PurposeThis study aimed to investigate the clinical utility of diverse aneuploid circulating tumor cell (CTC) subtypes and particularly CTC-associated white blood cell (CTC-WBC) clusters in predicting treatment response, prognosis and real-time monitoring disease progression in advanced driver gene-negative non-small lung cancer (NSCLC) patients.Materials and methodsA total of 74 eligible patients were prospectively enrolled and serial blood samples were collected at pre-treatment(t0), after two cycles of therapy (t1) and at post-four-to-six treatment cycles (t2). Co-detection of diverse subtypes of aneuploid CTCs and CTC-WBC clusters was conducted in advanced NSCLC patients receiving first-line treatment.ResultsAt baseline, CTCs were detected in 69 (93.24%) patients and CTC-WBC clusters were detected in 23 (31.08%) patients. Patients with CTCs < 5/6ml or with CTC-WBC clusters undetectable exhibited a better treatment response than patients with pre-therapeutic aneuploid CTCs ≥ 5/6ml or harboring CTC-WBC clusters (p= 0.034 andp= 0.012, respectively). Before treatment, patients bearing tetraploid CTCs ≥ 1/6ml showed significantly inferior progression-free survival (PFS) [hazard ratio (HR):2.420, 95% confidence interval (CI): 1.426–4.106;p= 0.001] and overall survival (OS) compared to patients with tetraploid CTCs < 1/6ml (HR:1.907, 95%CI: 1.119–3.251;p= 0.018). A longitudinal study demonstrated that post-therapeutic patients harboring CTC-WBC clusters displayed the reduced PFS and OS compared with those without CTC-WBC clusters, and subgroup analysis showed that the presence of CTC-WBC clusters indicated a worse prognosis in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients. After adjusting for multiple significant factors, post-therapeutic CTC-WBC clusters were the only independent predictor of both PFS (HR:2.872, 95% CI: 1.539–5.368;p= 0.001) and OS (HR:2.162, 95% CI: 1.168–4.003;p= 0.014).ConclusionsIn addition to CTCs, longitudinal detection of CTC-WBC clusters provided a feasible tool to indicate initial treatment response, dynamically monitor disease progression and predict survival in driver gene-negative advanced NSCLC patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-023-10985-1.