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

Author(s): Brungs D, Lynch D, Luk AW, Minaei E, Ranson M, Aghmesheh M, Vine KL, Carolan M, Jaber M, de Souza P, Becker TM

Publication: World J Gastroenterol, 2018, Vol. 24, Page 810-818

PubMed ID: 29467551 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the potential effects of PBMC frozen storage on circulating tumor cell (CTC) recovery. Associations between number of CTCs enriched from frozen PBMCs and clinical prognosis were also examined. Two whole blood specimens were collected in 7.5 mL EDTA tubes from 15 patients diagnosed with gastroesopheagal adenocarcinoma (5 post-chemotherapy treatment and 10 pre-chemotherapy treatment) and processed within 24 h to recover PBMCs using cell separation tubes and a density gradient medium. PBMCs from one specimen were immediately processed for CTC enrichment and PBMCs from the other were suspended in 1 mL of diluted patient-matched plasma and 7.5% DMSO and then stored at -80°C for 2 wk to 25.2 mo until processing. CTCs were enriched from fresh and frozen PBMC specimens by immunomagnetic capture using anti-EpCAM antibodies. Recovered cells were stained with anti-CD45, anti-urokinase plasminogen activator receptor (uPAR, CD87), and anti-cytokeratin antibodies and CTCs were enumerated by immunofluorescence microscopy. Cells were identified as CTCs based on CK+, CD45- immunostaining, confirmation of an intact nucleus, and morphology. CTCs isolated from an additional 43 frozen PBMC specimens from pre-chemotherapy treatment patients were examined for associations with survival outcomes. Variables for multivariate analysis included patient age (39-89, mean=64), sex (male, female), Eastern cooperative oncology group (ECOG) performance status (0-1, 2-4), TNM cancer stage (II, III, IV), primary tumor location (distal esophageal, gastroesophageal junction, gastric), and CTC count.

   Summary of Findings:

   Overall, the mean number of recovered CTCs were significantly lower when isolated from frozen compared to fresh PBMC specimens (34.4 versus 51.5, P=0.043); however, the magnitude of difference between CTC counts from fresh and frozen PBMC specimens was larger among specimens with CTC counts >50 (data not provided). Among specimens with ≤50 CTCs, the difference in the mean number of CTCs isolated from frozen and fresh samples was non-significant (10.7 versus 16.3, P=0.06). CTCs were recovered from 95.5% (42/43) of frozen PBMC specimens (median CTC count=17). Patients with metastatic disease (n=19) had a higher mean number of CTCs than those with resectable tumors (n=24) (53.8 vs 15.8, P=0.0013). A CTC count >17 in frozen PBMCs was associated with poorer overall patient survival (n=43, hazard ratio=4.4, 95%CI: 1.7-11.7, P=0.0013). These results were confirmed by multivariate analysis that accounted for sex, age, tumor stage, ECOG performance status, and primary tumor location (hazard ratio=3.7, 95%CI: 1.2-12.4, P=0.03). There was no significant association between the duration of frozen storage and the number of CTCs recovered (P=0.09).

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Fluorescent microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	39-89, mean=64
   Preaquisition	Patient gender	Female Male
   Storage	Storage duration	2 weeks - 25.2 months
   Biospecimen Acquisition	Biospecimen location	Distal esophageal Gastroesophageal junction Gastric
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Preaquisition	Prognostic factor	Eastern cooperative oncology group (ECOG) performance status 0-1 Eastern cooperative oncology group (ECOG) performance status 2-4 TNM cancer stage (II, III, IV)

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