Jiménez Cristina, Chillón María Del Carmen, Balanzategui Ana, Puig Noemí, Sebastián Elena, Alcoceba Miguel, Sarasquete María E, Conde Isabel P, Corral Rocío, Marín Luis A, Paiva Bruno, Ruano Montserrat, Antón Alicia, Maldonado Rebeca, San Miguel Jesús F, González Marcos, García-Sanz Ramón
*Department of Hematology, University Hospital of Salamanca †Center of Investigation in Cancer (CIC), Instituto Biosanitario de Salamanca (IBSAL), Salamanca, Spain.
Appl Immunohistochem Mol Morphol. 2014 Nov-Dec;22(10):768-73. doi: 10.1097/PAI.0000000000000020.
MYD88 L265P mutation has been reported in ∼90% of Waldenström's Macroglobulinemia (WM) patients and immunoglobulin M (IgM) monoclonal gammopathies of uncertain significance (MGUS), as well as in some cases of lymphoma and chronic lymphocytic leukemia. The present study aimed to develop a real-time allele-specific oligonucleotide PCR (ASO-RQ-PCR) to detect the MYD88 L265P mutation. We first evaluated the reproducibility and sensitivity of the technique with a diluting experiment of a previously known positive sample. Then, we evaluated the applicability of the methodology by analyzing 30 selected patients (10 asymptomatic WM, 10 symptomatic WM, and 10 IgM MGUS) as well as 10 healthy donors. The quantitative ASO-PCR assay could detect the MYD88 L265P mutation at a dilution of 0.25%, showing an inverse correlation between the tumor cell percentage and the cycle threshold (CT) value, thus allowing for tumor burden quantitation. In addition, mutated cases were distinguished from the unmutated by >10 cycles of difference between CTs. To sum up, ASO-RQ-PCR is an inexpensive, robust, and optimized method for the detection of MYD88 L265P mutation, which could be considered as a useful molecular tool during the diagnostic work-up of B-cell lymphoproliferative disorders.
据报道，约90%的华氏巨球蛋白血症（WM）患者以及意义未明的免疫球蛋白M（IgM）单克隆丙种球蛋白病（MGUS）中存在MYD88 L265P突变，一些淋巴瘤和慢性淋巴细胞白血病病例中也有该突变。本研究旨在开发一种实时等位基因特异性寡核苷酸PCR（ASO-RQ-PCR）方法来检测MYD88 L265P突变。我们首先通过对先前已知的阳性样本进行稀释实验来评估该技术的重复性和灵敏度。然后，我们通过分析30例选定患者（10例无症状WM、10例有症状WM和10例IgM MGUS）以及10名健康供体来评估该方法的适用性。定量ASO-PCR检测方法能够在0.25%的稀释度下检测到MYD88 L265P突变，显示肿瘤细胞百分比与循环阈值（CT）值呈负相关，从而能够对肿瘤负荷进行定量分析。此外，通过CT值之间大于10个循环的差异可将突变病例与未突变病例区分开来。总之，ASO-RQ-PCR是一种检测MYD88 L265P突变的廉价、可靠且优化的方法，可被视为B细胞淋巴增殖性疾病诊断检查过程中的一种有用分子工具。
