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Capsular inflammation after immediate breast reconstruction – Gene expression patterns and inflammatory cell infiltration in irradiated and non-irradiated breasts

  • A. Frisell
    Affiliations
    Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

    Dermatology, Theme Inflammation and Infection, Karolinska University Hospital, Stockholm, Sweden
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  • O. Bergman
    Affiliations
    Division of Cardiovascular Medicine, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Bioclinicum J8:20, Visionsgatan 4, Stockholm, Sweden
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  • A. Khan
    Affiliations
    Department of Plastic Surgery, The Royal Marsden Hospital NHS Foundation Trust, London, UK
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  • A. Gisterå
    Affiliations
    Division of Cardiovascular Medicine, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Bioclinicum J8:20, Visionsgatan 4, Stockholm, Sweden
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  • R.M. Fisher
    Affiliations
    Division of Cardiovascular Medicine, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Bioclinicum J8:20, Visionsgatan 4, Stockholm, Sweden
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  • J. Lagergren
    Affiliations
    Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

    Department of Surgery, Capio St. Göran's Hospital, Stockholm, Sweden
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  • J. de Boniface
    Affiliations
    Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

    Department of Surgery, Capio St. Göran's Hospital, Stockholm, Sweden
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  • M. Halle
    Correspondence
    Corresponding author at: Reconstructive Plastic Surgery, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.
    Affiliations
    Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

    Department of Reconstructive Plastic Surgery, Karolinska University Hospital, Stockholm, Sweden
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Open AccessPublished:October 15, 2022DOI:https://doi.org/10.1016/j.bjps.2022.10.011

      Summary

      Background

      Capsular contracture following post-mastectomy radiotherapy (PMRT) is commonly seen in patients undergoing implant-based immediate breast reconstruction (IBR). Further understanding of the underlying biology is needed for the development of preventive or therapeutic strategies. Therefore, we conducted a comparative study of gene expression patterns in capsular tissue from breast cancer patients who had received versus those who had not received PMRT after implant-based IBR.

      Methods

      Biopsies from irradiated and healthy non-irradiated capsular tissue were harvested during implant exchange following IBR. Biopsies from irradiated (n = 13) and non-irradiated (n = 12) capsules were compared using Affymetrix microarrays to identify the most differentially regulated genes. Further analysis using immunohistochemistry was performed in a subset of materials to compare the presence of T cells, B cells, and macrophages.

      Results

      Enrichment testing using Gene Ontology (GO) analysis revealed that the 227 most differentially expressed genes were mainly involved in an inflammatory response. Twenty-one GO biological processes were identified [p < 0.05, false discovery rate (FDR) < 5%], several with B-cell-associated inflammation. Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) analysis identified macrophages as the most common inflammatory cell type in both groups, further supported by immunostaining of CD68. Radiation remarkably increased B-cell infiltration in the capsular region of biopsies, as quantified by immunostaining of CD20 (p = 0.016).

      Conclusions

      Transcript analysis and immunohistochemistry revealed inflammatory responses in capsular biopsies regardless of radiotherapy. However, the radiation response specifically involved B-cell-associated inflammatory responses.

      Keywords

      Introduction

      Radiotherapy (RT) is an important part of adjuvant treatment for breast cancer because it significantly reduces the risk of local recurrence and death.
      • Kurtz J.
      The curative role of radiotherapy in the treatment of operable breast cancer.
      It does, however, also affect surrounding tissues such as underlying muscle and subcutaneous fat, which has detrimental consequences in breast reconstruction. The effects of RT involve chronic inflammatory changes, tissue remodelling, and fibrosis.
      • Stone H.B.
      • Coleman C.N.
      • Anscher M.S.
      • McBride W.H.
      Effects of radiation on normal tissue: Consequences and mechanisms.
      The tissue response can be substantial, resulting in capsular contracture around the implant, impaired cosmetic outcome, psychological distress, and pain.
      • Sheehan Joanne
      • Kerry A
      • Sherman T.L.
      • JB
      Association of information satisfaction, psychological distress and monitoring coping style with post-decision regret following breast reconstruction.
      In severe cases, the implant may have to be removed and replaced with a new implant after capsular revision or the breast may need to be re-reconstructed with autologous tissue. A Swedish study has estimated the 5-year failure rate in breast cancer patients who underwent implant-based reconstruction to be 28.2% for preoperatively and 25.2% for postoperatively irradiated patients (p < 0.001).
      • Eriksson M.
      • Anveden L.
      • Celebioglu F.
      • Dahlberg K.
      • Meldahl I.
      • Lagergren J.
      • et al.
      Radiotherapy in implant-based immediate breast reconstruction: Risk factors, surgical outcomes, and patient-reported outcome measures in a large Swedish multicenter cohort.
      The aetiology of capsular contracture is multifactorial, and additional factors, such as placement of the implant (submuscular versus prepectoral), implant surface texture, bacterial colonization, and seroma/haematoma, may affect the outcome.
      • Calobrace M.B.
      • Stevens W.G.
      • Capizzi P.J.
      • Cohen R.
      • Godinez T.
      • Beckstrand M.
      Risk factor analysis for capsular contracture: A 10-year sientra study using round, smooth, and textured implants for breast augmentation.
      Potential explanations of the underlying aetiology of radiation-induced fibrosis as a consequence of, for example, genetic variation are as yet inconclusive.
      • Wei J.
      • Wang B.
      • Wang H.
      • Meng L.
      • Zhao Q.
      • Li X.
      • et al.
      Radiation-induced normal tissue damage: Oxidative stress and epigenetic mechanisms.
      We have previously described variation in gene expression patterns both in irradiated recipient vessels from autologous flap reconstructions
      • Halle M.
      • Christersdottir T.
      • Bäck M.
      Chronic adventitial inflammation, vasa vasorum expansion, and 5-lipoxygenase up-regulation in irradiated arteries from cancer survivors.
      and in adipose tissue in the irradiated breast,
      • Lindegren A.
      • Schultz I.
      • Sinha I.
      • Cheung L.
      • Khan A.A.
      • Tekle M.
      • et al.
      Autologous fat transplantation alters gene expression patterns related to inflammation and hypoxia in the irradiated human breast.
      but, to our knowledge, no study has described gene expression patterns in irradiated implant capsules.
      Ameliorating capsular inflammation to reduce capsular contracture has been proposed in an aesthetic setting of breast augmentation.
      • Lille S.
      • Jacoby J.
      The potential benefit of preemptive leukotriene inhibitor treatment to breast augmentation/mastopexy surgery.
      Studies done after aesthetic breast augmentation have shown that in the absence of irradiation, capsular contracture is associated with inflammatory cell recruitment, together with an increased expression of toll-like receptor 4
      • Segreto F.
      • Carotti S.
      • Tosi D.
      • Pendolino A.L.
      • Marangi G.F.
      • Morini S.
      • et al.
      Toll-Like Receptor 4 expression in human breast implant capsules: Localization and correlation with estrogen receptors.
      and cysteine leukotriene receptor 2
      • Grella E.
      • Grella R.
      • Siniscalco D.
      • Fuccio C.
      • Rossi F.
      • De Novellis V.
      • et al.
      Modification of cysteinyl leukotriene receptors expression in capsular contracture: Follow-up study and definitive results.
      in fibroblasts within the capsular tissue. Leukotriene inhibitors have been suggested as a prophylactic treatment to reduce the risk of capsular contracture in aesthetic surgery.
      • Graf R.
      • Ascenço A.S.K.
      • Freitas R.D.S.
      • Balbinot P.
      • Peressutti C.
      • Costa D.F.B.
      • et al.
      Prevention of capsular contracture using leukotriene antagonists.
      Interleukin 8 and metallopeptidase 4 have been suggested as potential key diagnostic and prognostic biomarkers.
      • Kyle D.J.T.
      • Harvey A.G.
      • Shih B.
      • Tan K.T.
      • Chaudhry I.H.
      • Bayat A
      Identification of molecular phenotypic descriptors of breast capsular contracture formation using informatics analysis of the whole genome transcriptome.
      However, most studies have been restricted to aesthetic breast augmentation as opposed to breast cancer reconstruction, where RT may further enhance inflammatory cell recruitment and fibrosis formation. Lipa et al. showed that the Wnt signalling pathway, previously shown to be involved in the pathogenesis of radiation-induced fibroproliferation, may play an important role in capsular contracture after expander breast reconstruction.
      • Lipa J.E.
      • Qiu W.
      • Huang N.
      • Alman B.A.
      • Pang CY
      Pathogenesis of radiation-induced capsular contracture in tissue expander and implant breast reconstruction.
      However, that study was limited to only 3–5 patients and did not evaluate gene expression patterns.
      Given the limitations of the present data, we took an unbiased approach to evaluate gene expression patterns in irradiated and non-irradiated breasts in the search for future therapeutic targets. The latter could be of paramount importance to reduce the risk of developing RT-induced capsular contracture in the increasing number of breast cancer patients who undergo immediate breast reconstruction (IBR).

      Materials and methods

      Human tissue specimens

      A total of 40 patients undergoing implant exchange in 44 breasts after previous implant-based IBR were included after receiving informed consent. Patients who had a non-standard irradiation dose or who had recently undergone implant exchange for other reasons were excluded. Capsular biopsies from irradiated (n = 23) and healthy non-irradiated (n = 23) tissues were harvested during implant exchange at two breast centres. A full-thickness 10 × 10 mm capsular biopsy was harvested from the lateral lower quadrant of the breast. Biopsies were divided into two parts and stored in Allprotect Tissue Reagent® (Qiagen, Hilden, Germany) at –80 C for RNA purification and in formalin for immunohistochemistry. No samples were pooled. All samples were registered at the Stockholm Medical Biobank (no. 914). The study was approved by the regional ethical review board at the Karolinska Institutet in Stockholm (2017/1504–31/2) and was performed in agreement with the institutional guidelines and the principles of the Declaration of Helsinki.

      RNA extraction

      RNeasy Lipid Tissue kit® (Qiagen) was used according to the manufacturer's protocol to extract RNA. The quality of the RNA was assessed by microcapillary electrophoresis using an Agilent Bioanalyzer® with RNA 6000 Pico Kit and Agilent 2200 TapeStation with RNA ScreenTape (Agilent, Santa Clara, CA, USA). The quantity of RNA was measured by ultraviolet spectrophotometry with a NanoDrop® ND-1000 UV–Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Whole-transcriptome expression analysis was performed using the GeneChip® WT Pico Reagent Kit (Affymetrix, Santa Clara, CA, USA) by processing each sample of the RNA from the total RNA (50 ng). This kit produces amplified and biotinylated sense-strand DNA targets for hybridization to Clariom D (human) arrays.

      Gene expression profiling

      Microarray analysis was performed by the core facility for Bioinformatics and Expression Analysis at the Karolinska Institutet. Affymetrix® Clariom D human oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA) were used for gene expression profiling. CEL files from scanning were processed in the Transcriptome Analysis Console (TAC) using the SST-RMA method. A gene list was created by extracting transcripts registered as encoded genes according to the National center for Biotechnology Information's database for gene-specific information Entrez Gene (http://www.ncbi.nlm.nih.gov/gene) and present in at least one of the groups. Irradiated biopsies (RT+) were compared with non-irradiated biopsies (RT-) to extract the genes that were most differentially expressed. Enrichment analysis of differentially expressed genes was performed using the Molecular Signatures Database (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp) to identify different levels of gene sets and pathways associated with the RT-responsive genes.

      Estimating the composition of immune cells

      To estimate the immune cell composition in the sample based on the RNA expression pattern and quantify the relative levels of distinct immune cell types within a complex gene expression mixture, the analytical platform Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) (https://cibersort.stanford.edu/) was used. Different cell types were also compared in the RT- and RT+ groups using immunohistochemistry.

      Immunohistochemistry

      The formalin-fixed tissues were histoprocessed in an automated tissue processing machine (VIP 3000, Miles Scientific) and embedded in paraffin. Then, 4-μm-thick formalin-fixed, paraffin-embedded sections were mounted on glass slides (Superfrost+, Thermo Scientific) and heated for 3 h at 56 °C. After deparaffinization in xylene and rehydration in alcohol, heat-induced epitope retrieval was performed using a Decloaking Chamber (Biocare Medical) set for 5 min at 110 °C in citrate buffer, pH 6 (Sigma C-9999). For quenching of endogenous peroxidase, a 30-minute incubation in 0.15% hydrogen peroxidase was performed at room temperature, followed by a 30-minute blocking step using 1% bovine serum albumin (BSA). Primary antibodies were diluted 1/2000 (CD3), 1/600 (CD20), and 1/1000 (CD68) in 1% BSA and incubated at +4 °C overnight in a humidity chamber. The secondary biotinylated antibody (Vector Laboratories) was diluted 1/200 and incubated for 30 min at room temperature. Then, it was incubated for 30 min using the avidin–biotin enzyme complex (Vectastain) Elite ABC kit (HRP, Vector Laboratories). For visualization, the peroxidase substrate DAB was used (ImmPACT DAB SK-4105, Vector Laboratories) for 3 min. The sections were counterstained in Mayer's haematoxylin for 1 min, followed by dehydration with graded alcohols, xylene, and coverslipped with Mountex. Eight biopsies (four from each group) were analysed using one section per biopsy. Three equally sized high-power fields (× 20) were randomly selected and oriented horizontally to the most intact region of the capsular surface at a depth of 200 µm below the surface. The number of CD3-, CD20-, and CD68-positive cells was independently counted by two blinded evaluators, and the results are summarized and presented as a mean value.

      Statistical analysis

      Group analysis was used to test the differences between the irradiated and non-irradiated groups. Expression levels in the irradiated and non-irradiated sample groups were compared using moderated t-tests as implemented in the BioConductor Limma package. Only genes assessed by TAC to be expressed in at least one of the treatment conditions, to contain protein-coding exons, and to have an entry in Entrez Gene were included in further analysis. Genes with a p-value < 0.05 were selected for further enrichment testing. Gene Ontology (GO) biological functions and Reactome gene sets with a corrected p-value < 0.05 and false discovery rate (FDR) < 5% were considered to be significantly overrepresented.

      Results

      Human tissue specimens

      A total of 44 patients were enroled, of whom 2 had bilateral biopsies from each breast. Biopsies from 25 patients met the criteria for RNA quality and quantity and were included in the gene expression analysis (Figure 1). The median (range) time from completion of RT to capsular biopsy was 41 (18–304) months, and the median radiation dose was 50 (46–50) Gy (Table 1). All implants were submuscular and textured, with Mentor CPG and Becker 35 expanders the most common models. Body mass index, age at the time of biopsy, implant size, and the number of surgical re-entries into the implant cavity (e.g. haematomas and abdominal advancement) and non-surgical complications (e.g. haematomas and seromas) were evenly distributed across the irradiated and non-irradiated groups. However, four more patients had previously undergone an implant exchange in the irradiated group, and three more patients had a prior infection in the non-irradiated group (Table 1).
      Figure 1
      Figure 1Flow chart of the patient cohort and the final cohort of biopsies included in the gene expression analysis; 13 irradiated and 12 non-irradiated biopsies and the immunohistochemistry analysis; 4 irradiated and 4 non-irradiated biopsies. *RIN, RNA integrity number, is an algorithm for assigning integrity values to RNA measurements.
      Table 1Pre- and postoperative information per breast (n = 25).
      Irradiated (n = 13)Non-irradiated (n = 12)P-value
      Textured implants
      Yes13 (100)12 (100)
      No00
      Previous infection< 0.001
      Yes1 (7.7)4 (33.3)
      No12 (92.3)8 (66.7)
      Number of previous implant exchanges0.114
      07 (53.8)10 (83.3)
      ≥16 (46.2)2 (16.7)
      Previous surgical complications
      Surgical complication was defined as post-surgical intervention entering the implant cavity.
      < 0.001
      Yes1 (7.7)1 (8.3)
      No12 (92.3)11 (91.7)
      Previous non-surgical complications
      Non-surgical complication was defined as seromas/haematomas.
      < 0.001
      Yes1 (7.7)2 (16.7)
      No12 (92.3)10 (83.3)
      Irradiation dose in Gy/total fractions
      Median (range).
      50/25 (46/23 – 50/25)N.A.
      Time in months from irradiation to biopsy
      Median (range).
      41 (18 – 304)N.A.
      Body mass index
      Median (range).
      25 (20 – 30)25 (19–28)0.316
      Age at biopsy
      Median (range).
      52 (33–67)51 (32 – 65)0.479
      Implant size in cc
      Median (range).
      400 (120 – 500)420 (195 – 565)0.392
      Values in parentheses are percentages unless indicated otherwise.
      low asterisk Median (range).
      ^ Surgical complication was defined as post-surgical intervention entering the implant cavity.
      ^^ Non-surgical complication was defined as seromas/haematomas.

      RNA extraction

      Biopsies from 23 irradiated and 23 non-irradiated breasts were collected for RNA extraction. RNA from 13 irradiated and 12 non-irradiated biopsies had an RNA integrity number greater than 5 and a yield of more than 100 ng and were selected for gene expression analysis.

      Gene expression

      Microarray experiments were conducted at two time points and were therefore subjected to a batch-control analysis showing a negligible temporal effect, which further validated the reproducibility of the experiment. Altogether 3422 transcripts, registered as encoded genes according to Entrez Gene, were present in at least one of the groups. A set of the most differentially expressed transcripts, with a raw p-value < 0.05, was selected for enrichment testing (n = 227). GO analysis showed that the selected radiation-responsive genes were mainly involved in inflammatory response among the top 21 identified GO biological processes (p < 0.05, FDR < 5%). Both innate and adaptive immune responses were represented. The top three GO biological processes identified were humoral immune response mediated by immunoglobulins, followed by complement activation and B-cell-mediated immunity (Table 2). In the Reactome gene sets analysis, scavenging of haem from plasma, C2, and C4 activators and binding/uptake of ligands by scavenger receptors were the most dysregulated (Table 3).
      Table 2Biological pathways showing the most significant alterations in gene expression (GO biological processes).
      SetsP-valueFDR-corrected P
      Humoral immune response mediated by circulating immunoglobulin5.27e-133.87e-09
      Complement activation5.53e-122.03e-08
      B-cell-mediated immunity2.99e-107.33e-07
      Humoral immune response4.34e-107.99e-07
      Lymphocyte-mediated immunity1.35e-081.85e-05
      Phagocytosis recognition1.51e-081.85e-05
      Phagocytosis9.74e-080.0001
      B-cell receptor signalling pathway1.13e-070.0001
      Membrane invagination2.19e-070.0001
      Regulation of humoral immune response2.57e-070.0001
      Positive regulation of B-cell activation4.37e-070.0002
      Adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains5.19e-070.0003
      Defence response to bacterium3.16e-060.0017
      FC receptor-mediated stimulatory signalling pathway5.15e-060.0027
      Regulation of B-cell activation6.38e-060.0031
      FC epsilon receptor signalling pathway2.12e-050.0097
      Cell recognition2.37e-050.0102
      Positive regulation of cell activation3.69e-050.0151
      Activation of immune response5.34e-050.0206
      adaptive immune response8.17e-050.0300
      leucocyte migration0.00010.0482
      Table 3Biological pathways showing the most significant alterations in gene expression (Reactome).
      SetsP-valueFDR-corrected P
      Scavenging of haem from plasma5.06e-097.58e-06
      Creation of C2 and C4 activators1.00e-077.49e-05
      Uptake/binding of ligands by scavenger receptors1.56e-077.84e-05
      Initial triggering of complement2.55e-079.58e-05
      CD22-mediated BCR regulation3.99e-070.0001
      FCGR activation1.05e-060.0002
      Role of LAT2 NTAL lab on calcium mobilization1.31e-060.0002
      Role of phospholipids in phagocytosis3.94e-060.0007
      Antigen activates B-cell receptor BCR leading to generation of second messengers5.65e-060.0007
      FCERI-mediated CAPLUS2 mobilization5.65e-060.0007
      Complement cascade5.73e-060.0007
      FCERI-mediated MAPK activation6.16e-060.0007
      Regulation of actin dynamics for phagocytic cup formation5.73e-050.0066
      Cell surface interactions at the vascular wall7.21e-050.0077
      FCERI-mediated NF KB activation0.00010.0155
      FCGAMMA receptor FCGR-dependent phagocytosis0.00020.0205

      Estimating immune cell composition

      We used CIBERSORT to estimate the immune cell composition of the 25 samples (irradiated and non-irradiated combined) and quantify the relative levels of different cell types in a mixed cell population. As shown in Figure 2, macrophages and T cells were estimated to be the most common immune cells in the biopsy material, although without any significant differences in their numbers between the irradiated and non-irradiated groups. However, there was a significantly large number of γδT cells (p = 0.022) and a tendency towards more naïve B cells (p = 0.077) and activated NK cells (p = 0.077) in the irradiated group.
      Figure 2
      Figure 2CIBERSORT analysis of gene expression profiles showed that the ratios of macrophages and T cells were the most common cell types. There were no significant differences between the RT- and RT+ groups, except for γδT cells (p = 0.022).

      Immunohistochemistry

      Cell-specific markers of the most relevant cell types according to enrichment testing were analysed. Of the 25 biopsies that were included in gene expression analysis, only 8 had an intact morphology of the capsular surface and were of adequate staining quality to enable cell quantification. This was mainly due to surgical constraints, which resulted in uncertainty regarding orientation either during the sampling or paraffin embedding when biopsies were small. Two blinded evaluators obtained the cell counts. When all the stained sections were analysed as a whole, there were significantly more CD68+ cells than both CD3+ cells (p = 0.0062) and CD20+ cells (p = 0.0025), which corresponded to the results obtained using CIBERSORT. Further comparison of the RT+ and RT- groups revealed no differences apart from a large number of CD20+ cells in the RT+ biopsies (p = 0.016) (Figure 3). As shown in Figure 3, B-cell infiltration was restricted to irradiated capsules. Indeed, when bilateral capsular biopsies were obtained and stained for B cells (CD20+), there was an absence of B cells in the non-irradiated capsule, whereas there was an abundance of CD20+ B cells in the irradiated capsule (Figure 4).
      Figure 3
      Figure 3Cells were counted in three equally sized high-power fields (× 20) randomly orientated along the capsular surface to a depth of 200 µm below the surface. Four irradiated and four non-irradiated biopsies were analysed. Staining of capsular biopsies for T cells (CD3+), B cells (CD20+), and macrophages (CD68+) revealed that CD68+ cells were significantly more prevalent than CD3+ and CD20+ cells (p = 0.0062 and 0.0025, respectively, for RT- and RT+ groups combined). Comparison of staining in irradiated and non-irradiated groups identified differences for CD20+ cells, with significantly higher frequency in the irradiated material (p = 0.016). Indeed, B-cell infiltration was only present in irradiated biopsies.
      Figure 4
      Figure 4Staining of capsular biopsies for B cells (CD20+) in a patient from whom bilateral capsular biopsies were obtained. The non-irradiated right capsule displays an absence of B cells, whereas the irradiated left capsule reveals an abundance of CD20+ B cells.

      Discussion

      We conducted an exploratory study on the underlying human biology of breast reconstruction by comparing irradiated and non-irradiated capsular biopsies because IBR is an integral part of breast cancer treatment. Our results showed the occurrence of inflammatory responses irrespective of RT while B-cell-associated inflammatory responses appeared more specific for irradiated capsules.
      Our gene expression results support previous studies showing a sustained innate and adaptive immune response,
      • Halle M.
      • Gabrielsen A.
      • Paulsson-Berne G.
      • Gahm C.
      • Agardh H.E.
      • Farnebo F.
      • et al.
      Sustained inflammation due to nuclear factor-Kappa B activation in irradiated human arteries.
      lasting up to several years after RT exposure. Both innate and adaptive immune responses in capsular biopsies were confirmed using immunohistochemistry, although without significant differences in cell counts for CD3+ T cells and CD68+ macrophages. Somewhat unexpected was the difference in CD20+ B cell counts between irradiated and non-irradiated biopsies, which was further supported by differentially expressed B-cell-related genes. The top three GO biological processes that were most differentially expressed by RT were humoral immune response mediated by immunoglobulins, followed by complement activation and B-cell-mediated immunity. However, these results should be interpreted with caution because of the limited sample size and exploratory nature of the study.
      Regardless of irradiation status, a significant infiltration of inflammatory cells was seen around implants. It can be noted that all the implants used in both groups had Mentor's Siltex microtextured surface. Therefore, the influence of the surface is negligible regarding the comparison between the groups but is of general interest regarding the interaction at the interface between the implant and the patient's tissue. We believe that both the gene expression findings and the morphological evaluations reveal an interesting interplay between macrophages, T cells, and B cells at the capsular interface between the implant and the breast. This could be of particular importance because the infiltration of immune cells around breast implants has gained increasing attention in recent years following the World Health Organization's recognition of breast implant-associated anaplastic large-cell lymphoma in 2016.
      The results of this study need to be interpreted with caution because of a number of limitations. First, gene expression data need to be interpreted with caution because of the limited sample size. A generally low RNA quality was noted, which most likely reflects the challenges of handling sensitive tissues in a clinical setting. Compared with previous studies by our group in which irradiated tissue biopsies were compared with internal non-irradiated controls,
      • Halle M.
      • Christersdottir T.
      • Bäck M.
      Chronic adventitial inflammation, vasa vasorum expansion, and 5-lipoxygenase up-regulation in irradiated arteries from cancer survivors.
      ,
      • Christersdottir T.
      • Pirault J.
      • Gisterå A.
      • Bergman O.
      • Gallina A.L.
      • Baumgartner R.
      • et al.
      Prevention of radiotherapy-induced arterial inflammation by interleukin-1 blockade.
      this study showed differences of smaller magnitude between irradiated and non-irradiated samples. This may be due to decreased statistical power in the group (as opposed to paired) analysis but is more likely explained by considerable inter-individual differences. Paired, synchronous sampling of irradiated biopsies and non-irradiated internal controls offers the possibility to study the effect of radiation alone but was unfortunately not possible in this cohort. Another explanation for a comparably weak effect in the irradiated group in this study may relate to the fact that inflammation as a result of the implant per se was considerable in both groups. Further in-depth studies in a larger cohort, preferably with bilateral cases of one irradiated and one non-irradiated breast reconstructions, would be needed to elucidate the immune responses caused by RT.

      Conclusion

      Considering the growing population of breast cancer survivors, it is of paramount importance to better understand the biology underlying radiation-induced capsular contracture. We showed that inflammatory responses in capsular biopsies were present regardless of RT, whereas B-cell-associated inflammatory responses were specific to irradiated tissue. However, this study should be seen as a pilot study. Larger studies with more homogeneous material are needed in the future to continue elucidating the underlying biology of radiation-induced capsular contracture.

      Conflict of interest

      The authors declare no conflict of interest.

      Acknowledgements

      Anonymized data supporting the findings of this study are available upon reasonable request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

      Funding

      This study was funded by the Swedish Breast Cancer Society Research Funds of Radiumhemmet, The Swedish Breast Cancer Association in Stockholm, and the Swedish Society of Medicine.

      Ethical approval

      The study was approved by the regional ethical review board at the Karolinska Institutet in Stockholm (2017/1504–31/2) and was performed in agreement with the institutional guidelines and the principles of the Declaration of Helsinki.

      Appendix. Supplementary materials

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