HER2 amplification detected in the circulating DNA of patients with gastric cancer: a retrospective pilot study

Abstract

Background We used real-time quantitative polymerase chain reaction (rqPCR) to detect human epidermal growth factor receptor 2 (HER2) amplification in the circulating cell-free DNA (cfDNA) of patients with gastric cancer (GC), which shows the spatial and temporal intrinsic heterogeneity of HER2 expression/copy number during progression, for liquid biopsy and treatment monitoring. Methods We first enrolled 52 patients with advanced GC who underwent surgery and 40 healthy volunteers. For patients with GC, plasma cfDNA was obtained before surgery (43 patients) and during postoperative treatment (nine of 43 patients). After ribonuclease P RNA component H1 (RPPH1) had been selected as a reference gene for HER2 CN assessment by rqPCR in GC tumours and plasma, plasma HER2-to-RPPH1 ratios were determined retrospectively in a development cohort and an additional independent validation cohort.Results The HER2-to-RPPH1 ratio of GC tissues determined by rqPCR was concordant with routinely determined HER2 status. The plasma HER2-to-RPPH1 ratio was significantly higher for patients with HER2-positive tumours than for those with HER2-negative tumours. The sensitivity and specificity of the plasma HER2-to-RPPH1 ratio test were 0.539 and 0.967, respectively, in the development cohort, and 0.667 and 1.000, respectively, in the validation cohort. HER2 amplifications acquired and lost during tumour progression and treatment, respectively, were apparently detected by repeated assessments of plasma HER2-to-RPPH1 ratios during postoperative treatment. Conclusion Our preliminary data demonstrated the potential clinical use of circulating cfDNA to detect HER2 amplification as a therapeutic marker to detect and monitor HER2 CN status for effective molecular targeted therapy in patients with GC.

Monitoring the HER2 copy number status in circulating tumor DNA by droplet digital PCR in patients with gastric cancer

“…The same cutoff value (2.1) was obtained when we determined the best discriminating level of the HER2 ratio to separate HER2-positive patients from HER2-negative patients by generating a receiver operating characteristic curve and calculating the area under the curve. In 36 cases with plasma HER2 ratios determined by ddPCR in this study and rqPCR in our previous study [6], sensitivity and specificity were higher in the ddPCR-based method than in the rqPCR-based method (sensitivity and specificity were 0.700 and 1.000 in ddPCR, and 0.400 and 0.962 in rqPCR, respectively). Because an absolute CN is obtainable in digital PCR, which results in a more objective evaluation without sharing the calibrator sample between experiments and laboratories [21], these results may reflect the greater accuracy and reproducibility of ddPCR over rqPCR.…”

“…Preoperative plasma samples were collected from all patients. Of these, 36 patients were also included in our previous study [6]. Control plasma samples were obtained from 30 healthy adult volunteers by standard antecubital venous puncture.…”

“…Using ribonuclease P RNA component H1 (RPPH1) as an internal control to assess the CN of the HER2 gene [6], plasma HER2-to-RPPH1 ratios (the HER2 ratio) were determined using ddPCR. Each sample was partitioned into 20,000 droplets, with target and control (background) DNA being randomly, but uniformly distributed among the droplets.…”

“…A quantitative analysis of HER2 amplification using rqPCR was performed on the cell-free DNA (cfDNA) samples of 30 healthy volunteers as previously described elsewhere [6].…”

“…Recurrent tumors from HER2-positive cases may contain HER2-negative tumor cells as a major population, whereas HER2 amplification/overexpression may be acquired or HER2-positive cells existing as a minor population in resected tumors may be selected during treatment progress in HER2-negative cases. Liquid biopsy has recently attracted the interest of clinicians as a possible approach that may overcome the influence of heterogeneity [5,6], with a number of studies being conducted to investigate intratumoral heterogeneity [7][8][9]. The potential of circulating tumor DNA (ctDNA) as the source of a template in minimally invasive diagnostics is becoming increasingly apparent for monitoring the status of tumors with temporal and spatial heterogeneities [10][11][12].…”