PDF(Pubmed)
Abstract：
BACKGROUND: Gene rearrangements affecting KMT2A are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. KMT2A gene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termed KMT2A partial 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 of KMT2A. To deduce the presence of a KMT2A-PTD, we used the relative ratio of KMT2A exons 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 impacting KMT2A and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in KMT2A, 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 with KMT2A can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.
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 of KMT2A. To deduce the presence of a KMT2A-PTD, we used the relative ratio of KMT2A exons 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 impacting KMT2A and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in KMT2A, 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 with KMT2A can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.
摘要：
背景：影响KMT2A的基因重排在急性髓性白血病（AML）中很常见，并且通常与不良预后相关。KMT2A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证实。然而,小的基因内插入，称为KMT2A部分串联复制(KMT2A-PTD)，使用标准分子和细胞遗传学方法进行检测尤其具有挑战性。
方法：我们已经验证了使用定制的基于混合捕获的下一代测序（NGS）小组对在我们机构看到的AML患者进行全面分析。该NGS组靶向KMT2A的整个共有编码DNA序列。为了推断KMT2A-PTD的存在，我们使用KMT2A外显子覆盖率的相对比率。我们试图使用（1）多重连接探针扩增（MLPA）和（2）光学基因组作图（OGM）来证实KMT2A-PTDNGS结果。
结果：我们分析了932例AML病例，确定了41例携带KMT2A-PTD的患者。MLPA，NGS,和OGM证实了KMT2A-PTD在22例可能进行正交试验的病例中的存在。NGS的两个假阳性KMT2A-PTD调用可以通过影响KMT2A并干扰KMT2A-PTD分析的隐蔽结构变体的存在来解释。OGM揭示了KMT2A中这些先前未被发现的基因重排的性质，而MLPA产生的结果不确定。KMT2A-PTD的MLPA分析限于外显子4,而NGS和OGM分辨KMT2A-PTD大小和拷贝数水平。
结论：KMT2A-PTDs是复杂的基因重排，使用单一基因组平台无法完全确定。MLPA，NGS面板,和OGM是应用于AML患者的标准护理测试的补充技术。MLPA和NGS面板设计用于有针对性的拷贝数分析；然而，我们的结果显示,为了准确鉴定KMT2A-PTD,需要整合同时发生的基因组改变.与KMT2A重叠的不平衡染色体重排可干扰基于拷贝数的KMT2A-PTD检测方法的诊断灵敏度和特异性。
方法：我们已经验证了使用定制的基于混合捕获的下一代测序（NGS）小组对在我们机构看到的AML患者进行全面分析。该NGS组靶向KMT2A的整个共有编码DNA序列。为了推断KMT2A-PTD的存在，我们使用KMT2A外显子覆盖率的相对比率。我们试图使用（1）多重连接探针扩增（MLPA）和（2）光学基因组作图（OGM）来证实KMT2A-PTDNGS结果。
结果：我们分析了932例AML病例，确定了41例携带KMT2A-PTD的患者。MLPA，NGS,和OGM证实了KMT2A-PTD在22例可能进行正交试验的病例中的存在。NGS的两个假阳性KMT2A-PTD调用可以通过影响KMT2A并干扰KMT2A-PTD分析的隐蔽结构变体的存在来解释。OGM揭示了KMT2A中这些先前未被发现的基因重排的性质，而MLPA产生的结果不确定。KMT2A-PTD的MLPA分析限于外显子4,而NGS和OGM分辨KMT2A-PTD大小和拷贝数水平。
结论：KMT2A-PTDs是复杂的基因重排，使用单一基因组平台无法完全确定。MLPA，NGS面板,和OGM是应用于AML患者的标准护理测试的补充技术。MLPA和NGS面板设计用于有针对性的拷贝数分析；然而，我们的结果显示,为了准确鉴定KMT2A-PTD,需要整合同时发生的基因组改变.与KMT2A重叠的不平衡染色体重排可干扰基于拷贝数的KMT2A-PTD检测方法的诊断灵敏度和特异性。
