治疗研究

肿瘤免疫微环境研究之——肿瘤抗原发现对外显子测序的要求

分析背景

其明公司长达1年的准备,开发出了面向肿瘤免疫微环境的肿瘤抗原MHC复合物产品。由于相关产品上千种,究竟选择哪种肿瘤抗原作为淋巴细胞的激活物和流式分选四聚体呢?其中关键点是肿瘤抗原的检测平台——肿瘤全基因外显子测序的技术平台选择问题。为了解答这一问题,我们整理了从2013年至2020年以来顶级期刊【平均影响因子30分以上】发表的肿瘤抗原相关外显子测序深度信息、是否需要配对样本的问题。 由于其明公司的GCBI-MAS系统已完成25万份肿瘤外显子测序分析,该系统覆盖肿瘤抗原+HLA结合模式分析。经过十万例以上数据验证,我们推荐如下论文所代表的分析方法。

代表性论文

mRNA vaccine–induced neoantigen-specific T cell immunity in patients with gastrointestinal cancer. 2020年JCI

代表性段落“tumor and normal coverage of 10 or greater, a tumor variant read count of 4 or higher, a tumor variant frequency of 7% or higher, and 2 or more callers calling the variant”, 也就是肿瘤和正常样本大于10层即可,需配对血样,作为胚系变异的参比,用于发现真正的体细胞突变。

Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes. 2020 Nature Communication

代表性段落“At least 70x coverage was required for the melanoma cell lines and PBMCs/TILs. For tumor/normal lung tissues, at least 100x coverage was required". 细胞系测序为70层覆盖,肿瘤和正常组织测序为100层覆盖。

Neoantigen identification strategies enable personalized immunotherapy in refractory solid tumors. 2019年JCI

代表性段落“In brief, after sample preparation, DNA extraction, and library preparation, the enriched libraries were sequenced on HiSeq 4000 NGS platforms (Illumina) with coverage depths of at least 100×, 300×, and 3000× after removal of PCR duplicates for blood, FFPE/pleural effusion, and ctDNA, respectively. ” 血液标本100层,冰冻肿瘤组织标本300层,ctDNA3000层,需配对血样,作为胚系变异的参比,用于发现真正的体细胞突变。

Sensitive Detection and Analysis of Neoantigen-Specific T Cell Populations from Tumors and Blood. 2019年Cell Rep

代表性段落“Paired-end 2×100bp sequencing was carried out on the HiSeq 2000 platform (Illumina, San Diego, CA) following exon capture using the Nimblegen SeqCap EZ Human Exome Library v3.0 (Roche), which targets 65 Mb of genome. Each targeted base was covered by an average of 90–150 reads."  肿瘤样本每个碱基150层,需配对血样,作为胚系变异的参比,用于发现真正的体细胞突变。

Identification of unique neoantigen qualities in long term pancreatic cancer survivors. 2017年Nature

代表性段落 “ Paired End 100/100, using the TruSeq SBS Kit v3 (Illumina) with a target coverage of 150X for tumor samples and 70X for matched normal (MSKCC Center for Molecular Oncology).” 正常样本70层,肿瘤样本150层。

Immunogenicity of somatic mutations in human gastrointestinal cancers. 2015年Science

代表性段落 “ For patient 3971, whole-genome sequencing (WGS) was performed using the Illumina HiSeq 2000 with an average depth of 39.19 for the normal sample and 46.53 for the tumor sample. ” 正常样本40层,肿瘤样本45层。

Mining Exomic Sequencing Data to Identify Mutated Antigens Recognized by Adoptively Transferred Tumor-reactive T cells. 2013年Nat Med

代表性段落 “ Over 43 million bases of target DNA were analyzed in the tumor and normal samples, and an average of 42 to 51 reads were obtained at each base in the normal and tumor DNA samples.” 也就是说肿瘤组织测50层左右,需配对血样,作为胚系变异的参比,用于发现真正的体细胞突变。

总结分析

在肿瘤免疫微环境的肿瘤抗原分析部分,为了发现肿瘤抗原而进行的外显子测序,必须要同时检测肿瘤组织和正常组织,正常组织也可以用外周血样本替代。其目的是用正常或外周血组织作为胚系变异的参比,这些胚系变异将从肿瘤变异位点列表中剔除。

关于测序深度的问题,不同的文章答案不一,其主要决定因素是测序成本问题。如果使用Illumina Hiseq2000测序仪,肿瘤标本测序深度普遍不会超过150层。若使用更高通量的Hiseq4000甚至Xten、Novoseq测序仪,或者BGI测序仪,肿瘤标本的测序深度将超过300层。总之,相同的标本,随着测序仪的测序通量越大,测序单价越低的情况下,测序深度越深。

未尽事宜

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本文由 GCBI学院 作者:其明技术专家 发表,转载请注明来源!

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