Defining the role of seed and soil in peritoneal metastasis evolution - The peritoneum is the third most common site of metastasis in colon cancer. Despite the common occurrence of peritoneal metastasis and its dire prognosis, very little is known about its origins and characteristics. The lack of basic research into peritoneal metastasis has stymied treatment advances. We have spent the last 4 years studying the genetics of colorectal cancer peritoneal metastasis in a large international collaboration. Building on the findings from this effort, we here propose three aims to advance basic science and clinical care in this crucial area. All aims will take advantage of a large, retrospective cohort of peritoneal metastasis patients (N=103) that will undergo multi-region genomic analysis. The goal of the project is to rigorously define the evolutionary history and genetic characteristics of peritoneal metastases and to tie these features to clinical outcomes. In Specific Aim 1, we will determine how cancer cells reach the peritoneal cavity and how their seeding routes impact the genetic diversity of peritoneal metastases. Our preliminary data show profound differences in genetic diversity between synchronous and metachronous peritoneal metastases. The former are genetically homogeneous and closely related to the deep invading edge of the primary tumor, while the latter are heterogeneous and do not appear to originate at the invasive front, indicating that their seeding routes may be distinct (direct growth through the serosa vs. cancer cell release during primary tumor surgery). In this aim, we will map the routes by which peritoneal metastases are seeded and study how their origins shape genetic diversity, using a combination of high-efficiency phylogenetic reconstruction and deep exome sequencing. In Specific Aim 2, we will determine the genetic characteristics of cancer cells that succeed in colonizing the peritoneum. We have previously found that cancer cells disseminating to different host organs are exposed to environment-specific selective pressures. Our preliminary data indicate that genomically relatively intact cancer cells may be at an advantage over highly mutated or aneuploid cells during peritoneal colonization. We will use our extensively sampled cohort of peritoneal metastasis cases to compare driver mutations, somatic copy number alterations (SCNAs), as well as global measures of genetic stability (tumor mutation burden, SCNA burden) between primary tumors, locoregional, distant and peritoneal metastases. In Specific Aim 3, we will study how the characteristics of peritoneal metastases relate to survival outcomes. Prognostic factors to help clinicians estimate the likelihood of different survival outcomes are urgently needed. We will collect detailed follow-up data on our peritoneal metastasis patient cohort. In Aim 3A, we will study the association between survival and the genetic characteristics investigated in aims 1 and 2. In Aim 3B, we will broaden our inquiry to the microenvironment and examine via single-nucleus RNA sequencing (snRNAseq) and multi-color immunofluorescence whether the composition and transcriptome of stromal and immune cells present in peritoneal metastases correlate with survival.
