多位点取样，并不一定要结合NGS

前面我们介绍了大量探索肿瘤内部异质性的文献，主要是利用多位点取样的方式结合NGS技术，因为超高深度测序检测异质性的算法复杂，而且单细胞水平的检测技术并不常规，所以目前是多位点取样方案最流行。

不一定需要多位点取样才能看肿瘤内部异质性 (强烈推荐)

肿瘤异质性可粗略地分为

  • 时间异质性

  • 空间异质性

多位点取样方案就是为了探索空间异质性 ， 常表现为下述三种情况：

  • ①在未扩增的细胞背景中有成簇扩增细胞；

  • ②少量扩增背景中有未扩增的细胞；

  • ③孤立的细胞扩增

虽然多位点取样常常结合NGS技术，因为NGS技术已经常规化了。但是也可以单独考察某个基因，比如这篇文章， April 11, 2016https://doi.org/10.1371/journal.pone.0153278 病人分成2类：

  • tumor tissue (85 samples) from 12 patients (mean 4.2 tissue samples per patient) were analyzed (post-therapeutic intratumoral heterogeneity)

  • 114 biopsies from 34 patients (mean 3 biopsies per patient) were analyzed (pre-therapeutic intratumoral heterogeneity)

也就是说检测了 KRAS mutation status analyses were performed in 199 tumor samples from 47 patients with rectal cancer.

就不需要NGS技术了，普通的一代测序即可。

也可以考察多个指标

比如2017的研究主要检测Homologous recombination repair (HR) deficiency ，其纳入病人和样本数量：Eighty-one biopsies from 32 cancers were analyzed，检测指标包括：

  • Tumor BRCA status was available for all samples

  • HRD scores for 70

  • BRCA1 methylation values for 76 samples.

再比如文章 (2015). Intratumor heterogeneity in hepatocellular carcinoma.Clinical Cancer Research ：

In HCC of 23 patients without medical pretreatment, a total of 120 tumor areas were defined. Analyzed were cell and tissue morphology, expression of the liver cell markers CK7, CD44, AFP, EpCAM and glutamine synthetase along with mutations of TP53 and CTNNB1, assayed by both Sanger and next generation sequencing.

也可以使用超高深度测序

比如文章：Pancreas. 2016 ，使用 Deep sequencing of 47 pancreatic ductal adenocarcinoma cases was used to determine the fraction of KRAS mutant alleles.

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