Serum Heat Shock Protein Levels and Cytokine Profiles Are Altered In AML Patients Before Treatment and Following Disease Stabilizing Therapy,
Authors: Fredly Hanne , Reikvam Håkon , Gjertsen Bjorn T. , Bruserud Øystein 
Published: October 2019

Abstract
Abstract 3599
Heat shock proteins (HSPs) maintain cellular homeostasis and function as molecular chaperones assisting protein folding and translocation. Their intracellular levels are up-regulated in response to a wide variety of insults allowing the cell to survive otherwise lethal conditions. Heat shock proteins are also released to the extracellular space and lead to a more efficient cross-presentation to specific T cells and may also initiate release of proinflammatory cytokines, stimulate NK cells and facilitate DC maturation. Extracellular Heat shock protein (HSP) 70 and HSP90 are characteristics of immunogenic apoptosis, and primary human acute myeloid leukemia (AML) cells release these mediators during spontaneous in vitro apoptosis. Primary AML cells also show constitutive release of several cytokines; these heterogeneous mediators interact through local cytokine networks that regulate growth and differentiation of normal as well as malignant hematopoietic cells. In the present study we investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving disease-stabilizing treatment based on all-trans retinoic acid (ATRA) plus valproic acid.
Serum samples from 82 untreated AML patients, including 42 patients receiving AML-stabilizing therapy, and 20 healthy controls were analysed. HSP and cytokine levels were determined by ELISA and multiplex analyses.
Patients with untreated AML showed significantly increased HSP90 levels compared to healthy controls (Mann Whitney U-test, p<0.0001). The serum levels of HSP70 were significantly lower than the HSP90 levels both for the patients and for the healthy controls (p<0.0001), but the HSP70 serum levels did not differ significantly between patients and controls (Fig. 1A). HSP70 and HSP90 serum levels were significantly correlated for the AML patients (Fig. 1B, Spearman, p<0.01, R=0.425). HSP70 levels were not altered during AML-stabilizing palliative treatment, whereas HSP90 levels decreased during treatment with ATRA, valproic acid and low-dose cytarabine. High age and low differentiation of AML cells according to the FAB classification predicted lower HSP90 levels.Fig. 1Fig. 1.
AML patients showed an altered serum cytokine profile compared to healthy controls with significantly altered levels of several chemokines, interleukins, growth factors and immunomodulatory cytokines (Tab.1). These cytokine levels were usually increased compared to the healthy controls. Hierarchical cluster analysis of the untreated patients showed a close association between HSP70, HSP90 and Hepatocyte growth factor (HGF) levels, these mediators also showed a significant correlation (Spearman, p<0.01). Furthermore, disease-stabilizing therapy altered the serum cytokine profile, but the profiles differed when ATRA + valproic acid was combined with either theophyllin or low-dose cytarabine. The close association between HSP70, HSP90 and HGF levels was then maintained only in the cytarabine group.Tab.1.CytokinePatientsControlsP-valueIMMUNOMODULATORY CYTOKINES    TNF-α↑8.4 (<1.5–68.5)4.5 (2.3–13.4)0.016INTERLEUKINS    IL-1α↓0.5 (<0.4–7.0)2.0 (2.0–2.9)<0.0001    IL-4↓<4.5 (<4.5–53.9)11.8 (5–17.5)<0.0001    IL-5↑<0.7 (<0.7–39.1)0.9 (<0.7–0.9)0.0001    IL-6↑5.8 (<1.1–204.6)<1.1 (<1.1–7.7)<0.0001    IL-10↑1.3 (<0.3–238.0)0.3 (<0.3–0.8)0.0005CHEMOKINES    CXCL5↓128.4 (<4.1–3252)1283.5 (341–5307)<0.0001    CXCL8/IL-8↑30.5 (<2.0–1103)10.7 (2.2–22.8)0.0003    CXCL10↑56.7 (0.5–1958)15.25 (8.5–58.4)<0.0001GROWTH FACTORS    HGF↑797.3 (1–9703)262.4 (117.3–729.3)<0.0001    GM-CSF↑0.5 (<2.0–17.2)0.9 (0.9–3.6)0.0096
We conclude that both HSP levels and serum cytokine profiles are altered in untreated AML, and the modulation of these serum levels by AML-stabilizing chemotherapy may reflect a possible mechanism for the clinical effect of this treatment strategy.
The table shows serum levels for 82 AML patients and 20 healthy controls where significant differences were found (Mann-Whitney U-test, p<0.02). The results are presented as the cytokine analyzed, the alteration in the patients (↑ increased, ↓ decreased), levels in patients and healthy controls (median and range). All cytokine concentrations are presented as pg/ml, for cytokine levels not detected the values are set to < the detection limit.

Disclosures:
No relevant conflicts of interest to declare.