Background

In stage III colorectal cancer (CRC) patients, the extent of oxaliplatin-based adjuvant therapy remains uncertain. Approximately 25–50% of stage II–III CRC patients develop recurrence and metastasis even after comprehensive treatment, largely attributed to occult disease and minimal residual disease (MRD). Circulating tumor cells (CTCs) represent a bio-mechanistic source of extravasation leading to micro-metastatic disease. CRC patients receiving reduced adjuvant therapy (3–6 months) are known to exhibit increased CTC counts and positivity rates due to the emergence of resistant clones. Assays that detect CTCs and the expression of programmed death-ligand 1 (PD-L1) as a dynamic biomarker simultaneously have significant clinical implications, particularly when tissue biopsy samples are inadequate to identify molecular targets for immune checkpoint inhibitor (ICI) therapy.

Methods

In a retrospective study, 182 CRC patients were analyzed for the presence and distribution of CTCs at baseline and across follow-ups (0–4 follow-ups). Peripheral blood (1.5 ml) samples were analyzed using the CDSCO-approved OncoDiscover platform, which consists of a multifunctional magneto-nanosystem mediated by anti-epithelial cellular adhesion molecule (EpCAM) antibodies. CTCs were evaluated in patients with early-stage disease (pre- and post-treatment), progressive disease, disease-free status (DFS), and metastasis. Isolated cells were immunostained to detect CK18+, CD45-, DAPI+, and PD-L1+ expression. PD-L1 expression on CTCs was validated by analyzing the linear intensity gradients of fluorescence signals. CTCs were classified as PD-L1 negative when weak or no fluorescence signal was detected and PD-L1 positive when strong fluorescence signals were observed using automated image acquisition on a Zeiss fluorescence microscope.

Results

Among the cohort of 182 CRC patient samples, 128 (70.3%) showed the presence of CTCs. A fluorescence intensity-based assay was developed to evaluate PD-L1 expression as a robust functional biomarker for molecular characterization of CTCs. The distribution of CTCs ranged from 1 to 9 cells. The mean fluorescence intensity value and cut-off for PD-L1 expression in CTCs was approximately 1.02. Notably, 54 patients (42.2%) with CTCs showed positive PD-L1 expression. CTC-positive patients with PD-L1 expression were observed across all stages, including early-stage disease, progressive disease, and metastasis. Patients without detectable CTCs (n = 54, 29.7%) either had clinically stable disease or were in DFS with no radiographic evidence of disease.

Conclusions

PD-L1 overexpression on CTCs represents a dynamic blood-based biomarker indicating disease progression even in patients with DFS status. Enumeration of CTCs along with assessment of PD-L1 expression may enable more individualized treatment strategies for CRC patients and support better monitoring of disease progression and therapeutic response.