Ida Cristiane M, Butz Malinda L, Jenkins Robert B, Sarkaria Jann N, Kitange Gaspar J, Giannini Caterina, Kipp Benjamin R
Departments of Laboratory Medicine and Pathology.
Biochemistry and Molecular Biology.
Am J Clin Pathol. 2017 Oct 1;148(4):296-307. doi: 10.1093/ajcp/aqx073.
To develop and evaluate a real-time methylation-specific polymerase chain reaction (RT-MSP) MGMT assay, with a particular focus on small biopsies and indeterminate testing results.
We assessed formalin-fixed paraffin-embedded glioblastoma or gliosarcoma specimens (n = 641). A test-validation group (n = 51) with previously obtained reference laboratory (RL) results was used to determine performance characteristics of the RT-MSP assay. An indeterminate (equivocal) category was established for cases that could not be clearly classified as positive or negative.
Overall agreement of RT-MSP and RL results was 91% (41/45 nonindeterminate cases). Discordant cases were tested by pyrosequencing, and results were most concordant with RT-MSP. Among cases with limited amounts of tissue (n = 7), six yielded valid results by RT-MSP (all negative); the single invalid result consisted of a stereotactic biopsy specimen obtained 14 years prior. A subset of indeterminate cases obtained during clinical testing (n = 18/575 [3%]) was also evaluated by pyrosequencing and showed a heterogeneous pattern of methylation across the eight interrogated CpG sites.
The RT-MSP assay that we developed in-house is a robust clinical detection method for the heterogeneous process of MGMT promoter methylation in glioblastoma.
开发并评估一种实时甲基化特异性聚合酶链反应（RT-MSP）MGMT检测方法，尤其关注小活检标本和不确定的检测结果。
我们评估了福尔马林固定石蜡包埋的胶质母细胞瘤或胶质肉瘤标本（n = 641）。使用一个先前已获得参考实验室（RL）结果的测试验证组（n = 51）来确定RT-MSP检测方法的性能特征。对于无法明确分类为阳性或阴性的病例，设立了一个不确定（模棱两可）类别。
RT-MSP与RL结果的总体一致性为91%（41/45例非不确定病例）。对不一致的病例进行焦磷酸测序检测，结果与RT-MSP最为一致。在组织量有限的病例（n = 7）中，6例通过RT-MSP获得了有效的结果（均为阴性）；唯一无效的结果是一份14年前获得的立体定向活检标本。还对焦磷酸测序评估了临床检测期间获得的一部分不确定病例（n = 18/575 [3%]），结果显示在所检测的8个CpG位点上甲基化模式各异。
我们自行开发的RT-MSP检测方法是一种用于胶质母细胞瘤中MGMT启动子甲基化异质性过程的可靠临床检测方法。
