Intact interferon signaling in peripheral blood leukocytes of high-grade osteosarcoma patients

Abstract

High-grade osteosarcoma has a poor prognosis with an overall survival rate of about 60 percent. The recently closed European and American Osteosarcoma Study Group (EURAMOS)-1 trial investigates the efficacy of adjuvant chemotherapy with or without interferon-α. It is however unknown whether the interferon-signaling pathways in immune cells of osteosarcoma patients are functional. We studied the molecular and functional effects of interferon treatment on peripheral blood lymphocytes and monocytes of osteosarcoma patients, both in vivo and ex vivo. In contrast to other tumor types, in osteosarcoma, interferon signaling as determined by the phosphorylation of signal transducer and activator of transcription (STAT)1 at residue 701 was intact in immune cell subsets of 33 osteosarcoma patients as compared to 19 healthy controls. Also, cytolytic activity of interferon-α stimulated natural killer cells against allogeneic (n = 7 patients) and autologous target cells (n = 3 patients) was not impaired. Longitudinal monitoring of three osteosarcoma patients on interferon-α monotherapy revealed a relative increase in the CD16-positive subpopulation of monocytes during treatment. Since interferon signaling is intact in immune cells of osteosarcoma patients, there is a potential for indirect immunological effects of interferon-α treatment in osteosarcoma.

Fig. 1

PBMCs of osteosarcoma patients are normally responsive to IFN-α. a Flow cytometric evaluation of basal levels of phosphorylated STAT1 in immune cell subsets shows slightly lower levels of phosphorylated STAT1 in NK cells of osteosarcoma patients at diagnosis (n = 33) as compared to healthy donors (n = 19). b Following an in vitro stimulus with 1,000 IU/mL IFN-α, hyperphosphorylation of B and NK cells of osteosarcoma patients was observed. For both a and b, error bars denote standard error of the mean; student’s t-test P value <0.05 noted as *; <0.01 = **. c Lysis of the allogeneic NK cell target K562 by PBMCs of healthy donors (HD) and osteosarcoma patients (OS) increased significantly following overnight culture in 100 IU/mL IFN-α (IFN-α +). Effector to target (E:T) ratio was corrected for percentage of NK cells. Lysis was similar by healthy donor and osteosarcoma patient derived cells. Wilcoxon signed ranked test; P value <0.05 noted as *. d Following overnight culture of PBMCs in IFN-α, percentage-specific lysis correlated with fold change in pSTAT1 in NK cells (P value 0.06, Spearman r 0.52)

Fig. 2

Effect of in vivo IFN-α administration on monocytes and NK cells. a Following in vivo treatment with IFN-α of three osteosarcoma patients, the percentage of monocytes of PBMCs decreased. b The relative percentage of CD16-positive monocytes increased. c NK cells which had been exposed to IFN-α in vivo showed less phosphorylation of STAT1 in response to ex vivo IFN-α stimulation. d Cytolysis of the autologous osteosarcoma target cell line L2635 by PBMCs of patient 398 did not change significantly during in vivo treatment with IFN-α (solid lines). Following overnight culture in 100 IU/mL IFN-α, lysis by PBMCs collected at all time points increased (dashed lines). Effector to target (E:T) ratio was corrected for the percentage of NK cells. Error bars denote standard error of the mean of experiment performed in triplicate