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IntroductionHuman epidermal growth factor receptor 2 (HER2) amplification is present in almost 15%–20% of breast cancer tumors, making it an important parameter for testing. The present study was designed to evaluate a chip-based digital PCR (dPCR) system for assessing HER2 amplification from formalin-fixed paraffin-embedded breast carcinoma tissue and to compare this system with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).Materials and methodsA total of 84 breast carcinoma tissue samples were analyzed by IHC, FISH, and chip-based dPCR in a blinded manner.ResultsAll nine IHC-positive and 35 IHC-negative samples had equivalent results with dPCR, taking an amplification ratio threshold of 1.8 as a positive result. Of the 40 IHC equivocal samples, 10 were assessed as positive, 27 as negative, and three as equivocal by dPCR.ConclusionThese results demonstrate that chip-based dPCR is suitable for HER2 amplification detection in formalin-fixed paraffin-embedded samples in a clinical setting, providing the advantages of superior turnaround time, cost-effectiveness, and increased precision with absolute quantification compared with conventional tests such as FISH and IHC. This methodology was especially beneficial in tissue samples with low DNA concentration.

e18545 Background: Monitoring BCR-ABL1 fusion transcripts is the cornerstone of management in chronic myeloid leukemia (CML). Real Time PCR(RTPCR) has been the tool of choice for its sensitivity and dynamic range. The International Scale was recently introduced to allow for standardization across laboratories. Chip based Digital PCR(dPCR) which allows for absolute quantitation may obliviate the need for such standardization by giving absolute results.It has the potential to increase sensitivity for detection of minimal residual disease. This assumes great significance as we move into the era of treatment free remission. Methods: A total of 31 EDTA-blood samples of CML patients with known values ranging from 0.003-0.5 IS were processed via an accredited RTPCR protocol calibrated to the IS scale and a parallel dPCR workflow without any specific calibration . The analysis of digital PCR samples was performed on the Thermofisher Cloud Platform. Results: Both RTPCR and dPCR yielded extremely concordant results. The Pearson correlation between the two methods was r = 0.6043 (95%CI: 0.318 to 0.789; p = 0.0003). The calculated BCR-ABL/ABL ratios were comparable with a median of 0.098 for RT-PCR calibrated to the IS (range 0.003-0.55; n = 26) and 0.11 for dPCR (range 0.003-0.37; n = 29). The mean difference for the ratios between the two methods used for the detection was around 0.08. Conclusions: We demonstrate here the capability of dPCR to provide a parallel result to an IS scale calibrated protocol without any standardization. This approach required no specific calibrators or standards and resulted in extremely cost effective testing. This freedom from routine calibration provides for a significantly more robust workflow and greatly increased reliability. Limitations do persist in dPCR on account of limited chip densities which are the main parameter for the Poisson statistics. This has limited the dynamic range on dPCR to a maximum of Log 4 for accurate quantification. However as chip densities and emulsion concentrations increase in these technologies, they are poised to introduce a new era in the quest of accurate quantification.

e23120 Background: 15%-25% of breast cancer neoplasms exhibit Human epidermal growth factor receptor-2(HER2) amplification, as the driver mutation.Techniques to identify HER2 amplification include Immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH). Digital PCR (dPCR) has been increasingly explored in determining HER2 status in cases of indeterminate results on IHC, mainly in archived samples. In this study, we aim to demonstrate the clinical utility of the Quantstudio 3D Digital PCR system to evaluate HER2 levels from Formalin Fixed Paraffin Embedded (FFPE) tissue with RNaseP as a control target. Methods: 61 tissue samples were analyzed by IHC and dPCR in parallel in a double blinded manner. IHC equivocal samples were reflexed to FISH and compared to the results obtained from dPCR. Samples suboptimal for IHC or FISH were satisfactorily processed by dPCR. dPCR results were analyzed on the Thermofisher Cloud platform. The general turnaround time(TAT) was about 2 and 3 days for IHC and FISH respectively with that of dPCR being 24 hours. Results: All 9 IHC positive and 35 negative samples had similar results on dPCR using an amplification ratio threshold for a positive result of 1.8. Of 17 IHC-equivocal samples, 5 resulted as positive, 10 negative and 2 as equivocal by dPCR. There was 100% concordance between the dPCR and FISH results. Two IHC equivocal samples that were unanalyzable by FISH were negative on dPCR Conclusions: Our results demonstrate that the chip based dPCR was non-inferior for HER2 detection in FFPE samples in a clinical setting. Superior TAT's and objective results were obtained compared to more subjective techniques like FISH and IHC even with low sample input. dPCR requires controls but no standards for calibration as it gives absolute copy numbers. Further study is needed to understand dPCR interpretation in cases of chromosomal aneuploidy. [Table: see text]