Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 21, 2018; 24(7): 810-818
Published online Feb 21, 2018. doi: 10.3748/wjg.v24.i7.810
Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer
Daniel Brungs, David Lynch, Alison WS Luk, Elahe Minaei, Marie Ranson, Morteza Aghmesheh, Kara L Vine, Martin Carolan, Mouhannad Jaber, Paul de Souza, Therese M Becker
Daniel Brungs, Elahe Minaei, Marie Ranson, Morteza Aghmesheh, Kara L Vine, Martin Carolan, Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong 2500, Australia
Daniel Brungs, Elahe Minaei, Marie Ranson, Kara L Vine, School of Biological Sciences, University of Wollongong, Wollongong 2500, Australia
Daniel Brungs, Morteza Aghmesheh, Martin Carolan, Mouhannad Jaber, Illawarra Cancer Centre, Wollongong Hospital, Wollongong 2500, Australia
Daniel Brungs, David Lynch, Alison WS Luk, Elahe Minaei, Marie Ranson, Morteza Aghmesheh, Kara L Vine, Martin Carolan, Mouhannad Jaber, Paul de Souza, Therese M Becker, CONCERT-Translational Cancer Research Centre, New South Wales 2000, Australia
David Lynch, Paul de Souza, Therese M Becker, Centre for Circulating Tumor Cell Diagnostics and Research, Ingham Institute for Applied Medical Research, Liverpool Hospital, Sydney 2170, Australia
Paul de Souza, Therese M Becker, School of Medicine, University of Western Sydney, Sydney 2170, Australia
Paul de Souza, Therese M Becker, South Western Medical School, University of New South Wales, Sydney 2170, Australia
Institutional review board statement: This study was approved by South Western Sydney Local Health District Human Research Ethics Committee (Project Number 15/072). A written informed consent was obtained from each participant before sample collection.
Conflict-of-interest statement: All authors have no conflicts of interest to declare.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Daniel Brungs, BSc, MBBS, Doctor, Illawarra Cancer Care Centre, Wollongong Hospital, 348 Crown St, Wollongong NSW 2500, Australia.
Telephone: +61-2-42225200 Fax: +61-2-42225243
Received: November 6, 2017
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
Research background

A persisting challenge to the field of circulating tumor cell (CTC) research is the requirement for prompt analysis of samples at specialised centres. This has presented significant logistical challenges to researchers, compounded by the significant expertise, time and laboratory resources required for CTC analysis.

Research motivation

Current methods to overcome this issue, such as fixation of blood samples, extend the time for CTC processing for several days, but may interfere with downstream molecular analyses.

Cryopreservation of patient samples permits 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. However, up to now, there has been little research in how cryopreservation affects CTC recovery, and whether cryopreservation retains predictive value of CTCs.

Research objectives

The primary objective of our study was to investigate the feasibility and reliability of delayed CTC isolation from cryopreserved peripheral blood mononuclear cells (PBMCs) layer. This was determined by percentage of CTC loss during cryopreservation and thawing, and clinical validity of CTC enumeration from cryopreserved samples.

Research methods

CTCs were isolated from 7.5 mL blood samples collected from patients with gastroesophageal adenocarcinoma using EpCAM based immunomagnetic capture with the IsoFlux platform. CTC loss with cryopreservation was determined by comparing CTC enumeration from matched cryopreserved and freshly processed blood samples collected during the same blood draw. CTCs isolated from pre-treatment cryopreserved PBMCs were examined for association with clinicopathological variables and survival outcomes.

Research results

We found a minor loss of tumor cells in matched cryopreserved and freshly processed samples, mostly in samples with high CTC counts. A high CTC count isolated from cryopreserved PBMCs remained a statistically significant independent prognostic factor in gastroesophageal cancer.

Research conclusions

Our study demonstrates a feasible and robust protocol facilitating CTC isolation from cryopreserved PBMCs even after 2 years post freezing. Our results have immediate applicability in the design and conduct of translational studies, as it facilitates incorporation of CTC analysis in large international trials and biobanking projects.

Research perspectives

There is an increasing variety of techniques used for CTC isolation described in the literature. While the current work confirms the reliability of CTC isolation from cryopreserved samples using immunomagnetic separation, further work needs to be undertaken to confirm its suitability for other isolation approaches.