Pott C, Tiemann M, Linke B, Ott M M, von Hofen M, Bolz I, Hiddemann W, Parwaresch R, Kneba M
Department of Hematology and Oncology, Georg-August-University, Göttingen, Germany.
Leukemia. 1998 Oct;12(10):1630-7. doi: 10.1038/sj.leu.2401172.
Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.
套细胞淋巴瘤是恶性非霍奇金淋巴瘤（NHL）中一种临床病理特征独特的实体，其特征是特定的染色体易位t(11;14)(q13;q32)，涉及细胞周期蛋白D1基因（也称为11号染色体上的bcl-1/PRAD1基因）和14号染色体上的重链免疫球蛋白连接区。我们建立了一种聚合酶链反应（PCR）方法，通过bcl-1特异性引物与通用免疫球蛋白JH引物相结合，从新鲜冷冻和石蜡包埋组织的DNA中扩增t(11;14)连接序列。对总共65例组织学分类为套细胞淋巴瘤（MCL）的病例进行分析，以检测t(11;14)易位和单克隆IgH-CDR3重排的存在。26例经典MCL患者和3例MCL间变性变体患者的新鲜冷冻活检材料可用于DNA提取。我们在29个样本中的12个（41%）中检测到bcl-;JH重排。在36例中，石蜡包埋的淋巴结组织是唯一的DNA来源。在该材料中，我们在31个DNA完整的样本中的6个（20%）中发现了bcl-1/JH重排。为了确认PCR的特异性，并确定bcl-1/JH连接区序列作为个体患者的克隆特异性标志物，我们通过直接PCR测序对16例病例的连接DNA序列进行了特征分析。有趣的是，我们发现6个bcl-1/JH连接在其N区域含有DH片段，这表明bcl-1/JH重排可发生在B细胞个体发育的后期，在此期间发生完整的VH到DH-JH连接或VH替代。为了研究IgH-CDR3作为那些难以扩增t(11;14)易位的MCL患者的敏感分子标志物的适用性，我们另外通过IgH-CDR3-PCR分析了60例病例中单克隆重排IgH基因的存在情况。在29个新鲜冷冻样本中的24个（79%）中可鉴定出单克隆IgH-CDR3 PCR产物，而在31个石蜡来源DNA样本中只有11个（36%）呈阳性。我们证明，单克隆IgH-CDR3 PCR产物的自动荧光检测能够快速、灵敏地监测微小残留病，即使在缺乏可PCR扩增的t(11;14)易位的病例中也是如此。与等位基因特异性引物相结合，该方法可能会改进目前在初始分期检测隐匿性MCL细胞以及治疗期间和治疗后检测残留病的实验方法。
