[Transcriptome sequencing-based classification and quantification of IKZF1 transcript isoforms in B-cell acute lymphoblastic leukemia]

Objective: To classify and quantify IKZF1 mutant transcripts in B-cell acute lymphoblastic leukemia (B-ALL) by RNA sequencing (RNA-seq) and bioinformatics analysis. Methods: A cohort of 263 B-ALL cases was enrolled at Hebei Yanda Ludaopei Hospital from September 2018 to September 2020. An integrated bioinformatics pipeline was developed to adapt the classification and quantification of IKZF1 transcripts from RNA-seq and was applied to sequencing data of these cases. The IKZF1 mutant transcripts classified by RNA-seq analysis were compared with the qualitative reverse transcription PCR (RT-PCR). Results: IKZF1 mutant transcripts were identified in 53 B-ALL patients by RT-PCR and Sanger sequencing, among which IK6 and IK10 transcripts accounted for 67.9% (36/53) and 28.3% (15/53) respectively. Additionally, 2 patients were double positive for IK6 and IK10. RNA-seq analysis identified 51 patients with IKZF1 mutant transcripts. Compared with the RT-PCR result, the detection sensitivity and specificity of RNA-seq analysis reached 94.3% (50/53) and 99.5% (209/210), respectively. Among the 50 patients with IKZF1 mutant transcripts both in RNA-seq and RT-PCR analysis, the ratio of mutant transcripts to total IKZF1 transcripts in 6 patients was 0.14 (0.11, 0.35), which was significantly lower than that of the other 44 patients [0.88 (0.35, 0.97), Z=-3.945,P<0.001]. IKZF1 mutations mostly occurred in Ph⁺and Ph-like B-ALL, characterized by abnormal JAK-STAT pathway, and B-ALL with PAX5 translocation. Conclusions: Through the optimized bioinformatics analysis process, RNA-seq data can be used to classify and quantitatively analyze IKZF1 transcripts in B-ALL. Furthermore, the relative expression of mutant IKZF1 transcripts was found to cluster into two groups, and IKZF1 mutation was found often accompanied with PAX5 translocations.