Novella Elisabetta, Giaretta Ilaria, Elice Francesca, Madeo Domenico, Piccin Andrea, Castaman Giancarlo, Rodeghiero Francesco
Department of Cell Therapy and Hematology, San Bortolo Hospital, Vicenza, Italy.
Haematologica. 2002 Nov;87(11):1157-64.
Several polymerase chain reaction (PCR)-based techniques for tracking minimal residual disease (MRD) in B-lymphoproliferative disorders have been recently proposed. These procedures show significant variation in sensitivity and specificity. We describe an alternative assay based on fluorescent PCR combined with capillary electrophoresis and GeneScan analysis, to identify the monoclonal immunoglobulin heavy chain (IgH) rearrangement in multiple myeloma (MM) and to provide a semi-quantitative evaluation of MRD by limiting dilutions.
Different sets of family specific primers derived from the leader region and from the framework-1 of IgH were used, with a unique reverse fluorescent primer JH. The malignant clone was identified by GeneScan and sequenced. Two tumor primers, mapping in the complementarity determining regions CDRII and CDRIII, were designed for each patient. A comparison between the nested-PCR approach and direct fluorescent PCR was performed for three patients in complete clinical remission after autologous or allogeneic bone marrow transplantation.
Thirty-six consecutive patients with MM were screened and monoclonality was identified in about 70% of the cases. Molecular MRD evaluation was performed in 18 patients using tumor primers. This method allowed identification of 1 neoplastic cell among 10(4)-10(6) normal cells. In three cases, negative by nested-PCR and agarose gel electrophoresis, gene scanning showed persistence of the neoplastic clone, despite the negativity of the immunofixation.
Capillary electrophoresis of fluorescent fragments with gene scanning provides a simple, rapid and reproducible method to detect IgH rearrangement and to evaluate MRD. Furthermore, the sensitivity reached is up to 1 log higher than that of the conventional approach with nested-PCR, even though two steps of specificity are maintained.
最近提出了几种基于聚合酶链反应（PCR）的技术用于追踪B淋巴细胞增殖性疾病中的微小残留病（MRD）。这些方法在敏感性和特异性上存在显著差异。我们描述了一种基于荧光PCR结合毛细管电泳和基因扫描分析的替代检测方法，用于鉴定多发性骨髓瘤（MM）中的单克隆免疫球蛋白重链（IgH）重排，并通过有限稀释对MRD进行半定量评估。
使用了源自IgH引导区和框架1的不同家族特异性引物组，以及一个独特的反向荧光引物JH。通过基因扫描鉴定恶性克隆并进行测序。为每位患者设计了两个位于互补决定区CDRII和CDRIII的肿瘤引物。对3例自体或异基因骨髓移植后处于完全临床缓解的患者进行了巢式PCR方法与直接荧光PCR的比较。
对36例连续的MM患者进行了筛查，约70%的病例中鉴定出单克隆性。使用肿瘤引物对18例患者进行了分子MRD评估。该方法能够在10⁴ - 10⁶个正常细胞中鉴定出1个肿瘤细胞。在3例经巢式PCR和琼脂糖凝胶电泳检测为阴性的病例中，尽管免疫固定法检测为阴性，但基因扫描显示肿瘤克隆持续存在。
荧光片段的毛细管电泳结合基因扫描提供了一种简单、快速且可重复的方法来检测IgH重排并评估MRD。此外，即使保持了两步特异性，所达到的敏感性比传统巢式PCR方法高1个对数。
