国家重大科学研究计划项目“生物医学纳米材料对血细胞作用的研究”工作进展与讨论

基于纳米材料的白血病病因和病理学研究

苏州大学 尹斌 教授


1. 中枢神经系统浸润研究(陈子兴课题组)

最近尝试了应用对小鼠心内注射纳米磁铁标记的白血病SHI-1细胞制备小鼠中枢神经系统白血病的模型,发现比以往经尾静脉注射的方法效率提高。观察到一些在磁共振下的影像,但不是很清楚稳定。有些实验有待进一步证实。

2. Clonal origin and evolution of myelodysplastic syndrome analyzed by dysplastic morphology and fluorescence in situ hybridization(陈子兴课题组)

骨髓增生异常综合症(MDS)是源于造血干/祖细胞的克隆性疾病。细胞发育异常是一种显著的形态特征。然而,这种细胞发育异常代表的是异常的克隆还是仅仅是一种非特异性的表面现象,仍需要阐明。为了解决这个问题,本研究检测了97例患者的细胞发育异常特征,其中16例染色体异常的患者的骨髓涂片进行了荧光原位杂交来确定这些细胞的核型。此外,使用形态学评估结合荧光原位杂交对每个谱系的各个分化阶段的染色体异常核型的出现频率进行了评估。结果表明发育异常的细胞在各个WHO亚型中的存在比例无显著的差异,而巨核细胞谱系在各个亚型中显现出最频繁的细胞异常特征。细胞发育异常和荧光原位杂交检测的异常核型存在正相关性。但是,细胞发育异常的形态特征不是原位杂交检测到的异常克隆的特异性特征。荧光原位杂交检测的异常克隆可以分化为成熟的粒细胞和红细胞,与来源于正常克隆的细胞共存。

该工作已经在杂志International Journal of Hematology发表,Int J Hematol (2015) 101:58-66 DOI 10.1007/s12185-014-1700-1。主要参与该工作的有ChunMei Fu,ZiXing Chen,DanDan Liu,Jun Zhang, JinLan Pan,JianYing Liang。该文章具体内容介绍如下:

Abstract:

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem/progenitor cells. As bone marrow cells are extremely diverse in these disorders, the origin and evolution of MDS clones are difficult to identify and trace. Cellular dysplasia is a distinct morphologic feature; however, whether the dysplastic cells represent abnormal clones or only nonspecific superficial phenomena remains to be clarified. To address this question, 97 patients were examined for dysplasia features, among them bone marrow slides of 16 patients with chromosomal abnormalities were subjected to fluorescence in situ hybridization (FISH) to determine the karyotype of these dysplastic cells. Furthermore, the emerging frequencies of abnormal karyotypes in various differentiated stages of each lineage were also evaluated by a combination of morphological evaluation and FISH karyotyping. Our results indicate that the overall percentage of dysplastic cells does not differ significantly among the WHO subtypes, while the megakaryoid lineage presents the most frequent dysplasia in all subtypes. A positive correlation between dysplastic cells and FISHdetectable abnormal clones was observed, but the dysplastic morphology was not a specific feature of FISH-detectable abnormal clones. FISH-detectable abnormal clones can differentiate into mature granulocytes and erythrocytes, in coexistence with cells originating from the normal clones.

Results and Figures:

(1) The dysplastic morphological feature analysis

We have counted the cells with various dysplastic morphological features in each lineage separately and calculated three quantitative parameters in MDS patients with subtypes according to WHO classification (Table 3). No differences were found in the most frequent dysplastic features within these three lineages among all groups. The aberrant nuclear shape of normoblast, megaloblastoid and binuclearity with abnormality were the most frequently emerged dysplastic features within the erythroid lineage. Pseudo-Pelger–Huët (majority as II subtype), large banded or metamyelocyte bizarre nuclear shape were the most frequent dysplastic features observed in granulocytic lineage. All groups exhibited the large-mononuclear megakaryocyte, the megakaryocyte with small round separated nuclei and the micromegakaryocyte are the most frequent dysplasia features seen in megakaryocytoid lineage. As regarding to the cell dysplastic features related to karyotype abnormalities, we failed to find any distinct dysplasia pattern correlated to the karyotype changes. The most frequently occurring dysplasia changes in erythroid, granulocytic and megakaryocytoid lineage were megaloblastoid, aberrant nuclear shape of normoblast, pseudo-Pelger–Huët, large bands or metamyelocyte, large-mononuclear megakaryocyte, megakaryocyte with small round separated nuclei and micromegakaryocyte, respectively, regardless of normal or aberrant karyotype. However, interestingly as shown in Table 3, the PSDC of megakaryocytoid lineage was higher than the PSDC of erythroid and granulocytic lineage in all these groups. In addition, the ID of megakaryocytoid and erythroid lineage was higher than that in the granulocytic lineage in all four groups. Based on the criteria of dysplasia in MDS which defined that the dysplasia should occur in more than 10 % of each lineage, we found 6 patients with more than 10 % dysplasia in only one lineage, 17 cases of with dysplasia in two lineages, and 12 cases with dysplasia in three lineages, respectively, in the MDS-RAEB group.


(2) The karyotype of dysplastic cells analyzed by FISH on bone marrow smears

The FISH performed on the bone marrow slide provided fluorescent signals for the bone marrow cells in the smear which were previously treated with Wright–Giemsa staining, allowing to observe and identify the karyotype of the cells (Fig. 1). The colorful signals on the slides were usually as clear as that of conventional FISH. Depending on the cellularity of the bone marrow and the probes used for FISH, the optimal microscopic fields on the slides should be carefully selected to avoid the erroneous judgment for signals due to the cell overlapped or broken in the bone marrow smear. Overall, about 151–1134 bone marrow cells were analyzed for each patient, including 70–423 bone marrow cells with dysplasia.

The karyotypes of bone marrow dysplastic cells of erythroid and myeloid lineages visualized by FISH in these 16 patients are shown in Fig. 2. Both the dysplastic cells and the nondysplastic cells of the erythroid and myeloid lineages in these 16 MDS patients may exhibit abnormal karyotype, indicating a FISH-detectable abnormal clonal origin. However, the percentage of dysplasia cells with FISH-detectable abnormal clonality was higher than that of nondysplasia cells in all patients. The proportion of dysplasia cells with FISH-detectable abnormal clonal origin was positively correlated with that of nondysplasia cells which also demonstrated an abnormal karyotype. For example, in two patients both the dysplasia cells and the nondysplasia displayed a lower proportion of FISH-detectable abnormal clonal origin, while the other patients had a higher FISHdetectable abnormal clone proportion in cells regardless of dysplasia. Also, the cells identified as abnormal karyotype exhibited higher percentage of dysplasia than that in the cells with normal karyotype in all patients.

The dysplasia in megakaryocytic lineage, especially the emergence of micromegakaryocyte is practically an important marker for diagnosis of MDS. Therefore, we paid special attention on the clonality of these dysplastic megakaryocytes. As shown in Table 4, most of the dysplastic megakaryocytes were derived from FISH-detectable abnormal clone in MDS patients with abnormal karyotype, such as +8, −7, del(7q), del(5q), and del(20q). The proportion of the FISH-detectable abnormal clone of the dysplastic megakaryocytic cells was 84.4, 76.5, 94.8, 90.9 and 78.8 % in these patients, respectively. At the same time, we also analyzed the megakaryocytes with normal morphology in five MDS patients. The percentage of these “normal megakaryocytes” with an FISH-detectable abnormal clonal origin was 75.7, 80.0, 89.5, 75 and 81.0 %, in these patients, respectively.




(3) The clonal evolution during lineage differentiation in MDS

As the hematopoietic stem/progenitor cells of MDS, even after having undergone abnormal genetic changes, they still retained the capability of differentiation along various lineages before the MDS patient progressed toward acute myeloid leukemia. Thus, the cell population in the bone marrow of MDS patients contained heterogeneous cellular components, including cells of various lineages at all differentiation stages derived from either normal or abnormal progenitor clones. It was impossible to distinguish the cells with abnormal clonal origin from the normal one by morphology per se. To follow and trace the evolution of FISH-detectable abnormal clones during the cell differentiation in patients, cells of erythroid and myeloid lineages at various differentiation stages were firstly recognized by morphology with Wright–Giemsa staining, and the karyotypes were then analyzed by FISH. As shown in Fig. 3, the frequency of occurrence of abnormal clone displayed a reducing tendency in company with the hematopoietic cell differentiation from a relatively premature stage to their mature terminals in most patients, except three patients. In fact, the cell population in bone marrow demonstrated a situation of dynamic changes and balance between the offspring cells of normal and abnormal clones. Meanwhile we have counted 50 immature megakaryocytes totally in our 15 patients, finding that 92 % of these cells present FISHdetectable abnormal karyotype, which is higher than that in mature cells. In addition to these three lineages, FISHdetectable abnormal clones could also be detected in cells of lymphoid and monocytic lineages (as high as 40 % for lymphoid lineage and 90 % for monocytic lineage in some cases), suggesting that the MDS clones involved in these two lineages may initiate at the stage earlier than the common myeloid progenitor (CMP).