经典霍奇金淋巴瘤的芦苇细胞的流分选和外显子测序
1Innovation Laboratory, Center for Molecular Oncology,Memorial Sloan Kettering Cancer Center, 2Flow-Sorting Core Facility,Weill Cornell Medical College, 3Department of Laboratory Medicine,University of Washington, 4Department of Physiology and Biophysics, Institute for Computational Biomedicine,Weill Cornell Medical College, 5Department of Pathology and Laboratory Medicine,Weill Cornell Medical College, 6Department of Laboratory Medicine,Memorial Sloan Kettering Cancer Center
Summary
在这里，我们描述了一种组合流式细胞分选和低投入的下一代图书馆建设协议，旨在从古典霍奇金淋巴瘤（CHL）的霍奇金Reed-Sternberg（HRS）细胞产生高质量的全外显子数据。
Abstract
经典霍奇金淋巴瘤的霍奇金Reed-Sternberg细胞在炎性淋巴细胞的背景下稀疏分布，通常包含少于肿瘤块的1％。衍生自大块肿瘤的材料含有不足以表征的浓度的肿瘤含量。因此，使用八种抗体以及侧向和前向散射的荧光激活细胞分选在此被描述为一种快速分离和浓缩来自肿瘤的高纯度数千个HRS细胞的方法，用于随后的研究。同时，由于外源序列测序的标准协议通常需要100-1,000 ng的输入DNA，即使使用流分类方法也常常过高，因此我们还提供了优化的低输入库构建协议，能够生产高品质数据少于10 ng的输入DNA。这种组合能够生产适合于全e杂交捕获的下一代文库根据需要，xome诱饵或更专注于目标的面板。当与健康肿瘤T细胞或B细胞比较时，HRS细胞的外显子序列可以鉴定体细胞变异，包括突变，插入和缺失以及拷贝数变化。这些发现阐明HRS细胞的分子生物学，并可能揭示靶向药物治疗的途径。
Introduction
由于下一代测序，癌症基因组学的进展导致了治疗靶点的鉴定和许多血液学和非血液学肿瘤的预后的重大突破。基于特定基因组改变的新的个体化治疗策略正在迅速引入许多肿瘤类型（参考文献1,2 ）。尽管在淋巴瘤基因组学方面取得了显着的进展，经典霍奇金淋巴瘤（CHL）中肿瘤HRS细胞的基因组未被发现。研究受到反应性微环境中肿瘤性HRS细胞稀缺的阻碍，使得难以分离纯化的HRS细胞群体3 。
从原始肿瘤分离可行的HRS细胞的方法由Fromm 等人开发 如图4所示 ，该方法使用由CD30，CD15，CD40，CD95，CD45 CD20，CD5和CD64组成的八抗体混合物，从CHL肿瘤悬浮液中明确鉴定HRS细胞，使用这种方法，我们能够从由至少10 7个细胞（约10毫克组织）组成的肿瘤活组织检查的新鲜或冷冻细胞悬浮液中分离出至少1,000个可行的HRS细胞，通过流式细胞术分析纯度大于90％，估计为至少80％通过exome基因组分析连续十例。
我们已经完善了流式细胞分离技术，大大缓解了该过程，从而能够从原发性CHL肿瘤中快速分离成千上万个可行的HRS细胞。我们已经利用这种技术来产生被认为是霍奇金淋巴瘤初级病例中肿瘤细胞的第一个全部外显子序列。我们的研究证明了这一点高通量，全基因组研究个体CHL病例的可行性，已经导致鉴定新的基因组改变，具有解释CHL发病机理的潜力。
我们进一步开发了一条管道，以利用提取的DNA进行高通量基因组研究。为了从少至1,000个分选的HRS细胞（从顺序病例获得的最小值）获得可靠的结果，我们进一步开发了修改的下一代DNA文库构建程序8 ，其使得我们能够增加衔接子连接效率并产生DNA片段文库没有过多的放大。这种方法允许对常规临床样品的分析和复发性突变和染色体变异的检测7 。
Protocol
组织加工和冷冻收集磷酸盐缓冲盐水（PBS）或罗斯维尔公园纪念研究所培养基（RPMI）中的淋巴结组织，并收集24小时内的过程。将切除的淋巴结组织9转移到含有10mL具有2％胎牛血清（FCS）的RPMI的培养皿中，并用新鲜的手术刀刀片精细切碎。使用10 mL注射器柱塞的背面进一步研磨/解离组织。 将液体转移到一个50毫升锥形管通过一个100微米的细胞过滤器。用额外的10 mL…
Representative Results
在文库扩增和0.8倍珠粒清理后应采用生物分析仪。人们应该看到片段大小的“正常”分布在所需的范围内（ 图2a ）。与该形状的偏差，例如曲线中可见的“肩”表示存在高分子量或低分子量的假象。例如， 图2b -2d显示了包含可理想的未被排序的可见伪像的库的示例。如果图书馆受到严重影响，如果DNA可用和/或细?…
Discussion
掌握此技术后的未来应用或方向
该工作允许从含有至少10ng DNA的样品进行外显子测序。在临床情况下，由于材料不足，此限制不包括大多数细针抽吸样品，但包括足够的核心活检和切除活检样本。这将使得能够从更大的可能样本集中获取数据。
协议中的关键步骤
适当的冷冻和解离技术对于实验的成功至关?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
该项目方法的开发由威尔康奈尔医学院病理与实验室医学系资助。我们承认计算生物学和医学三部门培训计划的部分资助。我们要感谢与我们分享他们时间和知识的科学家，特别是玛丽亚·安德尔;丹·伯吉斯伊万卡·科扎雷瓦乍得Locklear以及来自威尔康奈尔医学院基因组学核心设施的所有人，包括张珍珍，肖波（肖恩）梁，董旭，魏章，惠民尚，塔蒂亚娜·贝森和托章。
Materials
| Petri or Cell Culture Dish (sterile) | |||
| RPMI-1640 Media | Roswell Park Memorial Institute | ||
| Fetal Calf Serum (FCS), (heat inactivated) | |||
| Freezing Media (RPMI, 20% FCS, 10% dimethylsulfoxide (DMSO))-make fresh and keep sterile | |||
| RPMI with 2% FCS (make fresh or store for up to 1 month) | |||
| scalpel with fresh blade | |||
| 10 ml syringe (no needle) | |||
| Cryogenic vials | |||
| 50 ml conical centrifuge tubes, force | |||
| Centrifuge | capable of handling 50 ml conical centrifuge tubes and providing 400g | ||
| Hepes buffer(1M, cell culture grade) | |||
| phosphate buffered saline (PBS) | |||
| Pluoronic-F68 | Thermo-Fisher | 24040-032 | |
| DNAase-I | Sigma-Aldrich, St. Louis, MO | D4527-10KU | store as 5mg/ml in RPMI in -200C |
| Bovine Serum Albumin (BSA) | |||
| Sort Media (PBS+2%BSA+25mM HEPES+ Pluoronic –F68 (1X)) | |||
| CD64-FITC (22) | Beckman Coulter, Miami, FL | 20 uL suggested starting volume; Titering is suggested | |
| CD30-PE (BerH83) | BD Biosciences, San Jose, CA | 20 uL suggested starting volume; Titering is suggested | |
| CD5-ECD (BL1a) | Beckman Coulter, Miami, FL | 10 uL suggested starting volume; Titering is suggested | |
| CD40-PerCP-eFluor 710 (1C10) | Ebiosciences, San Diego, CA | 5 uL suggested starting volume; Titering is suggested | |
| CD20-PC7 (B9E9) | Beckman Coulter, Miami, FL | 10 uL suggested starting volume; Titering is suggested | |
| CD15-APC (HI98) | BD Biosciences, San Jose, CA | 20 uL suggested starting volume; Titering is suggested | |
| CD45 APC-H7 (2D1) | BD Biosciences, San Jose, CA | Can be substituted with 10 uL suggested volume of CD45-Krome Orange (J.33, Beckman Coulter); Titering is suggested | |
| CD95-Pacific Blue (DX2) | Life Technologies, Grand Island, NY | 5 uL suggested starting volume; Titering is suggested | |
| CD2 (5 μg; clone RPA-2.10) | Biolegend, San Diego, CA | For optional protocol; Titering is suggested | |
| CD54 (10 μg; clone 84H10) | Serotec, Oxford, United Kingdom | For optional protocol; Titering is suggested | |
| CD58 (10 μg; clone TS2/9) | eBioscience, San Diego, CA | For optional protocol; Titering is suggested | |
| LFA-1 (12 μg; clone MHM23) | Novus Biologicals, Littleton, CO | For optional protocol; Titering is suggested | |
| BD CS&T Beads | BD Biosciences, San Jose, CA | ||
| BD Accudrop Beads | BD Biosciences, San Jose, CA | ||
| BC Versa Comp antibody capture beads | Beckman Coulter, Miami, FL | Compensation Beads | |
| BD-FACS ARIA special research order instrument using 5 lasers | BD Biosciences, San Jose, CA | any BD-FACS aria with capabilities to detect the fluorochromes in the antibody panel should be sufficient | |
| Wizard | Promega | A2360 | |
| 10 mM Tris-Cl buffer | NA | ||
| Qubit dsDNA HS Assay kit | Life Technologies, Carlsbad, CA | ||
| S2 Sonicator | Covaris, Woburn, MA | Alternatives may be substituted | |
| microTUBE | Covaris, Woburn, MA | ||
| Low-Throughput Library Preparation Kit | Kapa Biosystems, Wilmington, MA | KK8221 | |
| Sybr Green | Sigma-Aldrich, St. Louis, MO | S9430 | |
| Agencourt AMPure XP Beads | Beckman Coulter, Miami, FL | ||
| Bioanalyzer | Agilent Technologies, Santa Clara, CA | ||
| SeqCap EZ Exome v.3.0 | Roche Nimblegen | 6465684001 | |
| HiSeq | Illumina | ||
| TruSeq-style Universal adapter | Integrated DNA Technologies (IDT), Coralville, Iowa | HPLC purification; AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGAT*C*T | |
| TruSeq-style index adapter | Integrated DNA Technologies (IDT), Coralville, Iowa | HPLC purification; /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG | |
| TruSeq-style PCR primer 1 | Integrated DNA Technologies (IDT), Coralville, Iowa | AATGATACGGCGACCACCGAGA | |
| TruSeq-style PCR primer 2 | Integrated DNA Technologies (IDT), Coralville, Iowa | CAAGCAGAAGACGGCATACGAG | |
| Nuclease Free Duplex Buffer | Integrated DNA Technologies (IDT), Coralville, Iowa | ||
| BD FACSDIVA software | BD Biosciences, San Jose, CA | ||
| BD Falcon Tubes | BD Biosciences, San Jose, CA | ||
| BD Flow Tubes | BD Biosciences, San Jose, CA | ||
Riferimenti
- Abrams, J. National Cancer Institute’s Precision Medicine Initiatives for the new National Clinical Trials Network. American Society of Clinical Oncology educational book / ASCO. American Society of Clinical Oncology. Meeting. , 71-76 (2014).
- Gagan, J., Van Allen, E. M. Next-generation sequencing to guide cancer therapy. Genome medicine. 7, 80 (2015).
- Matsuki, E., Younes, A. Lymphomagenesis in Hodgkin lymphoma. Seminars in cancer biology. 34, 14-21 (2015).
- Fromm, J. R., Kussick, S. J., Wood, B. L. Identification and purification of classical Hodgkin cells from lymph nodes by flow cytometry and flow cytometric cell sorting. Am J Clin Pathol. 126 (5), 764-780 (2006).
- Fromm, J. R., Thomas, A., Wood, B. L. Flow cytometry can diagnose classical hodgkin lymphoma in lymph nodes with high sensitivity and specificity. Am J Clin Pathol. 131 (3), 322-332 (2009).
- Roshal, M., Wood, B. L., Fromm, J. R. Flow cytometric detection of the classical hodgkin lymphoma: clinical and research applications. Advances in hematology. 2011, 387034 (2011).
- Reichel, J., et al. Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells. Blood. 125 (7), 1061-1072 (2015).
- Kozarewa, I. A Modified Method for Whole Exome Resequencing from Minimal Amounts of Starting DNA. PLoS ONE. 7 (3), e32617 (2012).
- Brunicardi, F. C. . Schwartz’s Principles of Surgery. , (2014).
- Kantor, A. B., Roederer, M. FACS analysis of leukocytes. Handbook of Experimental Immunology. 2, (1996).
- Sanders, M. E. Molecular pathways of adhesion in spontaneous rosetting of T-lymphocytes to the Hodgkin’s cell line L428. Cancer Res. 48 (1), 37-40 (1988).
- Biosciences. . FACSAria II User’s Guide. Part No. 644832, Revision A. , (2009).
- . Qubit 3.0 Fluorometer, Catalog Number Q33216 Available from: https://www.thermofisher.com/order/catalog/product/Q33216 (2017)
- Roche Nimblegen. . SeqCap EZ Library SR User’s Guide ver. 4.1. , (2013).
- Li, H., Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 25 (14), 1754-1760 (2009).
- Li, H. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 25 (16), 2078-2079 (2009).
- Saunders, C. T. Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs. Bioinformatics. 28 (14), 1811-1817 (2012).
- Cingolani, P., et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 6 (2), 80-92 (2012).
- Dobin, A. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 29 (1), 15-21 (2013).
- Thorvaldsdottir, H., Robinson, J. T., Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Briefings in bioinformatics. 14 (2), 178-192 (2013).
- . DNA copy number data analysis. v.R package version 1.34.0 Available from: https://omictools.com/dnacopy-tool (2013)
Citazione di questo articolo
Reichel, J. B., McCormick, J., Fromm, J. R., Elemento, O., Cesarman, E., Roshal, M. Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma. J. Vis. Exp. (124), e54399, doi:10.3791/54399 (2017).