A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.
A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.
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通过单细胞和原位逆转录酶-聚合酶链反应测量，多发性骨髓瘤中循环 B 细胞与自体骨髓浆细胞共享克隆型 Ig 重链 VDJ 重排。
DOI：
10.1182/blood.v92.8.2844
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发表时间：
1998
期刊：
影响因子：
20.3
通讯作者：
L. Pilarski
中科院分区：
文献类型：
--
作者：
Agnieszka J. Szczepek;K. Seeberger;J. Wizniak;M. Mant;A. Belch;L. Pilarski
在多发性骨髓瘤 (MM) 中，MM 浆细胞表达的免疫球蛋白重链的 VDJ 重排提供了独特的克隆型标记。尽管在循环系统中发现了克隆型 MM 细胞，但它们的数量一直存在争议。我们的目标是使用单细胞检测为 18 名 MM 患者血液中克隆型细胞的频率提供直接证据，并确认其身份为 B 细胞。使用共有逆转录酶聚合酶链式反应 (RT-PCR)、亚克隆和测序从单个浆细胞中确定克隆型 Ig 重链 (IgH) VDJ。对于所有患者，使用患者特异性引物，从 10 个或更多个体浆细胞中扩增克隆型转录本。使用原位 RT-PCR，发现所有患者超过 80% 的浆细胞呈克隆型。采用 RT-PCR、单细胞 RT-PCR 和原位 RT-PCR 三种独立方法分析 MM 患者外周血单核细胞 (PBMC) 中的克隆型细胞。对从 MM 患者新鲜分离的 PBMC 进行有限稀释获得的单个细胞表达的 IgH 转录本的测序表明，所有 B 细胞都表达相同的 CDR3。这种克隆内同质性表明逃避了抗原选择，这是恶性 B 细胞的特征。对于该患者，克隆型 PBMC 的频率约为 25%，与 PBMC B 细胞的数量 (34%) 相当。由于 PBMC 包含少于 1% 的浆细胞，因此几乎所有克隆型 PBMC 都必须是 B 细胞。使用单细胞 RT-PCR，在血液中分选的单个 B 细胞中鉴定出克隆型 IgH 转录本。为了准确定量克隆型 B 细胞的数量，使用原位 RT-PCR 和患者特异性引物对来自 18 名 MM 患者（36 个样本）和 18 名健康供体（53 个样本）的分选 B 细胞进行了分析。在正常 B 细胞中检测不到克隆型转录本。对于 18 名 MM 患者，平均 66% +/- 4% (SE) 的血液 B 细胞是克隆型的（范围为 9% 至 95%），平均绝对数量为 0.15 +/- .02 x 10(9)/L 血液。随着时间的推移，传统化疗会暂时减少个别患者的循环克隆型 B 细胞。在一名患者的粒细胞集落刺激因子（G-CSF）动员血液中，它们的数量有所增加。然而，一名患者的克隆型 B 细胞在同种异体移植后检测不到，这与完全缓解相关。尽管可能对 MM 扩散和进展有贡献，但其恶性状态和影响尚未明确。它们在血液中的高频率以及它们对传统化疗的抵抗表明，应该对循环克隆型细胞的数量进行临床监测，并且针对这些 B 细胞的治疗目标可能有益于骨髓瘤患者。
In multiple myeloma (MM), the VDJ rearrangement of the immunoglobulin heavy chain expressed by MM plasma cells provides a unique clonotypic marker. Although clonotypic MM cells have been found in the circulation, their number has been controversial. Our objective was to provide direct evidence, using single-cell assays, for the frequency of clonotypic cells in blood of 18 MM patients, and to confirm their identity as B cells. The clonotypic Ig heavy-chain (IgH) VDJ was determined from single plasma cells using consensus reverse transcriptase-polymerase chain reaction (RT-PCR), subcloning, and sequencing. For all patients, using patient-specific primers, clonotypic transcripts were amplified from 10 or more individual plasma cells. Using in situ RT-PCR, for all patients greater than 80% of plasma cells were found to be clonotypic. Three separate methods, RT-PCR, single-cell RT-PCR, and in situ RT-PCR, were used to analyze clonotypic cells in peripheral blood mononuclear cells (PBMC) from MM patients. Sequencing of the IgH transcripts expressed by individual cells obtained by limiting dilution of freshly isolated PBMC from a MM patient showed that all B cells expressed an identical CDR3. This intraclonal homogeneity indicates an escape from antigenic-selection, characteristic of malignant B cells. For this patient, the frequency of clonotypic PBMC, about 25%, was comparable to the number of PBMC B cells (34%). Because the PBMC included less than 1% plasma cells, virtually all clonotypic PBMC must be B cells. Using single-cell RT-PCR, clonotypic IgH transcripts were identified in individual sorted B cells from blood. To accurately quantify the number of clonotypic B cells, sorted B cells derived from 18 MM patients (36 samples) and 18 healthy donors (53 samples) were analyzed using in situ RT-PCR with patient-specific primers. Clonotypic transcripts were not detectable among normal B cells. For the 18 MM patients, a mean of 66% +/- 4% (SE) of blood B cells were clonotypic (range, 9% to 95%), with mean absolute number of 0.15 +/- .02 x 10(9)/L blood. Over time in individual patients, conventional chemotherapy transiently decreased circulating clonotypic B cells. Their numbers were increased in granulocyte colony-stimulating factor (G-CSF)- mobilized blood of one patient. However, clonotypic B cells of a one patient became undetectable after allogeneic transplant, correlating with complete remission. Although contributions to MM spread and progression is likely, their malignant status and impact has yet to be clarified. Their high frequency in the blood, and their resistence to conventional chemotherapy suggests that the number of circulating clonotypic cells should be clinically monitored, and that therapeutic targeting of these B cells may benefit myeloma patients.
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使用单细胞聚合酶链反应分析人外周 B 细胞的重链库。
DOI：
--
发表时间：
1995
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Brezinschek,HP;Brezinschek,RI;Lipsky,PE
通讯作者：
Lipsky,PE
克隆循环细胞在浆细胞增殖性疾病中很常见：意义未明的单克隆丙种球蛋白病、冒烟型多发性骨髓瘤和活动性骨髓瘤的比较。
影响因子：
20.3
作者：
Billadeau,D;VanNess,B;Kimlinger,T;Kyle,RA;Therneau,TM;Greipp,PR;Witzig,TE
通讯作者：
Witzig,TE
影响因子：
20.3
作者：
B. Barlogie;J. Epstein;P. Selvanayagam;R. Alexanian
通讯作者：
B. Barlogie;J. Epstein;P. Selvanayagam;R. Alexanian
通过基于 PCR 的方法检测多发性骨髓瘤中的循环肿瘤细胞。
影响因子：
11.4
作者：
Corradini,P;Voena,C;Omede,P;Astolfi,M;Boccadoro,M;Dalla-Favera,R;Pileri,A
通讯作者：
Pileri,A
骨髓瘤的诊断和治疗进展。
影响因子：
3.6
作者：
Greipp,PR
通讯作者：
Greipp,PR