Longitudinal Analysis of Circulating Tumor Cells in Colorectal Cancer Patients by a Cytological and Molecular Approach: Feasibility and Clinical Application

Abstract

IntroductionLiquid biopsies allowing for individualized risk stratification of cancer patients have become of high significance in individualized cancer diagnostics and treatment. The detection of circulating tumor cells (CTC) has proven to be highly relevant in risk prediction, e.g., in colorectal cancer (CRC) patients. In this study, we investigate the clinical relevance of longitudinal CTC detection over a course of follow-up after surgical resection of the tumor and correlate these findings with clinico-pathological characteristics.MethodsIn total, 49 patients with histologically proven colorectal carcinoma were recruited for this prospective study. Blood samples were analyzed for CTC presence by two methods: first by marker-dependent immunofluorescence staining combined with automated microscopy with the NYONE® cell imager and additionally, indirectly, by semi-quantitative Cytokeratin-20 (CK20) RT-qPCR. CTC quantification data were compared and correlated with the clinico-pathological parameters.ResultsDetection of CTC over a post-operative time course was feasible with both applied methods. In patients who were pre-operatively negative for CTCs with the NYONE® method or below the cut-off for relative CK20 mRNA expression after analysis by PCR, a statistically significant rise in the immediate post-operative CTC detection could be demonstrated. Further, in the cohort analyzed by PCR, we detected a lower CTC load in patients who were adjuvantly treated with chemotherapy compared to patients in the follow-up subgroup. This finding was contrary to the same patient subset analyzed with the NYONE® for CTC detection.ConclusionOur study investigates the occurrence of CTC in CRC patients after surgical resection of the primary tumor and during postoperative follow-up. The resection of the tumor has an impact on the CTC quantity and the longitudinal CTC analysis supports the significance of CTC as a prognostic biomarker. Future investigations with an even more extended follow-up period and larger patient cohorts will have to validate our results and may help to define an optimal longitudinal sampling scheme for liquid biopsies in the post-operative monitoring of cancer patients to enable tailored therapy concepts for precision medicine.

Detection of Marker Associated with CTC in Colorectal Cancer in Mononuclear Cells of Patients with Benign Inflammatory Intestinal Diseases

“…In general, the detection of a biomarker in liquid biopsies, e.g., enumeration and characterization of circulating tumor cells (CTC), represents a powerful und reasonable strategy for early cancer detection as well as proof of minimal residual disease, as we demonstrated previously [ 9 ].…”

“…It was also shown that CK20 is a marker for response after neoadjuvant chemoradiation but not prognosis in patients with rectal cancer [ 12 , 13 , 14 ]. Furthermore, our recent study indicates that this molecular approach detecting CK20 expression in blood samples of CRC patients might also be a valuable tool for early detection of relapses [ 9 ]. Based on this finding it can be speculated that CK20 might also be suitable for early CRC detection.…”

Deep Phenotypic Characterisation of CTCs by Combination of Microfluidic Isolation (IsoFlux) and Imaging Flow Cytometry (ImageStream)

“…Low levels of CTCs are identified in pre-tumoural diseases such as colorectal polyps, and in colorectal cancer, the number of CTCs is associated with the tumour site, being greater in patients with sigmoid colon tumours compared with other locations [ 22 ]. Pre-operative CTC numbers in CRC patients subjected to surgery were associated with poorer overall survival [ 23 , 24 , 25 ], and it was shown to have diagnostic utility in early disease stages, as the number of CTCs was significantly greater in colorectal carcinoma compared with colorectal polyps patients [ 22 ]. However, most studies on CTCs have relied solely on the expression of epithelial markers (EpCAM/CK) versus the absence of CD45 expression [ 12 , 24 ], reducing the ability to detect/characterise CTCs and thus weakening their prognostic value [ 26 ].…”

“…Pre-operative CTC numbers in CRC patients subjected to surgery were associated with poorer overall survival [ 23 , 24 , 25 ], and it was shown to have diagnostic utility in early disease stages, as the number of CTCs was significantly greater in colorectal carcinoma compared with colorectal polyps patients [ 22 ]. However, most studies on CTCs have relied solely on the expression of epithelial markers (EpCAM/CK) versus the absence of CD45 expression [ 12 , 24 ], reducing the ability to detect/characterise CTCs and thus weakening their prognostic value [ 26 ]. Furthermore, poor prognosis is associated not only with CTC counts but also with CTC clusters (also called microemboli), as they increase with disease stage and are significantly associated with worse outcomes [ 27 ].…”

Circulating Tumor Cells: Technologies and Their Clinical Potential in Cancer Metastasis

“…Similarly, in NSCLC and [ 86 ] and castration-resistant prostate cancer [ 91 ], treatment resulted in decreased CTC counts associated with longer PFS in both studies. In another study, longitudinal monitoring of CTC counts in colorectal cancer preceded clinical symptoms and radiological imaging in a patient diagnosed with local tumor recurrence thirteen months after tumor resection [ 42 ]. Furthermore, in the phase III SWOG S0421 trial examining docetaxel-treated castration-resistant prostate cancer, CTC counts were significantly associated with baseline PSA, bone pain, liver disease, hemoglobin, alkaline phosphatase, and PSA and response evaluation criteria in solid tumors (RECIST) response, putting forth evidence that CTC counts could be used as a viable alternative index in determining treatment [ 91 ].…”