ORIGINAL RESEARCH published: 24 May 2022 doi: 10.3389/fonc.2022.825703 Cell Senescence-Related Pathways Are Enriched in Breast Cancer Patients With Late Toxicity After Radiotherapy and Low Radiation- Induced Lymphocyte Apoptosis Ester Aguado-Flor 1, Marı́a J. Fuentes-Raspall 2, Ricardo Gonzalo 3, Carmen Alonso 4, Teresa Ramón y Cajal 4, David Fisas 4, Alejandro Seoane 5, Álex Sánchez-Pla 3,6, Jordi Giralt 7,8, Orland Dı́ez 1,9 and Sara Gutiérrez-Enrı́quez 1* 1 Hereditary Cancer Genetics Group, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Edited by: Barcelona, Spain, 2 Radiation Oncology Department, Santa Creu i Sant Pau Hospital, Barcelona, Spain, 3 Statistics and Francesca Ballarini, Bioinformatics Unit, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona University of Pavia, Italy Hospital Campus, Barcelona, Spain, 4 Medical Oncology Department, Santa Creu i Sant Pau Hospital, Barcelona, Spain, Reviewed by: 5 Medical Physics Department, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Aidan D. Meade, Spain, 6 Genetics, Microbiology and Statistics Department, Universitat de Barcelona, Barcelona, Spain, 7 Radiation Oncology Technological University Dublin, Department, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain, 8 Radiation Ireland Oncology Group, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain, Dmitry Klokov, 9 Area of Clinical and Molecular Genetics, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, Institute of Radioprotection and Barcelona, Spain Nuclear Safety, France *Correspondence: Sara Gutiérrez-Enrı´quez Background: Radiation-induced late effects are a common cause of morbidity among sgutierrez@vhio.net cancer survivors. The biomarker with the best evidence as a predictive test of late reactions is the radiation-induced lymphocyte apoptosis (RILA) assay. We aimed to Specialty section: investigate the molecular basis underlying the distinctive RILA levels by using gene This article was submitted to Radiation Oncology, expression analysis in patients with and without late effects and in whom we had also a section of the journal first identified differences in RILA levels. Frontiers in Oncology Received: 30 November 2021 Patients and Methods: Peripheral blood mononuclear cells of 10 patients with late Accepted: 01 April 2022 severe skin complications and 10 patients without symptoms, selected from those Published: 24 May 2022 receiving radiotherapy from 1993 to 2007, were mock-irradiated or irradiated with Citation: 8 Gy. The 48-h response was analyzed in parallel by RILA assay and gene expression Aguado-Flor E, Fuentes-Raspall MJ, Gonzalo R, Alonso C, Ramón y Cajal T, profiling with Affymetrix microarrays. Irradiated and non-irradiated gene expression Fisas D, Seoane A, Sánchez-Pla Á, profiles were compared between both groups. Gene set enrichment analysis was Giralt J, Dı´ez O and Gutiérrez- Enrı´quez S (2022) Cell Senescence- performed to identify differentially expressed biological processes. Related Pathways Are Enriched in Results: Although differentially expressed mRNAs did not reach a significant adjusted p- Breast Cancer Patients With Late Toxicity After Radiotherapy value between patients suffering and not suffering clinical toxicity, the enriched pathways and Low Radiation-Induced indicated significant differences between the two groups, either in irradiated or non- Lymphocyte Apoptosis. Front. Oncol. 12:825703. irradiated cells. In basal conditions, the main differentially expressed pathways between doi: 10.3389/fonc.2022.825703 the toxicity and non-toxicity groups were the transport of small molecules, interferon Frontiers in Oncology | www.frontiersin.org 1 May 2022 | Volume 12 | Article 825703 Aguado-Flor et al. Radiotherapy-Induced Late Skin Toxicity signaling, and transcription. After 8 Gy, the differences lay in pathways highly related to cell senescence like cell cycle/NF-kB, G-protein-coupled receptors, and interferon signaling. Conclusion: Patients at risk of developing late toxicity have a distinctive pathway signature driven by deregulation of immune and cell cycle pathways related to senescence, which in turn may underlie their low RILA phenotype. Keywords: radiotherapy—adverse effects, radiation-induced lymphocyte apoptosis (RILA), gene set enrichment analysis (GSEA), late skin toxicity, breast cancer INTRODUCTION assay in radiotherapy-induced fibrosis was validated within the PHRC2005 (NCT00893035) prospective multicenter French study Radiotherapy (RT) is commonly given to breast cancer (BC) (21). However, cell mechanisms determining a low level of RILA are patients after surgery, and it has been shown to be effective in not well known. reducing recurrence and lowering BC mortality (1). Studies have We aimed to shed light on the molecular pathogenesis of low suggested that RT can benefit up to 83% of BC patients (2) and that RILA levels by characterizing RNA expression using microarrays breast-conserving surgery followed by whole-breast RT can provide on blood-derived cells for BC patients with and without fibrosis local control and survival rates equivalent to mastectomy (3–5). and/or telangiectasia induced by RT and in whom we had also However, early and late side effects caused by RT can negatively demonstrated different levels in RILA (20). Our study reveals affect patients’ quality of life (6). Late or chronic effects are typically that gene expression differences between patients with and expressed after latent periods of months to years and are highly without late toxicity are driven by deregulation of immune and relevant as they tend to be irreversible or even progressive in severity cell cycle pathways related to senescence, which may underlie the (7, 8). Chronic toxicity in BC patients mainly comprises radiation- low RILA phenotype. induced fibrosis, skin shrinkage or induration, atrophy, vascular damage, neural damage, lymphedema, myocardial infarction, secondary cancers, and a range of endocrine- and growth-related MATERIALS AND METHODS effects (6, 9). Treatment regimens are designed to ensure that the risk of severe late effects does not exceed 5%–10% (10). This means Patient Selection that a small fraction of radiosensitive patients imposes limits on the Twenty BC patients were selected from those prospectively dose that can be given to the entire patient population, although the receiving RT between 1993 and 2007 in the Hospital de la majority of patients could potentially tolerate a higher dose (11). A Santa Creu i Sant Pau of Barcelona. Ten patients with fibrosis more effective approach would be adjusting the treatment according and/or telangiectasias were matched according to treatment, to patient and tumor radiosensitivity. In non-sensitive patients, a demographic, and tumor characteristics with 10 patients with dose escalation could be considered, while in sensitive patients, no late reactions (Tables 1, 2). Patients were given whole breast interventions such as an alternative treatment (surgery), toxicity RT with conventional fractionation, 50 Gy, 2 Gy/fraction with reduction RT techniques (e.g., partial breast RT), therapeutic (with 60 Co unit. An additional tumorectomy electron boost (9– radioprotectors or radiation mitigators), and the omission of 16 MeV) from a linear accelerator was given, with doses postoperative RT in patients with a low risk for tumor recurrence ranging from 14 to 20 Gy. The dose was 20 Gy at 85% isodose. might be considered to reduce their risk of side effects (11–13). Planning was based on 2D simulation, including verification and Although the relation to BC radiotoxicity of clinical factors such quality assurance. The occurrence of acute and late effects of RT as smoking history, breast volume, additional boost delivery, and was monitored and documented by the physician during the diabetes (14, 15) is well-founded, it is hypothesized that standard patient follow-up for at least 6 years after RT, using the constitutional genetic factors are, in addition, key determinants of Radiation Therapy Oncology Group (RTOG) toxicity grading the wide interpersonal variability observed in clinical practice (16). system (22) (Table 1). We determined RILA values in these Accordingly, over the past two decades, there has been an increasing patients showing that those with late RT toxicity exhibited interest in deciphering the germline genetic susceptibility reduced apoptosis, and these findings were published elsewhere underlying radiation-induced toxicity after RT, with studies (20). The ethical committee of the hospital approved the study focused on common genetic variants through candidate gene or and all women participating signed an informed consent. genome-wide association studies (16–18). Likewise, different cellular and DNA markers after in-vitro irradiation of fibroblasts Collection of Samples, Irradiation, and lymphocytes have been tested to know if their response to and Incubation radiation is related to clinical toxicity (19). Some investigations Experiments for the RILA assay and microarray analysis were [including our work (20)] have shown that late toxicity due to RT is carried out in parallel with blood samples being collected significantly associated with low levels of radiation-induced between 2009 and 2010 (Table 1). For the microarray study, lymphocyte apoptosis (RILA) using 8 Gy of in-vitro irradiation peripheral blood mononuclear cells (PBMCs) were isolated using and an incubation time of 48 h. The predictive value of the RILA Lymphoprep™ (STEMCELL Technologies, Vancouver, Canada) Frontiers in Oncology | www.frontiersin.org 2 May 2022 | Volume 12 | Article 825703 Aguado-Flor et al. Radiotherapy-Induced Late Skin Toxicity TABLE 1 | Acute and late toxicity in the analyzed breast cancer patients [adapted from (20)]. Group Patient Acute toxicity (evaluated Late toxicity (evaluated >3 Year of end of Last year of Year of blood ID ≤3 months after months up to at least 6 years after radiotherapy toxicity confirmation extraction for RILA/ radiotherapy) radiotherapy) (years of follow-up) microarray studies Without late normal tissue reactions SP-13 Epithelitis None 2003 2014 (11) 2010 SP-18 None None 2005 2014 (9) 2010 SP-12 Erythema None 2000 2014 (14) 2010 SP-4 Epithelitis None 1999 – 2009 SP-16 Dermatitis None 2004 2014 (10) 2010 SP-7 None None 2002 2011 (9) 2009 SP-11 Erythema None 1994 2011 (17) 2010 SP-19 Erythema None 2004 2013 (9) 2010 SP-8 Epithelitis grade 2 None 1994 2013 (19) 2010 SP-6 Epithelitis grade 3 None 2002 2012 (10) 2009 With late normal tissue reactions SP-10 Erythema Telangiectasia grade 1 2004 2013 (9) 2010 SP-2 None Telangiectasia grade 2 bilateral and 1994 2012 (18) 2009 fibrosis grade 2 in right breast SP-1 Erythema and epithelitis Telangiectasia grade 2 and fibrosis 2005 2013 (8) 2009 grade 2 SP-5 Erythema Telangiectasia grade 1 2004 2011 (7) 2009 SP-14 Erythema and moist epithelitis Fibrosis grade 3 2007 2013 (6) 2010 grade 3 SP-17 Erythema Fibrosis grade 3 2002 2011 (9) 2010 SP-20 Erythema Telangiectasia grade 2 and fibrosis 2002 2012 (10) 2010 grade 3 SP-9 Erythema and edema Fibroadenoma grade 1 2006 2012 (6) 2010 SP-3 Erythema Telangiectasia grade 1 1998 2010 (12) 2009 SP-15 Epithelitis grade 2 in each Telangiectasia and fibrosis grade 3 in 1993 2012 (19) 2010 bilateral breast cancer each bilateral breast cancer TABLE 2 | Clinical and demographic characteristics of the analyzed patients [adapted from (20)]. Characteristic Non-toxicity group (n = 10) Toxicity group (n = 10) Age at radiotherapy (mean ± SD) 45.70 ± 10.01 52.80 ± 8.60a Age at study (mean ± SD) 55.20 ± 8.01 61.60 ± 8.63a BMI (mean ± SD) 25.42 ± 3.41 (n = 5) 26.90 ± 6.21a (n = 4) Smoking status Non-smoker 50% (n = 8) 70%b Current smoker 12.5% (n = 8) 10%b Former smoker 37.5% (n = 8) 20%b Histological type Invasive ductal 70% 80%b Invasive lobular 10% 0%b In situ 10% 10% Other 10% (mixed ductal and lobular) 10% (tubule-lobular carcinoma) Adjuvant treatment Neoadjuvant chemotherapy 10% 0%b None 10% 10%b Chemotherapy 0% 10%b, c Hormone therapy 20% 50%b Chemotherapy + hormone therapy 60% 20%b Radiotherapy Basic irradiation dose 50 Gy (n = 9), 46 Gy (n = 1) 50 Gy Boost dose 14–20 Gy 16–20 Gy Total prescribed dose Gy (mean ± SD) 65.80 ± 0.63a 67.00 ± 1.69a Boost with brachytherapy 10% 10% Radiotherapy concomitant to chemotherapy 30% 10%b a No statistically significant differences between both groups: t-test. b No statistically significant differences between frequencies of both groups: Yates’ corrected chi-square test. c One of them was also treated with a monoclonal antibody as adjuvant therapy. Frontiers in Oncology | www.frontiersin.org 3 May 2022 | Volume 12 | Article 825703 Aguado-Flor et al. Radiotherapy-Induced Late Skin Toxicity density gradient medium from whole blood. Two PBMC cultures Gene Set Enrichment Analysis were freshly set up for each of the 20 patients (with an average of Gene set enrichment analysis (GSEA) (version 3.0, Broad 8.5 × 106 cells in 20 ml of RPMI 1640 supplemented with 20% Institute http://software.broadinstitute.org/gsea/index.jsp) (26) fetal bovine serum): i) one culture was treated with 8 Gy at room was used to identify biological terms, pathways, and processes temperature at a dose rate of 3 Gy/min with 6 MV photon beam, that were up- or downregulated within each pairwise and ii) one culture was the control sham-irradiated. Both comparison. The genes obtained from the array were ranked cultures were then placed into a 37°C incubator with 5% CO2. using the logFC from the differential expression results. This The irradiation with 8 Gy was done at the same time for the ranked list was then used to perform a GSEA using the Reactome RILA assay and microarray cultures. gene sets database. The overlap between nodes was visualized by the generation of an enrichment map. The map was constructed Microarray Assay with an FDR cutoff of <0.05, except for the comparison in basal After 48 h of incubation (the same time as that used for the RILA conditions of patients with toxicity vs. non-toxicity, where the assay), total RNA was isolated using TRIzol isolation reagent FDR cutoff was <0.25. R statistical software v4.03 and (Invitrogen, Thermo Fisher Scientific, Waltham, MS, USA); see Bioconductor packages were used for the analysis. Finally, we Supplementary Information for more details on RNA searched the pathways in the Reactome website (www.reactome. purification. Gene expression was measured using an Affymetrix org) to label the clusters to broadly account for functions of GeneChip™ Human Exon 1.0 ST Array (Affymetrix, Thermo constitutive gene sets. Fisher Scientific, Santa Clara, CA, USA) for each RNA sample. One microgram of total RNA was used as starting material and Quantitative RT-PCR Validation ribosomal RNA was first removed using the RiboMinus Human/ To validate the microarray expression data, reverse Mouse Transcriptome Isolation Kit (Invitrogen, Thermo Fisher transcription-quantitative PCR (RT-qPCR) was performed Scientific, Waltham, MS, USA). Following the manufacturer’s using TaqMan probes (Applied Biosystems, Thermo Fisher instructions, treated RNA was then converted to cDNA and Scientific, Waltham, MS, USA) targeting selected genes as well subsequently processed and labeled, to detect the transcripts by as reference controls to normalize data. For a more detailed RT- hybridization onto the Exon arrays. After hybridization, each array qPCR methodology, see Supplementary Information. Paired t- was washed and stained according to the standard Affymetrix test was applied for statistical analysis, using GraphPad Prism 6 protocol. Finally, the stained array was scanned using an software. To deal with multiple testing issues, p-values were Affymetrix GeneChip Scanner 3000, and the signals were adjusted using the Benjamini and Hochberg method (25). processed by the GeneChip Operating System for each array. Statistical Analysis to Detect Differentially RESULTS Expressed Genes Raw expression values were obtained directly from .CEL files and Basal Expression: Toxicity vs. Non-Toxicity were preprocessed using the Robust Multiarray Average method Patients in Non-Irradiated Samples (23). Prior to any analysis, data were submitted to non-specific In basal non-irradiated cultures, no differentially expressed genes filtering to remove low signal genes and low variability genes. The with adjusted p <0.05 were found between the 10 patients with selection of differentially expressed genes between conditions was late skin toxicity and the 10 without (Supplementary Table 1). based on a linear model analysis with empirical Bayes moderation of The data visualization through heatmaps showed that mRNA the variance estimates following the methodology developed by expression clusters 13 of the 20 non-irradiated PBMC samples Smyth (24), by using the limma package from Bioconductor. To into two distinct groups depending on whether they are BC deal with multiple testing issues, p-values were adjusted to obtain patients with (left) or without (right) RT-induced late toxicity, strong control over the false discovery rate (FDR) using the except for patient SP-18 (Figure 1A). A third group (middle) of Benjamini and Hochberg method (25). seven patients did not discriminate between toxicity statuses. The lists of genes differentially expressed were generated for We performed a GSEA with the whole list of ranked genes, the following comparisons: since the analysis at the pathway level may be more biologically meaningful than considering individual genes, even if the i. Basal expression: toxicity vs. non-toxicity patients in non- changes in gene expression levels are minor or go undetected. irradiated samples In this case, 16 marginally significant enriched pathways were ii. 8 Gy-induced expression1: toxicity vs. non-toxicity patients observed with an adjusted p = 0.1117 (Supplementary Table 2). in irradiated samples The enrichment map shows that the two largest differentially iii. Overall in-vitro radiation response: irradiated vs. non- expressed gene clusters in toxicity patients compared irradiated samples, in all patients regardless of toxicity with normal responders are an overexpressed transport of small status and separately for each of the two groups molecules and an underexpressed interferon signaling, both with six Heatmaps for both basal and each radiation response comparisons were also generated. R statistical software v4.03 1 Net values were obtained by subtracting baseline RNA expression levels from and Bioconductor packages were used for all analyses. those obtained after irradiation. Frontiers in Oncology | www.frontiersin.org 4 May 2022 | Volume 12 | Article 825703 Aguado-Flor et al. Radiotherapy-Induced Late Skin Toxicity A B FIGURE 1 | Heatmap and GSEA of basal non-irradiated cells comparing patients with late skin toxicity to normal responders. (A) A different expression pattern between patients with toxicity (dark blue) and without toxicity (green) of baseline expressed genes with non-adjusted p-value < 0.05 is suggested by hierarchical clustering classification of patients (columns). (B) The enrichment map allows the visualization of the general expression landscape of the toxicity group compared with the non-toxicity group in basal conditions. The key is shown in the bottom right of the figure. The map is constructed with an FDR cutoff of <0.25. Each node corresponds to a gene set from the GSEA; its size represents the number of genes in the gene set and its color the p-value. The thickness of the lines connecting the nodes is proportional to the number of genes that overlap in the gene set. Clustering among the various gene sets is visualized with boxes over the nodes. Those sets that are upregulated in patients with toxicity are shown in red and those downregulated are shown in blue. CT, control non-irradiated sample in toxicity patients; CNT, control non-irradiated sample in non-toxicity patients; GSEA, gene set enrichment analysis. nodes, followed by an underexpression of transcription and most PBMC samples clustered into two distinct groups (non- lymphocyte costimulation clusters (Figure 1B). irradiated or irradiated), except for patients SP-11, SP-12, and SP-20 (Figure 3A). After clustering the differentially expressed 8 Gy-Induced Expression: genes between non-irradiated and irradiated cells of the 20 patients (Supplementary Table 6), the enrichment map Toxicity vs. Non-Toxicity Patients showed that the main significant upregulated pathway was in Irradiated Samples DNA repair with 22 nodes followed by nucleotide excision No differentially expressed genes were found with an adjusted repair and downregulated adaptive immune system with two p <0.05 when comparing irradiation-induced expression levels nodes each (Figure 3B). between the 10 patients with late skin toxicity and the 10 without To characterize the expression profile after in-vitro irradiation (Supplementary Table 3). The heatmap of the 8 Gy-induced for each group of patients, we generated separate lists of expression showed that most of the patients were grouped differentially expressed genes according to toxicity group according to their toxicity status (Figure 2A). After clustering (Supplementary Tables 7, 8). The RT-qPCR showed that the list of genes in pathways, significant differences could be APOBEC3H was significantly upregulated in both groups of observed (Supplementary Table 4). The main overrepresented patients (Supplementary Figure 2). Interestingly, the gene sets in late toxicity patients were an upregulated cell cycle/ heatmaps generated from each group of patients indicated that NF-kB cluster and interferon signaling pathway together with the clustering of expressed genes in non-irradiated and irradiated underexpressed signaling by the G-protein-coupled receptor cells was different between normal responders and sensitive (GPCR) cluster (Figure 2B). patients (Supplementary Figure 3). In line with these results, the map of significant enriched pathways was different between Overall In-Vitro Radiation Response: the two groups, with patients without side effects having two Irradiated vs. Non-Irradiated Samples clusters of pathways downregulated not present in the toxicity Microarray analysis of the 20 donors regardless of their toxicity group (Supplementary Figure 4). Looking at the common single status indicated that in-vitro irradiation led to a differential pathways that are differentially expressed after irradiation in the expression of 753 genes with an adjusted p <0.05 toxicity and non-toxicity patients, 107 pathways were found (Supplementary Table 5). RT-qPCR results validated the (Supplementary Tables 9, 10). On the contrary, 15 and 17 overexpression of APOBEC3H (adjusted p = 0.0002) pathways were unique to the non-toxicity and toxicity groups, (Supplementary Figure 1). A hierarchical clustering analysis respectively, mainly related to NF-kB and interferon signaling depicted in a heatmap showed that the mRNA expression of (Figures 4A, B). Frontiers in Oncology | www.frontiersin.org 5 May 2022 | Volume 12 | Article 825703 Aguado-Flor et al. Radiotherapy-Induced Late Skin Toxicity A B FIGURE 2 | Heatmap and GSEA of 8 Gy-induced expression comparing patients with late skin toxicity to normal responders. (A) A different expression pattern between patients without toxicity (orange) from those with toxicity (purple) of 8 Gy-induced expressed genes with non-adjusted p-value <0.05 is suggested by hierarchical clustering classification of patients (columns). (B) The enrichment map allows the visualization of the general expression landscape induced by 8 Gy of the toxicity group compared with the non-toxicity group. The key is shown in the bottom right of the figure. The map is constructed with an FDR cutoff of <0.05. Each node corresponds to a gene set from the GSEA; its size represents the number of genes in the gene set and its color the p-value. The thickness of the lines connecting the nodes is proportional to the number of genes that overlap in the gene set. Clustering among the various gene sets is visualized with boxes over the nodes. Those sets that are upregulated in patients with toxicity are shown in red and those downregulated are shown in blue. NetT, 8 Gy-induced expression in toxicity patients; NetNT, 8 Gy-induced expression in non-toxicity patients; GSEA, gene set enrichment analysis. A B FIGURE 3 | Heatmap and GSEA of overall in-vitro radiation response comparing irradiated to non-irradiated samples in all patients. (A) A clear different expression pattern is observed between non-irradiated (non-toxicity in green and toxicity in dark blue) and 8 Gy-irradiated (non-toxicity in yellow and toxicity in pink) PBMC samples. Hierarchical clustering of the differentially expressed genes with non-adjusted p <0.05 and absolute logFC >0.5 almost perfectly separates the two experimental conditions. (B) The enrichment map allows the visualization of the general expression landscape of the irradiated compared with the non-irradiated PBMCs. The key is shown in the bottom right of the figure. The map is constructed with an FDR cutoff of <0.05. Each node corresponds to a gene set from the GSEA; its size represents the number of genes in the gene set and its color the p-value. The thickness of the lines connecting the nodes is proportional to the number of genes that overlap in the gene set. Clustering among the various gene sets is visualized with boxes over the nodes. Those sets that are upregulated in irradiated samples are shown in red and those downregulated are shown in blue. Abs, absolute; CNT, control non-irradiated sample in non-toxicity patients; 8 Gy NT, 8 Gy-irradiated culture in non-toxicity patients; CT, control non-irradiated sample in toxicity patients; 8 Gy T, 8 Gy-irradiated culture in toxicity patients; PBMCs, peripheral blood mononuclear cells; GSEA, gene set enrichment analysis. Frontiers in Oncology | www.frontiersin.org 6 May 2022 | Volume 12 | Article 825703 Aguado-Flor et al. Radiotherapy-Induced Late Skin Toxicity A B FIGURE 4 | Involved pathways in cell response to 8 Gy comparing irradiated to non-irradiated samples in the toxicity and non-toxicity patients. (A) Gene sets exclusively downregulated (blue) and upregulated (red) in the non-toxicity group after 8 Gy. (B) Gene sets exclusively downregulated (blue) and upregulated (red) in the toxicity group after 8 Gy. NES, normalized enrichment score. DISCUSSION (Figure 1B). Landmark-Høyvik et al. (28) compared the whole blood basal gene expression of BC patients that developed The purpose of this study was to determine whether late clinical fibrosis after RT to those that did not, and they found toxicity after BC RT and a low level of RILA is associated with an differentially expressed pathways such as transcription and altered transcriptional profile of PBMCs. The analyzed patients intercellular transport/localization (28), which were also found were well defined by their toxicity and RILA status and were deregulated in our study in basal conditions (Figure 1B). In selected from a larger cohort treated with RT between 1993 and contrast, we found other differentially expressed pathways not 2007 with an average follow-up of 11 years (Tables 1, 2) (20). reported in these last two studies such as interferon signaling, Our comparison of the resulting microarray data for patients keratinization, and neutrophil degranulation (Figure 1B). with late toxicity vs. patients without shows that, after correcting Rødningen et al. found 60 differentially expressed genes after for multiple testing, no individual gene met the threshold for irradiation in fibroblast cultures from 31 BC patients with statistical significance. This may be because expression variable risk of radiation-induced fibrosis which were involved differences are modest relative to the possible noise resulting in processes like apoptosis, extracellular matrix remodeling/cell from a heterogeneous cell fraction included in PBMCs and to the adhesion, proliferation, and ROS scavenging (29). As opposed to intrinsic heterogeneity in patient samples. In contrast, our their study, we saw different responses between the toxicity and pathway analysis indicates a statistically significant enrichment non-toxicity groups after irradiation in the cell cycle/NF-kB of certain biological mechanisms that would contribute to cluster, signaling by GPCR, and interferon (Figure 2B). toxicity and low RILA levels. Variations in cell type, microarray platform, in-vitro Other microarray studies have been conducted in recent years irradiation protocol, RT protocol, and statistical strategy could to characterize baseline or irradiated expression profiles using influence the final outcome and explain why different molecular lymphocytes, whole blood, or skin fibroblasts from cancer pathways are highlighted between different reports. patients with adverse reactions in normal tissue after RT, and Our GSEA highlights a role in late toxicity for interferon all of these studies support that gene expression profiles might signaling both in basal and irradiated PBMCs, a finding not discriminate patients at risk [reviewed in (19, 27)]. In some cases, previously reported in similar studies (Figures 1B, 2B). patient classification was improved by considering the Interferons are key signaling molecules for innate and adaptive differential expression of functionally related genes belonging immunity also implicated in cell senescence with a complex to the same pathway, which might be more significant than the interplay (30). It has been described that there is an upregulation activity of individual genes and can also lead to a more relevant of interferon signaling in senescent cells and that the DNA biological interpretation of the results (27). damage process engages cell senescence through interferon Regarding BC studies, Henrı́quez Herná ndez et al. (18) signaling (31). Moreover, it is known that ionizing radiation identified 26 genes whose basal expression in lymphocytes can induce senescence in normal cells, which promotes normal segregated patients suffering severe late skin toxicity from tissue fibrosis and organ dysfunction [reviewed in (32, 33)]. A patients who did not. Their enrichment pathway analysis growing body of evidence supports that senescence may be at the shows similar pathways identified to those we have seen in our origin of radiation-induced pulmonary and skin fibrosis (31). study for basal conditions like T-cell receptor signaling, Likewise, we also found an overrepresentation of upregulated transcription, and lipid/cholesterol metabolism (18) NF-kB nodes in the group of patients with late skin toxicity in Frontiers in Oncology | www.frontiersin.org 7 May 2022 | Volume 12 | Article 825703 Aguado-Flor et al. Radiotherapy-Induced Late Skin Toxicity irradiated conditions (Figures 2B, 4B). After radiation-induced Amundson’s consensus list of radioresponsive genes (46–48) DNA damage, ATM protein activates DNA damage response such as CDKN1A, GADD45A, DDB2, XPC, and PCNA pathways which lead to the stimulation of NF-kB that induces (Supplementary Table 11). As expected, the biggest cluster of senescence-associated secretory phenotype (SASP) pro- differentially expressed pathways was DNA repair (Figure 3B), inflammatory expression including IL-1a/b, IL-6, TGF-b, which is consistent with previous studies (48, 49). Identifying TNF-a, and fibroblast growth factor, among others [reviewed this radiation signature was reassuring and supported in (31, 32)]. microarray experiment conditions and derived findings. In the irradiated PBMCs, we also observed a significant In conclusion, although the results indicate that patients with overrepresentation of a downregulated GPCR signaling and without toxicity share common cell responses, the enriched pathway in patients suffering toxicity (Figure 2B). GPCRs are pathways are different between both groups for basal and in-vitro involved in signal transduction and play important roles in irradiation conditions. The resulting pathways associated with nearly every physiological process including DNA damage late toxicity (such as interferon signaling, NF-kB, and signaling response and cell senescence (34, 35). Interestingly, a by GPCR) support a key role of cell senescence in RT-induced functional enrichment analysis of genes to detect loci for acute late toxicity and low RILA phenotype. post-RT pain in 1,112 BC patients showed that one of the most significant enriched biological processes was olfactory receptor activity, which are members of GPCR signaling (36). Thus, the co-occurrence of our findings in irradiated PBMC DATA AVAILABILITY STATEMENT of patients with toxicity of an upregulated NF-kB and interferon Microarray data for this study can be found in NCBI Gene signaling process as well as a downregulated GPCR pathway (all Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under known to be connected with cell senescence) might not be the accession number GSE178708. accidental and suggest their importance for RT-induced late toxicity phenotype. Our cohort of patients with late toxicities is characterized by a low level of RILA after 48 h post-irradiation with 8 Gy (20). A ETHICS STATEMENT time course RNA expression analysis adding for instance a 24-h The studies involving human participants were reviewed and post-irradiation time point would have led to a more approved by the Ethical Committee for Clinical Research Santa comprehensive profile. However, previous published evidence Creu i Sant Pau Hospital. The patients/participants provided showed that the correlation between low RILA values and their written informed consent to participate in this study. patients who did develop radiation-induced toxicity is seen both at 24 and 48 h post-irradiation (20, 37–40). Likewise, other reports point to an increase in radiation-induced apoptosis with radiation dose and time of incubation after in- AUTHOR CONTRIBUTIONS vitro irradiation in both sensitive and non-sensitive individuals (41, 42), which indicates that the apoptosis levels seen at 48 h EA-F, MF-R, CA, AS, OD, and SG-E participated in the would not be preceded by higher apoptosis at previous conception of the study. EA-F, MF-R, CA, TR, DF, AS, ASP, time points. and SG-E contributed to the data collection. Statistical analysis Our results of an overrepresentation of processes highly and interpretation of data were executed by EA-F, RG, ASP, and involved in senescence suggest that it would be interesting to SG-E. OD and SG-E contributed to supervision. SG-E further investigate its implication for low RILA levels shown by contributed to the study management. The first draft of the patients with late toxicity. However, it should be considered that manuscript was written by EA-F. EA-F, OD, and SG-E reviewed we quantified a bulk gene expression derived from a variety of and edited the manuscript. JG and SG-E acquired funding and cells included in the PBMC fraction (43), while a correlation of resources. All authors read the manuscript and approved the late toxicity with decreased values of RILA has been shown only final version. in T and B lymphocytes (20, 44, 45). In humans, the frequency of the fraction of comprised cells in PBMC populations varies across individuals, but typically, lymphocytes are in the range FUNDING of 70%–90%, monocytes from 10% to 20%, while dendritic cells are rare, accounting for only 1%–2% (43). The frequencies of cell This work has been funded by the Instituto de Salud Carlos III types within the lymphocyte population include 70%–85% CD3+ (ISCIII), an initiative of the Spanish Ministry of Economy and T cells, 5%–10% B cells, and 5%–20% NK cells (43). In Innovation partially supported by the European Regional consequence, the largest fraction of PBMCs corresponds to T Development FEDER Funds, Grant/Award Number: PI05/ lymphocytes, and therefore, the gene expression profile observed 2181. SG-E was supported by the ISCIII Miguel Servet II might be a reflection of this majority cell fraction. Program (CP16/00034). EA-F was supported by the When studying the effect of radiation in all patients, we saw ERAPerMed JTC2018, grant numbers: ERAPERMED2018-244 that irradiation of PBMCs led to the induction of Paul and and SLT011/18/00005. Frontiers in Oncology | www.frontiersin.org 8 May 2022 | Volume 12 | Article 825703 Aguado-Flor et al. Radiotherapy-Induced Late Skin Toxicity ACKNOWLEDGMENTS SUPPLEMENTARY MATERIAL The authors acknowledge the Cellex Foundation for providing The Supplementary Material for this article can be found online research facilities and equipment. 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(Springer, Cham) (2015) p. 161. doi: 10.1007/978-3- Fisas, Seoane, Sánchez-Pla, Giralt, Dıez ́ and Gutieŕ rez-Enrıquez. ́ This is an open- 319-16104-4_15 access article distributed under the terms of the Creative Commons Attribution 44. Bordó n E, Henrı́aquez-Herná ndez LA, Lara PC, Pinar B, Rodrı́aguez-Gallego License (CC BY). The use, distribution or reproduction in other forums is permitted, C, Lloret M. Role of CD4 and CD8 T-Lymphocytes, B-Lymphocytes and provided the original author(s) and the copyright owner(s) are credited and that the Natural Killer Cells in the Prediction of Radiation-Induced Late Toxicity in original publication in this journal is cited, in accordance with accepted academic Cervical Cancer Patients. Int J Radiat Biol (2011) 87:424–31. doi: 10.3109/ practice. No use, distribution or reproduction is permitted which does not comply with 09553002.2010.537433 these terms. Frontiers in Oncology | www.frontiersin.org 10 May 2022 | Volume 12 | Article 825703