Kantorova Barbara, Malcikova Jitka, Smardova Jana, Pavlova Sarka, Trbusek Martin, Tom Nikola, Plevova Karla, Tichy Boris, Truong Sim, Diviskova Eva, Kotaskova Jana, Oltova Alexandra, Patten Nancy, Brychtova Yvona, Doubek Michael, Mayer Jiri, Pospisilova Sarka
Central European Institute of Technology (CEITEC), Masaryk University Brno, Kamenice 5, CZ-625 00, Brno, Czech Republic.
Tumour Biol. 2015 May;36(5):3371-80. doi: 10.1007/s13277-014-2971-0. Epub 2014 Dec 20.
TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.
TP53基因缺陷是慢性淋巴细胞白血病（CLL）患者生存和治疗耐药的一个强有力的不良预后因素。尽管已经报道了多种TP53突变分析方法，但没有一种方法能够识别所有出现的序列变异，最合适的方法仍在讨论中。本研究的目的是确定CLL中常用的TP53突变检测方法的局限性，并提出一种最佳的检测方法。我们使用变性高效液相色谱（DHPLC）、酵母中分离等位基因的功能分析（FASAY）和AmpliChip p53研究检测法，对182例富集高危病例的CLL患者进行了平行检测。还使用超深度下一代测序（NGS）对69例患者的T53基因突变情况进行了评估。总共在57例（31%）患者中发现了79个TP53突变；其中，错义替换占主导（检测到的突变中有68%）。比较所使用方法的有效性，DHPLC和FASAY与直接桑格测序相结合均取得了最佳结果，分别识别出95%和93%的TP53突变患者。然而，我们表明，在携带低比例TP53突变的CLL患者中，更敏感的方法，如超深度NGS，可能更合适。使用DHPLC或FASAY进行TP53基因分析是一种合适的突变检测方法。超深度NGS有可能克服目前使用方法的缺点，能够检测低比例突变，因此是一种在不久的将来很有前景的方法。
