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

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.

SUBMITTER: Luk AWS

PROVIDER: S-EPMC5454959 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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

Luk Alison W S AWS Ma Yafeng Y Ding Pei N PN Young Francis P FP Chua Wei W Balakrishnar Bavanthi B Dransfield Daniel T DT Souza Paul de P Becker Therese M TM

International journal of molecular sciences 20170512 5

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 preserv ...[more]

PMID: 28498319

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Project description:BackgroundWe previously showed that pretreatment detection of androgen receptor splice variant-7 (AR-V7) in circulating tumor cells (CTCs) from men with castration-resistant prostate cancer is associated with resistance to abiraterone and enzalutamide, but not to taxane chemotherapies. Here, we conducted serial measurements of AR-V7 and evaluated patterns of longitudinal AR-V7 dynamics over the course of multiple sequential therapies.Patients and methodsMetastatic prostate cancer patients treated at Johns Hopkins with AR-directed therapies or taxane chemotherapies underwent serial liquid biopsies for CTC-based AR-V7 analysis at baseline, during therapy, and at progression. We used a CTC enrichment platform followed by multiplexed reverse-transcription polymerase chain reaction analysis to detect full-length androgen receptor and AR-V7 transcripts. Patients selected for inclusion in this report were those who provided ≥4 CTC samples, at least one of which was AR-V7 positive, over the course of ≥2 consecutive therapies.ResultsWe identified 14 patients who received a total of 37 therapies and contributed 70 CTC samples for AR-V7 analysis during a median follow-up period of 11 months. Three patients remained AR-V7 positive during the entire course of therapy. The remainder underwent transitions in AR-V7 status: there were eight instances of 'conversions' from AR-V7-negative to -positive status (during treatment with first-line androgen deprivation therapy, abiraterone, enzalutamide, and docetaxel), and six instances of 'reversions' from AR-V7-positive to -negative status (during treatment with docetaxel and cabazitaxel).ConclusionsAR-V7 is a dynamic marker, and transitions in AR-V7 status may reflect selective pressures on the tumor exerted by therapeutic interventions. While 'conversions' to AR-V7-positive status were observed with both AR-directed therapies and taxane chemotherapies, 'reversions' to AR-V7-negative status only occurred during taxane therapies. Serial blood-based AR-V7 testing is feasible in routine clinical practice, and may provide insights into temporal changes in tumor evolution.

Project description:BackgroundThe emergence of constitutively active androgen receptor (AR) splice variant AR-V7 poses a formidable challenge in treating prostate cancer, as it lacks the ligand binding region targeted by androgen-deprivation therapies such as enzalutamide and abiraterone. AR-V7 is critical for castration-resistant prostate cancer (CRPC) development and progression; however, the molecular mechanisms regulating its expression and biological function remain poorly understood. Here, we investigate the role of IGF2BP2 in regulating AR-V7 expression and CRPC progression.MethodsTo determine the clinical relevance of IGF2BP2 in CRPC, we analyzed the mRNA expression data for prostate cancer patients available in the Genomic Data Commons (GDC) Data Portal and cBioPortal. Next to investigate the role of IGF2BP2 in regulating AR-V7 expression and enzalutamide resistance, we performed shRNA-mediated IGF2BP2 knockdown and overexpression experiments followed by qRT-PCR, immunoblot, colony-formation, and MTT assays. Finally, we performed RIP-qPCR, actinomycin-D, and IGF2BP2 domain-deletion analysis to study the mechanism by which IGF2BP2 regulates AR-V7 stability, expression, and enzalutamide resistance in CRPC cells.ResultsOur analysis revealed that IGF2BP2 is upregulated in CRPC patients and its expression positively correlates with increasing Gleason score in patients with CRPC. We demonstrate that IGF2BP2 silencing leads to downregulation of AR-V7 and its downstream target genes without affecting AR levels. Additionally, IGF2BP2 knockdown also enhances the sensitivity of CRPC cells to enzalutamide while overexpression increases AR-V7 expression and confers increased resistance to enzalutamide. Mechanistically, our experiments demonstrate that IGF2BP2 binds to the intronic splicing enhancer (ISE) region of AR-V7, thereby enhancing its mRNA stability. Furthermore, our domain-deletion analysis pinpoints the role of KH3 and KH4 domains of IGF2BP2 in regulating AR-V7 stability and enzalutamide resistance.ConclusionsTaken together, our findings suggest that IGF2BP2 plays a critical role in regulating AR-V7 expression and stability, offering a novel target for developing therapeutic interventions for CRPC.

Dataset Information

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

Publications

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

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