Extrachromosomal DNA Dynamics Contribute to Intratumoral Receptor Tyrosine Kinase Genetic Heterogeneity and Drug Resistance in Gastric Cancer

Chromosomal instability in gastric cancer cells is associated with the amplification of oncogenes that encode receptor tyrosine kinases (RTK), such as HER2 and FGFR2; such gene amplification varies from cell to cell and manifests as genetic heterogeneity within tumors. The intratumoral genetic heterogeneity of RTK gene amplification causes heterogeneity in RTK protein expression, which has been suggested to be associated with therapeutic resistance to RTK inhibitors; however, the underlying mechanism is not fully understood. In this study, we show that extrachromosomal DNA (ecDNA) causes intratumoral genetic heterogeneity in RTKs and drug resistance due to diverse dynamic changes. We analyzed the dynamics of FGFR2 and MYC ecDNA in a gastric cancer cell line after single-cell cloning. Similar to those in parental cells, the copy numbers of FGFR2 and MYC in subclones differed significantly between cells, indicating intraclonal genetic heterogeneity. Furthermore, the ecDNA composition differed between subclones, which affected FGFR2 protein expression and drug sensitivity. Interestingly, clone cells that were resistant to the FGFR2 inhibitor AZD4547 presented diverse changes in ecDNA, including chimeric ecDNA, large ecDNA, and increased ecDNA numbers; these changes were associated with high expression and rephosphorylation of FGFR2. Conversely, when resistant clone cells were cultured under conditions that excluded AZD4547, the ecDNA status became similar to that of the original clone cells, and the inhibitory effect on cell growth was restored.

Implications: Our results show that dynamic quantitative and qualitative changes in ecDNA can drive the intratumoral genetic heterogeneity of RTKs and resistance to RTK inhibitors.