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
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.
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.
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.
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.
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.
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.
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.