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      CTC-mRNA (AR-V7) Analysis from Blood Samples—Impact of Blood Collection Tube and Storage Time

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          Abstract

          Circulating tumour cells (CTCs) are an emerging resource for monitoring cancer biomarkers. New technologies for CTC isolation and biomarker detection are increasingly sensitive, however, the ideal blood storage conditions to preserve CTC-specific mRNA biomarkers remains undetermined. Here we tested the preservation of tumour cells and CTC-mRNA over time in common anticoagulant ethylene-diamine-tetra-acetic acid (EDTA) and acid citrate dextrose solution B (Citrate) blood tubes compared to preservative-containing blood tubes. Blood samples spiked with prostate cancer cells were processed after 0, 24, 30, and 48 h storage at room temperature. The tumour cell isolation efficiency and the mRNA levels of the prostate cancer biomarkers androgen receptor variant 7 (AR-V7) and total AR, as well as epithelial cell adhesion molecule (EpCAM) were measured. Spiked cells were recovered across all storage tube types and times. Surprisingly, tumour mRNA biomarkers were readily detectable after 48 h storage in EDTA and Citrate tubes, but not in preservative-containing tubes. Notably, AR-V7 expression was detected in prostate cancer patient blood samples after 48 h storage in EDTA tubes at room temperature. This important finding presents opportunities for measuring AR-V7 expression from clinical trial patient samples processed within 48 h—a much more feasible timeframe compared to previous recommendations.

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          Most cited references25

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          Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer.

          A method for enumerating circulating tumor cells (CTC) has received regulatory clearance. The primary objective of this prospective study was to establish the relationship between posttreatment CTC count and overall survival (OS) in castration-resistant prostate cancer (CRPC). Secondary objectives included determining the prognostic utility of CTC measurement before initiating therapy, and the relationship of CTC to prostate-specific antigen (PSA) changes and OS at these and other time points. Blood was drawn from CRPC patients with progressive disease starting a new line of chemotherapy before treatment and monthly thereafter. Patients were stratified into predetermined Favorable or Unfavorable groups ( or =5 CTC/7.5mL). Two hundred thirty-one of 276 enrolled patients (84%) were evaluable. Patients with Unfavorable pretreatment CTC (57%) had shorter OS (median OS, 11.5 versus 21.7 months; Cox hazard ratio, 3.3; P 26 to 9.3 months). CTC are the most accurate and independent predictor of OS in CRPC. These data led to Food and Drug Administration clearance of this assay for the evaluation of CRPC.
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            Circulating tumor cells in patients with breast cancer dormancy.

            The purpose of this study was to test the hypothesis that circulating tumor cells (CTCs) are present in patients many years after mastectomy without evidence of disease and that these CTCs are shed from persisting tumor in patients with breast cancer dormancy. We searched for CTCs in 36 dormancy candidate patients and 26 age-matched controls using stringent criteria for cytomorphology, immunophenotype, and aneusomy. Thirteen of 36 dormancy candidates, 7 to 22 years after mastectomy and without evidence of clinical disease, had CTCs, usually on more than one occasion. Only 1 of 26 controls had a possible CTC (no aneusomy). The statistical difference of these two distributions was significant (exact P = 0.0043). The CTCs in patients whose primary breast cancer was just removed had a half-life measured in 1 to 2.4 hours. The CTCs that are dying must be replenished every few hours by replicating tumor cells somewhere in the tissues. Hence, there appears to be a balance between tumor replication and cell death for as long as 22 years in dormancy candidates. We conclude that this is one mechanism underlying tumor dormancy.
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              Molecular analysis of circulating tumour cells-biology and biomarkers.

              Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test-and readily amenable to serial sampling-have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch(®) system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial-mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                12 May 2017
                May 2017
                : 18
                : 5
                : 1047
                Affiliations
                [1 ]Centre for Circulating Tumour Cell Diagnostics and Research, Ingham Institute for Applied Medical Research, 1 Campbell St., Liverpool, NSW 2170, Australia; alisonluk@ 123456gmail.com (A.W.S.L.); yafeng.ma@ 123456unsw.edu.au (Y.M.); Pei.Ding@ 123456sswahs.nsw.gov.au (P.N.D.); francis.young@ 123456student.unsw.edu.au (F.P.Y.); P.DeSouza@ 123456westernsydney.ed u.au (P.d.S.)
                [2 ]Department of Medical Oncology, Liverpool Hospital, Elizabeth St & Goulburn St, Liverpool, NSW 2170, Australia; Wei.Chua2@ 123456sswahs.nsw.gov.au (W.C.); Bavanthi.Balakrishnar@ 123456sswahs.nsw.gov.au (B.B.)
                [3 ]Western Sydney University Clinical School, Elizabeth St, Liverpool, NSW 2170, Australia
                [4 ]South Western Clinical School, University of New South Wales, Goulburn St., Liverpool, NSW 2170, Australia
                [5 ]Tokai Pharmaceuticals, Inc., 255 State Street, 6th Floor, Boston, MA 02109, USA; dan@ 123456siamab.com
                Author notes
                [* ]Correspondence: t.becker@ 123456unsw.edu.au ; Tel.: +61-2-873-89033
                [†]

                Current address: Siamab Therapeutics, 90 Bridge Street, Suite 100, Newton, MA 02458, USA.

                Article
                ijms-18-01047
                10.3390/ijms18051047
                5454959
                28498319
                38739ca4-388c-4b73-92a6-7e8201084bc0
                © 2017 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 31 March 2017
                : 08 May 2017
                Categories
                Article

                Molecular biology
                circulating tumour cell,biomarker,androgen receptor,ar-v7,droplet digital polymerase chain reaction (ddpcr),blood storage tube

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