Abstract 4424: Heterogeneity in resistance mechanisms found in <i>KRAS</i> mutant and wild-type patient-derived organoids with anti-EGFR therapy panitumumab

Abstract Introduction: Tumor heterogeneity plays a major role in the development of resistance to therapies in colorectal cancer (CRC). The epidermal growth factor receptor (EGFR) antibodies, panitumumab (pani) and cetuximab are commonly used for the treatment of CRC. At resistance, very low frequency KRAS mutations can be detected by circulating tumor DNA (ctDNA). This raises the question if rare KRAS mutant clones can lead to EGFR inhibitor resistance. Here we utilized patient-derived cancer organoids (PDCOs) to investigate the impact of low frequency KRAS mutant clones on the development of pani resistance. Methods: PDCOs were obtained after surgery with patient consent under UW institutional review board protocol. Tissue samples were processed as described previously for PDCOs. Two CRC PDCO lines, with KRASG12V mutation (KR) and without (WT) were mixed in culture at multiple concentrations (v/v) (100% KR, 100% WT, 20% KR+80% WT, 10% KR+90% WT, 5% KR+95% WT, 1% KR+99% WT, 0.5% KR+99.5% WT, 0.25% KR+99.75% WT). Diameter analysis from day 0 to 4 was completed with increasing concentrations of pani. Once complete resistance to treatment was reached (&amp;gt;20% median growth in diameter under treatment at 100% physiologic Cmax for two consecutive weeks), PDCOs were collected for bulk RNA-Seq. Differential expression analysis was prepared by UW Madison Bioinformatics Research Core using edgeR. Gene Set Enrichment Analysis (GSEA) was performed on hallmark gene sets. Results: Despite ≤1% of KR PDCOs present in some populations, mixed conditions escalated to ≥ 40% Cmax within 10 weeks and reached complete resistance to pani by week 16, while 100% WT culture remained at 20% Cmax for 22 weeks. GSEA revealed 0.5% KR+99.5% WT condition resulted in (p &amp;lt; 0.05) up and downregulation of eight and 22 hallmark pathways (HP), respectively, compared to 100% WT at baseline. Upregulated HP of interest include MYC Targets V1/2 and MTORC1 Signaling, while downregulated HP of interest were KRAS Signaling Up, Apoptosis, and P53 Pathway. Comparing baseline mixed populations to resistant counterparts, the significant upregulation in HP was heterogenous across conditions. Some HP of note were KRAS Signaling Up and Apoptosis. In contrast, the significantly downregulated HP (p &amp;lt; 0.001) was relatively homogenous across only the three HP of E2F Targets, G2M Checkpoint, and MYC Targets V1. Conclusion: PDCO lines containing very low concentrations of resistant clones can lead to significant changes in HP regulation and treatment response. The heterogeneity in HP upregulation in fully resistant mixed populations indicate differing mechanisms of reaching resistance. To confirm these observations, further studies will evaluate the response of more PDCO lines to pani treatment and utilize patient ctDNA collected before and after treatment in clinic to see the effect of the presence of KRAS subclones on outcomes. Citation Format: Jordan N. Stoecker, Shirsa Udgata, Xingqi Shen, Cheri A. Pasch, Dustin A. Deming. Heterogeneity in resistance mechanisms found in KRAS mutant and wild-type patient-derived organoids with anti-EGFR therapy panitumumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4424.