Sreedharanunni Sreejesh, Thakur Venus, Balakrishnan Anand, Sachdeva Man Updesh Singh, Kaur Prabhjot, Raina Sudhanshi, Jamwal Manu, Singh Charanpreet, Sharma Praveen, Mallik Nabhajit, Naseem Shano, Rastogi Pulkit, Jain Arihant, Prakash Gaurav, Khadwal Alka, Malhotra Pankaj, Das Reena
Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India.
Department of Clinical Hematology and Medical Oncology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India.
Mol Diagn Ther. 2025 May;29(3):407-418. doi: 10.1007/s40291-025-00779-5. Epub 2025 Apr 5.
Recent World Health Organization (WHO) and International Consensus Classifications have introduced numerous molecular entities in B-lineage acute lymphoblastic leukemia (B-ALL), necessitating comprehensive genomic characterization by detecting gene fusions, expression, mutations, and exon deletions. While whole-genome plus transcriptome sequencing is the ideal strategy, it remains cost-prohibitive for routine use. This study reports a cost-effective and reasonably efficient alternate approach integrating a customized targeted hybrid capture RNA sequencing (RNAseq) into the routine workup.
A total of 95 consecutive adolescent/adult B-ALL cases negative for common chimeric gene fusions (CGF) (BCR::ABL1, KMT2A::AFF1, TCF3::PBX1, and ETV6::RUNX1) were analyzed using a customized 69-gene targeted RNAseq panel. In total, three fusion detection pipelines, the Trinity Cancer Transcriptome Analysis Toolkit (CTAT) Mutations pipeline, and the Toblerone alignment tool were employed, and the results were compared with fluorescence in situ hybridization (FISH)/multiplex ligation-dependent probe amplification (MLPA) testing.
RNAseq identified fusions in 43% of cases (including BCR::ABL1-like: 15.8% and IGH::DUX4: 10.5%), demonstrating superior detection of cryptic intrachromosomal rearrangements. Somatic variants were detected in 30% of cases (including rat sarcoma (RAS) pathway and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) variants in 18% and 5.3% respectively), and IKZF1 deletions were detected in 25% (77% concordance with MLPA). The integration of targeted RNAseq and comprehensive bioinformatic analysis with flow-cytometry-based ploidy analysis and FISH-based IGH rearrangements helped categorize 79% of common CGF-negative B-ALL. The BCR::ABL1/BCR::ABL1-like group showed a higher frequency of pathogenic IKZF1 deletions (50% versus 21.7%; p = 0.011), measurable residual disease (92% versus 51%; p = 0.009), and poorer overall survival (8.6 versus 22.8 months; p = 0.07).
Effective utilization of RNAseq data by comprehensive bioinformatic analysis to test fusions, mutations, and deletions, supported by only minimal supplementary FISH testing, provides a practical, cost-effective solution for the molecular characterization of B-ALL in real-world scenarios until a single alternative and cost-effective test is available.
世界卫生组织（WHO）和国际共识分类法最近在B淋巴细胞系急性淋巴细胞白血病（B-ALL）中引入了众多分子实体，因此需要通过检测基因融合、表达、突变和外显子缺失来进行全面的基因组特征分析。虽然全基因组加转录组测序是理想的策略，但对于常规使用来说成本仍然过高。本研究报告了一种将定制的靶向杂交捕获RNA测序（RNAseq）整合到常规检查中的经济高效的替代方法。
使用定制的69基因靶向RNAseq检测板对95例连续的青少年/成人B-ALL病例进行分析，这些病例的常见嵌合基因融合（CGF）（BCR::ABL1、KMT2A::AFF1、TCF3::PBX1和ETV6::RUNX1）均为阴性。总共采用了三种融合检测流程、Trinity癌症转录组分析工具包（CTAT）突变流程和Toblerone比对工具，并将结果与荧光原位杂交（FISH）/多重连接依赖探针扩增（MLPA）检测进行比较。
RNAseq在43%的病例中检测到融合（包括BCR::ABL1样：15.8%和IGH::DUX4：10.5%），显示出对隐匿性染色体内重排的卓越检测能力。在30%的病例中检测到体细胞变异（包括大鼠肉瘤（RAS）途径和Janus激酶（JAK）-信号转导和转录激活因子（STAT）变异，分别为18%和5.3%），并且在25%的病例中检测到IKZF1缺失（与MLPA的一致性为77%）。将靶向RNAseq和全面的生物信息学分析与基于流式细胞术的倍性分析和基于FISH的IGH重排相结合，有助于对79%的常见CGF阴性B-ALL进行分类。BCR::ABL1/BCR::ABL1样组显示出更高频率的致病性IKZF1缺失（50%对21.7%；p = 0.011）、可测量的残留疾病（92%对51%；p = 0.009）和更差的总生存期（8.6个月对22.8个月；p = 0.07）。
通过全面的生物信息学分析有效利用RNAseq数据来检测融合、突变和缺失，仅辅以最少的补充FISH检测，为现实场景中B-ALL的分子特征分析提供了一种实用、经济高效的解决方案，直到有单一的替代且经济高效的检测方法可用。
