VDJ-SEQ：重排免疫球蛋白重链基因的深度测序分析，以揭示的B细胞淋巴瘤克隆演变模式
Summary
This protocol describes an approach to interrogate the recombined immunoglobulin heavy chain VDJ regions of lymphomas by deep-sequencing and retrieve VDJ rearrangement and somatic hypermutation status to delineate clonal architecture of individual tumor. Comparing clonal architecture between paired diagnosis and relapse samples reveals lymphoma relapse clonal evolution modes.
Abstract
了解肿瘤的克隆是了解参与肿瘤的发生和疾病进展的机制是至关重要的。此外，理解内肿瘤发生响应于特定微环境或治疗方法，所述组合物克隆变化可能会导致更复杂的和有效的方法的设计消除肿瘤细胞。然而，跟踪肿瘤克隆亚种群已具有挑战性，由于缺乏可区分的标记。为了解决这个问题，一个VDJ-SEQ协议创建通过利用VDJ重组和体细胞突变（SHM），B细胞淋巴瘤两个独特的功能，以跟踪弥漫性大B细胞淋巴瘤（DLBCL）复发的克隆演变图案。
在这个协议中，新一代测序（NGS）库，索引潜在的扩增重排的免疫球蛋白重链（IgH重）VDJ区，构建由对初步诊断和复发DLBCL SAMP的LES。每个样品平均超过五十万的VDJ序列测序，同时含有的VDJ重排和SHM信息后获得。此外，自定义生物信息学管道开发，以充分利用序列信息FR3区域，VDJ剧目这些样品中的表征。此外，该管道允许重建和个体肿瘤的克隆构，这使得肿瘤的诊断和演绎的诊断和复发性肿瘤对之间克隆演变模式内的克隆异质性的检查的比较。当应用这一分析的几个诊断复发对，我们发现关键证据多个独特的肿瘤进化模式可能会导致DLBCL复发。此外，这种方法可以扩展为其它临床方面，如识别微小残留病，监测复发进展和治疗反应和免疫repertoir的调查ES在非淋巴瘤上下文。
Introduction
癌症是一种克隆性疾病。自从三十年前，当彼得C.诺维尔提出了癌症克隆演化模型1，许多研究都试图肿瘤样本中剖析克隆群体和重建克隆扩增和发展模式，背后的肿瘤发生过程中2。最近，全基因组测序，使调查人员深吸看克隆异质性和演化3,4。然而，由于缺乏在许多细胞类型易于处理的标记物，它是很难推断精确的克隆构和进化路径。幸运的是在成熟B细胞，许多淋巴组织的恶性肿瘤，包括DLBCL，源于自然的克隆性标志。响应于抗原刺激，每个B细胞可以从这些区段的大池通过加入为V H（可变的），一个D（多样性）形成单个生产性的IgH VDJ序列和歼轰（接合）片段一起。在这个公关ocess，原始序列的小部分可以被删除和附加非模板的核苷酸可以添加到创建一个唯一的VDJ重排。这个特定的VDJ重排可在该B细胞的所有子代继承，因此标注个体成熟的B细胞和其后代5。此外，SHM发生在随后的生发中心（GC）的反应重组的VDJ序列引入另外的突变的抗体池的膨胀和抗体亲和6的提高。因此，通过比较和对比已经经过这些过程淋巴瘤样品的VDJ和SHM图案，肿瘤内的异质性，可以划定及疾病的克隆演变路径可以被推断出来。
先前，VDJ重排和SHM可以通过PCR鉴定扩增重组的区域，克隆PCR产物，并随后Sanger测序来获得序列信息。这种方法我的低通量和低产量，检索整个重组的VDJ剧目只有一个非常小的部分，并阻碍给定样品中的克隆群的整体表现的表征。一种改进的方法是由来自VDJ PCR产物生成索引NGS测序文库并进行PE 2×150碱基序列，获得半数以上一万元左右的样品重组VDJ序列创建。此外，定制的管道的开发是为了进行质量控制（QC），对齐，滤波器的VDJ测序读数，以确定每次读取的重排和SHMS，以及执行系统发育分析每个样品的克隆构。此外，一种新的方法已经建立，以进一步表征克隆演变模式在不同疾病阶段收集的样本。
我们应用这种技术来DLBCL患者样本。 DLBCL是频繁复发的非霍奇金淋巴瘤的最多一个第一种侵袭性IRD的患者7。 DLBCL复发通常发生早，在2至3年的初步诊断，但也有一些确实发生5年8之后。愈后复发的患者差，只有10％实现3年无进展生存期，由于有限的治疗选择。这是基础，迫切需要新的方法来治疗DLBCL复发9,10。然而，DLBCL复发相关的分子机制仍知之甚少。具体地，克隆的异质性，在诊断和克隆演变的DLBCL复发发育过程中的作用是当前未表征，因此很难确定准确的和有用的生物标志物来预测复发。为了解决这些问题，我们运用我们的VDJ测序的方法对多对匹配的初步诊断复发DLBCL样本对。复发的两个不同的克隆的进化方案从克隆构的诊断和复发SAMP之间的比较出现莱这表明多个分子机制可能参与了DLBCL复发。
Protocol
Representative Results
Discussion
因为几乎无限数量的通过的VDJ重排和SHM在人B细胞的IGH轨迹编码序列信息迭代的，检查整个IGH剧目由高通量深测序证明是划定克隆一种有效和全面的方式和子克隆B细胞群。此外，该策略可用于通过比较沿该疾病的不同阶段收集的患者样本的克隆和子克隆构研究B细胞肿瘤的发展，缓解，复发的克隆演变路径。虽然很容易在实验室环境中使用可商购的引物进行从初级样品DNA重排的VDJ序列的扩增，扩增?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Dr Rita Shaknovich and members of Elemento lab, Melnick lab, and Tam lab for thoughtful discussions. We would also like to thank the Genomics Resources Core Facility at Weill Cornell Medical College for performing the VDJ-sequencing. YJ was supported by ASH Scholar Award. WT and OE are supported by Weill Cornell Cancer Center Pilot Grant. OE is supported by the NSF CAREER award, the Starr Cancer Consortium and the Hirschl Trust. We would also like to thank Katherine Benesch, JD, MPH for her generous support to this project.
Materials
| Ethanol | VWR | 89125-170 | 200 proof, for molecular biology |
| TE buffer | Life Technologies | 12090-015 | 10 mM Tris·Cl, pH 8.0; 10mM EDTA |
| Xylenes | VWR | EM-XX0055-6 | 500 ml |
| Proteinase K | Life Technonogies | 25530-015 | 100 mg |
| Deoxynucleotide triphosphate (dNTP) Solution Mix | Promega | U1515 | 10 mM each nucleotide |
| 10x PCR Buffer | Roche | 11699105001 | Without MgCl2 |
| AmpliTaq Gold DNA Polymerase with Gold Buffer and MgCl2 | Life Technonogies | 4311806 | 50 µl at 5 U/µl |
| Specimen Control Size Ladder | Invivoscribe Technologies | 2-096-0020 | 33 reactions |
| IGH Somatic Hypermutation Assay v2.0 – Gel Detection | Invivoscribe Technologies | 5-101-0030 | 33 reactions, Mix 2 for IGVHFR1 detection |
| IGH Gene Clonality Assay – Gel Detection | Invivoscribe Technologies | 1-101-0020 | 33 reactions, Tube B for IGVHFR2 detection |
| GoTaq Flexi DNA Polymerase | Promega | M8291 | 20 µl at 5 U/µl |
| 10X Tris-Borate-EDTA (TBE) buffer | Corning (cellgro) | 46-011-CM | 6×1 L |
| 50X TAE BUFFER | VWR | 101414-298 | 1 L |
| Ethidium bromide solution | Sigma-Aldrich | E1510 | 10 mg/ml |
| 25 bp DNA Ladder | Life Technologies | 10597011 | 1 µg/µl |
| 100 bp DNA Ladder | Life Technologies | 15628-019 | 1 µg/µl |
| UltraPure Agarose | Life Technologies | 16500-500 | 500 g |
| 40% Acrylamide/Bis Solution | Bio-Rad Laboratories | 1610144 | 500 ml |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | 50 columns |
| Agencourt AMPure XP | Beckman Coulter | A63881 | |
| Qubit dsDNA High Sensitivity Assay Kit | Life Technologies | Q32854 | |
| High Sensitivity DNA Kit | Agilent Technologies | 5067-4626 | |
| 2100 Bioanalyzer | Agilent Technologies | ||
| PhiX Control v3 | Illumina | FC-110-3001 | |
| MiSeq | Illumina | ||
| Qubit 2.0 Fluorometer | Life Technologies | Q32872 | |
| Resuspension buffer (Illumina TruSeq DNA Sample Preparation Kit v2) | Illumina | FC-121-2001 | |
| End repair mix (Illumina TruSeq DNA Sample Preparation Kit v2) | Illumina | FC-121-2001 | |
| A-tailing mix (Illumina TruSeq DNA Sample Preparation Kit v2) | Illumina | FC-121-2001 | |
| Ligation mix (Illumina TruSeq DNA Sample Preparation Kit v2) | Illumina | FC-121-2001 | |
| DNA adaptor index (Illumina TruSeq DNA Sample Preparation Kit v2) | Illumina | FC-121-2001 | |
| Stop ligation buffer (Illumina TruSeq DNA Sample Preparation Kit v2) | Illumina | FC-121-2001 | |
| PCR primer cocktail (Illumina TruSeq DNA Sample Preparation Kit v2) | Illumina | FC-121-2001 | |
| PCR master mix (Illumina TruSeq DNA Sample Preparation Kit v2) | Illumina | FC-121-2001 | |
| Magnetic stand | Life Technologies | 4457858 | |
| Gel imaging system | Bio-Rad Laboratories | 170-8370 |
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