples (for example, restriction enzyme digestion). Interlaboratory reproducibility (%CV) was less than 13% and 5% for copy number concentration and HER2/RPPH1 copy number ratio, respectively, demonstrating very high interlaboratory reproducibility in CNV measurements.
Successful participation in CCQM-K176 demonstrates the following measurement capabilities in determining copy number ratio (of a defined target sequence expressed relative to a reference gene sequence) in the range from 1.0 to 40.0 at a background of relatively high concentration of intact (non-fragmented) genomic DNA. The study will also support participants' claim for measurement of defined DNA sequence/gene copy number concentration in the range ~104-106 μL-1 (taking into account both reported reference gene and HER2 copy number concentration values).
To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/.
The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).