Low Dose Radiation Therapy Induces Long-Lasting Reduction of Pain and Immune Modulations in the Peripheral Blood - Interim Analysis of the IMMO-LDRT01 Trial

Abstract

The treatment of chronic inflammatory and degenerative diseases by low dose radiation therapy (LDRT) is promising especially for patients who were refractory for classical therapies. LDRT aims to reduce pain of patients and to increase their mobility. Although LDRT has been applied since the late 19th century, the immunological mechanisms remain elusive. Within the prospective IMMO-LDRT01 trial (NCT02653079) the effects of LDRT on the peripheral blood immune status, as well as on pain and life quality of patients have been analyzed. Blood is taken before and after every serial irradiation with a single dose per fraction of 0.5Gy, as well as during follow-up appointments in order to determine a detailed longitudinal immune status by multicolor flow cytometry. Here, we report the results of an interim analysis of 125 patients, representing half the number of patients to be recruited. LDRT significantly improved patients' pain levels and induced distinct systemic immune modulations. While the total number of leukocytes remained unchanged in the peripheral blood, LDRT induced a slight reduction of eosinophils, basophils and plasmacytoid dendritic cells and an increase of B cells. Furthermore, activated immune cells were decreased following LDRT. Especially cells of the monocytic lineage correlated to LDRT-induced improvements of clinical symptoms, qualifying these immune cells as predictive biomarkers for the therapeutic success. We conclude that LDRT improves pain of the patients by inducing systemic immune modulations and that immune biomarkers could be defined for prediction by improved patient stratification in the future.

Keywords: chronic degenerative and inflammatory diseases; immune status; immunophenotyping; low dose radiation therapy (LDRT); subjective pain level; x-rays.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KL declared a past co-authorship with authors UG and BF to the handling editor.

Copyright © 2021 Donaubauer, Becker, Weissmann, Fröhlich, Muñoz, Gryc, Denzler, Ott, Fietkau, Gaipl and Frey.

Figures

Figure 1

Study design of the prospective IMMO-LDRT01 trial. Examinations are performed before and after the first series of irradiation (tp 1 and 2), before and after the second series of irradiation (tp 3 and 4), as well as for the follow-up appointment three months later (tp 5). The blood drops indicate the time points of the blood withdrawals. Patients fill out questionnaires on life quality and pain levels at the same time points.

Figure 2

Overview of the analyzed immune cell subpopulations. The scheme presents all nine main immune cell types (neutrophilic, eosinophilic and basophilic granulocytes, monocytes, NK cells, NKT cells, T cells, B cells and DCs) that were identified by the expression of their specific pan marker (big arrows). Those main immune cells were subdivided by analyzing the expression of further surface markers (next to each cell). Finally, all cells are analyzed for the expression of specific activation markers (CD25, CD69, CD80, CD86, PD-L1, HLA-DR, CTLA-4, PD-1), as indicated in the boxes.

Figure 3

LDRT induces a significant pain reduction. The patients scored their pain level on a visual analogue scale (VAS) ranging from 0 (no pain) to 10 (worst pain imaginable) at the indicated time points (tp). A Spearman correlation and a simple linear regression were performed in order to correlate the pain levels of all patients to the time points (A–C). For the graphical illustration, the mean values of the pain levels were calculated for all five tp. (A) shows the correlation of the mean general pain level, (B) shows the correlation of the pain under load and (C) shows the correlation of the morning stiffness to the study tp (n = 100; ****p < 0.0001). (D) shows a box plot for the retrospective determination of the pain level before therapy and at least six months after therapy. A Wilcoxon test was used for the statistical analysis in graph (D) (n = 125; ****p < 0.0001).

Figure 4

LDRT modulates numbers of certain peripheral immune cells. In order to determine peripheral blood immune cell numbers, immunophenotyping was performed with patient’s blood. Blood withdrawals were performed before (tp 1) and after serial irradiation (tp 2). The absolute immune cell numbers were determined by flow cytometry. The Wilcoxon test was used for the statistical analysis. In the figure the immune cell numbers of the blood leukocytes (A), the eosinophilic and basophilic granulocytes (B, C), the plasmacytoid dendritic cells (D) and the B cells (E) are presented. (n = 125; *p < 0.05; **p < 0.01 ****p < 0.0001).

Figure 5

LDRT decreases the percentage of activated immune cells. The activation state of immune cells in the peripheral blood was determined by measuring the expression of distinct activation markers. Blood withdrawals were performed before (tp1) and after the serial irradiation (tp2). The expression levels of common activation markers were determined by flow cytometry. The Wilcoxon test was used for the statistical analysis. In the figure the percentage of CD25 positive and HLA-DR positive monocytes (A, B), as well as the percentage of PD1 positive T Helper cells (C) is presented. (n = 125; *p < 0.05; **p < 0.01).

Figure 6

Cells of the monocytic lineage predict the therapeutic outcome. Absolute immune cell numbers were determined by flow cytometry before and after every serial irradiation, as well as for the follow-up appointment. At the same time points, the patients scored their subjective pain level in different situations on a VAS. A Spearman correlation was performed to assess the relationship between the immunological variables and the pain related variables. A simple linear regression was performed afterwards. For the graphical illustration, the mean values of the immune cell numbers were calculated and plotted. In (A), the mean numbers of myeloid DCs (mDCs) are correlated to the general pain level and in (B) those numbers are correlated to the morning stiffness. In (C) the mean number of type 1 monocytes and in (D) the mean number of type 3 monocytes are correlated to the morning stiffness (n = 100; **p < 0.001; ***p < 0.001).