Acute toxicity in patients with oligometastatic cancer following metastasis-directed stereotactic body radiotherapy: An interim analysis of the E2-RADIatE OligoCare cohort.

AIM: To evaluate acute toxicity at 6 months after stereotactic body radiotherapy (SBRT) in patients with oligometastatic cancer within the OligoCare cohort. MATERIAL AND METHODS: OligoCare is a prospective, registry-based, single-arm, observational study that aims to report prospective real-world data of patients with oligometastases from solid cancer treated with SBRT (NCT03818503). Primary tumor included non-small cell lung cancer (NSCLC), breast cancer (BC), colorectal cancer (CRC), and prostate cancer (PC). This analysis addresses a secondary endpoint of the trial, acute toxicity within 6 months after SBRT. RESULTS: Out of 1,597registered patients, 1'468 patients were evaluated for acute toxicity. Globally, 290 (20 %) had NSCLC primary disease, 227 (16 %) had BC, 293 (20 %) had CRC, and 658 (45 %) had PC. Concomitant systemic treatment was administered in 527 (35.9 %) patients. According to the EORTC/ESTRO oligometastatic disease (OMD) classification, 828 (56 %) patients had de novo OMD, 464 (32 %) repeat OMD, and 176 (12 %) induced OMD. Acute grade ≥ 3 SBRT related adverse events were reported in 8 (0.5 %) patients, including 2 (0.1 %) fatal AEs. In particular, 6 (0.4 %) grade 3 events were: 1 empyema, 1 pneumonia, 1 radiation pneumonitis, 1 radiation skin injury, 1 decreased appetite, and 1 bone pain. Among those 2 occurred in NSCLC patients, 2 in BC patients, and 1 in CRC and PC patients each. The two (0.1 %) grade 5 toxicity were represented by: pneumonitis and cerebral hemorrhage. CONCLUSION: OligoCare is the largest prospective registry cohort on oligometastatic disease. Acute toxicity within 6 months was low, confirming the safety of SBRT in the treatment of oligometastases.

Dose-intensified stereotactic body radiotherapy for painful vertebral metastases: A randomized phase 3 trial.

BACKGROUND: The purpose of this randomised study was to determine whether dose-intensified stereotactic body radiotherapy (SBRT) for painful vertebral metastases results in increased rates of pain improvement compared with conventional external beam radiotherapy (cEBRT) (control) 6 months after treatment. METHODS: This randomized, controlled phase 3 trial was conducted between November 2016 and January 2023, when it was stopped early. Patients were eligible if they were aged 18 years or older; had one or two painful, stable, or potentially unstable vertebral metastases; and had a life expectancy of 1 year or longer according to the investigator's estimates. Patients received 48.5 grays (Gy) in 10 fractions (with epidural involvement) or 40 Gy in five fractions (without epidural involvement) in the SBRT group and 30 Gy in 10 fractions or 20 Gy in five fractions in the cEBRT group, respectively. The primary end point was an improvement in the pain score at the treated site by at least 2 points (on a visual analog scale from 0 to 10 points) at 6-month follow-up. Data were analyzed on an intention-to-treat and per-protocol basis. RESULTS: Of 214 patients who were screened for eligibility, 63 were randomized 1:1 between SBRT (33 patients with 36 metastases) and cEBRT (30 patients with 31 metastases). The median age of all patients was 66 years, and 40 patients were men (63.5%). In the intention-to-treat analysis, the 6-month proportion of patients who had metastases with pain reduction by 2 or more points was significantly higher in the SBRT group versus the control group (69.4% vs. 41.9%, respectively; two-sided p = .02). Changes in opioid medication intake relative to baseline were nonsignificant between the groups. No differences were observed in vertebral compression fracture or adverse event rates between the groups. CONCLUSIONS: Dose-intensified SBRT improved pain score more effectively than cEBRT at 6 months.

Cancer-specific dose and fractionation schedules in stereotactic body radiotherapy for oligometastatic disease: An interim analysis of the EORTC-ESTRO E2-RADIatE OligoCare study.

BACKGROUND AND INTRODUCTION: Optimal dose and fractionation in stereotactic body radiotherapy (SBRT) for oligometastatic cancer patients remain unknown. In this interim analysis of OligoCare, we analyzed factors associated with SBRT dose and fractionation. MATERIALS AND METHODS: Analysis was based on the first 1,099 registered patients. SBRT doses were converted to biological effective doses (BED) using α/ß of 10 Gy for all primaries, and cancer-specific α/ß of 10 Gy for non-small cell lung and colorectal cancer (NSCLC, CRC), 2.5 Gy for breast cancer (BC), or 1.5 Gy for prostate cancer (PC). RESULTS: Of the interim analysis population of 1,099 patients, 999 (99.5 %) fulfilled inclusion criteria and received metastasis-directed SBRT for NSCLC (n = 195; 19.5 %), BC (n = 163; 16.3 %), CRC (n = 184; 18.4 %), or PC (n = 457; 47.5 %). Two thirds of patients were treated for single metastasis. Median number of fractions was 5 (IQR, 3-5) and median dose per fraction was 9.7 (IQR, 7.7-12.4) Gy. The most frequently treated sites were non-vertebral bone (22.8 %), lung (21.0 %), and distant lymph node metastases (19.0 %). On multivariate analysis, the dose varied significantly for primary cancer type (BC: 237.3 Gy BED, PC 300.6 Gy BED, and CRC 84.3 Gy BED), and metastatic sites, with higher doses for lung and liver lesions. CONCLUSION: This real-world analysis suggests that SBRT doses are adjusted to the primary cancers and oligometastasis location. Future analysis will address safety and efficacy of this site- and disease-adapted SBRT fractionation approach (NCT03818503).

Health-related quality of life in men with oligometastatic prostate cancer following metastases-directed stereotactic body radiotherapy: Real-world data from the E2-RADIatE OligoCare cohort.

Objective: To evaluate the impact of metastases-directed stereotactic body radiotherapy (SBRT) on health-related quality of life (HRQoL) in men with oligometastatic prostate cancer (PCa) using real-world data from the OligoCare cohort. Materials and methods: OligoCare is a pragmatic, observational cohort designed to assess the impact of metastases-directed SBRT on patients with oligometastatic disease (OMD). We report an interim analyses of the secondary endpoint HRQoL, assessed using the EORTC QLQ-C30, within six months of metastases-directed SBRT for oligometastatic disease in men with PCa among the first 1600 registered patients. HRQoL data collection was optional within the OligoCare cohort. To compare HRQoL between baseline and first follow-up assessment, a Wilcoxon signed-rank test was used. A multiple linear regression model was used to explore the HRQoL associations with predefined factors. Results: Out of the 1600 registered patients, 658 were treated for oligometastatic PCa, of which 233 had baseline QoL data and 132 patients had both baseline and follow-up HRQoL data. At baseline, most patients had a WHO performance status of 0 or 1 (87 %), were de-novo oligometastatic (79 %), had one metastasis (90 %), and had a good overall global health status (mean 80.81, SD16.11, IQR 75-92). 51 % received hormonal therapy as concomitant systemic treatment. Patients with comorbidities as assessed by the Charlson Comorbidity index had a worse global health status at baseline (-4.88, 95 % CI:-9.35, -0.42). No clinically meaningful significant difference in global health status was observed at first assessment following SBRT (median 3.0 months) compared with baseline (mean difference 2.27, 95 % CI:-1.54, 6.08). Upon evaluating the proportions, meaningful clinically important differences (a 10-point or more difference) was observed in, 17 % and 11 % of the patients reporting deterioration and improvement of global health status, respectively. Conclusion: Metastases-directed stereotactic body radiotherapy had no negative impact on global HRQoL within the first six months after treatment.

Therapy Resistance of Glioblastoma in Relation to the Subventricular Zone: What Is the Role of Radiotherapy?

Glioblastoma is a highly heterogeneous primary malignant brain tumor with marked inter-/intratumoral diversity and a poor prognosis. It may contain a population of neural stem cells (NSC) and glioblastoma stem cells that have the capacity for migration, self-renewal and differentiation. While both may contribute to resistance to therapy, NSCs may also play a role in brain tissue repair. The subventricular zone (SVZ) is the main reservoir of NSCs. This study investigated the impact of bilateral SVZ radiation doses on patient outcomes. We included 147 patients. SVZs were delineated and the dose administered was extracted from dose-volume histograms. Tumors were classified based on their spatial relationship to the SVZ. The dose and outcome correlations were analyzed using the Kaplan-Meier and Cox proportional hazards regression methods. Median progression-free survival (PFS) was 7 months (range: 4-11 months) and median overall survival (OS) was 14 months (range: 9-23 months). Patients with an ipsilateral SVZ who received ≥50 Gy showed significantly better PFS (8 versus 6 months; p < 0.001) and OS (16 versus 11 months; p < 0.001). Furthermore, lower doses (<32 Gy) to the contralateral SVZ were associated with improved PFS (8 versus 6 months; p = 0.030) and OS (15 versus 11 months; p = 0.001). Targeting the potential tumorigenic cells in the ipsilateral SVZ while sparing contralateral NSCs correlated with an improved outcome. Further studies should address the optimization of dose distribution with modern radiotherapy techniques for the areas surrounding infiltrated and healthy SVZs.

Metastases-directed stereotactic body radiotherapy in combination with targeted therapy or immunotherapy: systematic review and consensus recommendations by the EORTC-ESTRO OligoCare consortium.

Stereotactic body radiotherapy (SBRT) for patients with metastatic cancer, especially when characterised by a low tumour burden (ie, oligometastatic disease), receiving targeted therapy or immunotherapy has become a frequently practised and guideline-supported treatment strategy. Despite the increasing use in routine clinical practice, there is little information on the safety of combining SBRT with modern targeted therapy or immunotherapy and a paucity of high-level evidence to guide clinical management. A systematic literature review was performed to identify the toxicity profiles of combined metastases-directed SBRT and targeted therapy or immunotherapy. These results served as the basis for an international Delphi consensus process among 28 interdisciplinary experts who are members of the European Society for Radiotherapy and Oncology (ESTRO) and European Organisation for Research and Treatment of Cancer (EORTC) OligoCare consortium. Consensus was sought about risk mitigation strategies of metastases-directed SBRT combined with targeted therapy or immunotherapy; a potential need for and length of interruption to targeted therapy or immunotherapy around SBRT delivery; and potential adaptations of radiation dose and fractionation. Results of this systematic review and consensus process compile the best available evidence for safe combination of metastases-directed SBRT and targeted therapy or immunotherapy for patients with metastatic or oligometastatic cancer and aim to guide today's clinical practice and the design of future clinical trials.

Feasibility of postoperative spine stereotactic body radiation therapy in proximity of carbon and titanium hybrid implants using a robotic radiotherapy device.

BACKGROUND AND PURPOSE: To assess the feasibility of postoperative stereotactic body radiation therapy (SBRT) for patients with hybrid implants consisting of carbon fiber reinforced polyetheretherketone and titanium (CFP-T) using CyberKnife. MATERIALS AND METHODS: All essential steps within a radiation therapy (RT) workflow were evaluated. First, the contouring process of target volumes and organs at risk (OAR) was done for patients with CFP-T implants. Second, after RT-planning, the accuracy of the calculated dose distributions was tested in a slab phantom and an anthropomorphic phantom using film dosimetry. As a third step, the accuracy of the mandatory image guided radiation therapy (IGRT) including automatic matching was assessed using the anthropomorphic phantom. For this goal, a standard quality assurance (QA) test was modified to carry out its IGRT part in presence of CFP-T implants. RESULTS: Using CFP-T implants, target volumes could precisely delineated. There was no need for compromising the contours to overcome artifact obstacles. Differences between measured and calculated dose values were below 11% for the slab phantom, and at least 95% of the voxels were within 5% dose difference. The comparisons for the anthropomorphic phantom showed a gamma-passing rate (5%, 1 mm) of at least 97%. Additionally the test results with and without CFP-T implants were comparable. No issues concerning the IGRT were detected. The modified machine QA test resulted in a targeting error of 0.71 mm, which corresponds to the results of the unmodified standard tests. CONCLUSION: Dose calculation and delivery of postoperative spine SBRT is feasible in proximity of CFP-T implants using a CyberKnife system.

Response assessment after stereotactic body radiation therapy for spine and non-spine bone metastases: results from a single institutional study.

BACKGROUND: The use of stereotactic body radiation therapy (SBRT) for tumor and pain control in patients with bone metastases is increasing. We report response assessment after bone SBRT using radiological changes through time and clinical examination of patients. METHODS: We analyzed retrospectively oligo-metastatic/progressive patients with bony lesions treated with SBRT between 12/2008 and 10/2018, without in-field re-irradiation, in our institution. Radiological data were obtained from imaging modalities used for SBRT planning and follow-up purposes in picture archiving and communication system and assessed by two independent radiologists blind to the time of treatment. Several radiological changes were described. Radiographic response assessment was classified according to University of Texas MD Anderson Cancer Center criteria. Pain response and the neurological deficit were captured before and at least 6 months after SBRT. RESULTS: A total of 35 of the 74 reviewed patients were eligible, presenting 43 bone metastases, with 51.2% (n = 22) located in the vertebral column. Median age at the time of SBRT was 66 years (range 38-84) and 77.1% (n = 27) were male. Histology was mainly prostate (51.4%, n = 18) and breast cancer (14.3%, n = 5). Median total radiation dose delivered was 24 Gy (range 24-42), in three fractions (range 2-7), prescribed to 70-90% isodose-line. After a median follow-up of 1.8 years (range < 1-8.2) for survivors, complete or partial response, stable, and progressive disease occurred in 0%, 11.4% (n = 4), 68.6% (n = 24), and 20.0% (n = 7) of the patients, respectively. Twenty patients (57.1%) died during the follow-up time, all from disease progression, yet 70% (n = 14) from this population with local stable disease after SBRT. From patients who were symptomatic and available for follow-up, almost half (44.4%) reported pain reduction after SBRT. CONCLUSIONS: Eighty percent of the patients showed local control after SBRT for bone metastases. Pain response was favorable. For more accurate response assessment, comparing current imaging modalities with advanced imaging techniques such as functional MRI and PET/CT, in a prospective and standardized way is warranted. Trial registration Retrospectively registered.

CCR5 is a potential therapeutic target for cancer.

INTRODUCTION: Chemokines and their cognate receptors play a major role in modulating inflammatory responses. Depending on their ligand binding, chemokine receptors can stimulate both immune activating and inhibitory signaling pathways. The CC chemokine receptor 5 (CCR5) promotes immune responses by recruiting immune cells to the sites of inflammation/tumor, and is involved in stimulating tumor cell proliferation, invasion and migration through various mechanisms. Moreover, CCR5 also contributes to an immune-suppressive tumor microenvironment by recruiting regulatory T cells and myeloid-derived suppressor cells facilitating tumor development and progression. In summary, cells expressing CCR5 modulate immune response and tumor progression. Expression of CCR5 is increased in various malignancies and associated with poor outcome. Experimental data show promising efficacy signals with CCR5 antagonists in preclinical tumor models. Therefore, CCR5 has been recognized as a potential therapeutic target for cancer. AREAS COVERED: In this review, we focus on the role of CCR5 in cancer progression and discuss its impact and potential as a therapeutic target for cancer. EXPERT OPINION: Beyond immune-checkpoint inhibitors, potentially synergistic immune-modulatory drugs such as CCR5 antagonists are a promising approach to enlarge our treatment armamentarium against cancer.

Evaluation of a new software prototype for frameless radiosurgery of arteriovenous malformations.

BACKGROUND: In order to locate an arteriovenous malformation, typically, a digital subtraction angiography (DSA) is carried out. To use the DSA for target definition an accurate image registration between CT and DSA is required. Carrying out a non-invasive, frameless procedure, registration of the 2D-DSA images with the CT is critical. A new software prototype is enabling this frameless procedure. The aim of this work was to evaluate the prototype in terms of targeting accuracy and reliability based on phantom measurements as well as with the aid of patient data. In addition, the user's ability to recognize registration mismatches and quality was assessed. METHODS: Targeting accuracy was measured with a simple cubic, as well as with an anthropomorphic head phantom. Clearly defined academic targets within the phantoms were contoured on the CT. These reference structures were compared with the structures generated within the prototype. A similar approach was used with patient data, where the clinically contoured target served as the reference structure. An important error source decreasing the target accuracy comes from registration errors between CT and 2D-DSA. For that reason, the tools in BC provided to the user to check these registrations are very important. In order to check if the user is able to recognize registration errors, a set of different registration errors was introduced to the correctly registered CT and 2D-DSA image data sets of three different patients. Each of six different users rated the whole set of registrations within the prototype. RESULTS: The target accuracy of the prototype was found to be below 0.04 cm for the cubic phantom and below 0.05 cm for the anthropomorphic head phantom. The mean target accuracy for the 15 patient cases was found to be below 0.3 cm. In the registration verification part, almost all introduced registration errors above 1° or 0.1 cm were detected by the six users. Nevertheless, in order to quantify and categorize the possibility to detect mismatches in the registration process more data needs to be evaluated. CONCLUSION: Our study shows, that the prototype is a useful tool that has the potential to fill the gap towards a frameless procedure when treating AVMs with the aid of 2D-DSA images in radiosurgery. The target accuracy of the prototype is similar to other systems already established in clinical routine.

Skin surface markers for stereotactic body radiation therapy of sternal metastasis.

Stereotactic body radiation therapy is an effective and safe treatment modality for bone metastasis which allows clinicians to accurately target lesions to high doses while minimizing dose to organs at risk. The commercially available CyberKnife® Xsight™ Spine Tracking System (Accuray, Inc., Sunnyvale, CA) tracks static skeletal structures and eliminates the need for implanted fiducial markers (FMs). However, the Xsight™ Spine Tracking system is not appropriate for bone metastases outside the spine, which are moving due to respiration and ,typically, FMs have to be implanted close to the lesion. These FMs will be used to track the dynamic target. For targets close to the surface, non-invasive fixation of the FMs to the patient's skin could be an option.

Part 1: Optimization and evaluation of dynamic trajectory radiotherapy.

PURPOSE: Although volumetric modulated arc therapy (VMAT) is a well-accepted treatment technique in radiotherapy using a coplanar delivery approach, VMAT might be further improved by including dynamic table and collimator rotations leading to dynamic trajectory radiotherapy (DTRT). In this work, an optimization procedure for DTRT was developed and the potential benefit of DTRT was investigated for different treatment sites. METHODS: For this purpose, a dedicated optimization framework for DTRT was developed using the Eclipse Scripting Research Application Programming Interface (ESRAPI). The contours of the target and organs at risk (OARs) structures were exported by applying the ESRAPI and were used to determine the fractional volume-overlap of the OARs with the target from several potential beam directions. Thereby, an additional weighting was applied taking into account the relative position of the OAR with respect to the target and radiation beam, that is, penalizing directions where the OAR is proximal to the target. The resulting two-dimensional gantry-table map was used as input for an A* path finding algorithm returning an optimized gantry-table path. Thereby, the process is also taking into account CT scan length and collision restrictions. The A* algorithm was used again to determine the dynamic collimator angle path by optimizing the area between the MLC leaves and the target contour for each gantry-table path leading to gantry-collimator paths. The resulting gantry-table and gantry-collimator paths are combined and serve as input for the intensity modulation optimization using a research VMAT optimizer and the ESRAPI resulting in dynamic trajectories. This procedure was evaluated for five clinically motivated cases: two head and neck, one lung, one esophagus, and one prostate. Final dose calculations were performed using the Swiss Monte Carlo Plan (SMCP). Resulting dose distributions for the DTRT treatment plans and for the standard VMAT plans were compared based on dose distributions and dose volume histogram (DVH) parameters. For this comparison, the dose distribution for the VMAT plans were recalculated using the SMCP. In addition, the suitability of the delivery of a DTRT treatment plan was demonstrated by means of gafchromic film measurements on a TrueBeam linear accelerator. RESULTS: DVHs for the target volumes showed similar or improved coverage and dose homogeneity for DTRT compared with VMAT using equal or less number of dynamic trajectories for DTRT than arcs for VMAT for all cases studied. Depending on the case, improvements in mean and maximum dose for the DTRT plans were achieved for almost all OARs compared with the VMAT plans. Improvements in DTRT treatment plans for mean and maximum dose compared to VMAT plans were up to 16% and 38% relative to the prescribed dose, respectively. The measured and calculated dose values resulted in a passing rate of more than 99.5% for the two-dimensional gamma analysis using 2% and 2 mm criteria and a threshold of 10%. CONCLUSIONS: DTRT plans for different treatment sites were generated and compared with VMAT plans. The delivery is suitable and dose comparisons demonstrate a high potential of DTRT to reduce dose to OARs using less dynamic trajectories than arcs, while target coverage is preserved.

Tumor stage, tumor site and HPV dependent correlation of perfusion CT parameters and [18F]-FDG uptake in head and neck squamous cell carcinoma.

BACKGROUND AND PURPOSE: This study investigated whether tumor perfusion, FDG uptake and their correlation depend on tumor stage, site and HPV in head and neck cancer. MATERIAL AND METHODS: 41/55 eligible patients with integrated FDG-PET/perfusion CT from 2 prospective studies were assessed. A GTV(CT) and GTV(PET) were created. Perfusion maps were calculated using singular value decomposition method. Blood volume (BV), blood flow (BF), mean transit time (MTT) and standardized uptake value (SUV) in the tumor were compared to the surrounding tissue using Wilcoxon test and Spearman correlation of perfusion and SUVmean in the tumor was studied (p=0.05). RESULTS: Perfusion parameters were significantly increased in the GTV(CT) of advanced tumors in comparison to the surrounding soft tissue (p<0.01). Oral cavity and oropharyngeal cancer showed a higher BF than laryngeal cancer (p<0.04). No correlation between perfusion and SUVmean was found, however SUVmean correlated significantly with BF for the HPV-positive tumors (r=0.86, p=0.04) and with BV for the oropharyngeal cancer (r=0.63, p=0.05). CONCLUSION: Tumor stage, site and HPV are associated with different perfusion or combined perfusion/SUV signatures. Further studies are needed to investigate if these signatures co-determine clinical outcome.

The pattern-recognition receptor nucleotide-binding oligomerization domain--containing protein 1 promotes production of inflammatory mediators in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-containing protein 2 (NOD-2), have been shown to contribute to the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to analyze the expression, regulation, and function of the PRR NOD-1 in RA synovial fibroblasts (RASFs), and to examine its interaction with other PRRs. METHODS: Expression of NOD-1 was analyzed by immunohistochemistry in synovial tissue from RA patients, psoriatic arthritis patients, gout patients, and osteoarthritis (OA) patients. RASFs and human monocyte-derived macrophages (HMDMs) were stimulated with L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid, palmitoyl-3-cysteine-serine-lysine-4, poly(I-C), lipopolysaccharide, heat-inactivated bacteria, tumor necrosis factor α (TNFα), or interleukin-1ß (IL-1ß). Expression levels of IL-6, CCL5, matrix metalloproteinases (MMPs), NODs, and TLRs were measured by real-time reverse transcription-polymerase chain reaction and/or enzyme-linked immunosorbent assay. NOD-1 and NOD-2 were silenced with target-specific small interfering RNA. Phosphorylation of IL-1 receptor-associated kinase 1 (IRAK-1) was measured by Western blotting. RESULTS: Expression of NOD-1 protein was significantly increased in RA synovium compared to OA synovium. The basal expression of NOD-1 was similar in RASFs, OASFs, healthy control peripheral blood mononuclear cells, and healthy control HMDMs. Stimulation of RASFs with TLR-3 up-regulated the expression of NOD-1. Expression of IL-6, CCL5, MMPs, TLR-2, and NOD-2 was significantly up-regulated in RASFs by stimulation with the NOD-1 ligand. A synergistic effect on IL-6 production was observed in cells stimulated with NOD-1 and TLR-2 ligands or NOD-1 and TLR-4 ligands. Silencing of NOD-1, but not NOD-2, decreased the levels of IL-6 in RASFs after stimulation with TLR-2 and IL-1ß, and blocked the phosphorylation of IRAK-1. CONCLUSION: NOD-1 is strongly expressed in different cell types in the synovial tissue of patients with RA. These results indicate that NOD-1, either alone or interacting with other inflammatory mediators, can play an important role in the chronic and destructive inflammation of the joints in RA.

7th meeting of the Global Arthritis Research Network.

Last October, the 7th meeting of the Global Arthritis Research Network was held in Zurich, Switzerland. European and American experts who have made major recent contributions to molecular biology got together to provide insights into novel technologies and approaches useful for biomedical research, especially for research on arthritis and related conditions.

Inhibition of fibroblast activation protein and dipeptidylpeptidase 4 increases cartilage invasion by rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP-4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage. METHODS: Expression of FAP and DPP-4/CD26 by RASFs was analyzed using fluorescence-activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L-glutamyl L-boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real-time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell-derived factor 1 (SDF-1 [CXCL12]), MMP-1, and MMP-3 protein levels were measured using enzyme-linked immunosorbent assay. The impact of FAP and DPP-4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry. RESULTS: Inhibition of serine protease activity of FAP and DPP-4/CD26 in vitro led to increased levels of SDF-1 in concert with MMP-1 and MMP-3, which are downstream effectors of SDF-1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP-expressing RASFs and marked by a significantly higher accumulation of MMP-1 and MMP-3, when compared with controls. CONCLUSION: Our results indicate a central role for the serine protease activity of FAP and DPP-4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.

Epigenetics and rheumatoid arthritis: the role of SENP1 in the regulation of MMP-1 expression.

The aggressive phenotype of RA synovial fibroblasts (RASF) is characterised by the increased expression of matrix metalloproteinase (MMP)-1 as well as the small ubiquitin like modifier (SUMO)-1 and decreased expression of SUMO-specific protease SENP1. Since we showed an increased activity of acetyltransferases in this autoimmune disease, we wanted to analyze whether this affects the expression of MMP-1 and can be reversed by the reconstitution of SENP1. In RASF, the acetylation of histone H4 was significantly increased in the distal region of the MMP-1 promoter by 274 +/- 36% compared to OASF. Most interestingly, overexpression of SENP1 in RASF decreased acetylation specifically in this region by 51 +/- 0.5% and globally by 73 +/- 11%. Furthermore, the overexpression of SENP1 resulted in a downregulation of MMP-1 at both the mRNA (58 +/- 7%) and protein levels (28 +/- 6%), significantly reduced the invasiveness of RASF (from 34 +/- 9% to 2 +/- 2%) and led to an accumulation of histone deacetylase 4 (HDAC4) on the MMP-1 promoter (197 +/- 36%). Interestingly, SENP1 failed to modulate the expression of MMP-1 in the cells silenced for HDAC4. This is the first study linking the SUMOylation pathway and the production of MMP-1 to an epigenetic control mechanism mediated through histone acetylation which has a functional consequence for the invasiveness of RASF.

Histone deacetylase 7, a potential target for the antifibrotic treatment of systemic sclerosis.

OBJECTIVE: We have recently shown a significant reduction in cytokine-induced transcription of type I collagen and fibronectin in systemic sclerosis (SSc) skin fibroblasts upon treatment with trichostatin A (TSA). Moreover, in a mouse model of fibrosis, TSA prevented the dermal accumulation of extracellular matrix. The purpose of this study was to analyze the silencing of histone deacetylase 7 (HDAC-7) as a possible mechanism by which TSA exerts its antifibrotic function. METHODS: Skin fibroblasts from patients with SSc were treated with TSA and/or transforming growth factor beta. Expression of HDACs 1-11, extracellular matrix proteins, connective tissue growth factor (CTGF), and intercellular adhesion molecule 1 (ICAM-1) was analyzed by real-time polymerase chain reaction, Western blotting, and the Sircol collagen assay. HDAC-7 was silenced using small interfering RNA. RESULTS: SSc fibroblasts did not show a specific pattern of expression of HDACs. TSA significantly inhibited the expression of HDAC-7, whereas HDAC-3 was up-regulated. Silencing of HDAC-7 decreased the constitutive and cytokine-induced production of type I and type III collagen, but not fibronectin, as TSA had done. Most interestingly, TSA induced the expression of CTGF and ICAM-1, while silencing of HDAC-7 had no effect on their expression. CONCLUSION: Silencing of HDAC-7 appears to be not only as effective as TSA, but also a more specific target for the treatment of SSc, because it does not up-regulate the expression of profibrotic molecules such as ICAM-1 and CTGF. This observation may lead to the development of more specific and less toxic targeted therapies for SSc.