Viswanatha D S, Chen I, Liu P P, Slovak M L, Rankin C, Head D R, Willman C L
Department of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, NM, USA.
Blood. 1998 Mar 15;91(6):1882-90.
The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFbeta-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFbeta-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR-negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFbeta-SMMHC fusion proteins. Molecular characterization of one RT-PCR-positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, -0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR-positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.
inv(16)(p13q22)和t(16;16)(p13;q22)细胞遗传学异常常见于急性髓系白血病（AML），通常与法美英（FAB）AML-M4Eo亚型相关。逆转录聚合酶链反应（RT-PCR）技术最近已被开发用于检测编码CBFβ-平滑肌肌球蛋白重链（SMMHC）融合蛋白的CBFB-MYH11融合基因几种变体的存在。我们现已确定一种针对最常见类型的CBFβ-SMMHC融合蛋白（A型）连接表位的多克隆抗体[抗inv(16)抗体]的临床应用，该融合蛋白存在于90%的inv(16)/t(16;16) AML病例中。使用流式细胞术，开发了可重复的方法用于检测通透细胞中的CBFβ-SMMHC蛋白；然后将流式细胞术结果与细胞遗传学和RT-PCR检测方法进行关联。在对42例具有各种细胞遗传学异常的白血病病例和几个正常对照的分析中，抗inv(16)抗体特异性检测到所有23例细胞遗传学上inv(16)或t(16;16)阳性的病例，包括1例RT-PCR阴性的AML病例。除了检测所有A型融合体，抗inv(16)抗体还意外地鉴定出C型和D型CBFβ-SMMHC融合蛋白。对1例RT-PCR阳性且抗体阳性的t(16;16)病例的非A型产物进行分子特征分析，显示出一种新的此前未报道的CBFB-MYH11融合（CBFB第455位核苷酸-MYH11第1893位核苷酸）。使用柯尔莫哥洛夫-斯米尔诺夫统计D值分析流式细胞术结果，阳性样本的中位数为0.65（范围为0.35至0.77），而阴性组为0.07（范围为-0.21至0.18）（P <.0001）。细胞遗传学与RT-PCR之间的总体一致性为97%，而流式细胞术与细胞遗传学之间的一致性为100%。因此，使用抗inv(16)抗体，可以快速鉴定所有细胞遗传学阳性且RT-PCR阳性的inv(16)或t(16;16) AML病例。本研究证明了该抗体作为inv(16)/t(16;16) AML研究工具的用途，并表明开发此类试剂可能具有潜在的临床诊断用途。
