Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection.

IF 2.8 4区 医学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
OncoTargets and therapy Pub Date : 2025-07-19 eCollection Date: 2025-01-01 DOI:10.2147/OTT.S526628
Jordi Martínez-Serra, Aser Alonso-Carballo, Ángel Horrillo, Paula Gómez, Oliver Vögler, Antonio Gutiérrez, Antonia Sampol
{"title":"Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal Detection.","authors":"Jordi Martínez-Serra, Aser Alonso-Carballo, Ángel Horrillo, Paula Gómez, Oliver Vögler, Antonio Gutiérrez, Antonia Sampol","doi":"10.2147/OTT.S526628","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Internal tandem duplications (FLT3-ITDs) in the FLT3 gene constitute a key driver mutation in acute myeloid leukemia (AML), strongly associated with poor prognosis and therapeutic resistance. Although general-purpose structural variant callers such as Sniffles have been used to detect FLT3-ITDs, their limitations in resolving clonal diversity and low-frequency variants can lead to underrepresentation of minor clones. These shortcomings highlight the need for a dedicated bioinformatics pipeline.</p><p><strong>Materials and methods: </strong>We developed a custom clustering-based pipeline to overcome the constraints of generic SV callers, leveraging Oxford Nanopore's MinION for sequencing. Our method focuses on FLT3-ITDs by grouping near-identical insertions into biologically meaningful subclones, thereby allowing accurate variant detection of even low-frequency events. The pipeline was benchmarked against capillary electrophoresis (CE) and Sniffles at various thresholds (including 10%, 20%, and 50% allele-frequency cutoffs), with results validated via IGV inspection and cross-mapping.</p><p><strong>Results: </strong>The pipeline successfully detected FLT3-ITDs across all tested samples, including low-frequency variants and diverse subclones that Sniffles overlooked. Analyses uncovered complex multiclonal architectures composed of dominant clones (~20-25% of reads) plus multiple minor subclones differing in length, sequence, and breakpoint. Crucially, our approach identified duplications as short as 15 bp-events often dismissed by conventional SV callers. Comparative analyses showed that Sniffles failed to call several biologically validated ITDs detected by our custom pipeline.</p><p><strong>Conclusion: </strong>Third-generation sequencing combined with a tailored clustering strategy enhances the detection of FLT3-ITDs and clonal diversity in AML compared to generic variant callers. This method provides critical insights into subclonal populations driving relapse and therapeutic resistance-particularly in relapsed/refractory AML-underscoring the importance of specialized pipelines for precision medicine in leukemia.</p>","PeriodicalId":19534,"journal":{"name":"OncoTargets and therapy","volume":"18 ","pages":"811-820"},"PeriodicalIF":2.8000,"publicationDate":"2025-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12288229/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"OncoTargets and therapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.2147/OTT.S526628","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Background: Internal tandem duplications (FLT3-ITDs) in the FLT3 gene constitute a key driver mutation in acute myeloid leukemia (AML), strongly associated with poor prognosis and therapeutic resistance. Although general-purpose structural variant callers such as Sniffles have been used to detect FLT3-ITDs, their limitations in resolving clonal diversity and low-frequency variants can lead to underrepresentation of minor clones. These shortcomings highlight the need for a dedicated bioinformatics pipeline.

Materials and methods: We developed a custom clustering-based pipeline to overcome the constraints of generic SV callers, leveraging Oxford Nanopore's MinION for sequencing. Our method focuses on FLT3-ITDs by grouping near-identical insertions into biologically meaningful subclones, thereby allowing accurate variant detection of even low-frequency events. The pipeline was benchmarked against capillary electrophoresis (CE) and Sniffles at various thresholds (including 10%, 20%, and 50% allele-frequency cutoffs), with results validated via IGV inspection and cross-mapping.

Results: The pipeline successfully detected FLT3-ITDs across all tested samples, including low-frequency variants and diverse subclones that Sniffles overlooked. Analyses uncovered complex multiclonal architectures composed of dominant clones (~20-25% of reads) plus multiple minor subclones differing in length, sequence, and breakpoint. Crucially, our approach identified duplications as short as 15 bp-events often dismissed by conventional SV callers. Comparative analyses showed that Sniffles failed to call several biologically validated ITDs detected by our custom pipeline.

Conclusion: Third-generation sequencing combined with a tailored clustering strategy enhances the detection of FLT3-ITDs and clonal diversity in AML compared to generic variant callers. This method provides critical insights into subclonal populations driving relapse and therapeutic resistance-particularly in relapsed/refractory AML-underscoring the importance of specialized pipelines for precision medicine in leukemia.

Abstract Image

Abstract Image

Abstract Image

使用MinION测序对AML中FLT3-ITD进行多克隆分析:一种定制的聚类方法来增强亚克隆检测。
背景:FLT3基因的内部串联重复(FLT3- itds)是急性髓性白血病(AML)的一个关键驱动突变,与不良预后和治疗耐药性密切相关。尽管通用结构变异调用器(如Sniffles)已被用于检测FLT3-ITDs,但它们在解决克隆多样性和低频变异方面的局限性可能导致次要克隆的代表性不足。这些缺点突出了建立专门的生物信息学管道的必要性。材料和方法:我们开发了一个定制的基于聚类的管道,以克服通用SV调用者的限制,利用牛津纳米孔的MinION进行测序。我们的方法侧重于FLT3-ITDs,通过将几乎相同的插入分组到生物学上有意义的亚克隆中,从而可以准确地检测低频事件的变异。在不同阈值(包括10%、20%和50%等位基因频率截止值)下,对管道进行毛细管电泳(CE)和Sniffles基准测试,并通过IGV检查和交叉定位验证结果。结果:该管道在所有测试样本中成功检测到FLT3-ITDs,包括Sniffles忽略的低频变体和各种亚克隆。分析揭示了复杂的多克隆结构,包括优势克隆(约20-25%的reads)和多个在长度、序列和断点上不同的次要亚克隆。至关重要的是,我们的方法识别了短至15 bp的重复事件,这些事件通常被传统的SV调用者忽略。对比分析表明,Sniffles无法调用我们定制管道检测到的几个经过生物学验证的过渡段。结论:与泛型变异呼叫者相比,第三代测序结合定制的聚类策略可以增强AML中FLT3-ITDs的检测和克隆多样性。该方法提供了亚克隆群体驱动复发和治疗耐药性的关键见解-特别是在复发/难治性aml中-强调了白血病精准医学专业管道的重要性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
OncoTargets and therapy
OncoTargets and therapy BIOTECHNOLOGY & APPLIED MICROBIOLOGY-ONCOLOGY
CiteScore
9.70
自引率
0.00%
发文量
221
审稿时长
1 months
期刊介绍: OncoTargets and Therapy is an international, peer-reviewed journal focusing on molecular aspects of cancer research, that is, the molecular diagnosis of and targeted molecular or precision therapy for all types of cancer. The journal is characterized by the rapid reporting of high-quality original research, basic science, reviews and evaluations, expert opinion and commentary that shed novel insight on a cancer or cancer subtype. Specific topics covered by the journal include: -Novel therapeutic targets and innovative agents -Novel therapeutic regimens for improved benefit and/or decreased side effects -Early stage clinical trials Further considerations when submitting to OncoTargets and Therapy: -Studies containing in vivo animal model data will be considered favorably. -Tissue microarray analyses will not be considered except in cases where they are supported by comprehensive biological studies involving multiple cell lines. -Biomarker association studies will be considered only when validated by comprehensive in vitro data and analysis of human tissue samples. -Studies utilizing publicly available data (e.g. GWAS/TCGA/GEO etc.) should add to the body of knowledge about a specific disease or relevant phenotype and must be validated using the authors’ own data through replication in an independent sample set and functional follow-up. -Bioinformatics studies must be validated using the authors’ own data through replication in an independent sample set and functional follow-up. -Single nucleotide polymorphism (SNP) studies will not be considered.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信