通过有针对性的测序方法进行比较病变分析
Summary
本文介绍了一种识别特定患者不同标本之间的克隆和亚克隆变化的方法。虽然此处描述的实验侧重于特定的肿瘤类型，但该方法广泛适用于其他实体肿瘤。
Abstract
评估肿瘤内异质性（ITH）对于预测靶向疗法的失败并相应地设计有效的抗肿瘤策略至关重要。尽管由于样品处理和覆盖深度的差异，人们经常引起关注，但下一代实体肿瘤测序已经解开了肿瘤类型中高度可变的ITH程度。通过识别克隆和亚克隆种群来捕捉原发性病变和转移性病变之间的遗传关联性，对于设计早期疾病的治疗方法至关重要。在这里，我们报告一种比较病变分析的方法，该方法允许在同一患者的不同标本中识别克隆和亚克隆种群。本文描述的实验方法集成了三种成熟的方法：组织分析、高覆盖率多病变测序和免疫表位分析。为了尽量减少通过不适当的样品处理对亚克隆事件检测的影响，我们让组织进行仔细的病理检查和肿瘤细胞富集。然后，对肿瘤病变和正常组织进行质量控制的DNA，以409个相关癌症基因的编码区域为目标进行高覆盖率测序。虽然只看有限的基因组空间，我们的方法能够评估体细胞变化（单核苷酸突变和拷贝数变异）与给定患者不同病变的异质程度。通过对测序数据的比较分析，我们可以区分克隆与亚克隆的变化。大多数 ITH 通常归因于乘客突变;因此，我们还使用免疫组化学来预测突变的功能后果。虽然此协议已应用于特定的肿瘤类型，我们预计此处描述的方法广泛适用于其他实体肿瘤类型。
Introduction
下一代测序（NGS）的出现彻底改变了癌症的诊断和治疗方式。与多区域测序结合的NGS在实体肿瘤2中暴露了高度的肿瘤内异质性（ITH），这在一定程度上解释了靶向治疗的失败，因为存在具有不同药物敏感性的亚克隆2.全基因组测序研究提出的一个重要挑战是必须区分个别癌症的乘客（即中性）和驾驶员突变3。几项研究确实表明，在某些肿瘤中，乘客突变占ITH的大部分，而驾驶员的改变往往在同一个体的病变中保存。同样重要的是要注意，大突变负担（如肺癌和黑色素瘤所示）并不一定意味着一个大的亚克隆突变负担2。因此，在突变性低的肿瘤中可以发现高度的ITH。
转移导致全世界90%以上的癌症相关死亡5;因此，在原发性病变和转移性病变中捕捉驱动基因的突变异质性，对于设计治疗晚期疾病的有效疗法至关重要。临床测序一般对固定组织的核酸进行，由于DNA质量差，使得全基因组的探索变得困难。另一方面，临床测序的目的是识别可操作的突变和/或突变，这些突变可能预测对给定治疗方案的响应/反应能力。就目前情况而言，测序可以限制在基因组的较小部分，以便及时提取临床相关信息。从低通量DNA分析（例如，桑格测序）到NGS的转变使得在高深度下分析数百个与癌症相关的基因成为可能，从而能够检测亚克隆事件。在这里，我们报告一种比较病变分析的方法，该方法允许在同一个体的不同标本中识别克隆和亚克隆种群。此处描述的方法集成了三种成熟的方法（组织分析、高覆盖率多病变测序和免疫表位分析），以预测所识别变异的功能后果。该方法在图1中进行了原理图描述，并应用于胰腺5个固体伪皮毛肿瘤（SPN）转移病例的研究。虽然我们描述正式固定石蜡嵌入 （FFPE） 组织标本的处理和分析，但同样的过程可以应用于来自新鲜冷冻组织的遗传物质。
Protocol
研究中使用的材料是根据当地道德委员会批准的一项特定协议收集的。所有患者均可获得书面知情同意。 1. 组织标本的组织学和免疫本型修正 注：专家病理学家负责下面描述的活动。 根据既定的诊断标准对选定病例进行组织病理学修订。 使用微体从代表性的FFPE组织块中切割4⁄5μm厚的组织部分，并将部分安装在标准组织学幻灯片上。 …
Representative Results
研究工作流如图1所示。针对409个癌症相关基因的编码序列的5个SPN病例的多病变（n = 13）测序共确定了8个基因中的27个体细胞突变（CTNNB1，KDM6A，BAP1，TET1，SMAD4，TP53）， FLT1和FGFR3）。当突变在给定患者的所有病变之间共享时，突变被定义为创始人/克隆，当在给定患者的某些（但不是所有病变）中检测到进展者/子克隆…
Discussion
我们的方法通过整合垂直数据（即形态学、DNA测序和免疫组织化学）与特定患者的不同病变，识别实体肿瘤进展中涉及的分子变化。通过查询409个癌症相关基因8的编码序列，我们证明了我们的方法检测突变沉默肿瘤类型（即胰腺的SPN、固体伪皮毛肿瘤）的克隆和亚克隆事件的能力。此处使用的基于放大素的靶向测序方法的一个优点是覆盖均匀性（90% 的目标碱基覆盖 100 倍，95% ?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了意大利癌症基因组计划的支持。FIRB RBAP10AHJB），意大利协会，意大利皇家空军（AIRC;第12182号给AS，18178号给VC），FP7欧洲共同体赠款（Cam-Pac No 602783到AS）。供资机构在收集、分析和解释数据或编写手稿方面没有作用。
Materials
| 2100 Bioanalyzer Instrument | Agilent Technologies | G2939BA | Automated electrophoresis tool |
| Agencourt AMPure XP Kit | Fisher Scientific | NC9959336 | Beads technology for the purification of PCR products; beads-based purification reagent |
| Agilent High Sensitivity DNA Kit | Agilent Technologies | 5067-4627 | Library quantification |
| Anti-BAP1 | Santa Cruz Biotechnology | sc-28383 | Antibody |
| Anti-GLUT1 | Thermo Scientific | RB-9052 | Antibody |
| Anti-KDM6A | Cell Signaling | #33510 | Antibody |
| Anti-p53 | Novocastra | NCL-L-p53-DO7 | Antibody |
| Anti-βcatenin | Sigma-Aldrich | C7207 | Antibody |
| Blocking Solution | home made | – | 5 % Bovine serum albumin (BSA) in TBST |
| Endogenous peroxidases inactivation solution | home made | – | 3% H2O2 in Tris-buffered saline (TBS) 1x |
| Leica CV ultra | Leica | 70937891 | Entellan mountin media |
| Epitope Retrieval Solution 1 | Leica Biosystems | AR9961 | Citrate based pH 6.0 epitope retrieval solution |
| Epitope Retrieval Solution 2 | Leica Biosystems | AR9640 | EDTA based pH 9.0 epitope retrieval solution |
| Eppendorf 0.2 ml PCR Tubes, clear | Eppendorf | 951010006 | Tubes |
| Eppendorf DNA LoBind Tubes, 1.5 mL | Eppendorf | 22431021 | Tubes |
| Ethanol | DIAPATH | A0123 | IHC deparaffinization reagent |
| ImmEdge Pen Hydrophobic Barrier Pen | Vector Laboratories | H4000 | Hydrophobic Pen |
| ImmPACT DAB Peroxidase | Vector Laboratories | SK4105 | HRP substrate |
| ImmPRESS AntiRabbit Ig Reagent Peroxidase | Vector Laboratories | MP740150 | Secondary antibody |
| ImmPRESS AntiMouse Ig Reagent Peroxidase | Vector Laboratories | MP740250 | Secondary antibody |
| Integrative Genomics Viewer (IGV) | Broad Institute | – | https://software.broadinstitute.org/software/igv/home |
| Ion AmpliSeq Comprehensive Cancer Panel | Thermofisher Scientific | 4477685 | Multiplexed target selection of 409 cancer related gene. https://assets.thermofisher.com/TFS-Assets/CSD/Reference-Materials/ion-ampliseq-cancer-panel-gene-list.pdf |
| Ion AmpliSeq Library Kit 2.0 | Thermofisher Scientific | 4480441 | Preparation of amplicon libraries using Ion AmpliSeq panels |
| Ion Chef Instrument | Thermofisher Scientific | 4484177 | Automated library preparation, template preparation and chip loading |
| Ion PI Chip Kit v3 or Ion 540 Chip | Thermofisher Scientific | A26771 or A27766 | Barcoded chips for sequencing |
| Ion PI Hi-Q Chef Kit or Ion 540 Kit-Chef | Thermofisher Scientific | A27198 or A30011 | Template preparation |
| Ion PI Hi-Q Sequencing 200 Kit or Ion S5 Sequencing Kit | Thermofisher Scientific | A26433 or A30011 | Sequencing |
| Ion Proton or Ion GeneStudio S5 System | Thermofisher Scientific | 4476610 or A38196 | Sequencing system |
| Ion Reporter Software – AmpliSeq Comprehensive Cancer Panel tumour-normal pair | Thermofisher Scientific | 4487118 | Workflow |
| Ion Reporter Software – uploader plugin | Thermofisher Scientific | 4487118 | Data analysis tool |
| Ion Torrent Suite Software – Coverege Analysis plugin | Thermofisher Scientific | 4483643 | Plugin that describe the level of sequance coverage produced |
| Ion Torrent Suite Software – Variant Caller plugin | Thermofisher Scientific | 4483643 | Plugin able to identify single-nucleotide polymorphisms (SNPs), insertions and deletions in a sample across a reference |
| Ion Xpress Barcode Adapters 1-96 Kit | Thermofisher Scientific | 4474517 | Unique barcode adapters |
| NanoDrop 2000/2000c Spectrophotometers | Thermofisher Scientific | ND-2000 | DNA purity detection |
| NCBI reference sequence (RefSeq) database | NCBI | – | https://www.ncbi.nlm.nih.gov/refseq/ |
| Platinum PCR SuperMix High Fidelity | Fisher Scientific | 12532-016 or 12532-024 | SuperMix for PCR amplification; high-fidelity PCR mix |
| QIAamp DNA Blood Mini Kit | Quiagen | 51106 0r 51104 | DNA blood extraction kit |
| QIAamp DNA FFPE Tissue | Quiagen | 56404 | DNA FFPE tissue extraction kit |
| Qubit 2.0 Fluorometer | Thermofisher Scientific | Q32866 | DNA quantification |
| Qubit dsDNA BR Assay Kit | Thermofisher Scientific | Q32850 | Kit for DNA quantification on Qubit 2.0 Fluorometer |
| TBST | home made | – | Tris-buffered saline (TBS) and 0.1% of Tween 20 |
| Tissue-Tek Prisma Plus & Tissue-Tek Film | Sakura Europe | 6172 | Automated tissue slide stainer instrument |
| Variant Effect Predictor (VEP) software | EMBI-EBI | – | http://grch37.ensembl.org/Homo_sapiens /Tools/VEP |
| Xilene, mix of isomeres | Carlo Erba | 492306 | IHC deparaffinization reagent |
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Tags
Targeted SequencingGenetic RelatednessMolecular AlterationsDisease ProgressionIntratumor HeterogeneityAnti-tumor StrategiesSolid Tumor TypesSequencing RunIon Proton SystemIon Gene Studio S5 SystemIon AmpliSeq 2.0 Library KitIon ChefCoverage Analysis PluginGRCH37HG19 GenomeBarcode SetClonal AmplificationNext Generation Sequencing
Citazione di questo articolo
Vicentini, C., Mafficini, A., Simbolo, M., Fassan, M., Delfino, P., Lawlor, R. T., Rusev, B., Scarpa, A., Corbo, V. Comparative Lesions Analysis Through a Targeted Sequencing Approach. J. Vis. Exp. (153), e59844, doi:10.3791/59844 (2019).