Abstract A005: Ultra-rapid and precise measurement of HER2 copy number alteration by next-generation digital PCR capable of real-time analysis in patients with breast cancer: A multicenter retrospective study
Authors: Chang Jin hyuk , Kim YoonSik , Choi Hee-Joo , Park Soo Young , Park Ji-Hye , Won Hee-Young , Song Min Ji , Kim Da Sol , Kim Hayeon , Yang Sohyeon , Heo Nam Hun , Song Minsik , Shin Seung-Shick , Lee Do Young , Ryu Han Suk , Jang Si-Hyong , Lee Jeong-Yeon 
Published: February 2024

Abstract
The HER2 diagnostics is necessary for selection of patients harboring HER2 gene amplification or protein overexpression who will benefit from anti-HER2 therapies in breast cancer. However, HER2 testing is still challenging due to the subjective natures of immunohistochemistry (IHC) and in situ hybridization (ISH), standard methods for determining HER2 status. Thus, a new method is needed to accurately quantify HER2 levels. Here, we developed a clinically reliable HER2 testing method enabling ultra-fast detection of HER2 gene amplification with high accuracy by using the digital real-time PCR (drPCR) system, a potential new diagnostic platform with improved performance by integrating both real-time and digital PCR technologies. For drPCR-based HER2 copy number (CN) measurement, primer-probe sets specific to HER2 gene and a genomic region adjacent to chromosome 17 centromere (CEP17) were designed, and the optimal drPCR condition was determined in clinical breast tumor specimens. To test the clinical validity and standardize procedures of drPCR-based HER2 status evaluation, three independent breast cancer cohorts from different institutions were enrolled, which assigned as a training (SCHU hospital, n = 103) and two validation sets (SNU hospital, n = 170; CNUH hospital, n = 45), and the drPCR assay was compared with current standard HER2 testing methods. In the training cohort, the HER2/CEP17 ratio values from FISH and drPCR tests were highly correlated (r2 = 0.81; P &lt; 0.001), and the drPCR results displayed 98.1% concordance to HER2 status defined by IHC and/or FISH with 92.6% sensitivity and 100% specificity. Eight samples further verified by targeted NGS showed 100% concordance of dPCR to NGS. Consistently, two validation cohorts also showed high concordance of drPCR to IHC and/or ISH results (accuracy = 97.1% and 97.8% in SNU and CNUH cohorts, respectively). The optimal cutoff for HER2 positivity in the drPCR assay was set as a HER2/CEP17 ratio ≥ 1.9 with AUC of 0.963 based on the results from training cohort, and the same cut-off for drPCR was applicable to two independent validation cohorts, supporting the clinical validity of our drPCR-based HER2 assessment. In some discordant cases, low tumor purity (≤ 25%) was observed and microdissection partly improved the drPCR results. The discordance between drPCR and ISH results was also found in marginal HER2+ cases with HER2/CEP17 ratio 2-3, but these cases showed inter-observer variability when re-evaluating the ISH/IHC data due to intratumoral HER2 heterogeneity. Of note, in HER2 IHC3+ cases with negative drPCR results, re-evaluation of IHC using an artificial intelligence (AI)-based HER2 scoring system revised the HER2 IHC 3+ score to 2+, and ISH assessment also confirmed that these cases are indeed HER2-negative, proving the high accuracy of HER2 CN drPCR assay. In conclusion, given the advantages of drPCR-based HER2 assessment with high accuracy, sensitivity, and simplicity, the drPCR assay could be a complementary or alternative method to IHC and ISH to greatly improve current HER2 testing.
Citation Format: Jin hyuk Chang, YoonSik Kim, Hee-Joo Choi, Soo Young Park, Ji-Hye Park, Hee-Young Won, Min Ji Song, Da Sol Kim, Hayeon Kim, Sohyeon Yang, Nam Hun Heo, Minsik Song, Seung-Shick Shin, Do Young Lee, Han Suk Ryu, Si-Hyong Jang, Jeong-Yeon Lee. Ultra-rapid and precise measurement of HER2 copy number alteration by next-generation digital PCR capable of real-time analysis in patients with breast cancer: A multicenter retrospective study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A005.