Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, IN, USA.
Oncogene. 2018 Jan 11;37(2):185-196. doi: 10.1038/onc.2017.322. Epub 2017 Sep 11.
Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics, detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here, in order to identify rare mutations, we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach, we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells, a subset of which has been reported in brain metastatic but not primary breast tumors. In addition, whole-genome sequencing identified mutations enriched in liver metastases of various cancers, including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases, irrespective of cancer types. Mutations/rearrangements in FHIT, involved in purine metabolism, were detected in 4/5 liver metastases, and the same four liver metastases shared mutations in 32 genes, including mutations of different HLA-DR family members affecting OX40 signaling pathway, which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B, which are mutated in >50% of hepatocellular carcinomas, were also mutated in liver metastases. Thus, irrespective of cancer types, organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors, the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable, metastasis-specific genomic aberrations.
肿瘤复杂性和肿瘤内异质性导致亚克隆多样性。尽管下一代测序（NGS）和生物信息学取得了进展，但检测导致亚克隆多样性的原发性肿瘤和转移灶中的罕见突变仍是精准基因组学的挑战。在这里，为了鉴定罕见突变，我们采用了最近描述的上皮细胞重编程测定法，用于从原发性和转移性肿瘤中短期扩增上皮细胞。使用这种方法，我们扩增了次要克隆，并获得了上皮细胞特异性 DNA/RNA，用于定量 NGS 分析。对未处理的乳腺肿瘤和从同一肿瘤中扩增的肿瘤细胞的 DNA 进行了比较 Ampliseq 综合癌症panel 序列分析。我们鉴定了以前在培养的肿瘤细胞中仅存在的未表征的突变，其中一部分已在脑转移瘤中报道，但在原发性乳腺癌中不存在。此外，全基因组测序鉴定了各种癌症肝转移中富集的突变，包括 Notch 通路突变/染色体倒位，在 5/5 个肝转移中均存在，而与癌症类型无关。在 4/5 个肝转移中检测到参与嘌呤代谢的 FHIT 基因突变/重排，而这 4 个肝转移共享 32 个基因中的突变，包括不同 HLA-DR 家族成员的突变，影响 OX40 信号通路，这可能影响对转移细胞的免疫反应。所有肝转移中突变基因的通路分析显示转移性细胞中肿瘤坏死因子和转化生长因子信号异常。在肝转移中也发生了表观遗传调节剂的突变，包括 KMT2C/MLL3 和 ARID1B，它们在超过 50%的肝细胞癌中发生突变。因此，无论癌症类型如何，器官特异性转移可能具有共同的基因组异常。由于最近的研究表明原发性肿瘤和转移灶的独立进化，并且在大多数情况下转移灶中的突变负担高于原发性肿瘤，因此这里描述的方法可能允许早期检测与转移进展相关的亚克隆体细胞改变，并可能鉴定出治疗上可行的、转移特异性的基因组异常。
