Jann Johann-Christoph, Nowak Daniel, Nolte Florian, Fey Stephanie, Nowak Verena, Obländer Julia, Pressler Jovita, Palme Iris, Xanthopoulos Christina, Fabarius Alice, Platzbecker Uwe, Giagounidis Aristoteles, Götze Katharina, Letsch Anne, Haase Detlef, Schlenk Richard, Bug Gesine, Lübbert Michael, Ganser Arnold, Germing Ulrich, Haferlach Claudia, Hofmann Wolf-Karsten, Mossner Maximilian
III Medizinische Klinik, Hämatologie und Onkologie, Universitätsmedizin Mannheim, Mannheim, Germany.
Medizinische Klinik und Poliklinik I, Universitatsklinikum Carl Gustav Carus, Dresden, Germany.
J Med Genet. 2017 Sep;54(9):640-650. doi: 10.1136/jmedgenet-2017-104528. Epub 2017 Jun 9.
Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings.
For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers.
Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77-0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring.
In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.
细胞遗传学异常，如5号染色体缺失（del(5q)），是血液系统恶性肿瘤常规临床诊断的关键要素。目前已确立的方法，如中期细胞遗传学、荧光原位杂交（FISH）或基于芯片的方法，由于依赖活细胞、成本高或具有半定量性质而存在局限性。重要的是，它们不能用于低丰度DNA。因此，我们旨在建立一种克服这些缺点的强大且定量的技术。
为了精确测定del(5q)细胞分数，我们开发了一种廉价的多重PCR检测方法，该方法仅需纳克级DNA，可同时检测12个独立短串联重复序列标记的等位基因失衡情况。
将该方法应用于来自260名个体的1142份样本，结果显示各标记间具有很强的一致性（R²=0.77 - 0.97）和可重复性（平均标准差：1.7%）。值得注意的是，即使使用亚纳克量的DNA，该检测方法通过标准曲线评估仍显示出准确的定量结果（R²>0.99），并且与配对的FISH测量结果高度一致（R²=0.92）。此外，在接受来那度胺治疗的骨髓增生异常综合征del(5q)患者中，可靠地证实了细胞遗传学反应。虽然该检测方法在受试者工作特征分析中显示出良好的诊断准确性（曲线下面积：0.97），但我们进一步观察到骨髓样本与外周血样本之间具有很强的相关性（R²=0.79），这表明其可能适用于侵入性较小的克隆监测。
总之，我们提出了一种适用于定量染色体畸变的工具，特别是在有问题的样本中，该工具应易于应用于更多肿瘤实体。
