van Wezel Esther M, Zwijnenburg Danny, Zappeij-Kannegieter Lily, Bus Erik, van Noesel Max M, Molenaar Jan J, Versteeg Rogier, Fiocco Marta, Caron Huib N, van der Schoot C Ellen, Koster Jan, Tytgat Godelieve A M
Department of Experimental Immunohematology, Sanquin Research, Amsterdam, Academical Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Landsteiner Laboratory, Department of Pediatric Oncology, Emma Children's Hospital, Academical Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
Department of Oncogenomics, Academical Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
J Mol Diagn. 2015 Jan;17(1):43-52. doi: 10.1016/j.jmoldx.2014.09.005. Epub 2014 Oct 24.
PCR-based detection of minimal residual disease (MRD) in neuroblastoma is currently based on RNA markers; however, expression of these targets can vary, and only paired-like homeobox 2b has no background expression. We investigated whether chromosomal breakpoints, identified by whole-genome sequencing (WGS), can be used as patient-specific DNA MRD markers. WGS data were used to develop large numbers of real-time PCRs specific for tumors of eight patients. These PCRs were used to quantify chromosomal breakpoints in primary tumor and bone marrow samples. Finally, the DNA breakpoints with the highest abundance were compared with a panel of RNA markers. By WGS we identified 42 chromosomal breakpoints in tumor samples from eight patients and developed specific quantitative real-time PCRs for each breakpoint. The tumor-specific breakpoints were all present in bone marrow at diagnosis. For one patient slight clonal selection was observed in response to treatment. Positivity of DNA MRD markers preceded disease progression in four of five patients; in one patient the RNA markers remained negative. For 16 of 22 samples MRD levels determined by RNA and DNA were comparable and in 6 of 22 samples higher MRD levels were detected by DNA markers. DNA breakpoints used as MRD targets in neuroblastoma are reliable and stable markers. In addition, this technique might be applicable for detecting tumor cells in other types of cancer.
基于聚合酶链反应（PCR）检测神经母细胞瘤微小残留病（MRD）目前是基于RNA标志物；然而，这些靶点的表达可能会有所不同，只有配对样同源盒2b没有背景表达。我们研究了通过全基因组测序（WGS）确定的染色体断点是否可以用作患者特异性DNA MRD标志物。WGS数据用于开发大量针对8名患者肿瘤的实时PCR。这些PCR用于定量原发性肿瘤和骨髓样本中的染色体断点。最后，将丰度最高的DNA断点与一组RNA标志物进行比较。通过WGS，我们在8名患者的肿瘤样本中鉴定出42个染色体断点，并为每个断点开发了特异性定量实时PCR。肿瘤特异性断点在诊断时均存在于骨髓中。一名患者在治疗后观察到轻微的克隆选择。在五名患者中的四名患者中，DNA MRD标志物的阳性先于疾病进展；一名患者的RNA标志物仍为阴性。在22个样本中的16个样本中，通过RNA和DNA测定的MRD水平相当，在22个样本中的6个样本中，DNA标志物检测到更高的MRD水平。用作神经母细胞瘤MRD靶点的DNA断点是可靠且稳定的标志物。此外，该技术可能适用于检测其他类型癌症中的肿瘤细胞。
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