The purpose of this pilot study was to determine whether blood-borne microvesicles from newly diagnosed glioblastoma patients could be used as biomarkers. Researchers at UPENN collected 2.8 mL blood from 16 post-operative patients at the time that they were being simulated for chemoradiation therapy (radiation with concurrent temozolomide). Two additional samples were collected during chemoradiation therapy and a final sample was collected at the end of chemoradiation therapy. Patients continued with the therapy suggested by their physicians, based on tumor conference consensus and were followed for recurrence and overall survival.
Microvesicles were isolated using serial centrifugation and stained for surface markers (Annexin V for phosphotidyl serine, CD41 for platelets, anti-EGFR for tumor cells, and CD235 for red blood cells). Flow cytometry analysis was performed. The researchers findings provide initial evidence that increases in Annexin V positive microvesicle levels during chemoradiation therapy are associated with earlier recurrence and shorter overall survival in newly diagnosed glioblastoma patients. The effect is dramatic, with over a four-fold increase in the hazard ratio for an individual at the 75th versus the 25th percentile. Moreover the pattern of Annexin V positive microvesicles remain significant after adjustment for confounding clinical variables that have previously been shown to be prognostic for recurrence and survival. Inclusion of neutrophil levels at the start of chemoradiation therapy in the model yielded the largest attenuation of the observed association. Further studies will be needed to verify and further investigate the association between these two entities.
Examples of data in two patients with opposite trends in number of AnnV + MV
Blood samples were taken before, twice during and at the end of CRT. The y-axis is the raw number of MV as recorded by FCM. This value can be multiplied by the dilution factor, 50 and then by ≈2 to account for plasma versus whole blood, in order to estimate the number of MV/mL in blood. Graphs include the timing of critical clinical events (*). Note that numerical value of the axes are not the same in both patients. Patient 1, (a) has a decreasing slope during CRT and had a recurrence at 580 days and was alive at 818 days. Patient 7, (b) has an increasing slope with first recurrence at 199 days and an overall survival of 274 days