多位点取样探索肿瘤异质性的研究集锦

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2012-新英格兰-4个ccRCC病人的26个肿瘤部位

Endesfelder, D., Math, D., Gronroos, E., Ph, D., Martinez, P., Ph, D., … Ph, D. (2012). Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing. New England Journal of Medicine.
只有4个ccRCC病人，26个肿瘤组织测序，平均测序深度74而已，是clear cell renal cell carcinoma，取样如下：
image-20190610224408114
说明了Branched tumor evolution，那些trunk突变更趋向于是标志物或者治疗靶点。
image-20190610224444953
Genomics analyses from single tumor-biopsy specimens may underestimate the mutational burden of heterogeneous tumors. Reconstructing tumor clonal architectures and the identification of common mutations located in the trunk of the phylogenetic tree may contribute to more robust biomarkers and therapeutic approaches.

2014-science-7个NSCLC病人的25个部位

Voss, F. K., Ullrich, F., Münch, J., Lazarow, K., Lutter, D., Andrade-navarro, M. a, … Thomas, J. (2014). Spatial and temporal diversity in genomic instability processes defines lung cancer evolution. Science, 251(October). https://doi.org/10.1126/science.1252826
研究癌症是：non–small cell lung cancer (NSCLC) 实验测序策略是：25 spatially distinct regions from 7 operable NSCLCs and found evidence of branched evolution，如下图所示：
Total number of nonsilent mutations is provided below each tumor with percentage of heterogeneous mutations in brackets.
可以看到肿瘤异质性范围很大。
image-20190610224631790
重要集中在对抽烟与否分组的病人进行不同的比较！
A large fraction of the genome had undergone alterations in all tumors, and genomic profiles were more similar within tumors than between different tumors

2014-science-11个LUAD病人的48个肿瘤部位

Bell, D. W., Settleman, J., Haber, D. A., Nussenzweig, A., Leibowitz, M. L., Pellman, D., … Human, N. (2014). Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing. Science.
研究策略是：多位点取样的全外显子测序，48 tumor regions from 11 resected lung adenocarcinomas
同样是构建不同肿瘤部位的进化关系树，发现On average, 76% of all mutations were detected in all regions of the same tumors. 也就是说 肿瘤异质性很小。
image-20190610230136069
跟2012的新英格兰发表的关于ccRCC研究不同的是，本研究说明了对肿瘤病人的单个部位采样是足够检查到大部分重要突变的。因为 ITH patterns may be different between cancer types. 需要更大样本量的临床队列来证实不同癌症不同亚型病人 异质性到底处于什么范围。
而且提高测序深度，肿瘤异质性会降低。
image-20190610230248380

2015-oncogenesis-2个ESCC病人的11个肿瘤部位

Cao, W., Wu, W., Yan, M., Tian, F., Ma, C., Zhang, Q., … Biddle, F. G. (2015). Multiple region whole-exome sequencing reveals dramatically evolving intratumor genomic heterogeneity in esophageal squamous cell carcinoma. Oncogenesis, 4(11). https://doi.org/10.1038/oncsis.2015.34
研究策略：使用WES和(aCGH),技术对2个ESCC病人的11个肿瘤部位进行探索，其中芯片数据在GEO数据库，但是测序数据是在EGA。
同样说明是branching进化模型，但是ITH高达90%，其它癌症普遍都是30%~60%的异质性。
image-20190610230642892
测序深度有点小：average target exome coverage of 50× in neoplastic DNA and 60 × in reference tissue using 76- bp paired-end reads
因为病人数量少，所以每个病人的受突变影响的基因集拿去GO/KEGG功能富集注释，
从SNV和CNV角度来量化，结果两个病人的：
In PtA, we classified 158 non-silent mutated genes into 17 trunk gene mutations, 54 branch gene mutations and 87 private mutations.
In PtB, 27 of 203 mutant genes were in the trunk, 76 mutant genes were located in the branch section and 100 genes were private mutations.

2015-cancer-Discovery-8个EAC患者的40个肿瘤部位

Murugaesu, N., Wilson, G. A., Birkbak, N. J., Watkins, T. B. K., McGranahan, N., Kumar, S., … Swanton, C. (2015). Tracking the genomic evolution of esophageal adenocarcinoma through neoadjuvant chemotherapy. Cancer Discovery, 5(8), 821–832. https://doi.org/10.1158/2159-8290.CD-15-0412
实验策略是：40 tumor regions from 8 EAC患者 ， before and after platinum-containing NAC.
image-20190610231024443
用到了 intratumor heterogeneity (ITH) index.这个概念，而且看到了它跟NAC疗效显著相关。
也是和公共数据集比较 ：EAC tumors from the Cancer Genome Atlas (TCGA) dataset

2015-nature-genetics-15个CRC病人的349个肿瘤样品

Sottoriva, A., Kang, H., Ma, Z., Graham, T. A., Salomon, M. P., Zhao, J., … Curtis, C. (2015). A Big Bang model of human colorectal tumor growth. Nature Genetics, 47(3), 209–216. https://doi.org/10.1038/ng.3214
研究策略是： 349 individual glands from 15 colorectal tumors，采样区分左右肿瘤部分，每个部分十多个样品测序，最后说明是大爆炸的进化模型。
image-20190610231422966
包括WES, whole-exome sequencing; TS, targeted sequencing; 两个技术。
most detectable ITH occurs early after the transition to an advanced tumor.
Although private alterations continuously occur, only those that occur early have time for the corresponding clone to expand to a detectable size.
这个大爆炸模型的临床意义
image-20190610231621805

2016-nature-genetics-13个ESCC病人的51个肿瘤样品

Hao, J. J., Lin, D. C., Dinh, H. Q., Mayakonda, A., Jiang, Y. Y., Chang, C., … Koeffler, H. P. (2016). Spatial intratumoral heterogeneity and temporal clonal evolution in esophageal squamous cell carcinoma. Nature Genetics, 48(12), 1500–1507. https://doi.org/10.1038/ng.3683
测序策略：13个ESCC病人的51个部位的WES数据，然后其中3个病人还是有了甲基化芯片。
image-20190610231802248
平均是35.8%的异质性，跟前面的2个ESCC病人的11个肿瘤部位进行探索的研究的90%异质性相反，作者推测是测序深度的原因。
然后可以看到驱动突变的异质性要低。

2016-Molecular Oncology-3个膀胱癌转移患者的22个部位

Thomsen, M. B. H., Nordentoft, I., Lamy, P., Høyer, S., Vang, S., Hedegaard, J., … Dyrskjøt, L. (2016). Spatial and temporal clonal evolution during development of metastatic urothelial carcinoma. Molecular Oncology, 10(9), 1450–1460. https://doi.org/10.1016/j.molonc.2016.08.003
平均测序深度对肿瘤硬盘是68X (range 27-215X) ，对正常对照组织是167X (95-301X)，部分突变位点，做了超过3000的高深度测序验证。
image-20190610232050520
因为样本量很小，所以作者重点放在了药物基因数据库，发现大部分可能的药物靶点突变都发生在branch，而不是trunk。

2016-Cancer-Research-8个皮肤癌的41个肿瘤部位

Harbst, K., Lauss, M., Cirenajwis, H., Isaksson, K., Rosengren, F., Törngren, T., … Jönsson, G. (2016). Multiregion whole-exome sequencing uncovers the genetic evolution and mutational heterogeneity of early-stage metastatic melanoma. Cancer Research, 76(16), 4765–4774. https://doi.org/10.1158/0008-5472.CAN-15-3476
发现3~38%的异质性，如下所示：
image-20190610232319238
trunk和branch突变的突变特征比率有变化，就是突变频谱
不同部位的肿瘤的VAF的相关性散点图，还有DNA和RNA的VAF的散点图。
不同组学数据的肿瘤异质性并不一致。

2016-PLoS Genetics-9个CRC病人的75个肿瘤部位

Uchi, R., Takahashi, Y., Niida, A., Shimamura, T., Hirata, H., Sugimachi, K., … Mimori, K. (2016). Integrated Multiregional Analysis Proposing a New Model of Colorectal Cancer Evolution. PLoS Genetics, 12(2), 1–24. https://doi.org/10.1371/journal.pgen.1005778
做WES和甲基化芯片，如下：
image-20190610232524507
During this process generating ITH, mutation accumulations, CN alterations, and methylation alterations appeared to occur in a correlated manner.
提出中性进化模型。
image-20190610232632953

2017新英格兰杂志的TRACERx计划关于NSCLC的研究

Jamal-Hanjani, M., Wilson, G. A., McGranahan, N., Birkbak, N. J., Watkins, T. B. K., Veeriah, S., … Swanton, C. (2017). Tracking the Evolution of Non–Small-Cell Lung Cancer. New England Journal of Medicine, 376(22), 2109–2121. https://doi.org/10.1056/NEJMoa1616288
队列：100 early-stage NSCLC 病人（62 Men, 38 Women）的 327个肿瘤样品进行外显子测序，平均测序深度超400X
然后发现SNV的异质性比例是30% (range, 0.5 to 93) ，变化范围很大，生存分析不显著。
而CNV的异质性比例是8% (range, 0.3 to 88) ，生存分析显著。
image-20190610232941577

然后795个驱动突变事件里面，219个都是亚克隆的，多位点取样测序能显著提高发现驱动事件的比例。

2017-BMC Medical Genomics-1个61岁男性胃癌患者的6个肿瘤样品

Zhou, Z., Wu, S., Lai, J., Shi, Y., Qiu, C., Chen, Z., … Chen, S. (2017). Identification of trunk mutations in gastric carcinoma: A case study. BMC Medical Genomics, 10(1), 1–8. https://doi.org/10.1186/s12920-017-0285-y
首先指出和肯定ITH在精准医疗时代的重要性，所以测了1个61岁男性胃癌患者的6个肿瘤样品的全外显子，发现382个ns点突变，其中：

  • 35 trunk mutations (54.97%, 210/382)
  • 17 branch mutations (16.84%, 64/382),
  • 108 private mutations (28.27%, 108/382).
    image-20190610233140367
    最后的结论是trunk的VAF比branch高
    值得一提的是作者下载了2014发表在science的LUAD的多位点取样原始数据走他自己的数据分析流程，个人觉得，纯粹是因为自己的数据太少了。

    2017- Journal of Hepatology-5个HCC患者的32个肿瘤部位

    Huang, A., Zhao, X., Yang, X. R., Li, F. Q., Zhou, X. L., Wu, K., … Zhou, J. (2017). Circumventing intratumoral heterogeneity to identify potential therapeutic targets in hepatocellular carcinoma. Journal of Hepatology, 67(2), 293–301. https://doi.org/10.1016/j.jhep.2017.03.005
    包括Whole exome sequencing (WES) and targeted deep sequencing (TDS) were 两个测序技术，异质性如下：
    image-20190610233424133
    最后文章的亮点可能是：Circulating cell-free DNAs (cfDNAs)
    image-20190610233623249
    外显子实验的平均测序深度超200，这5个病人共1220个体细胞突变，涉及581个基因，比较了Whole exome sequencing (WES) and targeted deep sequencing (TDS)技术的差异。
    image-20190611100323631

    2017-Annals of Oncology-4个CRC病人的28个肿瘤部位

    Wei, Q., Ye, Z., Zhong, X., Li, L., Wang, C., Myers, R. E., … Yang, H. (2017). Multiregion whole-exome sequencing of matched primary and metastatic tumors revealed genomic heterogeneity and suggested polyclonal seeding in colorectal cancer metastasis. Annals of Oncology, 28(9), 2135–2141. https://doi.org/10.1093/annonc/mdx278
    对原位癌症和转移癌症部分都取样测序，进化树如下：
    image-20190610233821660
    说明了CRC的转移是polyclonal seeding机制，

    2017-BMC Cancer-2个肾癌患者的9个肿瘤部位

    Liu, M., Liu, Y., Di, J., Su, Z., Yang, H., Jiang, B., … Su, X. (2017). Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer. BMC Cancer, 17(1), 1–11. https://doi.org/10.1186/s12885-017-3777-4
    测序策略：9 tumor regions and 88 single cells from 2 rectal cancer patients，重点应该是单细胞基因组测序看拷贝数变异。
    image-20190610234035879
    从 single nucleotide variations (SNVs) and somatic copy number alterations (SCNAs) 角度来看异质性
    然后单细胞基因组测序用的是multiple an- nealing and looping-based amplification cycles (MALBAC)。

  • a Clustered heatmap of 24 single tumor cells with SCNA profiles in patient 1 based on Euclidean distance and ward.D method.
  • b Clustered heat map and PCA of 35 single tumor cells of patient 2 based on SCNA profiles. Single tumor cells were grouped into two clusters.
  • c Subclonal SCNAs of patients 1 and 2 divided single tumor cells into two subpopulations, which was in accordance with two clusters identified by PCA. The
    image-20190610234204732
    因为只有两个病人，所以肯定会细致的比较两个病人的区别。

    2017-Oncotarget-两个MANEC病人的多位点

    两个病人分开分析，因为样本量太小，所以非常细致的探索了它们的方方面面，外显子测序平均测序深度高达350，而且还针对目标突变进行高深度的捕获测序，大于1000X的，拷贝数变异的量化使用的是Illumina Human
    OmniZhongHua-8 BeadChips。 测序数据都是可以下载的SRP079168
    取样如下：
    image-20190611100459382
    突变比较如下：
    image-20190611100530676
    绘制进化关系图如下：
    image-20190611100547099

    2018-NC-16个LUAD病人的79个肿瘤区域

    Nahar, R., Zhai, W., Zhang, T., Takano, A., Khng, A. J., Lee, Y. Y., … Tan, D. S. W. (2018). Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing. Nature Communications, 9(1). https://doi.org/10.1038/s41467-017-02584-z
    LUAD的多位点取样早在2014的science文章就发表了，而且2017的新英格兰关于TRACERx计划也涉及到了LUAD。 所以本文的重点是Asian EGFR-mutant LUAD
    纳入16个病人的79个肿瘤区域样品，分析肿瘤异质性高达60%，远超于之前2014科学杂志的研究报道的30%。
    image-20190611101034217
    还比较了吸烟和不吸烟的LUAD病人的肿瘤进化区别：
    image-20190611101219961

    2018-Journal of Hepatology-6个ICC病人的69个PDPC部位

    前面已经有发表在2017- Journal of Hepatology-的5个HCC患者的32个肿瘤部位的研究，本研究侧重于ICC，取PDPCs: patient-derived primary cancer cells; 进行 WES 测序。病人来源于复旦大学中山医院。
    结果 肿瘤异质性高达60%，而且超过85%的驱动突变都是branch的.
    image-20190611101650668

    2018-89个ccRCC患者的178个肿瘤样品

    Moore, A. L., Kuipers, J., Singer, J., Burcklen, E., & Schraml, P. (2018). Intra-tumor heterogeneity and clonal exclusivity in renal cell carcinoma. BioRxiv, 1–43. https://doi.org/10.1101/305623
    测序策略：89个ccRCC患者的178个肿瘤样品测序。
    第一期纳入16个病人，每个病人取2个肿瘤样品进行全外显子测序和转录组测序，近40%的异质性。
    第二期对全部的89个病人，捕获826个基因进行高深度测序。
    image-20190611103637788
    文章提到了一个 GeneAccord 的算法可能有帮助。
    image-20190611103700894

    2018-NC-10个早期肠癌患者的53个肿瘤部位

    Saito, T., Niida, A., Uchi, R., Hirata, H., Komatsu, H., Sakimura, S., … Mimori, K. (2018). A temporal shift of the evolutionary principle shaping intratumor heterogeneity in colorectal cancer. Nature Communications, 9(1), 1–11. https://doi.org/10.1038/s41467-018-05226-0
    作者在2016有ACRC的队列的多位点看异质性的研究，这次纳入10个早期肠癌患者的53个肿瘤部位进行外显子测序，命名为PCRCs队列继续看肿瘤异质性。
    A是advance，P是precancerous lesions
    两个队列区别很大：
    image-20190611104428036
    提出CRC的进化模型：
    image-20190611104509082

    2018-CCR-11个TNBC病人的78个肿瘤样品

    Barry, P., Vatsiou, A., Spiteri, I., Nichol, D., Cresswell, G. D., Acar, A., … Sottoriva, A. (2018). The spatiotemporal evolution of lymph node spread in early breast cancer. Clinical Cancer Research, 24(19), 4763–4770. https://doi.org/10.1158/1078-0432.CCR-17-3374
    对11个TNBC病人取样78进行肿瘤测序，包括原位癌和淋巴结转移的样品，如下：
    image-20190611104712933
    3种测序手段都用上了：

  • whole-exome sequencing (WES),
  • whole-genome sequencing (WGS),
  • targeted deep sequencing
    高达75%的异质性，如下：
    image-20190611104751754
    同样的也纳入了ctDNA相关数据

    2019-NC-39个ESCC患者的185个肿瘤样品

    Yan, T., Cui, H., Zhou, Y., Yang, B., Kong, P., Zhang, Y., … Cui, Y. (2019). Multi-region sequencing unveils novel actionable targets and spatial heterogeneity in esophageal squamous cell carcinoma. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-09255-1
    测序策略：纳入 39个ESCC患者的185个肿瘤样品，平均测序深度高达300，发现ITH平均高达65%
    image-20190611104941151
    多位点取样这个策略，可以发现的驱动事件要多于单个样品。
    然后进化模型每个病人都不一样：
    image-20190611105101368

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