Novel Chip-Based Digital Real-Time PCR Platform with Lab on an Array for the Sensitive Detection of BCR::ABL1 Transcripts in Chronic Myeloid Leukemia

Introduction: With the advent various BCR::ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable disease and current therapeutic goal is treatment-free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) is one of the prerequisite requirements.

Since undetectable BCR::ABL1 fusion transcripts by conventional Taqman™-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of the CML clone, it requires the development of more sensitive technologies.

Currently, to overcome some limitations of conventional qPCR, such as the rigorous standardization process and variations in sensitivity between laboratories, digital PCR (dPCR) methods have been developed for accurate and precise detection and its use in the routine clinical practice is gradually expanding.

However, current dPCR methods may have limitations due to end-point interpretation and a narrow dynamic range. Therefore, we have developed a novel technology, High Dynamic Range Digital Real-time PCR (HDR-drPCR) to achieve wider dynamic range, which allows more stable, accurate and sensitive monitoring of BCR::ABL1 in a timely manner.

Methods: The Genotizer™ HDR BCR::ABL1 Major IS kit, utilizing HDR-drPCR for detecting BCR::ABL1, employs the GUSB gene as a control gene to determine %IS values. The correction factor (CF) was calculated using the 1^st WHO International Genetic Reference Panel.

For the linearity assessment, a sample panel was prepared by mixing the BCR::ABL1 positive (e14a2) K562 cell line with the BCR::ABL1 negative HL60 cells. The panel was constructed using a 10-fold serial dilution across seven points, spanning a range from 25 %IS to 2.5x10⁻⁵ %IS.

Based on the cell line results, we evaluated the correlation/equivalence between conventional qPCR and HDR-drPCR using 219 clinical specimens. Among these, 100 specimens were identified as DMR (<0.01 %IS) according to the qPCR test results. The correlation and equivalence assessments were performed using correlation plots and Bland-Altman plots.

In detail, conventional qPCR was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) for cDNA synthesis and Real-Q BCR-ABL Quantification Kit (Biosewoom, South Korea) for qPCR.

HDR-drPCR was performed using the Genotizer™ HDR BCR::ABL1 Major IS kit (Optolane Technologies, Korea), which includes disposable digital real-time PCR chips, and the LOAA Analyzer (Optolane Technologies, Korea). The total RNA used for the evaluation was measured with GUSB gene concentrations ranging from 10⁶ to 10⁷ copies per reaction.

Results: Linearity was assessed by the dilution of RNA of K562 with RNA of HL60 using 10-fold serial dilution of up to - 7 log. The result showed accurate linearity (R² = 0.9972) at the range of BCR::ABL1/GUSB ratio of 10¹ to 10^-5(%). Notably, HDR-drPCR technology allowed to detect the level of BCR::ABL1 transcript of MR 6.34 (0.00005 %IS).

The correlation analysis between the measured values of conventional qPCR and HDR-drPCR for 209 clinical samples showed a Pearson correlation coefficient (R²) of 0.9731, indicating a very high correlation. The Bland-Altman plot for equivalence assessment showed a mean bias of 0.159, with the limit of agreement at the 95% confidence interval (LOA95) calculated to be between -0.387 and 0.705. Additionally, the difference in measurements between the two methods was within 2-fold in 72.25% of cases and within 5-fold in 96.17% of cases, indicating a high level of agreement.

Of the 100 clinical specimens that were determined to be <0.01 %IS by conventional qPCR, HDR-drPCR successfully detected BCR::ABL1 transcripts in 96 specimens, while conventional qPCR detected BCR::ABL1 transcripts in only 90 specimens. This result indicates that HDR-drPCR has higher sensitivity compared to conventional qPCR, demonstrating its ability to detect very low levels of BCR::ABL1 transcripts below MR 5.0 even in clinical specimens.

Conclusion: We believe that high dynamic range real-time digital PCR (HDR-drPCR) can minimize false positive results and maximize the analytical sensitivity for detecting BCR::ABL1 fusion transcripts. Therefore, it could be highly useful for accurately assessing treatment in routine clinical practice and facilitating the application of TFR.

Study support: Funded by Korea Research Foundation

Song:Optolane: Current Employment. Song:Optolane: Ended employment in the past 24 months. Shin:Optolane: Ended employment in the past 24 months. Kim:Enliven: Honoraria, Research Funding; BMS: Honoraria, Research Funding, Speakers Bureau; Pharmaessencia: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Il-Yang: Honoraria, Research Funding, Speakers Bureau; Korea Otsuka: Honoraria, Research Funding, Speakers Bureau.