Comparative enumeration of circulating tumor cells with PD-L1 overexpression using anti-EpCAM antibody to N-cadherin in solid cancers.

Abstract

e14530 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. Difference in phenotypic specificity, as EpCAM likely misses CTCs that have undergone EMT, while N-cadherin enables 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, breast, rectal, colon, prostate, lung, etc. cancer patients. CTCs were detected using an affinity marker-independent isolation platform to avoid EpCAM bias. CTCs were isolated using a marker-independent, anti EpCAM+ ve, and N-cadherin +ve platform OncoDiscover platform approved by CDSCO using in 1.5 mL of peripheral blood. CTCs were confirmed with CK18+, DAPI+ and CD45− and PD-L1+ expression using automated Zeiss microscopy. Anti EpCAM+ and N-cadherin +ve CTCs were classified using validated intensity thresholds, and concordance/discordance rates, cluster frequency, and 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% CTCs expressed PD-L1, whereas PD-L1 positivity was lower and present in 30.30% of N-Cadherin–positive CTCs. Importantly, 04 N-Cadherin+/EpCAM– PD-L1 positive CTCs were identified, which were not captured by EpCAM affinity; conversely, 06 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 +ve 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 indicative for the progression of micro-metastasis.