Published online Feb 21, 2018. doi: 10.3748/wjg.v24.i7.810
Peer-review started: November 7, 2017
First decision: November 30, 2017
Revised: December 11, 2017
Accepted: December 19, 2017
Article in press: December 19, 2017
Published online: February 21, 2018
To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells (PBMCs) for prognostic circulating tumor cell (CTC) detection in gastroesophageal cancer.
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 (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.
While 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).
PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.
Core tip: This study demonstrates a novel and robust protocol for the cryopreservation and thawing of patient blood samples, demonstrating reliable circulating tumor cell isolation and characterisation after the long term storage of patient samples. Using the largest patient cohort reported to date, we validated our method by confirming the independent prognostic association of circulating tumor cell (CTC) enumeration from cryopreserved peripheral blood mononuclear cells. Cryopreservation may assist with the wider incorporation of CTC collection and analysis in biobanking, retrospective studies, and large international clinical trials, by facilitating specimen storage, bulk transporting, and batch processing.