FLT3-内部串联重复发生在20-30%的急性髓细胞性白血病中,其等位基因比例是关键的风险分层指标,因此预后不良。美国食品和药物管理局最近批准了FLT3抑制剂midostaurin和gilteritinib用于FLT3突变阳性的急性髓细胞性白血病。历史上,FLT3通过片段分析进行测试,已成为国际准则认可的标准方法。然而,由于下一代测序能够同时评估多种临床信息标志物,因此越来越多地用于急性髓细胞性白血病的诊断.由于FLT3内部串联重复检测被称为下一代测序具有挑战性,其结果具有深远的预后和治疗意义,彻底检查其在FLT3内部串联重复检测和等位基因比率分类中的性能非常重要。在片段分析的比较研究中,我们使用定制设计的产品回顾了我们的经验,基于杂交捕获,靶向的下一代测序组。在7902个案例中,在335中检测到FLT3内部串联重复,具有可变大小(3-231bp)和插入位点。402例也进行了碎片分析,在FLT3内部串联重复检测中显示100%一致性。在136个双重测试中,阳性病例,128/136(94%)表现出一致的高/低等位基因比分类。剩余的6%通过下一代测序显示临界低等位基因比率。两种方法在通过片段分析靶向的热点D835/I836处检测FLT3-酪氨酸激酶结构域突变方面是一致的。此外,通过下一代测序检测到可能受益于FLT3抑制剂治疗的七个突变,在片段分析未覆盖的区域。我们的研究表明,使用基于杂交捕获的化学和优化的生物信息学管道,下一代测序可以可靠地检测FLT3内部串联重复,并对其等位基因比率进行分类,以进行急性髓系白血病风险分层.下一代测序还在FLT3突变检测中表现出优越的全面性,并可能进一步改善个性化,急性髓系白血病的靶向治疗.
FLT3-internal tandem duplication occurs in 20-30% of acute myeloid leukemia and confers an adverse prognosis with its allelic ratio being a key risk stratifier. The US Food and Drug Administration recently approved FLT3 inhibitors midostaurin and gilteritinib in FLT3 mutation-positive acute myeloid leukemia. Historically, FLT3 was tested by fragment analysis, which has become the standard method endorsed by international guidelines. However, next generation sequencing is increasingly used at acute myeloid leukemia diagnosis given its ability to simultaneously evaluate multiple clinically informative markers. As FLT3-internal tandem duplication detection was known to be challenging by next generation sequencing and the results carry profound prognostic and therapeutic implications, it is important to thoroughly examine its performance in FLT3-internal tandem duplication detection and allelic ratio classification. In a comparative
study with fragment analysis, we retrospectively reviewed our experience using a custom-designed, hybridization capture-based, targeted next generation sequencing panel. Among 7902 cases, FLT3-internal tandem duplication was detected in 335 with variable sizes (3-231 bp) and insertion sites. Fragment analysis was also performed in 402 cases, demonstrating 100% concordance in FLT3-internal tandem duplication detection. In 136 dual-tested, positive cases, 128/136 (94%) exhibited concordant high/low allelic ratio classifications. The remaining 6% showed borderline low allelic ratio by next generation sequencing. The two methods were concordant in FLT3-tyrosine kinase domain mutation detection at the hotspot D835/I836 targeted by fragment analysis. Furthermore, seven mutations which may benefit from FLT3 inhibitor therapy were detected by next generation sequencing, in regions not covered by fragment analysis. Our
study demonstrates that using a hybridization capture-based chemistry and optimized bioinformatics pipeline, next generation sequencing can reliably detect FLT3-internal tandem duplication and classify its allelic ratio for acute myeloid leukemia risk stratification. Next generation sequencing also exhibits superior comprehensiveness in FLT3 mutation detection and may further improve personalized, targeted therapy in acute myeloid leukemia.