Elucidating subclonal evolution and fitness dynamics in oral carcinogenesis - PROJECT SUMMARY Seventy million people worldwide have an oral premalignant lesion (OPL) at risk of transformation into oral squamous cell carcinoma (OSCC). Once transformed, 1/3 will die of OSCC. Timely identification and removal of high-risk lesions is of critical importance. Histopathologic assessment of tissue biopsies is the clinical gold- standard for estimating risk. However, diagnosis is limited by inter-rater variability among pathologists where diagnostic and subtype definitions are often inconsistent. Better strategies to improve risk stratification are needed. Genomic alterations are the primary drivers of transformation of OSCC. Current knowledge of genomic drivers has predominantly focused on early inactivation events including the deletion of chromosomal segments (e.g., loss of heterozygosity at 9p encompassing tumor suppressor CDKN2A) and the loss of tumor suppressors such as TP53. However, these inactivation events alone are insufficient for transformation into OSCC. There exists an important knowledge gap regarding how subsequent genomic events lead to selection of high-risk subclones that ultimately become OSCC. The overarching goal of this proposal is to identify and interrogate the functional importance of additional genomic events beyond early events that drive subclonal selection in oral carcinogenesis. Our primary hypothesis is that while inactivation alterations initiate clonal expansion, copy number gains and activation of oncogenes promote an increase in subclonal diversity and competition to drive transformation. This proposal leverages orthogonal approaches to comprehensively interrogate the evolution of OPL into OSCC, which include (1) the use of serial brush biopsy for OPLs, which we have shown as a non-invasive method of faithfully reconstructing subclonal architecture in both patients and mice, (2) our capability to detect persistent OPL subclones that evolve into OSCC through analysis of single cell CNAs from FFPE, (3) our ability to functionally test key mutations and chromosomal changes in primary oral epithelial cell lines, and (4) our animal models featuring spatial and temporal control of Cdkn2a loss or Trp53 mutation, crucial for studying subclonal evolution subsequent to these driver mutations. Using these innovative approaches and resources, the aims of this project are: 1. To construct the subclonal evolutionary timeline of oral carcinogenesis in patients with OPL 2. To perform functional analysis of convergent genomic alterations identified in dominant subclones 3. To understand the functional impact of additional driver mutations occurring after Cdkn2a loss or Trp53 mutation in promoting OSCC This project will provide a comprehensive analysis of subclone evolution in oral carcinogenesis, with the goal of leveraging this knowledge to improve risk stratification and prevention strategies.
