Abstract 1148: Longitudinal monitoring of circulating tumor cell burden and single-cell RNA profiling in ALK-positive NSCLC patients

Abstract Introduction: Anaplastic lymphoma kinase-positive non-small cell lung cancer (ALK+ NSCLC) is a NSCLC subtype driven by oncogenic ALK gene rearrangements. While targeted tyrosine kinase inhibitors (TKIs) have improved patient outcomes, resistance to treatment inevitably develops. This highlights the need for longitudinal monitoring. Circulating tumor cells (CTCs) have been previously reported as a predictive and prognostic biomarker. In this study, we employed liquid biopsy for longitudinal monitoring, with CTCs to track disease progression and molecular changes in ALK+ patients. Methods: Blood samples from NSCLC patients were processed with a microfluidic device, Labyrinth, to isolate CTCs. CTCs were immunofluorescence stained for DAPI, CD45, Cytokeratin, EpCAM, and vimentin (Vim) markers to identify epithelial and mesenchymal (EM) phenotypes. Parallel single-cell RNA sequencing (scRNA-seq) was performed to analyze the gene expression profiles and population dynamics of CTCs and other circulating cells. Results: We collected 67 blood samples from 38 ALK+ patients under treatment. Most patients (58%) were Stage IV, with a median age of 57 (28-81). The median CTC count was 77/ml of blood (4-228), with 57.6% of CTCs exhibiting a hybrid EM phenotype. Notably, older patients (>66 years) had higher CTC counts and a greater percentage of Vim+ CTCs (p=0.0196; p=0.00445). Female patients (n=20) had higher percentages of CTCs across all phenotypes (p=0.0462). Additionally, patients with ≥2 treatment changes showed a higher percentage of Ep+ CTCs compared to those with ≤1 treatment change (p<0.02), showing an association between CTC phenotypes and treatment resistance. A subset of 12 patients contributed 40 samples during follow-up visits, 50% of patients having stable disease and 42% showing disease progression. CTC burden changes in this group were associated with clinical status, with two distinct phenotypes changing patterns. ScRNA analysis was performed on 3 Stage IV patients: 2 on Alectinib and 1 on Lorlatinib. In CTCs’ scRNA, we detected ALK expression and IGFBP2, a gene associated with TKI resistance in NSCLC. Additionally, one patient who progressed showed a shift in immune cell populations, with fewer innate immune cells (e.g., monocytes) and more adaptive immune cells (e.g., CD8+ and CD4+ T cells). One patient, with two treatment changes, exhibited downregulation of ITGB3 and ITGB2B in their CTCs. These genes are involved in the EM phenotype shift, which was linked to observations from previous CTC burden analyses. Another patient with consistently stable disease showed upregulation of IGKC and B2M, may reflect an immune response potentially associated with disease stability, consistent with findings from literature. Conclusion: We show the utility of longitudinal CTC analysis in ALK+ NSCLC for non-invasive monitoring, providing insights into tumor dynamics. Citation Format: Yuru Chen, Shamileh Fouladdel, Leah Kidder, Harrison Ball, Zhaoping Qin, Abert Liu, Xu Cheng, Liwei Bao, Habib Serhan, Varun Kathawate, Laura Goo, Mary Horn, Stacy Fry, Nathan Merrill, Aaron Udager, Peter Ulintz, Angel Qin, Sofia Merajver, Sunitha Nagrath. Longitudinal monitoring of circulating tumor cell burden and single-cell RNA profiling in ALK-positive NSCLC patients [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 1148.