[Improved detection methods and sequence characteristics of PML::RARA variant transcripts in patients with acute...

A retrospective analysis was conducted on the European Collaboration Against Cancer-Real-time Fluorescence Quantitative polymerase chain reaction (EAC-qPCR) test results of 39 patients diagnosed with PML::RARA-V variant positive acute promyelocytic leukemia (APL) at Hebei Yandalu Daopei Hospital from April 2012 to September 2024. Gene sequence determination was also performed to analyze the sequence characteristics of the PML::RARA-V variant transcript. Based on the sequencing results, new detection primers for the PML::RARA-V variant were designed. A total of 39 patients with the PML::RARA-V variant were included, including 16 males and 23 females, aged 10 to 73 years. Nine cases (23.1%) could not be accurately detected by the EAC-qPCR protocol, among which 4 cases were misclassified and had abnormally low quantitative values, and 5 cases were false negatives; the remaining 30 cases could be accurately detected. Among the 39 patients, 20 completed gene sequence determination. The results showed that 7 cases had no intron sequence insertion, and 13 cases had insertion of the second intron sequence of the RARA gene. The PML gene breaks in 20 patients occurred in exon 6 (with different specific break sites), and the RARA gene of the fusion transcript started from exon 3, and all were in-frame fusions. The new detection primers VNF1 and VNF2 replace the upstream primer for the V variant in the EAC-qPCR protocol and can effectively identify all V variant transcripts. The sequence of the PML::RARA-V variant transcript is highly variable, leading to approximately 23.1% of V variant cases being missed or misidentified by the EAC-qPCR protocol. The new primers can improve the detection accuracy of V variant transcripts.