A Pilot Clinical Study on the Prognostic Relevance of Plasmatic Exosomes Levels in Oral Squamous Cell Carcinoma Patients

Abstract

Background: To evaluate the relationship between the plasmatic CD63 and CAV1 positive exosome levels, in patients with OSCC before and after surgical treatment and to correlate it with their overall survival.

Methods: A double-blind pilot study over 10 patients OSCC and T4 stage without distant metastases or local bone invasion has been performed. The average follow-up period was 37.64 months (34.3⁻40.84). We obtained 2 plasma tubes of 1 mL each before surgery and 7 days after surgery. Before performing the immunocapture-based analysis, EVs (Extracellular Vesicles) were isolated from the plasma and characterized with western blot analysis.

Results: Mean values of CD63 positive plasmatic exosomes (EXO-CD63) after surgery decreased from 750.88 ± 286.67 to 541.71 ± 244.93 (p = 0.091). On the other hand, CAV-1 positive plasmatic exosomes (EXO-CAV-1) increased after surgery from 507 ± 483.39 to 1120.25 ± 1151.17 (p = 0.237). Patients with EXO-CD63 levels lower than the mean global value before the surgery had a survival of 36.04 months compared with the group with EXO-CD63 higher than the average who only survived 12.49 ± 1.67 months from the diagnosis, p = 0.225. When EXO-CAV-1 levels before surgery was lower than the average (813.94 ± 801.21) overall survival was 24.69 ± 22.23 months in contrast when it was higher that was only 11.64 months, p = 0.157. Patients with lower EXO-CD63 levels after surgery lived an average of 23.84 ± 23.9 months, while those with higher plasmatic levels of EXO-CD63 live 13.35 months, p = 0.808. When EXO-CAV-1 levels after surgery were lower, the average overall survival was 20.344 ± 15.40 months, in contrast when the EXO-CAV-1 levels were higher showing rather an estimate survival expectation of 1.64 months.

Conclusions: Surgical treatment induced a dramatic reduction of the plasmatic levels of exosomes expressing CD63 as early as 1 week after resection. This first result suggests that the tumour mass is responsible of the high levels of circulating exosomes detected in cancer patients. At the same time point exosome expressing CAV-1 increased, possibly due to the inflammatory reaction immediately after surgery. Lastly, statistical analysis showed that lower levels of plasmatic exosomes both before and after surgery correlated with a better life expectancy of OSCC patients. Hopefully, this approach will prove useful in the clinical follow-up of cancer patients.

Keywords: CAV-1; CD63; OSCC; exosomes; oral squamous cell carcinoma; overall survival.

Figure 1

Protein quantification and characterization by Western blot analysis for housekeeping markers of exosomes. Western blot analyses of tumour susceptibility gene 101 (Tsg101). HD1 and 2 are healthy donors; PT 1 and 2 are two patients.

Figure 2

Box plot shows how CD63 positive plasmatic exosomes decrease after surgery from 750.88 ± 286.67 to 541.71 ± 244.93 (p = 0.091).

Figure 3

The graph shows the cumulative survival of patients according to their average levels of exosomes with CD-63 (left) and CAV-1 (right) before resective surgery.

Figure 4

The graph shows the cumulative survival of patients according to their average levels of exosomes with CD-63 (left) and CAV-1 (right) after resective surgery.