Date of Graduation

5-2016

Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Nicholas E Navin PhD

Committee Member

Elsa R Flores PhD

Committee Member

E Scott Kopetz MD PhD

Committee Member

Ralf Krahe PhD

Committee Member

Kenneth Y Tsai MD PhD

Abstract

Metastasis is the primary cause of human cancer deaths. Patients with metastatic colorectal cancer (mCRC) show only an 11% 5-year survival rate, compared to those without local or distant metastases (92% 5-year survival rate). Understanding the CRC tumor evolution may provide valuable insights on how to improve treatment in patients with mCRC. However, the genomic basis of metastasis has been difficult to study, in part due to the extensive intratumor heterogeneity at both the primary and metastatic tumor sites, and the low frequency of subclones with metastatic potential. Previous studies have applied conventional bulk next-generation sequencing (NGS) methods, which have limited ability to resolve intratumor heterogeneity.

To address this problem, we have developed a highly-multiplexed single cell DNA sequencing method that combines flow-sorting of single nuclei, multiple-displacement-amplification using Φ29 polymerase, low-input library preparation, library barcoding, targeted capture and NGS to generate high-coverage data from single cells. We validate this method by generating high coverage sequencing data from single human cells, with low allelic dropout and high detection efficiencies for single nucleotide variants.

Using this method, we sequenced 186 single cells from primary tumor and liver metastases from two mCRC patients to delineate the clonal architecture of the tumor and reconstruct their phylogenetic lineages. We also performed exome sequencing on the bulk tumor tissues. Our data identified a large number of nonsynonymous mutations that evolved in the root node during the earliest stage of primary tumor evolution and were maintained in all single cells during the clonal expansion of the tumor mass. We also identified a small number of mutations that were specific to the liver metastases, which are likely to play an important role in metastatic dissemination. Furthermore, we found three diploid cells with only APC mutations in CO5, which may represent the progenitor clones that gave rise to the primary and metastatic tumors. Using the single cell data, we construct phylogenetic trees, which revealed branched evolution in metastasis. Our data suggest that both mCRC patients are consistent with the late-dissemination model, in which the primary tumors evolved for a long period of time prior to the dissemination of clones to distant organ sites.

In summary, we have developed novel methods for single cell DNA sequencing, and applied these methods to gain unprecedented understanding of clonal evolution during metastasis in colorectal cancer.

Keywords

single cell sequencing, cancer genomics, tumor evolution, intratumor heterogeneity, next generation sequencing, colorectal cancer

Available for download on Saturday, April 22, 2017

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