Table 1.
Primary characteristics of 319 patients with leukemia.
Fig 1.
Primers and probe selection based on the breakpoint of PML-RARa.
(A) The sequence of breakpoint of PML-RARa bcr1, bcr2 and bcr3. (B) Location of primers and probes for detection of all PML-RARa with respect to PML breakpoint. Forward primer 1 (bcr1 and bcr2) and Forward primer 2 (bcr3) were used in combination with the common Reverse primer and probe to detect three PML-RARa transcripts. Details of primers and probe sequences are provided in Table 2. The short red arrow on Fig. 1A indicates breakpoints of PML-RARa bcr1, bcr2 and bcr3, respectively. The long red arrow on Fig. 1B indicates breakpoint of PML-RARa bcr2. Abbreviations: S, short isoform; L, long isoform; V, variable breakpoint. The PML gene and RARA gene are positioned according to accession numbers M73778 (PML) and X06538 (RARA).
Table 2.
Primers and probes for 3-plex RT-qPCR of PML-RARa and ABL.
Fig 2.
Standard curve for PML-RARa bcr1, bcr2, bcr3 and ABL control gene.
(A) Standard curve for PML-RARa bcr1. (B) Standard curve for PML-RARa bcr2. (C) Standard curve for PML-RARa bcr3. (D) Standard curve for ABL control. The range on all curves is from 106 to 10 copies of corresponding plasmids. The efficiencies of PML-RARa bcr1, bcr2 and bcr3 were found to be similar to that of ABL, with slope values much closer to -3.32 (the slope associated with a PCR reaction with maximal efficiency).
Table 3.
Comparative results for 3-plex RT-qPCR and other methods from 319 patients with leukemia for transcripts of PML-RARa.
Fig 3.
The analytical sensitivity of the PML-RARa 3-plex RT-qPCR assay.
(A) Amplification plot for PML-RARa of serial plasmid dilutions with analytical sensitivity of 10 copies per reaction. (B) Amplification plot for ABL of serial plasmid dilutions with analytical sensitivity of 10 copies per reaction. (C) Amplification plot for no template control (NTC). (D) Amplification plot for 1μg RNA from PML-RARa positive NB4 cell line diluted in control RNA sample with analytical sensitivity of 10-4 (1 in 104) dilution. Dilutions of PML-RARa and ABL ranged from 106 to 100 copies, and dilutions of NB4 cell RNA ranged from 10-1 to 10-5.
Fig 4.
Intra- and inter-assay reproducibility of PML-RARa bcr1, bcr2 and bcr3 for 3-plex RT-qPCR assay.
(A) The intra-assay reproducibility for bcr1, bcr2 and bcr3. (B) The inter-assays reproducibility for bcr1, bcr2 and bcr3. The inter- and intra-assay coefficients of variation (CVs) for serial dilution of plasmid standards of PML-RARa bcr1, bcr2, and bcr3 (from 106 to 10 copies/2 μL). All inter-assay and intra-assay CVs were less than 3%.
Fig 5.
The results of 3-plex RT-qPCR in patients with leukemia at diagnosis.
(A) Ct values of ABL for patients with leukemia at diagnosis and healthy controls. (B) PML-RARa/ABL NCN for patients with leukemia at diagnosis and healthy controls. (C) Ct values for APL patients with PML-RARa bcr1, bcr2 and bcr3. (D) PML-RARa/ABL NCN of APL patients with PML-RARa bcr1, bcr2 and bcr3. There was no significant difference among bcr1, bcr2 and bcr3 patients in the Ct values of PML-RARa (P > 0.05) and PML-RARa/ABL NCN regardless of age, gender, white blood cell count (WBC), hemoglobin, platelet count and percentage of promyelocytes (P > 0.05).
Fig 6.
The representative amplification plots of PML-RARa bcr1, bcr2, bcr3 and control by 3-plex RT-qPCR.
(A) Amplification plot for PML-RARa bcr1 positive samples. (B) Amplification plot for PML-RARa bcr2 positive samples. (C) Amplification plot for PML-RARa bcr3 positive samples. (D) Amplification plot for PML-RARa negative samples. The red arrow indicates the amplification curve of PML-RARa, whereas black arrow indicates the amplification curve of ABL control.
Fig 7.
Comparison of 3-plex RT-qPCR assay with single RT-qPCR assay for detection of PML-RARa positive samples from 60 APL patients.
(A) Correlation of PML-RARa/ABL NCN between 3-plex RT-qPCR assay and single RT-qPCR assay. (B) Sensitivity of detection for 60 APL samples by 3-plex RT-qPCR assay and single RT-qPCR assay.
Fig 8.
3-plex RT-qPCR for MRD detection in patients with APL treated with ATRA-based therapy after induction and 3 cycles of consolidation.
(A) Percentage of APL patients with PML-RARa detectable after induction and consolidation. (B) PML-RARa/ABL NCN of patients with APL after induction and consolidation. (C) MRD assessment of patients with APL after induction and consolidation. (D) Assay sensitivity of APL samples after induction and consolidation. Post consolidation 1, after the 1st cycle of consolidation; Post consolidation 2, after the 2nd cycle of consolidation; Post consolidation 3, after the 3rd cycle of consolidation.