Nanopore-based DNA methylation profiling for rapid molecular classification of NOS/NEC CNS tumors: multi-institutional evaluation using FFPE archives and frozen tissues.

IF 6.5 2区医学 Q1 NEUROSCIENCES

Acta Neuropathologica Communications Pub Date : 2026-06-30 DOI:10.1186/s40478-026-02359-y

Wanming Hu, Jin Zhu, Hang Li, Qiubo Yu, Song Chen, Qinglan Ren, Jingxian Wu, Youde Cao, Yanghao Hou

{"title":"Nanopore-based DNA methylation profiling for rapid molecular classification of NOS/NEC CNS tumors: multi-institutional evaluation using FFPE archives and frozen tissues.","authors":"Wanming Hu, Jin Zhu, Hang Li, Qiubo Yu, Song Chen, Qinglan Ren, Jingxian Wu, Youde Cao, Yanghao Hou","doi":"10.1186/s40478-026-02359-y","DOIUrl":null,"url":null,"abstract":"Background: DNA methylation profiling has become crucial for accurate classification of CNS tumors, yet Illumina EPIC arrays are restricted by long turnaround times, batching requirements, and limited accessibility in non-specialized centers. Rapid, affordable alternatives are urgently needed, particularly in resource-constrained settings. We aimed to evaluate nanopore sequencing as a real-world diagnostic tool for resolving diagnostically challenging CNS tumors.Methods: We applied the ROBIN pipeline to 34 consecutive CNS tumors that remained NOS/NEC after routine histopathological and molecular workup. Samples comprised 16 routine FFPE blocks and 18 frozen tissues (9 fresh-frozen, 9 pre-extracted DNA) from three pathology centers. Nanopore sequencing was performed on PromethION R10.4.1 flow cells, targeting definitive methylation-based classification report within 24 h.Results: A confident methylation class was assigned in 28/34 cases (82.4%), including 14/16 (87.5%) methylation calls of FFPE samples with median target coverage of 0.03×. Integration of simultaneously generated CNV profiles rescued an integrated glioblastoma diagnosis in two methylation-unclassifiable cases via 7+/10- signatures. The nanopore-based classifications also achieved 84.6% (11/13) concordance with paired result from Illumina Methylation EPIC arrays. In high-coverage samples, diagnostic-relevant SNVs (BRAF V600E, PTEN, NF1/TP53) were recovered from the same run. SNV detection was strongly coverage-dependent, however: it was not achievable in the low-coverage FFPE and fresh-frozen specimens.Conclusions: Nanopore sequencing enables rapid, accurate, multidimensional molecular classification of diagnostically intractable CNS tumors using routine FFPE and frozen tissue, achieving high diagnostic resolution in comparison with reference methods while drastically reducing turnaround time and cost, making precision neuropathology feasible beyond quaternary centers.","PeriodicalId":6914,"journal":{"name":"Acta Neuropathologica Communications","volume":" ","pages":""},"PeriodicalIF":6.5000,"publicationDate":"2026-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Acta Neuropathologica Communications","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1186/s40478-026-02359-y","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"NEUROSCIENCES","Score":null,"Total":0}

引用次数: 0

Abstract

Background: DNA methylation profiling has become crucial for accurate classification of CNS tumors, yet Illumina EPIC arrays are restricted by long turnaround times, batching requirements, and limited accessibility in non-specialized centers. Rapid, affordable alternatives are urgently needed, particularly in resource-constrained settings. We aimed to evaluate nanopore sequencing as a real-world diagnostic tool for resolving diagnostically challenging CNS tumors.

Methods: We applied the ROBIN pipeline to 34 consecutive CNS tumors that remained NOS/NEC after routine histopathological and molecular workup. Samples comprised 16 routine FFPE blocks and 18 frozen tissues (9 fresh-frozen, 9 pre-extracted DNA) from three pathology centers. Nanopore sequencing was performed on PromethION R10.4.1 flow cells, targeting definitive methylation-based classification report within 24 h.

Results: A confident methylation class was assigned in 28/34 cases (82.4%), including 14/16 (87.5%) methylation calls of FFPE samples with median target coverage of 0.03×. Integration of simultaneously generated CNV profiles rescued an integrated glioblastoma diagnosis in two methylation-unclassifiable cases via 7+/10- signatures. The nanopore-based classifications also achieved 84.6% (11/13) concordance with paired result from Illumina Methylation EPIC arrays. In high-coverage samples, diagnostic-relevant SNVs (BRAF V600E, PTEN, NF1/TP53) were recovered from the same run. SNV detection was strongly coverage-dependent, however: it was not achievable in the low-coverage FFPE and fresh-frozen specimens.

Conclusions: Nanopore sequencing enables rapid, accurate, multidimensional molecular classification of diagnostically intractable CNS tumors using routine FFPE and frozen tissue, achieving high diagnostic resolution in comparison with reference methods while drastically reducing turnaround time and cost, making precision neuropathology feasible beyond quaternary centers.

查看原文本刊更多论文

基于纳米孔的DNA甲基化分析用于NOS/NEC CNS肿瘤的快速分子分类：使用FFPE档案和冷冻组织的多机构评估

背景：DNA甲基化分析已成为中枢神经系统肿瘤准确分类的关键，然而Illumina EPIC阵列受到周转时间长、批量要求高以及非专业中心有限的可及性的限制。迫切需要快速、负担得起的替代方案，特别是在资源有限的情况下。我们的目的是评估纳米孔测序作为一种现实世界的诊断工具来解决诊断上具有挑战性的中枢神经系统肿瘤。方法：我们对34例经常规组织病理学和分子检查仍为NOS/NEC的连续中枢神经系统肿瘤应用ROBIN管道。样本包括来自三个病理中心的16个常规FFPE块和18个冷冻组织（9个新鲜冷冻组织，9个预提取DNA）。对PromethION R10.4.1流式细胞进行纳米孔测序，在24小时内获得明确的甲基化分类报告。结果：28/34例（82.4%）被确定为甲基化类别，其中14/16例（87.5%）FFPE样本的甲基化通话，中位目标覆盖率为0.03×。整合同时生成的CNV谱通过7+/10-特征在两个甲基化无法分类的病例中挽救了胶质母细胞瘤的综合诊断。基于纳米孔的分类与Illumina甲基化EPIC阵列的配对结果也达到了84.6%（11/13）的一致性。在高覆盖率的样本中，诊断相关的snv （BRAF V600E， PTEN, NF1/TP53）从同一次运行中恢复。然而，SNV检测强烈依赖于覆盖度：在低覆盖度的FFPE和新鲜冷冻标本中无法实现。结论：利用常规FFPE和冷冻组织，纳米孔测序能够快速、准确、多维度地对难治性中枢神经系统肿瘤进行分子分类，与参考方法相比，实现了高诊断分辨率，同时大大减少了周转时间和成本，使精确的神经病理学超越了四元中心。

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

Acta Neuropathologica Communications Medicine-Pathology and Forensic Medicine

CiteScore

11.20

自引率

2.80%

发文量

162

审稿时长

8 weeks

期刊介绍： "Acta Neuropathologica Communications (ANC)" is a peer-reviewed journal that specializes in the rapid publication of research articles focused on the mechanisms underlying neurological diseases. The journal emphasizes the use of molecular, cellular, and morphological techniques applied to experimental or human tissues to investigate the pathogenesis of neurological disorders. ANC is committed to a fast-track publication process, aiming to publish accepted manuscripts within two months of submission. This expedited timeline is designed to ensure that the latest findings in neuroscience and pathology are disseminated quickly to the scientific community, fostering rapid advancements in the field of neurology and neuroscience. The journal's focus on cutting-edge research and its swift publication schedule make it a valuable resource for researchers, clinicians, and other professionals interested in the study and treatment of neurological conditions.