Requisite Analytic and Diagnostic Performance Characteristics for the Clinical Detection of BRAF V600E in Hairy Cell Leukemia: A Comparison of 2 Allele-specific PCR Assays
Requisite Analytic and Diagnostic Performance Characteristics for the Clinical Detection of BRAF V600E in Hairy Cell Leukemia: A Comparison of 2 Allele-specific PCR Assays
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DOI：
10.1097/pai.0000000000000024
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发表时间：
2015-09-01
影响因子：
1.6
通讯作者：
Betz, Bryan L.
中科院分区：
文献类型：
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作者：
Brown, Noah A.;Weigelin, Helmut C.;Betz, Bryan L.
在毛细胞白血病（HCL）中检测高频BRAF V600 E突变具有重要的诊断实用性。然而，用于常规检测HCL中BRAF V600 E突变的临床试验的必要分析性能尚未明确定义。在这项研究中，我们试图确定福尔马林固定，石蜡包埋（FFPE）和冷冻样本所需的分析灵敏度水平，并比较2个等位基因特异性聚合酶链反应（PCR）检测的性能。采用实验室开发的等位基因特异性PCR检测法和市售的等位基因特异性定量PCR检测法myT BRAF Ultra对29例经典HCL（包括22例FFPE骨髓穿刺液和7例血液或骨髓冷冻标本）进行了评价。还包括6例HCL变异型和40例非HCL B细胞淋巴瘤。2例经典HCL，1例显示CD 5表达，尽管涉及高水平白血病，但基于PCR和测序的阴性结果，确实为BRAF V600 E阴性。在其余27份标本中，实验室开发和商业检测试剂分别在88.9%（17/20份FFPE; 7/7份冷冻标本）和81.5%（15/20份FFPE; 7/7份冷冻标本）中检出V600 E突变。在46例非HCL淋巴瘤中未检测到突变。两种检测方法均显示，冷冻标本中的分析灵敏度为0.3%，FFPE组织中为5%。基于这些结果，临床检测HCL标本中的V600 E突变需要具有高分析灵敏度的检测。两种等位基因特异性PCR检测在冷冻和FFPE骨髓穿刺液中表现良好，尽管FFPE组织中的检测需要5%或更多的参与。
Detection of high-frequency BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. However, the requisite analytic performance for a clinical assay to routinely detect BRAF V600E mutations in HCL has not been clearly defined. In this study, we sought to determine the level of analytic sensitivity needed for formalin-fixed, paraffin-embedded (FFPE) and frozen samples and to compare the performance of 2 allele-specific polymerase chain reaction (PCR) assays. Twenty-nine cases of classic HCL, including 22 FFPE bone marrow aspirates and 7 frozen specimens from blood or bone marrow were evaluated using a laboratory-developed allele-specific PCR assay and a commercially available allele-specific quantitative PCR assaymyT BRAF Ultra. Also included were 6 HCL variant and 40 non-HCL B-cell lymphomas. Two cases of classic HCL, 1 showing CD5 expression, were truly BRAF V600E-negative based on negative results by PCR and sequencing despite high-level leukemic involvement. Among the remaining 27 specimens, V600E mutations were detected in 88.9% (17/20 FFPE; 7/7 frozen) and 81.5% (15/20 FFPE; 7/7 frozen), for the laboratory-developed and commercial assays, respectively. No mutations were detected among the 46 non-HCL lymphomas. Both assays showed an analytic sensitivity of 0.3% involvement in frozen specimens and 5% in FFPE tissue. On the basis of these results, an assay with high analytic sensitivity is required for the clinical detection of V600E mutations in HCL specimens. Two allele-specific PCR assays performed well in both frozen and FFPE bone marrow aspirates, although detection in FFPE tissue required 5% or more involvement.