Abstract 5929: Individual organoid level analyses reveal hidden tumor molecular heterogeneity

Abstract

Abstract Introduction: Tumor heterogeneity is a major cause of resistance to therapies in cancer patients occurring during disease progression or secondary to therapeutic intervention due to clonal selection. Mapping tumor heterogeneity is crucial for developing therapeutics for cancer patients. Currently there are no reliable models for identifying heterogeneity in vitro. Patient-derived cancer organoids (PDCOs) from tissue samples of patients capture clonal and subclonal mutations. Here we use PDCOs from colorectal cancer (CRC) patients to study molecular heterogeneity across individual organoids and how the heterogeneity changes with passages. Methods: Fresh CRC tissues from 4 patients undergoing endoscopy were obtained following consent on an IRB-approved protocol. PDCOs were cultured using Matrigel and previously published CRC organoid media. Following maturation, individual organoids (spikes) were separated into Matrigel domes and expanded. Parent cultures from high/low passage numbers and expanded spikes were collected for next generation sequencing (NGS). A variant allele frequency (VAF) was determined for each nonsynonymous variant and considered subclonal if the VAF was between 10%-30%. Variants were annotated pathogenic or not using ClinVar. Results: A total of 49 spikes were collected from 4 CRC PDCO lines and compared to the parent cultures using NGS. For low passage parent cultures, a median of 4 (range 3-8) subclonal alterations were identified. At high passages the number of subclonal variants decreased (median: 2 (range 2-4)). The individual organoid spikes maintained the founder (clonal) alterations and most also contained subclonal alterations, though at a lower frequency. Of those subclonal variants in the parent cultures, 31% were found in the spikes and an additional 54% found in the spikes were not identified in the parent. In a high tumor mutation burden line (MTB111), 12/17 spikes showed subclonal alterations in APCA896V, F1515C, Q1544* that were not present in the parent culture. Two PDCO lines (RC46A and RC46B) obtained from distinct sites of the same tumor were generated. One contained a subclonal mutation in MYCN26S in the high passage parent culture and 7/9 spikes. This mutation was not present in any RC46A samples. The high passage RC46A parent culture and 2/7 spikes showed an alteration in ARID1AG1037fs while it was present only in a single spike of RC46B. Conclusion: PDCOs identified subclonal alterations at the single-organoid level and are an exciting tool to study tumor heterogeneity. The spikes presented less subclonal variants than the parent but were largely not clonal. Future implications of using heterogeneity data from single organoids in therapeutic decisions are warranted. Citation Format: Shirsa Udgata, Aishwarya Sunil, Katherine A. Johnson, Cheri A. Pasch, Dustin A. Deming. Individual organoid level analyses reveal hidden tumor molecular heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5929.