RESUMO
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.
Assuntos
Biomarcadores Tumorais/sangue , Preservação de Sangue/efeitos adversos , Coleta de Amostras Sanguíneas/efeitos adversos , Equipamentos Descartáveis/normas , Receptores Androgênicos/sangue , Biomarcadores Tumorais/normas , Preservação de Sangue/instrumentação , Preservação de Sangue/normas , Coleta de Amostras Sanguíneas/instrumentação , Coleta de Amostras Sanguíneas/normas , Estudos de Casos e Controles , Linhagem Celular Tumoral , Citratos/química , Ácido Edético/química , Molécula de Adesão da Célula Epitelial/sangue , Feminino , Humanos , Masculino , Células Neoplásicas Circulantes/metabolismo , Plásticos/efeitos adversos , Plásticos/química , Neoplasias da Próstata/sangue , Fatores de TempoRESUMO
INTRODUCTION: Intravesical delivery of chemotherapy agents is used very commonly for the treatment of superficial bladder cancer. We recently completed a phase II study of intravesical gemcitabine in which an alkaline adjusted gemcitabine preparation was used initially, based on very early phase I studies. However, crystallization was noted in some of the pre-prepared syringes, which prompted us to investigate the conditions under which gemcitabine crystallized. METHODS: Gemcitabine was prepared in syringes in triplicate and conditions were varied with respect to pH, temperature, and duration. Samples were observed for up to 48 h for the development of crystallization. High-performance liquid chromatography analysis of gemcitabine concentrations was undertaken for all samples. RESULTS: Crystallization of gemcitabine was favored under conditions of bicarbonate treatment and lowering of temperature. However, the process was reversible, as demonstrated by recovery of gemcitabine concentrations in samples brought back to room temperature. Crystallization resulted in reduction of gemcitabine concentrations in the pre-prepared syringes. CONCLUSIONS: Gemcitabine solutions may be associated with crystallization if the native pH is increased with the addition of sodium bicarbonate, and samples are stored in a cold environment.