Seto Andrew, Downs Gregory, King Olivia, Salehi-Rad Shabnam, Baptista Ana, Chin Kayu, Grenier Sylvie, Nwachukwu Bevoline, Tierens Anne, Minden Mark D, Smith Adam C, Capo-Chichi José-Mario
Genome Diagnostics & Cancer Cytogenetics Laboratories, Laboratory Medicine Program, University Health Network, Toronto, ON M5G 2C4, Canada.
Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada.
Cancers (Basel). 2024 Apr 26;16(9):1693. doi: 10.3390/cancers16091693.
Gene rearrangements affecting are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. gene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termed partial tandem duplication (KMT2A-PTD), are particularly challenging to detect using standard molecular and cytogenetic approaches.
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 of . To deduce the presence of a KMT2A-PTD, we used the relative ratio of exons coverage. We sought to corroborate the KMT2A-PTD NGS results using (1) multiplex-ligation probe amplification (MLPA) and (2) optical genome mapping (OGM).
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 impacting and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in , 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.
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 with can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.
影响[基因名称]的基因重排在急性髓系白血病（AML）中很常见，且常与不良预后相关。[基因名称]基因融合通常通过染色体显带分析检测，并通过荧光原位杂交确认。然而，使用标准分子和细胞遗传学方法检测称为KMT2A部分串联重复（KMT2A-PTD）的小基因内插入尤其具有挑战性。
我们验证了使用基于定制杂交捕获的下一代测序（NGS） panel对在我们机构就诊的AML患者进行全面分析的用途。该NGS panel靶向[基因名称]的整个共有编码DNA序列。为了推断KMT2A-PTD的存在，我们使用了[基因名称]外显子覆盖的相对比率。我们试图使用（1）多重连接探针扩增（MLPA）和（2）光学基因组图谱（OGM）来证实KMT2A-PTD的NGS结果。
我们分析了932例AML病例，鉴定出41例携带KMT2A-PTD的个体。在可以进行正交测试的22例分析病例中，MLPA、NGS和OGM证实了KMT2A-PTD的存在。NGS的两个假阳性KMT2A-PTD检测结果可以通过影响[基因名称]并干扰KMT2A-PTD分析的隐匿结构变异的存在来解释。OGM揭示了这些先前未检测到的[基因名称]基因重排的性质，而MLPA产生了不确定的结果。KMT2A-PTD的MLPA分析仅限于外显子4，而NGS和OGM解析了KMT2A-PTD的大小和拷贝数水平。
KMT2A-PTD是复杂的基因重排，不能使用单一基因组平台完全确定。MLPA、NGS panel和OGM是应用于AML患者标准护理检测的互补技术。MLPA和NGS panel设计用于靶向拷贝数分析；然而，我们的结果表明，准确鉴定KMT2A-PTD需要整合同时发生的基因组改变。与[基因名称]重叠的不平衡染色体重排会干扰基于拷贝数的KMT2A-PTD检测方法的诊断敏感性和特异性。
