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The limited sensitivity of circulating tumor cell (CTC) detection in pancreatic adenocarcinoma (PDAC) stems from their extremely low concentration in the whole circulating blood, necessitating enhanced detection methodologies. This study sought to amplify assay-sensitivity by employing diagnostic leukapheresis (DLA) to screen large blood volumes. Sixty patients were subjected to DLA, with a median processed blood volume of ~ 2.8 L and approximately 5% of the resulting DLA-product analyzed using CellSearch (CS). Notably, DLA significantly increased CS-CTC detection to 44% in M0-patients and 74% in M1-patients, yielding a 60-fold increase in CS-CTC enumeration. DLA also provided sufficient CS-CTCs for genomic profiling, thereby delivering additional genomic information compared to tissue biopsy samples. DLA CS-CTCs exhibited a pronounced negative prognostic impact on overall survival (OS), evidenced by a reduction in OS from 28.6 to 8.5 months (univariate: p = 0.002; multivariable: p = 0.043). Additionally, a marked enhancement in sensitivity was achieved (by around 3-4-times) compared to peripheral blood (PB) samples, with positive predictive values for OS being preserved at around 90%. Prognostic relevance of CS-CTCs in PDAC was further validated in PB-samples from 228 PDAC patients, consolidating the established association between CTC-presence and reduced OS (8.5 vs. 19.0 months, p < 0.001). In conclusion, DLA-derived CS-CTCs may serve as a viable tool for identifying high-risk PDAC-patients and aiding the optimization of multimodal treatment strategies. Moreover, DLA enables comprehensive diagnostic profiling by providing ample CTC material, reinforcing its utility as a reliable liquid-biopsy approach. This high-volume liquid-biopsy strategy presents a potential pathway for enhancing clinical management in this malignancy.

Abstract Background Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model. Methods We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested. Results We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods. Conclusions This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS).

Background Circulating tumour cells (CTCs) and tumour-derived extracellular vesicles (tdEVs) have great potential for monitoring therapy response and early detection of tumour relapse, facilitating personalized adjuvant therapeutic strategies. However, their low abundance in peripheral blood limits their informative value. In this study, we explored the presence of CTCs and tdEVs collected intraoperatively from a tumour-draining vein (DV) and via a central venous catheter (CVC) prior to tumour resection. Methods CellSearch analyses of 395 blood samples from 306 patients with gastrointestinal tumours and 93 blood samples from healthy donors were used to establish and validate gates for the automated detection of CTCs and tdEVs with ACCEPT software and R scripts. The selected gate settings were applied to 227 samples of 142 patients with colorectal cancer (CRC) from two independent collectives. Phenotypic features were obtained via numeric analysis of their fluorescence signals (e.g. size, shape, and intensity) and were used for calculating diversity using Shannon index (SI) of clusters generated via the k-means algorithm after Uniform Manifold Approximation and Projection (UMAP) pre-processing, and standard deviation (SD). Results CTCs and tdEVs were more abundant in the DV samples compared to CVC samples ( p  < 0.05). tdEVs were detected in higher numbers than CTCs in both compartments. Importantly, tdEVs in CVCs were associated with tumor spread, whereas CTCs in DVs were linked to tumor size. In both compartments, the prognostic value of tdEVs for overall survival (OS) surpassed that of CTCs, as demonstrated by univariate, multivariate, and Kaplan-Meier analyses. CTCs and tdEVs in DVs were phenotypically distinct, being larger, more eccentric, and displaying stronger cytokeratin intensities ( p  < 0.05) compared to those in CVC samples. Furthermore, increased diversity in CTC and tdEV phenotypes was significantly associated with shorter survival, validating the prognostic relevance of the SD-diversity metric. Conclusion Our study demonstrates that DV sampling significantly enhances the detection of prognostically relevant CTCs and tdEVs in CRC patients, underscoring the superior prognostic significance of tdEVs compared to CTCs. Importantly, the combined phenotypic diversity of both markers emerges as a more powerful biomarker than their enumeration alone. These findings suggest that comprehensive, automated analysis of CTCs and tdEVs in DVs may open new avenues for tailoring individualized therapies in CRC patients.

Cement augmentation via percutaneous vertebroplasty or kyphoplasty for treatment of spinal metastasis is a well-established treatment option. We assessed whether elevated intrametastatic pressure during cement augmentation results in an increased dissemination of tumour cells into the vascular circulation. We prospectively collected blood from patients with osteolytic spinal column metastases and analysed the prevalence of circulating tumour cells (CTCs) at three time-points: preoperatively, 20 minutes after cement augmentation, and 3–5 days postoperatively. Enrolling 21 patients, including 13 breast- (61.9%), 5 lung- (23.8%), and one (4.8%) colorectal-, renal-, and prostate-carcinoma patient each, we demonstrate a significant 1.8-fold increase of EpCAM+/K+ CTCs in samples taken 20 minutes post-cement augmentation ( P  < 0.0001). Despite increased mechanical CTC dissemination due to cement augmentation, follow-up blood draws demonstrated that no long-term increase of CTCs was present. Array-CGH analysis revealed a specific profile of the CTC collected 20 minutes after cement augmentation. This is the first study to report that peripheral CTCs are temporarily increased due to vertebral cement augmentation procedures. Our findings provide a rationale for the development of new prophylactic strategies to reduce the increased release of CTC after cement augmentation of osteolytic spinal metastases.

Here, we tested two magnetic-bead based systems for the enrichment and detection of rare tumor cells in concentrated blood products. For that, the defined numbers of cells from three pancreatic cancer cell lines were spiked in 108 peripheral blood mononuclear cells (PBMNCs) concentrated in 1 mL, mimicking diagnostic leukapheresis (DLA) samples, and samples were processed for circulating tumor cells (CTC) enrichment with the IsoFlux or the KingFisher systems, using different types of magnetic beads from the respective technology providers. Beads were conjugated with different anti-EpCAM and MUC-1 antibodies. Recovered cells were enumerated and documented by fluorescent microscopy. For the IsoFlux system, best performance was obtained with IsoFlux CTC enrichment kit, but these beads compromised the subsequent immunofluorescence staining. For the KingFisher system, best recoveries were obtained using Dynabeads Biotin Binder beads. These beads also allowed one to capture CTCs with different antibodies and the subsequent immunofluorescence staining. KingFisher instrument allowed a single and streamlined protocol for the enrichment and staining of CTCs that further prevented cell loss at the enrichment/staining interface. Both IsoFlux and KingFisher systems allowed the enrichment of cell line cells from the mimicked-DLA samples. However, in this particular experimental setting, the recovery rates obtained with the KingFisher system were globally higher, the system was more cost-effective, and it allowed higher throughput.

In Ovarian Cancer (OC), the analysis of single circulating tumor cells (sCTCs) might help to investigate genetic tumor evolution during the course of treatment. Since common CTC identification features failed to reliably detect CTCs in OC, we here present a workflow for their detection and genomic analysis. Blood of 13 high-grade serous primary OC patients was analyzed, using negative immunomagnetic enrichment, followed by immunofluorescence staining and imaging for Hoechst, ERCC1, CD45, CD11b and cytokeratin (CK) and sCTC sorting with the DEPArrayTM NxT. The whole genome of single cells was amplified and profiled for copy number variation (CNV). We detected: Type A-cells, epithelial (Hoechstpos, ERCC1pos, CD45neg, CD11bpos, CKpos); Type B-cells, potentially epithelial (Hoechstpos, ERCC1pos, CD45neg, CD11bpos, CKneg) and Type C-cells, potentially mesenchymal (Hoechstpos, ERCC1pos, CD45neg, CD11bneg, CKneg). In total, we identified five (38.5%) patients harboring sCTCs with an altered CN profile, which were mainly Type A-cells (80%). In addition to inter-and intra-patient genomic heterogeneity, high numbers of Type B- and C-cells were identified in every patient with their aberrant character only confirmed in 6.25% and 4.76% of cases. Further identification markers and studies in the course of treatment are under way to expand sCTC analysis for the identification of tumor evolution in OC.

Epigenetics contributes to molecular mechanisms leading to tumor cell transformation and systemic progression of cancer. However, the dynamics of epigenetic remodeling during metastasis remains unexplored. In this context, circulating tumor cells (CTCs) might enable a direct insight into epigenetic mechanisms relevant for metastasis by providing direct access to systemic cancer. CTCs can be used as prognostic markers in cancer patients and are regarded as potential metastatic precursor cells. However, despite substantial technical progress, the detection and molecular characterization of CTCs remain challenging, in particular the analysis of DNA methylation. As recent studies have started to address the epigenetic state of CTCs, we discuss here the potential of such investigations to elucidate mechanisms of metastasis and to develop tumor biomarkers.