Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer

Abstract

AIMTo demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells (PBMCs) for prognostic circulating tumor cell (CTC) detection in gastroesophageal cancer.METHODSUsing 7.5 mL blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the IsoFlux platform. Paired specimens taken during the same blood draw (n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo (median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs (n = 43) were examined for associations with clinicopathological variables and survival outcomes.RESULTSWhile there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens (mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected (P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count (> 17) was associated with poorer overall survival (OS) (n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS (HR = 3.7, 95%CI: 1.2-12.4, P = 0.03).CONCLUSIONPBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.

Experimental and Clinical Evidence Supports the Use of Urokinase Plasminogen Activation System Components as Clinically Relevant Biomarkers in Gastroesophageal Adenocarcinoma

“…Brungs et al assessed 43 patients from whom CTCs were isolated using the standard EpCAM isolation methods at any clinical stage of GOC. In 93% of patients, where EpCAM selected CTCs were identified, a proportion also co-expressed surface uPAR (CK+/Ep-CAM+/DAPI+/CD45-/uPAR+ CTCs) [130]. In further analyses, we found that where more than 60% of these EpCAM selected uPAR+ CTCs also co-expressed uPAR, histological tumour uPAR IHC scoring was also increased (Figure 3).…”

The Diagnostic and Prognostic Value of a Liquid Biopsy for Esophageal Cancer: A Systematic Review and Meta-Analysis

“…Eleven studies were enrolled for OS analysis, with a total of 854 EC patients [ 13 , 25 , 26 , 27 , 29 , 32 , 54 , 75 , 82 , 83 , 84 , 85 ]. In this cohort, the average age was 63.6 years (61.5–66) and the median CTC positivity rate was 46.4% (18.0–79.7).…”

Establishment of novel long-term cultures from EpCAM positive and negative circulating tumour cells from patients with metastatic gastroesophageal cancer

“…This hypothesis is also supported by the clinical picture. CTC numbers are known to increase with disease burden 40 , and at the time of blood draw patient 20 had high volume and rapidly progressive disease. It is reasonable to suggest this patient www.nature.com/scientificreports www.nature.com/scientificreports/ had a high number of biologically relevant CTCs which were missed using standard CTC isolation techniques.…”

“…A separate matched 7.5 ml EDTA blood sample was collected at the same blood draw for EpCAM based capture for CTC enumeration using the IsoFlux system (Fluxion Biosciences), and processed as per manufacturer instructions 43 . Enumerated CTCs were defined by the standard EpCAM/ Cytokeratin/DAPI positive and CD45 negative phenotype 40 .…”