Transcriptional Inflammatory Signature in Healthy Donors and Different Radiotherapy Cancer Patients

Abstract

Cancer and ionizing radiation exposure are associated with inflammation. To identify a set of radiation-specific signatures of inflammation-associated genes in the blood of partially exposed radiotherapy patients, differential expression of 249 inflammatory genes was analyzed in blood samples from cancer patients and healthy individuals. The gene expression analysis on a cohort of 63 cancer patients (endometrial, head and neck, and prostate cancer) before and during radiotherapy (24 h, 48 h, ~1 week, ~4-8 weeks, and 1 month after the last fraction) identified 31 genes and 15 up- and 16 down-regulated genes. Transcription variability under normal conditions was determined using blood drawn on three separate occasions from four healthy donors. No difference in inflammatory expression between healthy donors and cancer patients could be detected prior to radiotherapy. Remarkably, repeated sampling of healthy donors revealed an individual endogenous inflammatory signature. Next, the potential confounding effect of concomitant inflammation was studied in the blood of seven healthy donors taken before and 24 h after a flu vaccine or ex vivo LPS (lipopolysaccharide) treatment; flu vaccination was not detected at the transcriptional level and LPS did not have any effect on the radiation-induced signature identified. Finally, we identified a radiation-specific signature of 31 genes in the blood of radiotherapy patients that were common for all cancers, regardless of the immune status of patients. Confirmation via MQRT-PCR was obtained for BCL6, MYD88, MYC, IL7, CCR4 and CCR7. This study offers the foundation for future research on biomarkers of radiation exposure, radiation sensitivity, and radiation toxicity for personalized radiotherapy treatment.

Keywords: biomarkers; cancer patients; gene expression; human blood; inflammation; radiotherapy.

Figure 1

The study design of the 63 cancer patients and 23 healthy donors involved in this study and the experimental pathway of nCounter analysis, bioinformatic analysis, and MQRT-PCR validation.

Figure 2

Bioinformatical analysis of health donors’ gene expression profiles. (A) Normal variability of inflammatory genes. nCounter expression analysis of human inflammation genes in 4 healthy human blood donors (H20, H61, H64, and H65) repeated 3 times at 3-week intervals. Unsupervised clustering and Spearman correlation as the distance method were performed on the z-score normalized data, and a complete hierarchical clustering method was used. The negative row z-score (below −2) is shown in dark blue, and the positive row z-score (above +2) is shown in dark red. (B) Scatterplot showing a comparison of expression changes in the blood from 7 healthy donors treated ex vivo with LPS (y-axis) and in the blood from 7 healthy donors who received a flu vaccine (x-axis). The thresholds obtained in the analysis of the Gaussian mixture model are shown in the figure with a dashed line. Up-regulated genes are shown in light grey, with similar responses shown in dark grey. N represents the number of genes in each category, and the percentage of all genes in a given group is provided in parentheses. The uniform dispersion of points suggests no significant difference between expressions from different samples. (C) UMAP analysis of nCounter inflammatory expression in human healthy donor controls vs cancer patient samples taken before radiotherapy. The preexposure samples of each cancer type (10 endometrium, 31 head and neck, and 22 prostate) and control samples from 12 healthy donors were used. For the collected nCounter data, a batch effect correction was performed. The first 54 PCA components, which explained about 99% of the observed variance, were selected for UMAP-based dimensionality reduction. Each point represents a different sample.

Figure 3

MQRT-PCR expression of the genes (A) BCL6, (B) MYD88, (C) MYC, (D) IL7, (E) CCR4, and (F) CCR7 in the whole blood of endometrial cancer patients (blue), head and neck cancer patients (orange), and prostate cancer patients (red) preexposure, 24 h after the 1st fraction and 48 h after the 1st fraction, 5th/6th fraction (~1 week), and last fraction (4–8 weeks) and 1 month after the end of the treatment. The box plot shows expression analysed in 10 endometrial cancer patients, 31 head and neck cancer patients, and 11 prostate cancer patients treated with IMRT. Expression levels are normalised to HPRT. Statistical analysis was performed on log-transformed data. * Significantly different (t-test, p < 0.05).

Figure 4

MQRT-PCR expression of the genes (A) BCL6, (B) MYD88, (C) MYC, (D) IL7, (E) CCR4, and (F) CCR7 in the whole blood of prostate cancer patients undergoing IMRT (pink) and CK (red) radiotherapy treatment taken preexposure, 24 h after the 1st fraction, 5th/6th fraction (~1 week), and last fraction (4–8 weeks) and 1 month after the end of treatment. The box plot shows expression analysed in 11 IMRT and 11 CK prostate cancer patients. Expression levels are normalised to HPRT. Statistical analysis was performed on log-transformed data. * Significantly different (p < 0.05).

Figure 5

Patient radiotherapy timeline and blood collection. The section in grey illustrates the CyberKnife prostate cancer patients not included in bioinformatics analysis. MCSI refers to the Maria Sklodowska-Curie Institute and FNHK refers to Fakultní Nemocnice Hradec Králové.