Background: Gene rearrangements affectingKMT2Aare frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis.KMT2Agene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termedKMT2Apartial tandem duplication (KMT2A-PTD), are particularly challenging to detect using standard molecular and cytogenetic approaches. Methods: We have validated the use of a custom hybrid-capture-based next-generation sequencing (NGS) panel for comprehensive profiling of AML patients seen at our institution. This NGS panel targets the entire consensus coding DNA sequence ofKMT2A. To deduce the presence of a KMT2A-PTD, we used the relative ratio ofKMT2Aexons coverage. We sought to corroborate the KMT2A-PTD NGS results using (1) multiplex-ligation probe amplification (MLPA) and (2) optical genome mapping (OGM). Results: We analyzed 932 AML cases and identified 41 individuals harboring a KMT2A-PTD. MLPA, NGS, and OGM confirmed the presence of a KMT2A-PTD in 22 of the cases analyzed where orthogonal testing was possible. The two false-positive KMT2A-PTD calls by NGS could be explained by the presence of cryptic structural variants impactingKMT2Aand interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements inKMT2A, while MLPA yielded inconclusive results. MLPA analysis for KMT2A-PTD is limited to exon 4, whereas NGS and OGM resolved KMT2A-PTD sizes and copy number levels. Conclusions: KMT2A-PTDs are complex gene rearrangements that cannot be fully ascertained using a single genomic platform. MLPA, NGS panels, and OGM are complementary technologies applied in standard-of-care testing for AML patients. MLPA and NGS panels are designed for targeted copy number analysis; however, our results showed that integration of concurrent genomic alterations is needed for accurate KMT2A-PTD identification. Unbalanced chromosomal rearrangements overlapping withKMT2Acan interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.
背景：影响KMT2A的基因重排常见于急性髓系白血病（AML），且通常与不良预后相关。KMT2A基因融合常通过染色体显带分析检测，并经荧光原位杂交确认。然而，称为KMT2A部分串联重复（KMT2A-PTD）的小型基因内插入，使用标准分子和细胞遗传学方法检测尤为困难。方法：我们验证了一种基于定制杂交捕获的新一代测序（NGS）panel，用于对本机构收治的AML患者进行全面基因分析。该NGS panel靶向KMT2A的全部共识编码DNA序列。为推断KMT2A-PTD的存在，我们采用KMT2A外显子覆盖度的相对比值。我们试图通过（1）多重连接探针扩增（MLPA）和（2）光学基因组图谱（OGM）对KMT2A-PTD的NGS结果进行验证。结果：我们分析了932例AML病例，发现41例携带KMT2A-PTD。在可进行正交验证的病例中，MLPA、NGS和OGM共同确认了22例存在KMT2A-PTD。NGS检测中出现的两例假阳性KMT2A-PTD结果，可归因于存在影响KMT2A并干扰KMT2A-PTD分析的隐匿性结构变异。OGM揭示了这些先前未检出的KMT2A基因重排的本质，而MLPA则得出不确定结果。MLPA对KMT2A-PTD的分析仅限于第4外显子，而NGS和OGM可解析KMT2A-PTD的片段大小和拷贝数水平。结论：KMT2A-PTD是复杂的基因重排，无法通过单一基因组平台完全确认。MLPA、NGS panel和OGM是AML患者标准诊疗检测中互为补充的技术。MLPA和NGS panel专为靶向拷贝数分析设计；然而，我们的结果表明，需要整合并发的基因组改变才能实现准确的KMT2A-PTD鉴定。与KMT2A重叠的不平衡染色体重排可能干扰基于拷贝数的KMT2A-PTD检测方法的诊断敏感性和特异性。
肿瘤(癌症)患者之家