Abstract
Background/Aim: The aim of the present study was to determine whether the early systemic markers of inflammation, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), respond to high dose-rate (HDR) brachytherapy, and their possible correlation with radiation-induced liver injury of patients with liver metastases. Patients and Methods: This prospective study included 20 tumor patients (TP) undergoing irradiation-based interstitial HDR brachytherapy (iBT) of liver metastases, who received total radiation ablative doses to the planning target volume ranging from 15 to 25 Gy, depending on the tumor entity. Hepatobiliary magnetic resonance imaging (MRI) was performed 6 weeks after iBT to assess the maximum extent of focal radiation-induced liver injury (fRILI). Furthermore, blood samples for the pro-inflammatory cytokine response were taken one day prior to and 6 weeks after irradiation. IL-6 and TNF-α were measured by flow cytometry. Ten healthy volunteers (HV) were used as control group. Results: Compared to HV, TNF-α was significantly enhanced in TP before and after therapy (p<0.05 for both comparisons), while IL-6 increase at baseline was not statistically significant. HDR brachytherapy significantly reduced IL-6 levels after 6 weeks in TP (p<0.05). IL-6 levels after 6 weeks have shown a significant negative correlation with the tumor volume (r=−0.5606; p=0.0261), while no significant correlation was observed between baseline IL-6 or follow-up IL-6 levels with the fRILI. Baseline TNF-α levels positively correlated with the tumor volume (r=0.4342; p=0.0492), and post treatment TNF-α levels showed a significant correlation with the fRILI (r=0.7404; p=0.0022). Conclusion: TNF-α was correlated with both tumor volume and radiation-induced liver injury; thus, representing a promising biomarker for focal radiation-induced liver injury.
Locoregional therapies, defined as imaging-guided liver tumor-directed procedures, are emerging treatment options for primary and secondary liver malignancies (1, 2). In the last decades several techniques have been developed including thermal and irradiation-based ablation methods, latter comprising Yttrium90 radioembolization (Y90 RE), stereotactic-ablative body radiotherapy and interstitial high dose rate (HDR) brachytherapy (iBT) (3-6). However, the irradiation-based iBT of liver metastases bears a risk for radiation-induced liver disease (RILD), which originates from sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD) (7, 8).
The development of SOS/VOD, as a potentially life-threatening complication, has been mostly associated with high-intensity chemotherapies upon allogeneic or autologous hematopoietic stem cell transplantation, thus, not necessarily representing the iBT-induced SOS/VOD (9). It may occur rapidly, and since it remains unpredictable, it is of great importance to identify risk factors and biomarkers to facilitate prompt diagnosis and subsequent timely optimal treatment of this complication. The histopathological findings of this condition show deposits of extracellular matrix leading to a congestion of sinusoids and central veins with subsequent hepatocellular necrosis (10, 11).
Irradiation-induced liver dysfunction emerges upon exceeding a tolerable radiation limit, and after four to twelve weeks, clinical symptoms of RILD, such as weight gain and jaundice by hyperbilirubinemia can be observed (12, 13). Since the iBT provides high-conformal irradiation of liver malignancies, focal radiation-induced liver injury (fRILI) can be quantified by fusing the hepatobiliary magnetic resonance imaging (MRI) with the 3D irradiation plan (14). Thus, merging the acquired MRI data set with the dose distribution of administered iBD enables the quantification of fRILI, and can be applied for the analyses of pathomechanistically involved factors, as well as for the assessment of biomarkers and inflammatory factors, as novel diagnostic techniques to indicate fRILI.
Liver tissue damage generally leads to the induction of endogenous damage or danger molecules, which constitute the hallmark of the acute-phase response, as an early defense mechanism, maintaining homeostasis and initiating tissue repair (15). Pro-inflammatory cytokines, including the pleiotropic tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, are major players of the acute phase reaction (15-17). They are actively involved in cellular injury processes via the generation of reactive oxygen species, as well as apoptosis, in both acute and chronic liver inflammation (16, 18). Senescent and apoptotic cells also secrete cytokines that can contribute to long-term vascular dysfunction (19).
TNF-α is produced by several cell types including Kupffer cells, natural killer cells, T lymphocytes, and also by other hepatic cell types (20). It plays a crucial role in the regulation of inflammatory effects and host defense against microbial pathogens. TNF-α constitutes as a circulatory mediator of innate immunity capable of inducing hemorrhagic necrosis in tumors (20). Similar to TNF-α, dysregulation of IL-6 signaling has been associated with chronic inflammation, infectious diseases and cancer, where it often acts as a diagnostic or prognostic indicator of disease activity and therapy response (21, 22). IL-6 is considered as a key cytokine linking inflammation and cancer (23). Accordingly, elevated TNF-α, as well as IL-6, levels in various animal models of selective liver irradiation and acute toxin-induced liver damage have been reported (19, 24, 25). Late radiation-induced cytokine changes in Hiroshima atomic bomb survivors included a significant increase in C-reactive protein and IL-6, which may lead to increased risk of cardiovascular disease and other non-cancer diseases in this population (26). Thus, identifying biological factors, that play a pathomechanistical role, and may serve as reliable biomarkers for early fRILI detection, is crucial for maximizing the treatment efficacy.
The aim of the present study was to determine whether early systemic markers of inflammation, IL-6 and TNF-α respond to HDR brachytherapy and correlate with radiation-induced liver injury of patients with liver metastases. For this purpose, healthy volunteers as controls and tumor patients undergoing iBT of liver metastases were included. Baseline TNF-α levels were correlated with the tumor volume, and post treatment TNF-α levels showed a significant correlation with the fRILI.
Patients and Methods
Patient cohort. In this prospective study, 20 patients (11 males and 9 females) with mean age 63.1 years (SD=10.8) undergoing interstitial HDR brachytherapy of liver metastases (colorectal carcinoma n=15, breast cancer n=3, neuroendocrine tumor n=1, lymphangiosarcoma n=1) were included. Patients with ongoing infections were excluded. Ten healthy volunteers (HV) served as controls. The study was conducted in accordance with the Declaration of Helsinki. All patients included gave written informed consent, prospective data collection and analysis was approved by the local ethics committee (nr. 13/09) and competent authority of the University Hospital of the Otto-von-Guericke University, Magdeburg, Germany.
Treatment by interstitial brachytherapy. The technique of interstitial HDR brachytherapy of liver malignancies utilizing 192Iridium source is described in detail elsewhere (4). In summary, liver lesions were punctured under computed tomography fluoroscopy guidance in local anaesthesia supplemented by conscious sedation with fentanyl and midazolame. Then, 6F angiographic catheter sheaths (Terumo Radifocus® Introducer II, Terumo Europe, Leuven, Belgium) were placed in Seldinger’s technique over a stiff guide wire (Amplatz SuperStiff™, Boston Scientific, Marlborough, MA, USA). Finally, 6F brachytherapy catheters were inserted in the sheaths (afterloading catheter, Primed® Medizintechnik GmbH, Halberstadt, Germany) and the catheter system was fixed by a single suture. Depending on the lesion size and its geometry, multiple catheters may be required to facilitate an optimal distribution of irradiation and to spare organs at risk close to the target lesion (e.g., stomach, duodenum, large bowel). After successful catheter insertion, a 3D treatment plan was generated based on a final CT scan of the treated region utilizing a dedicated software (Oncentra® Brachy, Elekta Instrument AB, Stockholm, Sweden). Ablative irradiation was then achieved in afterloading technique in a single fraction with doses to the liver lesions tailored for tumor entity (Table I); for example, 25 Gy in liver metastases of colorectal carcinoma (27). After complete ablation, catheters were removed stepwise with application of gelatin sponge to the catheter track for the prevention of post-interventional bleeding. An exemplary case is illustrated in Figure 1.
Patient and treatment characteristics (N=20).
Patient with hepatocellular carcinoma in liver cirrhosis. (A) Peri-interventional CT depicting two catheters inserted into the lesion (green line) in the left liver lobe, (B) Irradiation planning with corresponding isodoses, (C) and (D) MRI with hepatobiliary contrast agent Gd-EOB-DTPA 6 weeks and 3 months.
Data acquisition and follow-up. Prior to treatment, patients underwent a baseline CT scan, as well as hepatobiliary MRI, while laboratory parameters of liver function and coagulation status were determined. The follow-up consisted of imaging (CT/MRI) and laboratory evaluation every 3 months. To assess the radiation-induced liver injury, additional hepatobiliary MRI was scheduled 6 weeks after irradiation according to the assumed maximum of irradiation damage at that time (28). Correspondingly, blood samples were taken for study-specific parameters. Determination of threshold isodoses matching fRILI was performed by semi-automatic image fusion and point-to-point registration of the 3D irradiation plan with 3D T1 gradient echo sequences in hepatobiliary MRI in a 1.5 T scanner (Achieva, Philips, Best, the Netherlands) after administration of Gd-EOB-DTPA (Primovist, Bayer Healthcare, Leverkusen, Germany). Liver function was assessed prior and after iBT by the determination of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyltransferase (GGT), albumin and bilirubin.
Laboratory analysis of cytokines. Plasma samples were obtained one day prior to and six weeks after irradiation. Blood samples were collected in prechilled citrate tubes (BD Vacutainer, Becton Dickinson Diagnostics, Aalst, Belgium) and kept on ice. Blood was centrifuged at 2,000 × g for 15 min at 4°C. Plasma was stored at −80°C until sample analysis. Blinded specimens were used for measurements of IL-6 and TNF-α by the laboratory of the Experimental Radiology at the University Hospital of the Otto-von-Guericke University Magdeburg, Germany, using the highly specific, commercially available, LEGENDplex Multi-Analyte Flow Assay Kit (BioLegend, San Diego, CA, USA) according to manufacturer’s instructions. The measurement and analyses were performed with BD FACSCanto II™ (BD Heidelberg, Germany).
Statistical analysis. Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). Besides descriptive statistics, changes of cytokine levels between HV (measured once) and baseline (pre), as well as follow up (post), in tumor patients (TP pre-iBT and TP post-iBT, respectively) were analyzed by the unpaired nonparametric Mann-Whitney test. Statistical differences between pre-iBT and post-iBT cytokine values in TP were tested by nonparametric Wilcoxon signed rank test for paired data. Furthermore, Spearman correlation analysis was performed to explore possible relationships between IL-6 and TNF-α and the main characteristics of treatment (liver volume, tumor volume, liver function, isodose and volume of fRILI), age and sex. Data are presented as the mean±standard error of the mean (SEM) unless indicated otherwise. Results were considered as statistically significant when the p-value was <0.05.
Ethical approval. The study was conducted in accordance with the Declaration of Helsinki. All patients included gave written informed consent, prospective data collection and analysis were approved by the local ethics committee and competent authority of the University Hospital of the Otto-von-Guericke University, Magdeburg, Germany.
Results
Treatment characteristics. Twenty patients with 28 liver lesions (up to 3 per patient) underwent single-fraction interstitial brachytherapy (Table I). Tumor volume as defined by CTV was 36±45 ml in a mean liver volume of 1421±368 ml. After 6 weeks, initial follow-up included hepatobiliary MRI and laboratory evaluation of inflammatory cytokines IL-6 and TNF and liver specific parameters. Matched-pair analysis of pre-iBT vs. post-iBT ALT levels in TP showed a significant ALT increase after iBT (TP pre-iBT: 42.14±3.22 vs. TP post-iBT: 66.75±12.84 U/I; p<0.05; Table II). AST also increased significantly after iBT compared to pre-iBT levels (TP pre-iBT: 51.00±3.71 vs. TP post-iBT: 64.50±4.90 U/I; p<0.05; Table II). Similar data were found for GGT, as it was significantly increased after iBT vs. pre-iBT (TP pre-iBT: 190.70±60.19 vs. TP post-iBT: 272.10±72.27 U/I; p<0.05; Table II). Total albumin was reduced after iBT; however, this was not significant (Table II). Matched-pair analysis of total bilirubin levels in TP pre-iBT vs. post-iBT has shown a significant decrease after iBT (TP pre-iBT: 81.20±6.93 vs. TP post-iBT: 72.96±5.78 μmol/l; p<0.05; Table II).
Liver function was assessed in tumor patients (TP) before (pre) and post interstitial high dose rate brachytherapy (iBT) by determination of alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyltransferase (GGT), albumin and bilirubin.
fRILI was observed in 18 patients (90%) with a mean volume of 99±100 ml. The corresponding isodose of fRILI (equalling the mean hepatic tolerance dose) as determined by image fusion with the 3D irradiation plan was 13.5±5.3 Gy. Treatment and patient data is summarized in Table I.
Pre- and post- iBT pro-inflammatory response in tumor patients versus healthy volunteer controls. Systemic levels of the pro-inflammatory cytokine IL-6 were enhanced in TP pre-iBT vs. HV; however, this result was not significant. Matched-pair analysis of IL-6 levels in TP pre-iBT vs. post-iBT has shown a significant decrease in IL-6 after iBT (TP pre-iBT: 21.05±5.66 vs. TP post-iBT: 11.68±4.43 pg/ml; p<0.05; Figure 2A). Baseline systemic levels of IL-6 have shown a significant positive correlation with the IL-6 levels post-iBT in TP (r=0.5518, p=0.0290; Table III). A significant negative correlation was found between the IL-6 values post-iBT and baseline albumin values pre-iBT in TP (r=−0.5484, p=0.0279).
Cytokine levels in tumor patients (TP) pre and post interstitial high dose rate brachytherapy (iBT) versus healthy volunteer controls (HV). Systemic levels of (A) interleukin-6 (IL-6), and (B) tumor necrosis factor-alpha (TNF-α) are shown. Plasma levels of cytokines were compared at baseline (pre-iBT) and after 6 weeks (post-iBT) in TP (n=20) and HV (n=10). Data are presented as mean±standard error of the mean. *p<0.05 between the indicated group.
The correlation analyses between interleukin 6 (IL-6), as well as tumor necrosis factors-alpha (TNF-α), with the different clinical parameters were performed by determining the Spearman correlation significance and Spearman r. Analyses were performed before (pre) and post interstitial high dose rate brachytherapy (iBT).
Circulatory levels of the pro-inflammatory cytokine TNF-α were significantly increased in both TP pre-iBT, as well as post-iBT, vs. HV (TP pre-iBT: 18.51±4.04 and TP post-iBT: 15.34±2.89 pg/ml vs. HV: 2.29±0.76 pg/ml; p<0.05; Figure 2B). There were no significant differences between baseline and post-iBT TNF-α levels. Baseline systemic levels of TNF-α have shown a significant positive correlation with the TNF levels post-iBT in TP (r=0.7301, p=0.0013).
Correlation of pro-inflammatory markers in tumor patients pre- and post- iBT with tumor volume and fRILI. Baseline circulatory levels of IL-6 did not correlate with the tumor volume (Figure 3A). However, post-iBT IL-6 levels have shown a significant negative correlation with the hepatic tumor volume (r=−0.5606, p=0.0261; Figure 3B, Table III). Neither baseline nor post-iBT levels of IL-6 did correlate with the fRILI (Figure 3C and D, Table III).
Correlation analyses of interleukin (IL)-6 in tumor patients (TP) before the interstitial high dose rate brachytherapy (pre-iBT) or six weeks after the iBT (post-iBT) with the tumor volume (A-B) or with the focal radiation-induced liver injury (fRILI) (C-D). Spearman correlation analyses were performed. Tumor volume was correlated with the IL-6 levels at baseline before iBT (A), or IL-6 levels assessed at six weeks after iBT (B). FRILI was correlated with the IL-6 levels at baseline before iBT (C), or IL-6 levels assessed at six weeks after iBT (D).
Baseline systemic levels of TNF-α have shown positive correlation with the tumor volume (r=0.4342, p=0.049; Figure 4A, Table III), while post-iBT TNF levels did not correlate with the hepatic tumor volume (Figure 4B). While the baseline systemic levels of TNF did not correlate with the fRILI (Figure 4C), post-iBT TNF levels have shown a significant and positive correlation with the fRILI (r=0.7404, p=0.002; Figure 4D, Table III).
Correlation analyses of tumor necrosis factor-alpha (TNF-α) in tumor patients (TP) before the interstitial high dose rate brachytherapy (pre-iBT) or six weeks after the iBT (post-iBT) with the tumor volume (A-B) or with the focal radiation-induced liver injury (fRILI) (C-D). Spearman correlation analyses were performed. Tumor volume was correlated with the TNF-α levels at baseline before iBT (A), or TNF-α levels assessed at six weeks after iBT (B). fRILI was correlated with the TNF-α levels at baseline before iBT (C), or TNF-α levels assessed at six weeks after iBT (D).
Discussion
Imaging-guided irradiation-based tumor-directed therapies are emerging treatment options for primary and secondary liver malignancies that bear a risk for RILD. The current knowledge regarding the key biological factors, playing a pathomechanistical role in RILD, which might serve as potentially reliable biomarkers for early fRILI detection, is limited. Thus, in the present study we determined whether early systemic markers of inflammation IL-6 and TNF-α respond to HDR brachytherapy and correlate with radiation-induced liver injury of patients with liver metastases. We found a significant TNF-α increase in pre- and post- iBT TNF-α levels of tumor patients before compared to those of the control group. Moreover, pre-therapy TNF-α levels positively correlated with the tumor volume, while post treatment TNF-α levels significantly correlated with the fRILI. The levels of IL-6 in tumor patients were reduced after therapy, compared to the baseline levels. In addition, IL-6 negatively correlated with the tumor volume; however, showed no correlation with the fRILI.
Technically, the irradiation-based iBT as therapeutic technique for primary and secondary liver malignancies delivers a high-conformal radiation to the target, while surrounding healthy liver tissue is mostly preserved. Thus, the radiation therapy for cancer implies an optimal balance between tumor control and normal tissue complications (19). However, RILD is still a significant limiting factor in large tumors and/or multifocal disease and might lead to a dose reduction with subsequent risk of non-ablative treatment. The histopathology of RILD is well described, and changed levels of pro-inflammatory cytokines in blood suggest that radiation evokes an inflammatory state responsible for side effects (10, 11, 19). Although, the molecular mechanisms implicated in RILD are still not fully understood, further studies might allow to identify patients at risk or pose a target to preventive medication. The major drivers of radiation-induced injury are oxidative stress, deoxyribonucleic acid (DNA) damage, and inflammation (29, 30). Several reports have shown persisting radiation-induced activation and expression of the genotoxic stress-induced pathways, following oxidative stress, that induce an inflammatory state with enhanced expression of cytokines including IL-6 and TNF-α, which have been assessed in the underlying study (19, 31, 32). Cytokines play a key role in immune feasibility and repair mechanisms via further activation of immune, as well as non-immune cells (e.g., endothelial or epithelial cells) (16, 33). It is well-described that senescent and apoptotic cells secrete cytokines that can contribute to long-term vascular dysfunction (19). In our study, baseline levels of IL-6 and notably TNF-α were enhanced in tumor patients, suggesting their potential involvement in the pathology. In vivo studies have shown that a whole-body irradiation with 10 Gy induced a cascade of inflammatory responses that involved increased levels of IL-6 and TNF-α (34). Comparatively, the effects of low doses (≤ 2 Gy) of ionizing radiation on endothelial activation are still under critical debate (30, 35). Such immune activation can be beneficial or detrimental because in the low dose range, both pro- and anti- cancerogenic or organ-damaging side effects are possible (32). In general, tolerogenic immune responses in the radiotherapy-relevant dose range should be avoided. Thus, to reduce late radiation-induced effects and long-term treatment-induced toxicity, as well as to improve therapy outcomes, clinical experimental studies are of greatest importance. In the present study, increased IL-6 and TNF-α levels were found in TP before the interstitial HDR brachytherapy of liver metastases, while in TP IL-6 levels were significantly decreased six weeks after iBT. Our data are in line with previously reported clinical findings showing that at diagnosis, oligometastatic breast cancer patients present with higher levels of serum IL-6 compared to healthy donors, and maintained this difference also after stereotactic body radiation therapy (SBRT) (36). Based on increased IL-6 release after radiation therapy in their in vitro breast cancer models the authors suggested that SBRT may favor the induction of this endogenous pyrogen responsible of inflammatory response (36, 37). Yet, iBT induced a significant decrease in our cohort. It is known that cytokines from the IL-6 family play fundamental roles in mediating tumor-promoting inflammation within the tumor microenvironment and cancer progression (23, 38). Thus, it is currently under discussion of how the immunobiology of IL-6 may influence the clinical decisions, and more specifically, how and when an inhibition of this cytokine might improve disease outcome and patient wellbeing. The data from the baseline measurements as well as the significant negative correlation of IL-6 levels post-iBT with the tumor volume in our study underline the current approaches of a combinatory immunotherapy with radiation therapy, affording an opportunity to prevent, for example, endothelial injury and its consequences (39, 40).
Interestingly, TNF-α that is involved in the acute-phase reaction did not markedly change after iBT, yet the early baseline levels of TNF-α positively correlate with the tumor volume. Experimental studies provide evidence that TNF-α plays a pivotal role in radiation-induced hepatic damage (41). A single-dose liver irradiation (25 Gy) results in increased TNF-α expression together with an elevation of serum liver transaminases six to 24 hours after therapy (41). The authors provide evidence that these early observed effects can be prevented by TNF-α inhibition (42). Furthermore, experimental data suggest that the intracellular defense system of hepatocytes is reduced when irradiation is administered in combination with inflammatory mediators, such as TNF-α (43). Our data demonstrate a decrease in TNF-α, six weeks after therapy. The differences regarding the timelines in the study design are of utmost importance, since an early acute-phase protein response exerted by increased TNF-α levels is expected. In a recent clinical study, the association between circulating lymphocyte populations before and after SBRT and the one-, two- and three- year overall survival rates in patients with hepatocellular carcinoma demonstrated that TNF-α was an independent factor for inferior overall survival (44). Studies regarding RILD in this context are sparse. However, a RILD prophylaxis with ursodeoxycholic acid had an independent positive impact on overall survival in patients with metastatic breast cancer and reduced the frequency and severity of RILD (45). In patients with liver metastases from colorectal carcinoma who were scheduled for iBT, post-therapeutic administration of ursodeoxycholic acid reduced the extent and incidence fRILI at six weeks after therapy (46). Interestingly, ursodeoxycholic acid is known to reduce the concentration of potentially hepatotoxic bile acids and presumably down-regulate pro-inflammatory cytokines including TNF-α (47). Interestingly, in our study, the follow-up TNF-α levels detected after six weeks positively correlated with the iBT-induced fRILI, suggesting that TNF-α is an efficient marker of irradiation-induced liver injury in our patient cohort that should be further elaborated in larger clinical studies.
The major limitation of the present study is the low number of patients that have been included. Also, their treatment was heterogeneous since different radiation doses were applied according to different tumor entities treated. This makes it rather difficult to draw reliable conclusions and requires further studies with larger cohorts of patients. In addition, evaluation of dose-response interrelationship could not be performed due to the restricted number of patients. Moreover, the inclusion criteria did not account for natural tolerance or sensitivity to radiation in patients. Therefore, in future studies, correlating the therapy outcome with biomarkers of liver radiation sensitivity would be advantageous. In addition, further clinical outcomes, such as recurrence of the metastasis, have not been assessed. Regarding the measured cytokines TNF-α and IL-6, we cannot draw any conclusions regarding the half-life of both cytokines, that certainly might be affected by the brachytherapy. Thus, in future studies, a more frequent time course for the assessment of biomarkers after therapy should be included. However, since both IL-6 and TNF-α have a quite short half-life (2-6 h and 30 min, respectively), we assume that their levels at six weeks after therapy are not directly affected by the brachytherapy itself.
This exploratory study describes that IL-6 and TNF-α correlate with the tumor volume, and that HDR brachytherapy significantly reduced IL-6 levels. Specifically, TNF-α levels assessed post HDR brachytherapy significantly correlated with the fRILI. Therefore, results from this study suggest that the investigation of inflammatory cytokines, notably TNF-α in blood samples from tumor patients may be an appropriate tool to predict side effects after interstitial HDR brachytherapy of liver metastases, such as fRILI. Since a pre-therapy TNF-α increase has also been observed, in a perspective study, the patient cohort should be expanded and adapted to homogeneous criteria of the pathology, as well as examinations of tumor biopsy material to characterize the status of the pathology should be performed.
Acknowledgements
This research was funded by the Federal Ministry of Education and Research (BMBF), The STIMULATE Research Campus, grant number 13GW0473A.
Footnotes
Authors’ Contributions
Conceptualization, R.D., M.S. and B.R.; methodology, R.D., R.S., J.R., M.S., F.H.; validation, R.D., and B.R.; formal analysis, R.D., F.H., and B.R.; investigation, R.D., F.H..; resources, M.P., J.R., and B.R.; data curation, R.D., B.R.; writing – original draft preparation, R.D., and B.R.; writing – review and editing, P.C., F.H., S.G., J.O.; visualization, R.D., and B.R.; supervision, R.D., and B.R.; funding acquisition, B.R. All authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received May 27, 2022.
- Revision received July 12, 2022.
- Accepted August 9, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.