Measure of minimal residual burden on circulating tumor cells with over-expression of programmed death-ligand 1 as a dynamic biomarker in patients with colorectal cancer.
Authors: Aravindan Vasudevan, Manoj Dongare, Sreeja Jayant, Gourishankar Aland, Ganesh Khutale, Prathamesh Patil, Tanvi Deshpande, Vikas Leelavati Balasaheb Jadhav, Sujata Mali, Nayan Maktedar, Suhas Mitkari, Amrut Ashturkar, Rick Kamble, Nirmal Vivek Raut, Atul Bharde, Jayant Khandare
Journal: Journal of Clinical Oncology (2024)

e14553 Background: In stage III colorectal cancer (CRC) patients, the extent of oxaliplatin-combination as an adjuvant therapy is still undetermined. Approx. 25-50% Stage II-III CRC patients are known to develop recurrence and metastasis even after comprehensive treatment, attributed to occult disease and Minimal Residual Disease (MRD). Circulating Tumor Cells (CTCs) are bio-mechanistic extravasation source for micro-metastatic disease. CRC patients, with lower adjuvant therapy from 3 to 6 months are known to exhibit increased CTCs and positivity rate due to the emergence of resistant clones. Assays to detect CTCs and expression of Programmed Cell Death-Ligand 1 (PD-L1) as a dynamic biomarker simultaneously has wide clinical implications. Especially, when the tissue biopsy is not inadequate to detect molecular targets for immune checkpoint inhibitor (ICT) therapy. Methods: Retrospectively, 182 CRC cancer patients were analyzed for the presence of CTC and distribution at baseline and follow-ups (up to 0-4 follow ups). The peripheral blood 1.5 ml was analyzed using the OncoDiscover platform approved by CDSCO, India consisting multifunctional magneto-nanosystem mediated by anti-epithelial cellular adhesion molecule (EpCAM) antibody. CTCs were analyzed in patients with early stages- pre and post treatment, progressive disease, Disease Free Status (DFS) and in metastasis. The isolated cells were immune-stained to detect CK-18 +ve, CD45 -ve, DAPI +ve and PD-L1 +ve expression. PD-L1 expression in CTCs was validated by analyzing the linear intensity gradients of the fluorescence signals. CTCs were termed as PD-L1 -ve based on a weak or no detectable fluorescence signal and termed +ve based on a high fluorescence signal using automated image acquisition Zeiss microscope. Results: In a cohort of 182 CRC patient samples, n = 128 (70.3 %) showed the presence of CTCs. The fluorescence intensity for PD-L1 expression as a robust functional assay on CTCs for molecular characterization was developed. The distribution of CTCs ranged from 1-9. The mean fluorescence intensity value and cut-off for the expression of PD-L1 in CTCs was accounted to be ~1.02. Interestingly, in n = 54 (42.2 %) patients, CTCs showed positive expression of PD-L1. CTC +ve patients with PD-L1 expression were associated at all stages and even in early stage, progressive disease, and metastasis. Patients without any CTCs n = 54 (29.7%) either had clinically stable disease or were in DFS with no radio-graphical image. Conclusions: The presence of PD-L1 over-expression on CTCs is a dynamic biomarker in blood, showing the progression of disease even in patients with DFS status. CTC enumeration and assessing the PD-L1 expression on CTCs could offer highly individualized treatment plans in CRC patients.