van Belzen N, Hupkes P E, Doekharan D, Hoogeveen-Westerveld M, Dorssers L C, van't Veer M B
Department Hematology, Dr Daniel den Hoed Cancer Center, Erasmus University, Rotterdam, The Netherlands.
Leukemia. 1997 Oct;11(10):1742-52. doi: 10.1038/sj.leu.2400797.
Rearranged immunoglobulin heavy chain (IgH) genes provide unique clonal markers for B cells. Since amplification of the rearranged gene by polymerase chain reaction (PCR) and demonstrating that the amplified sequence is indeed derived from tumor cells is more problematic in non-Hodgkin's lymphoma (NHL) than in other B cell malignancies, we used a comprehensive PCR primer set and formulated stringent selection criteria to identify tumor-specific rearranged IgH genes. Rearranged IgH genes amplified from lymphoma DNA were considered to be of tumor origin if they were monoclonal, and if the same rearrangement was amplified with at least two independent VH-specific primers. From 11 of 13 (85%) intermediate- and high-grade malignant NHL, IgH rearrangements were isolated. Intraclonal IgH sequence heterogeneity was studied in four lymphomas, and detected in two of them. PCR using a lymphoma-specific primer followed by Southern hybridization of PCR product with a specific probe allowed detection of lymphoma DNA after 10,000-fold dilution. Circulating lymphoma cells were detected in patient blood and bone marrow samples which were negative by morphological and immunological criteria. Thus, also in intermediate- and high-grade malignant lymphoma, sensitive minimal disease detection using the rearranged IgH gene as a marker appears feasible.
重排的免疫球蛋白重链（IgH）基因可为B细胞提供独特的克隆标志物。由于通过聚合酶链反应（PCR）扩增重排基因并证明扩增序列确实源自肿瘤细胞在非霍奇金淋巴瘤（NHL）中比在其他B细胞恶性肿瘤中更具问题，我们使用了一套全面的PCR引物并制定了严格的选择标准来鉴定肿瘤特异性重排的IgH基因。如果从淋巴瘤DNA扩增的重排IgH基因是单克隆的，并且用至少两种独立的VH特异性引物扩增出相同的重排，则认为其源自肿瘤。从13例中高级恶性NHL中的11例（85%）分离出IgH重排。在4例淋巴瘤中研究了克隆内IgH序列异质性，其中2例检测到。使用淋巴瘤特异性引物进行PCR，然后用特异性探针进行PCR产物的Southern杂交，可在10000倍稀释后检测到淋巴瘤DNA。在形态学和免疫学标准为阴性的患者血液和骨髓样本中检测到循环淋巴瘤细胞。因此，在中高级恶性淋巴瘤中，使用重排的IgH基因作为标志物进行敏感的微小疾病检测似乎是可行的。
