Cryopreservation for Delayed Circulating Tumor Cell Isolation is a Valid Strategy for Prognostic Association of Circulating Tumor Cells in Gastroesophageal Cancer

Aim: To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells (PBMCs) for prognostic circulating tumor cell (CTC) detection in gastroesophageal cancer.

Methods: Using 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 ( = 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 ( = 43) were examined for associations with clinicopathological variables and survival outcomes.

Results: While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens (mean number of CTCs 34.4 51.5, = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected ( = 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) ( = 43, HR = 4.4, 95%CI: 1.7-11.7, = 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, = 0.03).

Conclusion: PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.