用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA
Summary
从肿瘤组织获得高质量的基因组 DNA 是利用下一代测序分析基因改变的重要第一步。本文提出一种简便、快速的方法来丰富肿瘤细胞, 从接触印记细胞学标本中获得完整的 DNA。
Abstract
在癌症患者的特定分子靶向药物的治疗前, 确定肿瘤的突变状态是至关重要的。在临床环境中, 福尔马林固定石蜡 (FFPE) 组织广泛用于基因检测。然而, FFPE DNA 在福尔马林固定过程中通常会受到损伤和分裂。因此, 由于 dna 质量和数量的降低, FFPE dna 有时不足以进行基因检测。在这里, 我们提出了触摸印记细胞学 (TIC) 的方法, 以获得肿瘤细胞的基因组 DNA, 可以在显微镜下观察到。细胞形态学和癌细胞数量可以用 TIC 标本来评估。此外, 从 TIC 样品中提取基因组 DNA 可以在两天内完成。利用该方法获得的 TIC dna 的总量和质量均高于 FFPE dna。这种快速简便的方法使研究人员能够获得高质量的基因检测 DNA (例如、下一代测序分析、数字 pcr 和定量实时 pcr), 并缩短报告结果的周转时间。
Introduction
下一代测序技术为研究人员在基因变异、孟德尔病、遗传性倾向和癌症方面的基因组信息分析提供了重要的进展1,2,3.癌症基因图谱 (TCGA) 和国际癌症基因组联合会 (ICGC) 一直在寻找几种常见癌症的基因改变的鉴定4。数以百计的基本癌症驱动基因已被成功地确定, 其中一些分子被瞄准药物开发1,5,6。
在临床环境中, FFPE 标本通常用于各种疾病, 包括癌症的病理诊断和分子检测。然而, 在福尔马林固定过程中, dna 蛋白或 dna dna 交联发生, 导致 dna 分裂。因此, FFPE dna 样本并不总是适合遗传分析, 因为质量和数量的 dna7,8,9。此外, 准备 FFPE 标本需要数天的时间, 技术技能是准确准备剖面的必要条件。因此, 建立一种简便、快速的获取高质量完整 DNA 的方法是可取的。
细胞学是病理诊断的一种替代方法。与 FFPE 准备10相比, 细胞学样品制备方法更简单、成本更低、更快速。采用 TIC 技术对乳腺癌患者的前哨淋巴结和边缘组织进行了一些年的术中快速诊断, 其方法为11,12。然而, 很少有报告研究了高质量的基因组 DNA 是否可以从 TIC 标本中提取, 并用于后续的遗传分析。细胞学标本通常染色阴道 (Pap) 或姬姆萨染色, 我们以前报告说, 从 TIC 标本 (特别是姬姆萨染色样品) 提取的 DNA 的数量和质量优于从 FFPE 获得的样本组织13。与 Pap 染色相比, 姬姆萨染色在要求较少染色的过程中具有优势。在 Pap 染色, 样品被固定和染色后, 必须安装在安装培养基 (例如, Malinol), 以区分样本内容, 如肿瘤细胞, 正常细胞和炎症细胞在显微镜下。如果在没有安装步骤的情况下制备 Pap 标本, 在显微镜下观察细胞几乎是不可能的, 因为试样是干的。相比之下, 姬姆萨染色可以观察到干燥状态, 因此, 安装步骤是不必要的快速细胞评估。对于显微切割, 姬姆萨染色更适合, 因为它需要干燥标本。
在本报告中, 我们介绍了一个简单快速的方法来制备 tic 标本与姬姆萨染色, 并表明 TIC 是一个更好的来源的 DNA 比较 FFPE 标本。
Protocol
1. 利用普通玻片进行快速显微评估的 TIC 准备 在临床病理组织材料可用后, 尽快完成 TIC 的准备工作。如果 TIC 标本不能立即准备好, 保持用盐水湿不育纱布覆盖的组织材料, 并贮存在冰箱中, 以防止组织的干燥。 准备5毫米3组织材料, 如实体肿瘤 (如, 肝, 肺和乳腺组织), 临床上通过手术或内窥镜获得。 如果组织表面有大量血液, 用无菌纱布涂上生理盐水并清除血?…
Representative Results
图 1显示了从准备 TIC 标本到 DNA 提取的整个过程。值得注意的是, 该手术仅需两天时间才能从 TIC 样品中获得基因组 DNA。我们评估了在滑动处理前肿瘤贮存的任何影响。我们发现, 当组织标本立即触及到幻灯片上时, 肿瘤细胞附着在玻璃滑梯上, 当组织被保存在生理盐水湿润的无菌纱布中时, 1 小时 (图 2)。然而, 当组织保持在室温…
Discussion
在本研究中, 我们提出了一种从临床病理标本中获取肿瘤 DNA 的替代方法。TIC 准备是非常简单的, 需要较少的时间与 FFPE 方法相比, 没有特殊仪器的要求10。从 TIC 准备到 DNA 提取的所有程序都可以在两天内完成 (图 1)。因此, 这种方法缩短了进行基因检测的周转时间。值得注意的是, 这为缩短分子分析所需的天数提供了重要的优势。这一短暂的周转时间使我们?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢医院和病人的所有医务和辅助人员同意参加。我们感谢加布里埃尔白狼, 博士, 从 Edanz 集团 (www.edanzediting.com/ac) 编辑本报告草稿。这项研究得到了来自山梨州 (Y.H. 和作案手法) 的基因组研究项目的资助, 以及 YASUDA 医学基金会 (Y.H.) 的赠款。
Materials
| FINE FROST white20 micro slide glass | Matsunami Glass ind, Ltd | SFF-011 | |
| Arcturus PEN Membrane Glass Slides | Thermo Fisher Scientific | LCM0522 | |
| Cyto Quick A solution | Muto Pure Chemicals | 20571 | |
| Cyto Quick B solution | Muto Pure Chemicals | 20581 | |
| May-Grunwald Solution | Muto Pure Chemicals | 15053 | |
| Giemsa solution | Muto Pure Chemicals | 15002 | |
| QIAamp DNA FFPE tissue kit | Qiagen | 56404 | |
| TaqMan Fast Advanced Master Mix | Thermo Fisher Scientific | 4444557 | |
| TaqMan RNase P Detection Reagents Kit | Thermo Fisher Scientific | 4316831 | |
| TaqMan Assay from FFPE DNA QC Assay v2 | Thermo Fisher Scientific | 4324034 | |
| MicroAmp Fast Optical 96-Well Reaction Plate | Thermo Fisher Scientific | 4346907 | |
| MicroAmp optical Adhesive Film | Thermo Fisher Scientific | 4311971 | |
| MicroMixer E36 | TITEC | 0027765-000 | |
| ViiA 7 Real-Time PCR System | Thermo Fisher Scientific | VIIA7-03 | |
| Himac CF16RXII | Hitachi-koki | CF16RII | |
| Ion Library TaqMan Quantitation Kit | Thermo Fisher Scientific | 4468802 | |
| Ion AmpliSeq Cancer Hotspot Panel v2 | Thermo Fisher Scientific | 4475346 | |
| Ion AmpliSeq Library Kit 2.0 | Thermo Fisher Scientific | 4480442 | |
| Ion Xpress Barcode Adapters 1-16 Kit | Thermo Fisher Scientific | 4471250 | |
| Ion PGM Hi-Q View Sequencing Kit (200 base) | Thermo Fisher Scientific | A30044 | |
| Ion Chef System | Thermo Fisher Scientific | 4484177 | |
| Veriti 96-well Thermal Cycler | Thermo Fisher Scientific | Veriti200 | |
| Ion 318 Chip Kit v2 BC | Thermo Fisher Scientific | 4488150 | |
| Ion PGM System | Thermo Fisher Scientific | PGM11-001 | |
| Ion PGM Wash 2 Bottle kit | Thermo Fisher Scientific | A25591 | |
| Agencourt™ AMPure™ XP Kit | Beckman Coulter | A63881 | |
| 16-position Magnetic Stand | Thermo Fisher Scientific | 4457858 | |
| Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube) | Thermo Fisher Scientific | AM12450 | |
| Nuclease-free water | Thermo Fisher Scientific | AM9938 | |
| MicroAmp™ Optical 96-well Reaction Plates | Thermo Fisher Scientific | 4306737 | |
| MicroAmp™ Clear Adhesive Film | Thermo Fisher Scientific | 4306311 | |
| Agencourt™ AMPure™ XP Kit | Beckman Coulter | A63881 | |
| Ethanol(99.5) | Nacalai Tesque | 08948-25 | |
| Sodium hydroxide (10M) | Sigma | 72068 | |
| DTU-Neo | TAITEC | 0063286-000 | |
| E-36 | TAITEC | 0027765-000 | |
| ECLIPSE Ci-L | Nikon | 704354 | |
| Pipet-Lite LTS Pipette L-2XLS+ | METTLER TOLEDO | 17014393 | |
| Pipet-Lite LTS Pipette L-10XLS+ | METTLER TOLEDO | 17014388 | |
| Pipet-Lite LTS Pipette L-20XLS+ | METTLER TOLEDO | 17014392 | |
| Pipet-Lite LTS Pipette L-100XLS+ | METTLER TOLEDO | 17014384 | |
| Pipet-Lite LTS Pipette L-200XLS+ | METTLER TOLEDO | 17014391 | |
| Pipet-Lite LTS Pipette L-1000XLS+ | METTLER TOLEDO | 17014382 | |
| petit-change | WAKEN | MODEL8864 | Mini centrifuge |
| petit-incubator | WAKEN | WKN-2290 | Air incubator |
| SensiCare Powder-free Nitrile Exam Gloves | MEDLINE | SEM486802 | |
| Sterile gauze | Osaki | 11138 | |
| Refrigerator MediCool | SANYO | MPR-312DCN-PJ | |
| FEATHER TRIMMING BLAD | FEATHER | No.130 | |
| FEATHER TRIMMING BLAD | FEATHER | No.260 | |
| FEATHER S | FEATHER | FA-10 | |
| Vortex Genius 3 | IKA | 41-0458 | Vortex mixer |
| Pincette | NATSUME | A-5 | |
| 1.5 mL microtube | BIOBIK | RC-0150 |
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Cite This Article
Amemiya, K., Hirotsu, Y., Oyama, T., Omata, M. Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology. J. Vis. Exp. (133), e56943, doi:10.3791/56943 (2018).