Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression

• Published in: European journal of haematology Vol. 101; no. 3; pp. 291 - 296
• Main Authors: Wang, Wen‐Jun, Zheng, Chao‐Feng, Liu, Zhuang, Tan, Yan‐Hong, Chen, Xiu‐Hua, Zhao, Bin‐Liang, Li, Guo‐Xia, Xu, Zhi‐Fang, Ren, Fang‐Gang, Zhang, Yao‐Fang, Chang, Jian‐Mei, Wang, Hong‐Wei
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.09.2018
• Subjects: Abl gene; Adolescent; Adult; Aged; Aged, 80 and over; BCR gene; BCR protein; BCR/ABL(P210); Cell suspensions; Chronic myeloid leukemia; CML; Disease Progression; droplet digital PCR; Enzyme inhibitors; Female; Fusion protein; Fusion Proteins, bcr-abl - genetics; Gene Expression Regulation, Leukemic; Humans; K562 Cells; Kinases; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - diagnosis; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - genetics; Leukocytes (mononuclear); Male; Middle Aged; Minimal residual disease; Myeloid leukemia; Neoplasm, Residual - diagnosis; Neoplasm, Residual - genetics; Patients; Peripheral blood mononuclear cells; Protein-tyrosine kinase; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Reproducibility of Results; RT‐qPCR; Sensitivity and Specificity; Statistical analysis; Young Adult; minimal residual disease; CML; droplet digital PCR; RT-qPCR; BCR/ABL(P210)
• ISSN: 0902-4441; 1600-0609
• DOI: 10.1111/ejh.13084

Objective This study intended to establish a droplet digital PCR (dd‐PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)‐positive chronic myeloid leukemia (CML), thereby achieving deep‐level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment. Methods Using dd‐PCR and RT‐qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML‐chronic phase (CML‐CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow‐ups. By RT‐qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd‐PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods. Results At the cellular level, consistency of results of dd‐PCR and RT‐qPCR reached R2 ≥ 0.99, with conversion equation of Y = 33.148X1.222 (Y: dd‐PCR results; X: RT‐qPCR results). In the dd‐PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P < .01). In the follow‐up of 10 CML patients who were in MR4.5. All patients were loss of MR4.0, and 4 were tested positive by dd‐PCR 3 months earlier than by RT‐qPCR. Conclusion In contrast with RT‐qPCR, dd‐PCR is more sensitive, thus enabling accurate conversion of dd‐PCR results into internationally standard RT‐qPCR results by conversion equation, to achieve a deeper molecular biology‐based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic.