Quantitative assessment of minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction
Quantitative assessment of minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction
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DOI：
10.1016/j.vetimm.2008.09.004
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发表时间：
2008-12-15
影响因子：
1.8
通讯作者：
Tsujimoto, Hajime
中科院分区：
文献类型：
--
作者：
Yamazaki, Jumpei;Baba, Kenji;Tsujimoto, Hajime
淋巴瘤是犬最常见的造血系统恶性肿瘤。尽管大部分患有淋巴瘤的狗可以通过初始化疗实现临床缓解，但大多数狗因肿瘤复发而死亡。我们利用实时聚合酶链反应（PCR）建立了犬淋巴瘤微小残留病（MRD）定量检测系统。使用犬 T 细胞淋巴瘤衍生细胞系 UL-1 来检查 MRD 检测系统的特异性和敏感性。根据从狗基因组数据库中获得的 UL-1 细胞 T 细胞受体伽马链 (TCR gamma) 基因片段序列及其下游序列，设计等位基因特异性寡核苷酸引物和探针。对含有来自 UL-1 细胞的 TCR gamma 基因的质粒 DNA 和 UL-1 细胞的基因组 DNA 进行的实时 PCR 系统显示，该系统每次反应的准确度为 10-100,000 个拷贝，其灵敏度为每 10,000 个细胞 1 个细胞。为了监测犬淋巴瘤中肿瘤细胞数量的动力学，我们对7只接受化疗的淋巴瘤犬外周血中的MRD水平进行了定量。由于免疫球蛋白重链（IgH）基因克隆重排的发现表明7名患者的淋巴瘤细胞是B细胞来源，因此根据每个病例重排的IgH基因的序列制备等位基因特异性寡核苷酸引物和探针。在2例V期淋巴瘤中，通过实时PCR测量的外周血肿瘤细胞数量与通过常规血液学检查估计的数量相当。即使在完全缓解（CR）阶段，所有 7 例患者的外周血中均检测到 MRD。在7只淋巴瘤犬中，外周血MRD水平的变化通常与淋巴结体积的变化平行。在这 7 名接受化疗的患者中，均未观察到 MRD 水平低于检测限的分子 CR。通过此处描述的实时 PCR 方法检测到的 MRD 水平将有助于研究犬淋巴瘤患者肿瘤细胞生长的动力学及其消退。 (C) 2008 Elsevier B.V. 保留所有权利。
Lymphoma is the most common hematopoietic malignancy in dogs. Although a large proportion of dogs with lymphoma can achieve clinical remission by initial chemotherapy, most dogs die as a consequence of tumor relapse. We established a quantitative detection system for minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction (PCR). A canine T-cell lymphoma-derived cell line, namely, UL-1, was used to examine the specificity and sensitivity of the MRD detecting system. Allele-specific oligonucleotide primers and probes were designed based on the sequence of T-cell receptor gamma chain (TCR gamma) gene fragment of UL-1 cells in conjunction with its downstream sequence, which were obtained from the dog genome database. The real-time PCR system for plasmid DNA containing the TCR gamma gene derived from UL-1 cells and the genomic DNA of UL-1 cells revealed that the system was accurate for 10-100,000 copies per reaction and its sensitivity was 1 cell per 10,000 cells. In order to monitor the kinetics of tumor cell number in canine lymphoma, we quantified the level of MRD in the peripheral blood of 7 dogs with lymphoma under chemotherapy. Since the lymphoma cells from the 7 patients were shown to be B-cell origin from the finding of clonal rearrangement of immunoglobulin heavy chain (IgH) gene, allele-specific oligonucleotide primers and probes were prepared based on the sequence of rearranged IgH gene in each case. The number of peripheral blood tumor cells measured by the real-time PCR was comparable to that estimated by conventional hematological examination in 2 cases of stage V lymphoma. MRD in the peripheral blood was detectable in all 7 cases, even in the complete remission (CR) phase. In the 7 lymphoma dogs, changes in the MRD levels of peripheral blood generally paralleled with the changes in the Volumes of lymph nodes. Molecular CR, in which the MRD level was below the detection limit, was not observed in any of these 7 patients under chemotherapy. The MRD level detected by the real-time PCR method described here would be useful for investigating the kinetics Of tumor cell growth and its regression in canine lymphoma patients. (C) 2008 Elsevier B.V. All rights reserved.