Abstract

Background

Minimal cellular residual disease (MCRD) with PD-L1 expression on circulating tumor cells (CTCs) is highly evidenced for possible aggressive diseases systemically. CTCs captured using N-cadherin—a calcium-dependent transmembrane glycoprotein—targets epithelial-mesenchymal transition (EMT) tumor cells. There is a difference in phenotypic specificity, as EpCAM likely misses CTCs that have undergone EMT, while N-cadherin enables the detection of these aggressive, invasive cells. Thus, N-cadherin-based CTC capture is more effective for identifying metastasis-prone CTCs. Using both markers together may improve overall CTC capture efficiency to provide a more comprehensive landscape of tumor heterogeneity and disease progression. We show the comparative and paired outcome of CTC capture using both anti-EpCAM antibodies versus N-cadherin across solid cancers.

Methods

Retrospectively, we compared 33 patients with different stages of breast, rectal, colon, prostate, lung, and other cancers. CTCs were detected using an affinity marker-independent isolation platform to avoid EpCAM bias. CTCs were isolated using a marker-independent, anti-EpCAM-positive, and N-cadherin-positive OncoDiscover platform evaluating PD-L1+ expression using automated Zeiss microscopy. Anti-EpCAM-positive and N-cadherin-positive CTCs were classified using validated intensity thresholds, concordance/discordance rates, cluster frequency, and the mean distribution of CTCs.

Results

OncoDiscover platform EpCAM and N-cadherin expression showed an overall concordance of 60.61% and a discordance of 39.39%, indicating EMT-related phenotypic divergence. The mean CTC counts were comparable between anti-EpCAM-positive and N-cadherin-positive samples (0.66 and 0.70 per sample, respectively). Among EpCAM-positive CTCs, 42.85% expressed PD-L1, whereas PD-L1 positivity was lower and present in 30.30% of N-cadherin-positive CTCs. Importantly, 4 N-cadherin+/EpCAM- PD-L1-positive CTCs were identified, which were not captured by EpCAM affinity; conversely, 6 EpCAM+/PD-L1+ CTCs lacked N-cadherin expression. Notably, CTC clusters were found in 12.12% of EpCAM+ cases and 6.06% of N-cadherin-positive cases. Collectively, these findings demonstrate that dual-marker profiling improves detection sensitivity relative to single-marker interrogation.

Conclusions

Using both EpCAM and N-cadherin together improved CTC capture efficiency. However, N-cadherin-based CTC capture is more implicative in identifying metastasis-prone CTCs. The dual affinity accounts for CTCs for MCRD surveillance for the presence of disease systemically and is indicative of the progression of micro-metastasis.