Kneba M, Eick S, Herbst H, Willigeroth S, Pott C, Bolz I, Bergholz M, Neumann C, Stein H, Krieger G
Department of Hematology, University Clinics, Göttingen, Germany.
Cancer Res. 1991 Jun 15;51(12):3243-50.
We have examined 165 unselected cases of non-Hodgkin's lymphomas for rearrangements involving the t(14;18) major breakpoint region using a polymerase chain reaction (PCR) and direct sequencing of amplified major breakpoint region bcl-2/JH junctional regions. The lymphomas, diagnosed according to the updated Kiel classification, consisted of 33 centroblastic-centrocytic, 37 centroblastic, 27 immunocytic, 10 immunoblastic, 10 centrocytic, 2 lymphoblastic, 2 Ki-1-positive anaplastic large cell, 14 peripheral T-cell, and 4 unclassified lymphomas. In addition 18 chronic lymphocytic leukemias, 2 hairy cell leukemias, and 6 plasmacytomas were studied. In 17 cases a bcl-2/JH gene fusion sequence was amplified by PCR. A bcl-2/JH gene fusion was detected only in three lymphoma subgroups: 13 of 33 centroblastic-centrocytic (39%), 2 of 37 centroblastic (6%), and 2 of 27 immunocytic (8%) were positive. In two cases, major breakpoint region bcl-2 rearrangements verified by genomic Southern analysis were not detected by PCR. Direct sequencing of all 17 PCR-amplified, previously uncharacterized t(14;18) junctional regions provided corroborating evidence for the specificity of the assay. The procedure gave sequencing results even from limited amounts of lymphoma cells as obtained by fine needle aspiration of lymph nodes or from clinically uninvolved sites. Clone-specific sequences were identified due to the involvement of different JH segments, the variations among the exact JH and bcl-2 breakpoint positions, and the extensive incorporation of junctional region (D-) N-nucleotides. These clone-specific sequences allow accurate identification of clinically occult lymphoma cells and reduce the threat of false positive results. The finding of exceptionally long intervening sequences in some of the junctions and the partial homology with published DH segments in three cases support the view that some of the putative N-regions harbor DH regions.
我们使用聚合酶链反应（PCR）及对扩增的主要断裂点区域bcl-2/JH连接区进行直接测序，检测了165例未经选择的非霍奇金淋巴瘤病例，以确定是否存在涉及t(14;18)主要断裂点区域的重排。这些淋巴瘤根据更新后的基尔分类法进行诊断，包括33例中心母细胞-中心细胞型、37例中心母细胞型、27例免疫细胞型、10例免疫母细胞型、10例中心细胞型、2例淋巴母细胞型、2例Ki-1阳性间变性大细胞型、14例外周T细胞型以及4例未分类淋巴瘤。此外，还研究了18例慢性淋巴细胞白血病、2例毛细胞白血病和6例浆细胞瘤。通过PCR扩增出17例bcl-2/JH基因融合序列。仅在三个淋巴瘤亚组中检测到bcl-2/JH基因融合：33例中心母细胞-中心细胞型中的13例（39%）、37例中心母细胞型中的2例（6%）以及27例免疫细胞型中的2例（8%）呈阳性。在两例中，通过基因组Southern分析证实的主要断裂点区域bcl-2重排未被PCR检测到。对所有17个PCR扩增的、先前未鉴定的t(14;18)连接区进行直接测序，为该检测方法的特异性提供了确证。即使是通过细针穿刺淋巴结获得的少量淋巴瘤细胞或来自临床未受累部位的细胞，该方法也能给出测序结果。由于不同JH片段的参与、JH和bcl-2确切断裂点位置的差异以及连接区（D-）N-核苷酸的广泛掺入，鉴定出了克隆特异性序列。这些克隆特异性序列能够准确鉴定临床隐匿的淋巴瘤细胞，并减少假阳性结果的风险。在一些连接区发现了异常长的间隔序列，且在三例中与已发表的DH片段存在部分同源性，这支持了一些假定的N区含有DH区的观点。
