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Non-invasive HER2 status diagnosis in gastric cancer using surrogate DNA methylation markers
Background
Gastric cancer (GC) is the fifth most common and fourth most lethal cancer worldwide. Unlike other cancer types, e.g., lung or breast cancer, very few targeted therapeutics have been developed for GC. HER2 (ERBB2) status is an essential biomarker for guiding the trastuzumab (Herceptin) therapy, which is the only molecularly targeted drug accepted as a first-line therapy, for the treatment of patients with advanced HER2-overexpressing GC. HER2 detection in GC often requires repeated testing to improve the accuracy of the result due to its high degree of heterogeneity. Moreover, HER2 status is dynamic during the clinical course due to genetic differentiation accompanied by neoplastic progression and clonal selection via various factors including chemo- and radiotherapies. Thus, the assessment of HER2 gene copy number in liquid biopsy recently gained a lot of interest for its non-invasiveness, suitability for repeat testing and homogeneity compared to tissue biopsy; however, the limited signal-to-noise ratio (cirdulating tumor DNA (ctDNA) represents a very small fraction in cell-free DNA, which may be less than 0.1%) poses a great challenge for the accuracy and robustness of the tests (either targeted sequencing or droplet digital PCR).
Methods
Targeted bisulfite sequencing using an enriched panel with pre-selected GC-associated CpG sites was performed on 74 FFPE tissue samples (44 IHC0/1+ and 30 IHC3+) to identify HER2-overexpression-specific methylation markers. Then we verified the performance of these markers for HER2 status determination using methylation-specific quantitative PCR (qMSP) in 71 independent tissue samples, as well as three GC cell lines (N-87 and MKN-7 (Her2+), and MKN-28 (Her2-)). We further validated the performance of the markers on 110 GC plasma samples collected before surgery. A HER2-status diagnostic model was built and the performance was evaluated.
Results
We first discovered 105 statistically significant methylation markers for inferring HER2 status in tissue based on the results from targeted sequencing. 69 out of the 105 markers (66%) are located in chromosome 17. qMSP assays were designed for these candidate markers and validated on 110 GC plasma samples. A 3-marker diagnostic model was built and demonstrated sensitivity of 86.7% and specificity of 96.9%, which discriminates HER2-positive from HER2-negative GC patients. The overall plasma-tissue concordance of this liquid biopsy test was 95.5%. Furthermore, the HER2-status test can stratify HER2 IHC2+ patients into either HER2-negative or HER2-positive status, which was confirmed by conventional FISH test.
Conclusions
We have developed a novel, accurate and noninvasive qMSP test for determining HER2 status in GC patients. The high concordance with IHC/FISH results of this blood test holds great promise as an auxiliary method to guide HER2-targeted therapy in GC patients.
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The author.
Funding
This study was supported by Scheme of Guangzhou Economic and Technological Development District for Leading Talents in Innovation and Entrepreneurship (Grant NO.2017-L152); Scheme of Guangzhou for Leading Talents in Innovation and Entrepreneurship (Grant NO.2016007); Scheme of Guangzhou for Leading Team in Innovation (Grant NO.201909010010); Science and Technology Planning Project of Guangdong Province, China (Grant NO.2017B020226005).
Disclosures
M. Bibikova: Shareholder / Stockholder / Stock options: AnchorDx, Illumina; Full / Part-time employment: AnchorDx, Illumina; Officer / Board of Directors: AnchorDx. Z. Chen: Full / Part-time employment: AnchorDx Medical Co., Ltd., AnchorDx, Inc.. All other authors have declared no conflicts of interest.
