Matsuo Hidemasa, Shiga Shuichi, Imai Tsuyoshi, Kamikubo Yasuhiko, Toki Tsutomu, Terui Kiminori, Ito Etsuro, Adachi Souichi
Department of Human Health Sciences, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho, Syogoin, Sakyoku, Kyoto, 606-8507, Japan.
Department of Clinical Laboratory, Kyoto University Hospital, Kyoto, Japan.
Int J Hematol. 2017 Jul;106(1):55-59. doi: 10.1007/s12185-017-2227-z. Epub 2017 Apr 13.
In treatment of acute myeloid leukemia (AML), prognostic factors, including gene mutation and abnormal gene expression, enable risk stratification of patients. However, in the case of a small proportion of leukemic blast cells, such as AML associated with Down syndrome (AML-DS), it is not possible to examine prognostic factors precisely due to the large proportion of normal cells. Here, we present a novel method for examining prognostic factors by making a smear on a membrane slide glass from a small amount of diagnostic specimen and collecting highly pure leukemic blast cells by laser microdissection (LMD). We verified the effectiveness of this method using 10% KPAM1 cell line suspension and peripheral blood containing 20% blast cells obtained from a patient with transient abnormal myelopoiesis (TAM). After making blood smears, approximately 100 cells were collected and analyzed by direct sequencing. Frameshift mutations (2 bp deletion and 17 bp duplication, respectively) in GATA-1 were detected in each sample, suggesting KPAM1 and TAM blast cells were accurately purified. This novel method enables us to precisely examine prognostic factors in many cases, even in cases with a small proportion of leukemic blast cells or small specimens to preserve.
在急性髓系白血病（AML）的治疗中，包括基因突变和基因表达异常在内的预后因素可对患者进行风险分层。然而，对于一小部分白血病原始细胞，如唐氏综合征相关的AML（AML-DS），由于正常细胞比例较大，无法精确检测预后因素。在此，我们提出一种新方法，即从少量诊断标本在膜载玻片上制作涂片，并通过激光显微切割（LMD）收集高度纯化的白血病原始细胞，以检测预后因素。我们使用10%的KPAM1细胞系悬液和从一名短暂异常髓系造血（TAM）患者获得的含20%原始细胞的外周血验证了该方法的有效性。制作血涂片后，收集约100个细胞并通过直接测序进行分析。在每个样本中均检测到GATA-1的移码突变（分别为2bp缺失和17bp重复），表明KPAM1和TAM原始细胞被准确纯化。这种新方法使我们能够在许多情况下精确检测预后因素，即使是在白血病原始细胞比例小或标本量小需要保存的情况下。
