<table><caption>**Table 1.** *Cont.*</caption><thead><tr><th>Variable</th><th>TPMT Non-NM (n = 50)</th><th>TPMT NM (n = 1000)</th><th>p-Value</th></tr></thead><tbody><tr><td>Biochemistry</td><td></td><td></td><td></td></tr><tr><td>WBC count (U/L)</td><td>3920 (3600–5400)</td><td>3900 (3500–5660)</td><td>0.73</td></tr><tr><td>ALT (U/L)</td><td>13.5 (11–27)</td><td>13 (10–21)</td><td>0.16</td></tr><tr><td>Medication</td><td></td><td></td><td></td></tr><tr><td>MTX</td><td>6 (12.0)</td><td>125 (12.5)</td><td>0.92</td></tr><tr><td>Cyclophosphamide</td><td>5 (10.0)</td><td>137 (13.7)</td><td>0.46</td></tr><tr><td>Comorbidity</td><td></td><td></td><td></td></tr><tr><td>Hepatitis B</td><td>6 (12.0)</td><td>36 (3.6)</td><td>0.01</td></tr><tr><td>SLE</td><td>30 (60.0)</td><td>647 (64.7)</td><td>0.50</td></tr><tr><td>SS</td><td>24 (48.0)</td><td>456 (45.6)</td><td>0.74</td></tr><tr><td>DM and polymyositis</td><td>7 (14.0)</td><td>129 (12.9)</td><td>0.82</td></tr><tr><td>Pemphigus</td><td>3 (6.0)</td><td>25 (2.5)</td><td>0.14</td></tr><tr><td>Other autoimmune diseases ^</td><td>12 (24.0)</td><td>244 (24.4)</td><td>0.95</td></tr><tr><td>Division</td><td></td><td></td><td></td></tr><tr><td>IMRH</td><td>42 (84)</td><td>864 (86.4)</td><td></td></tr><tr><td>NEPH</td><td>5 (10)</td><td>59 (5.9)</td><td></td></tr><tr><td>NEUR</td><td>2 (4)</td><td>24 (2.4)</td><td>0.52</td></tr><tr><td>DERM</td><td>0 (0)</td><td>27 (2.7)</td><td></td></tr><tr><td>Others &amp;</td><td>1 (2)</td><td>26 (2.6)</td><td></td></tr></tbody></table>

Data are expressed as *n* (%) or the mean ± SD or the median (interquartile range). ^ Other autoimmune diseases: RA, MCTD, vasculitis, SSc, Behcet's disease, IBD. & Other divisions: CM, CV, GI, HEMA, META, GS, CS, CRS, ENT, OPH, PNEP. TPMT, thiopurine S-methyltransferase; NM, normal metabolizer; AZA, azathioprine; WBC, white blood cell; ALT, alanine transaminase; MTX, methotrexate; SLE, systemic lupus erythematosus; SS, Sjogren's syndrome; DM, dermatomyositis; RA, rheumatoid arthritis; MCTD, mixed connective tissue disease; SSc, systemic sclerosis; IBD, inflammatory bowel disease; IMRH, immunology and rheumatology; NEPH, nephrology; NEUR, neurology; DERM, dermatology; CM, chest medicine; CV, cardiology; GI, gastroenterology and hepatology; HEMA, hematology; META, metabolism, endocrinology, and nutrition; GS, gastrointestinal surgery; CS, thoracic surgery; CRS, colorectal surgery; ENT, otolaryngology; OPH, ophthalmology; PENP, pediatrics.

### 3.3. TPMT Phenotypes and Risks of Hepatotoxicity and Leukopenia

The average dosage of AZA was not different between the TPMT non-NM and NM groups. However, more than half of the patients took less than 25 mg daily. A hepatotoxicity event was defined by an ALT level of more than 150 U/L, which showed no difference between the TPMT non-NM and NM groups. However, the onset time of hepatotoxicity was shorter in the TPMT non-NM group compared with the TPMT NM group (Table 2). Cox proportional hazards regression analysis was applied to evaluate the independent risk factors for hepatotoxicity following AZA treatment. Among 1050 AZA users, male gender (hazard ratio (HR): 1.77, 95% confidence interval (CI): 1.07–2.91), non-NM genotype (HR: 3.85, 95% CI: 1.83–8.10), and MTX use (HR: 1.62, 95% CI: 1.03–2.57) were associated with hepatotoxicity compared with the NM genotype. Age, AZA dosage, and hepatitis B carrier status were not associated with hepatotoxicity (Table 3). The cumulative incidence of hepatotoxicity was significantly higher in the first three years in the TPMP non-NM group than in the NM group (*p* < 0.001). In the TPMP non-NM group, the 1-year cumulative incidence rate was 8.5%, the 2-year cumulative incidence rate was 18.6%, and the 3-year cumulative incidence rate was 18.6% (Figure 2).

<table><caption>**Table 2.** Outcome post-AZA treatment.</caption><thead><tr><th>Variable</th><th>TPMT non-NM<br>(n = 50)</th><th>TPMT NM<br>(n = 1000)</th><th>p-Value</th></tr></thead><tbody><tr><td></td><td>AZA dose (mg)</td><td></td><td></td></tr><tr><td>≤25</td><td>27 (54.0)</td><td>563 (56.3)</td><td></td></tr><tr><td>25–50</td><td>21 (42.0)</td><td>333 (33.3)</td><td></td></tr><tr><td>>50</td><td>2 (4.0)</td><td>104 (10.4)</td><td>0.21</td></tr></tbody></table>
<table><caption>**Table 2.** *Cont.*</caption><thead><tr><th>Variable</th><th>TPMT non-NM<br>(n = 50)</th><th>TPMT NM<br>(n = 1000)</th><th>p-Value</th></tr></thead><tbody><tr><td colspan="4">Outcome postAZA exposure</td></tr><tr><td>Leukopenia ^ cases</td><td>28 (59.5)</td><td>611 (63.3)</td><td>0.60</td></tr><tr><td>Hepatitis &amp; cases</td><td>10 (20.0)</td><td>150 (15.0)</td><td>0.33</td></tr><tr><td>Lowest WBC (U/L)</td><td>4375 (3210–5420)</td><td>4200 (3150–5390)</td><td>0.52</td></tr><tr><td>Highest ALT (U/L)</td><td>38 (22–84)</td><td>39 (26–73)</td><td>0.68</td></tr><tr><td>Onset of leukopenia ^ (days)</td><td>1359.3 ± 1709.7</td><td>1597.3 ± 1606.6</td><td>0.44</td></tr><tr><td>Onset of hepatitis &amp; (days)</td><td>676.3 ± 837.9</td><td>2395.8 ± 1911.0</td><td>&lt;0.0001</td></tr></tbody></table>

Data are expressed as *n* (%) or the mean ± SD or the median (interquartile range). ^ WBC count ≤ 4000 (U/L). & ALT level ≥ 150 (U/L). TPMT, thiopurine S-methyltransferase; NM, normal metabolizer; AZA, azathioprine; WBC, white blood cell; ALT, alanine transaminase.

<table><caption>**Table 3.** Cox regression analysis of risk factors for hepatotoxicity following AZA treatment.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Hepatotoxicity ^</th><th rowspan="2">p-Value</th></tr><tr><th>HR</th><th>95% CI</th></tr></thead><tbody><tr><td>Age</td><td>0.99</td><td>0.98–1.01</td><td>0.60</td></tr><tr><td>Gender (reference<br>female)</td><td>1.77</td><td>1.07–2.91</td><td>0.03</td></tr><tr><td>TPMT non-NM<br>genotype</td><td>3.85</td><td>1.83–8.10</td><td>0.0004</td></tr><tr><td>AZA dose (mg)</td><td>1.00</td><td>1.00–1.01</td><td>0.31</td></tr><tr><td>MTX</td><td>1.62</td><td>1.03–2.57</td><td>0.04</td></tr><tr><td>Hepatitis B carrier</td><td>1.18</td><td>0.40–3.48</td><td>0.76</td></tr></tbody></table>

^ Development of an ALT level ≥ 150 U/L after AZA treatment. ALT, alanine transaminase; TPMT, thiopurine S-methyltransferase; NM, normal metabolizer; AZA, azathioprine; MTX, methotrexate; HR: hazard ratio; CI: confidence interval.

<figure><img src="image_101.png" /><figcaption>**Figure 2.** Three-year cumulative incidence of hepatotoxicity between the TPMT non-NM and NM groups. TPMT, thiopurine S-methyltransferase; NM, normal metabolizer.</figcaption></figure>
**4. Discussion**

This was a hospital-wide retrospective case-control study. The patients were selected from the TPMI cohort in which the TPMT gene status was tested. We enrolled all AZA users. Among 1050 AZA users, the non-NM genotype, male gender, and MTX usage were independent risk factors for hepatotoxicity. The cumulative incidence of hepatotoxicity was significantly higher in the first three years in the TPMT non-NM group than in the NM group. Our result shows that pre-emptive TPMT genotyping before starting AZA therapy may minimize the risk of hepatotoxicity.

Two meta-analyses by Liu et al. [19,20] reported that the incidence of TPMT polymorphisms was not associated with AZA-induced hepatotoxicity in patients with autoimmune diseases and IBD. Unlike the previous studies, our study revealed an association between TPMT polymorphisms and thiopurine-induced hepatotoxicity. First, our study design was different from those of the previous studies. We classified patients by TPMT genotyping before evaluating hepatotoxicity events. However, the earlier studies classified patients with hepatotoxicity events before assessing the TPMT genotype. Our study showed no difference in the first hepatotoxicity events between the TPMT non-NM and the NM groups. However, as the onset time of hepatotoxicity was considered and eliminating the interaction effect of variables by Cox regression analysis, the TPMT non-NM group was more prominent. Our study design was closer to the prospective concept. Second, our study population was different. Our patients had various autoimmune diseases, but the previous studies mainly analyzed patients with IBD. The subjects in the earlier studies discussing AZA-induced hepatotoxicity were primarily Caucasians. The Asian population was limited, but our study population was all Asians and was larger. As genetic testing is becoming less expensive and more accessible, pre-emptive TPMT testing is suggested in Asians to prevent post-AZA hepatotoxicity.

Previous studies revealed a significant association between TPMT polymorphisms and bone marrow toxicity. However, in our study, this was not noted. The previous studies primarily discussed Caucasians with IBD. The median dose of AZA in the previous studies was more than 1–2 mg/kg/day [23–28]. In contrast, our study enrolled patients with various autoimmune diseases, particularly SLE and SS. Our average AZA dosage was 36.4 mg in the TPMT non-NM group and 39.6 mg in the TPMT NM group. Approximately 90% of patients took less than 50 mg per day. We followed the AZA treatment strategy of starting with a lower dose and increasing the dose at a slow pace. Such a strategy seemed to keep the patients safe from myelosuppression even in those with the TPMT non-NM genotype. However, our study results show that taking AZA for a longer duration still has risks of hepatotoxicity.

In addition to the TPMT genotype, methotrexate usage and male gender were independent risk factors for hepatotoxicity. Clinical studies have widely reported the hepatotoxicity of MTX [29]. According to the FAERS database, females were less likely to develop liver injury than males [30]. A cross-sectional community study in Taiwan revealed that hepatitis with an elevated ALT was more common in men than in women [31]. According to our research, male patients with the TPMT non-NM genotype using MTX would have the highest risk of hepatotoxicity. These patients may take benefit from pre-emptive genotyping. This finding provides insights into the potential utilization of pharmacogenomics in individualized medical care.

There are several limitations to this study. First, the study design was retrospective. Missing data were inevitable. After three years of observation, the cumulative incidence of hepatotoxicity related to the TPMT non-NM genotype was noted. However, it is impossible to define a causal relationship between AZA and hepatotoxicity. The result was still significant. Second, we may have underestimated the number of cases of hepatotoxicity. There are three types of AZA-related hepatotoxicity: hepatocellular, cholestatic, and mixed. However, we only enrolled patients with hepatocellular-type hepatotoxicity. According to research by Siramolpiwat et al., the AZA-related hepatotoxicity type was predominantly a mixed type [32]. Evaluating only the ALT level may not miss patients with mixed-type
hepatotoxicity. Thus, our hepatotoxicity event number was still reliable. Third, we only discussed the TMPT genotype. In addition to the TPMT genotype, our study did not discuss many other genetic polymorphisms, such as nucleoside diphosphate-liked moiety X motif 15 (NUDT15). NUDT15 was reported to be associated with more frequent adverse events than TPMT polymorphisms in the Asian population [33]. Further studies are needed to verify our results.

**5. Conclusions**

This hospital-wide retrospective case-control study supported the hypothesis that the TPMT non-NM genotype is associated with hepatotoxicity following AZA therapy. Pre-emptive testing for TPMT polymorphisms could help to individualize AZA therapy by minimizing the risk of hepatotoxicity, especially in male patients taking MTX.

**Author Contributions:** H.-S.S. was involved in the conceptualization of this study, methodology, original draft preparation, and reviewing and editing of the manuscript. Y.-M.C. was involved in the conceptualization of this study, methodology, data generation, curation, resource acquisition, original draft preparation, and reviewing and editing of the manuscript. Y.-J.L. was involved in the methodology, data analysis, and review and editing of the manuscript editing. C.-Y.W. was involved in the methodology, data analysis, and reviewing and editing of the manuscript. J.-P.C. was involved in data curation, statistical analysis, and reviewing, and editing of the manuscript. H.-J.L. was involved in the data analysis. W.-T.H. was involved in the conceptualization of this study, methodology, data generation, curation, resource acquisition, original draft preparation, and reviewing and editing of the manuscript. W.-N.H. was involved in interpreting the results, resource acquisition, and reviewing and editing of the manuscript. Y.-H.C. participated in the study design, methodology, data interpretation, resource acquisition, and reviewing and editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by Academia Sinica 40-05-GMM and AS-GC-110-MD02.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Taichung Veterans General Hospital (SF19153A).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent was obtained from the patients to publish this paper.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank all the participants and investigators of the Taiwan Precision Medicine Initiative.

**Conflicts of Interest:** The authors declare that they have no conflict of interest to declare.

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<figure><img src="image_102.png" /></figure>

Article

# Short- and Long-Term Effects of a Prebiotic Intervention with Polyphenols Extracted from European Black Elderberry—Sustained Expansion of *Akkermansia* spp.

Simon Reider <fnref n="1" />,<fnref n="2" /> ID, Christina Watschinger <fnref n="1" />,<fnref n="2" />, Julia Längle <fnref n="1" />, Ulrike Pachmann <fnref n="3" /> ID, Nicole Przysiecki <fnref n="1" />,<fnref n="4" />, Alexandra Pfister <fnref n="1" />,<fnref n="4" />, Andreas Zollner <fnref n="1" />,<fnref n="4" />, Herbert Tilg <fnref n="4" />, Stephan Plattner <fnref n="5" /> and Alexander R. Moschen <fnref n="1" />,<fnref n="2" />,*

**Abstract:** (1) Background: The intestinal microbiome has emerged as a central factor in human physiology and its alteration has been associated with disease. Therefore, great hopes are placed in microbiota-modulating strategies. Among various approaches, prebiotics, substrates with selective metabolization conferring a health benefit to the host, are promising candidates. Herein, we studied the prebiotic properties of a purified extract from European black elderberries, with a high and standardized content of polyphenols and anthocyanins. (2) Methods: The ELDERGUT trial represents a 9-week longitudinal intervention study divided into 3 distinct phases, namely a baseline, an intervention and a washout period, three weeks each. The intervention consisted of capsules containing 300 mg elderberry extract taken twice a day. Patient-reported outcomes and biosamples were collected weekly. Microbiome composition was assessed using 16S amplicon metagenomics. (3) Results: The supplementation was well tolerated. Microbiome trajectories were highly individualized with a profound shift in diversity indices immediately upon initiation and after termination of the compound. This was accompanied by corresponding changes in species abundance over time. Of particular interest, the relative abundance of *Akkermansia* spp. continued to increase in a subset of participants even beyond the supplementation period. Associations with participant metadata were detected.

**Keywords:** black elderberry; polyphenols; prebiotic; microbiota

## 1. Introduction

Although to date no distinct pattern of a healthy microbiota has emerged, certain alterations of microbiota composition and function are described as markers of disease-associated states. Collectively, these changes are summarized as dysbiosis, although a clear definition of the term is lacking and it has grown to be regarded increasingly unhelpful by experts in the field [1]. Nevertheless, loss of microbial diversity, a reduction in the number of beneficial microbes and an expansion of pathobionts are regarded as hallmarks of dysbiosis [2,3].

Because dysbiotic states of the microbiota are observed in and have been associated with numerous disease conditions [4], the field of microbiota modulation is receiving utmost attention. One potential means to influence this important determinant of health and disease are dietary interventions. The influence of long-term nutritional patterns on the microbiota has been studied and described extensively [5,6]. However, evidence suggests that drastic and sustained modifications of the diet are necessary to achieve long-term

**Citation:** Reider, S.; Watschinger, C.; Längle, J.; Pachmann, U.; Przysiecki, N.; Pfister, A.; Zollner, A.; Tilg, H.; Plattner, S.; Moschen, A.R. Short- and Long-Term Effects of a Prebiotic Intervention with Polyphenols Extracted from European Black Elderberry—Sustained Expansion of *Akkermansia* spp. *J. Pers. Med.* **2022**, *12*, 1479. https://doi.org/10.3390/jpm12091479

Academic Editor:
Anne-Marie Caminade

Received: 15 August 2022

Accepted: 6 September 2022

Published: 9 September 2022

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<figure><img src="image_103.png" /></figure>

**Copyright:** © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

<footnote n="1">¹ Christian Doppler Laboratory for Mucosal Immunology, Faculty of Medicine, Johannes Kepler University, 4020 Linz, Austria</footnote>

<footnote n="2">² Department of Internal Medicine 2, Faculty of Medicine, Johannes Kepler University, 4020 Linz, Austria</footnote>

<footnote n="3">³ VASCage (Research Centre on Vascular Ageing and Stroke) GmbH, 6020 Innsbruck, Austria</footnote>

<footnote n="4">⁴ Division of Internal Medicine I (Gastroenterology, Hepatology, Endocrinology, and Metabolism), Department of Medicine, Medical University Innsbruck, 6020 Innsbruck, Austria</footnote>

<footnote n="5">5 IPRONA AG/SPA, 39011 Lana, Italy</footnote>

<footnote>* Correspondence: alexander.moschen@jku.at</footnote>
effects [7]. In a more focused approach, prebiotic supplementation could address this gap. The International Association for Probiotics and Prebiotics (ISAPP) defines prebiotics as nutritional compounds or substances which undergo selective metabolization by specific members of the intestinal microbiota, ultimately conferring a health benefit on the host [8]. While traditionally, non-digestible carbohydrates such as inulin, fructooligosaccharides and galactooligosaccharides have been considered prebiotics, the ISAPP has pointed out that some plant polyphenols might also fulfill these criteria and therefore might be considered prebiotics [8]. However, this is currently not reflected in the legal and regulatory status of these compounds within the European Union.

Polyphenols are a heterogenous class of secondary plant metabolites present in fruit and vegetables. Phenolic compounds possess one or more aromatic rings with two or more hydroxyl groups. They can be present in free forms or conjugated with sugars, acid and other biomolecules [9–11].

Plant polyphenols are not readily taken up in the small intestine and reach the colon in considerable amounts, where they can serve as substrate for the colonic bacterial microbiota [9]. As potential health effects of polyphenols seem to depend on the resulting metabolites from degradation by the colonic microbiota [12], the individual microbiome configuration seems to be a key determinant of potential effects of polyphenol-rich black elderberry extracts. The bioavailability of polyphenols is variable and depends on the nutritional setting in which the substance is presented and an extensive secondary metabolism, largely by the intestinal microbiome [13–19]. Thus, microbiome composition might influence polyphenol effects in given individuals through diverse modification [20,21]. Bacterial secondary metabolization of dietary polyphenols improves bioavailability and hydrophilicity of these compounds and enables detection in the urine [20]. One systematic review identified data hinting at potential antibacterial and anti-inflammatory effects, although underlying mechanisms remained elusive and the overall level of evidence was classified as weak [22]. Anthocyanins of black elderberry extract are taken up and incorporated into endothelial cell membranes and cytosol, hinting at potential antioxidative benefits [23]. On the other hand, microbiota-modifying properties of polyphenols have been reported and include an expansion of Bifidobacteria [24] and Lactobacilli [12,25] and alterations of the Bacteroides–Firmicutes ratio, possibly reflecting an adaption to a higher glycan load [26]. Besides direct prebiotic effects on the microbiota, an environmental mechanism has also been proposed for polyphenol-associated microbiota modulation. Certain polyphenols have been shown to exert anti-microbial activity in vitro [27]. Therefore, broad antimicrobial activity of certain polyphenol compounds could serve to open up niches in the gastrointestinal tract and allow for expansion of other taxa. To include this dualistic effect, the term “duplibiotics” has been coined [28,29].

Food-derived anthocyanins exhibit a low bioavailability in their native form [15] but are partly metabolized and taken up in the intestine and can be recovered from the urine [13–19,30,31].

Effectively, the polyphenol–microbiota axis exerts influence on host physiology. Certain polyphenol metabolites of microbial origin serve to strengthen barrier function and boost secretion of antimicrobial peptides [32] and effects on secondary bile acid metabolism have been described [33]. In a mouse model, food rich in polyphenols attenuated detrimental metabolic and inflammatory responses [34]. Short-chain fatty acids are also produced from polyphenols by the intestinal microbiota [35] and may serve as regulators of colonic mucosal homeostasis [36].

European black elderberries (Sambucus nigra) and their extracts have been used traditionally in parts of Europe for centuries as a remedy for common colds and influenza-like symptoms. Recently, an alleviating effect of an extract of black elderberries on symptoms and duration of influenza infections has been shown [37–39]. As research has mostly focused on the effects of black elderberry on upper respiratory tract infections, little is known on its microbiota-directed effects. Black elderberries are rich in polyphenols [27] and also contain various immunomodulating polysaccharides such as pectin [40,41]. The main
group of polyphenols in black elderberry fruits are anthocyanins (cyanidin 3-sambubioside-5-glucoside, cyanidin 3,5-diglucoside, cyanidin 3-sambubioside, cyanidin 3-glucoside). Additional polyphenols present in black elderberry are chlorogenic acids, rutin, and isoquercitrin [42].

This study aimed to provide a comprehensive in depth characterization of the microbiota-modulating effects of a 3-week intervention with black elderberry extract, standardized to anthocyanin and polyphenol levels.

## 2. Materials and Methods

### 2.1. *Trial Design and Study Cohort*

The ELDERGUT trial was a 9-week longitudinal intervention study with 3 distinct study phases of 3-week duration each. A baseline phase serving to obtain robust initial measurements was followed by a 3-week intervention period during which a daily dose of 600 mg of highly purified black elderberry extract (300 mg twice daily) was consumed by the participants. Participants logged digestive symptoms, bowel movements and possible adverse effects in a study diary provided by the investigational team. Participants could be either male or female and had to be 18 to 50 years old. These individuals had to report a history of vaginal delivery and being breast-fed for at least 3 months after birth. Exclusion criteria were recent antibiotic treatment within 3 months of the beginning of the study, a history of gastrointestinal disease or other acute or chronic medical conditions with the potential to affect the intestinal microbiome, a history of major abdominal surgery (appendectomy excluded), baseline consumption of dietary supplements or probiotics, a strictly vegan diet, relevant pathologic abnormalities in baseline clinical biochemistry tests and a known allergy to black elderberry.

The ELDERGUT trial (Figure 1A,B) included n = 30 healthy individuals (15 females, 15 males) that volunteered to participate in this study. All study subjects completed the 9-week study period and provided biological samples and clinical information at weekly study visits as indicated in Figure 1A. No study drop-outs occurred. All patients underwent a thorough clinical and biochemical evaluation at the baseline. Fecal calprotectin—a marker of intestinal inflammation—was within the normal range in all study subjects. Notably, two study participants happened to have a positive screening test for IgA antibodies against tissue transglutaminase and the diagnosis of celiac disease was confirmed histologically in both participants. These were excluded from the analysis as celiac disease was a pre-defined exclusion criterion. During conduction of the trial, 2 male participants contracted SARS-CoV2 during week 5 of the study period. The disease course was mild and the infection did not result in exclusion from the study or patients not providing samples.

The mean age of the trial population was 23.7 years (±3.05 years, range 19–31 years) and the mean body mass index was 22.3 kg/m² (±2.49, range 17.7–27.3). Baseline questionnaire-based nutritional assessment revealed a well-balanced nutritional pattern with differences between female and male participants. Male participants reported less intake of dietary fiber (*p* < 0.01, chi-square test), and a considerable but not significantly larger proportion of daily intake of animal fat (Figure 1C).

### 2.2. *Intervention*

Capsules containing 300 mg European black elderberry extract (ElderCraft®) were provided by IPRONA AG/SPA (Lana, Italy). ElderCraft® is a full spectrum water extract standardized to 14% anthocyanins (primarily cyanidin 3-sambubioside-5-glucoside, cyanidin 3,5-diglucoside, cyanidin 3-sambubioside, cyanidin 3-glucosid) and 18% polyphenols (primarily chlorogenic acids, rutin, isoquercitrin).
<figure><img src="image_104.png" /></figure>

<figure><img src="image_105.png" /></figure>

<figure><img src="image_106.png" /></figure>

**Figure 1.** ELDERGUT trial design and baseline nutrition data: (A) Timeline of interventions and sampling during the 9-week study period, depicting the baseline, the intervention and the washout phase of the study. During the study, biological samples including urine, faeces, and saliva were collected once a week, blood samples were drawn once per study phase (i.e., in weeks 1, 6 and 9). Questionnaires on well-being and gastrointestinal symptoms were completed by the participants during weekly visits. (B) Overview of the inclusion and screening strategy. (C) Data from questionnaires at baseline, indicating nutritional patterns within the cohort as well as gender-associated differences in food intake.

## 2.3. Biosample Acquisition and Biobanking

During the trial period, weekly biospecimen sampling (feces, urine, saliva) was performed. Additionally, blood samples were drawn once per study phase (at weeks 1, 6 and 9). Fecal samples were collected simultaneously into tubes pre-filled with nucleic acid stabilizing solution (INVITEK, Berlin, Germany) and standard fecal collection tubes (Sarstedt, Nümbrecht, Germany). Stabilized fecal samples were stored at -20 °C, unstabilized samples were stored in aliquots at -80 °C. Urine samples were centrifuged and the supernatant stored at -80 °C. Saliva samples were directly stored at -80 °C. Blood samples were collected into EDTA- and Serum-Tubes (Sarstedt, Nümbrecht, Germany), centrifuged and the supernatants were stored at -80 °C.
## 2.4. Fecal DNA Extraction

Fecal DNA was extracted from stabilized samples, according to the instructions provided by the manufacturer using a spin-column based kit (Qiagen Fast DNA Stool Kit, Qiagen, Germany). Briefly, homogenized suspensions were heated to 95 °C for 10 min, centrifuged and treated with Proteinase K. Then, samples were incubated with the buffer provided in the kit and pure ethanol. After vortexing, the preparations were transferred onto spin columns and centrifuged, followed by multiple washing steps on the column using the washing buffer from the isolation kit. Finally, DNA was eluated in 100 µL of a TE-based buffer. Nucleic acid concentration was measured using a Nanodrop 1000 spectrophotometer (Eppendorf, Hamburg, Germany).

## 2.5. Metagenomic Analysis

Fecal DNA isolates were analyzed using 16S amplicon based metagenomics in cooperation with a commercial provider. DNA concentration and purity was monitored on 1% agarose gels and DNA was amplified using a PCR with primers targeting the V3-V4 variable regions of the bacterial 16S rRNA gene (primer pair 314F-806R) All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). PCR products were mixed at equal ratios and purified using Qiagen Gel extraction kit (Qiagen, Germany). Libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina and quantification was done using Qubit and qPCR. Sequencing was performed on the Illumina Miseq platform with PE250 chemistry.

Paired end reads were assigned to samples using specific barcodes added in the PCR amplification step. Barcode sequences were removed and reads were merged using FLASH (v.1.2.7; Baltimore, MD, USA) [43]. Resulting raw tags were quality filtered using QIIME (v.1.7, Boulder, CO, USA [44]). Chimera detection and removal was performed using UCHIME (Boulder, CO, USA) against its Gold reference database [45,46]. OTU clustering was performed using UPARSE version 7 [47] using effective tags at a clustering level of 97%. Representative sequences were annotated for every OTU using mothur [48] and the SILVA database [49]. Further downstream analysis including alpha- and beta-diversity and analysis of differential abundance is detailed in the statistics section.

## 2.6. Statistics

Data from study diaries were summarized using descriptive statistics and significance was tested using ANOVA with the post hoc Tukey test and the Kruskal-Wallis/Wilcoxon test for continuous numeric variables depending on the underlying distribution. The frequency of symptoms and data from the nutrition questionnaire was assessed using chi-square statistics. Amplicon-based metagenomic data were imported into R v. 4.1.2 (R Core Team, Vienna, Austria) [50] and RStudio using phyloseq [51] and then analyzed according to published workflows. Permutational analysis of variance (PERMANOVA) was performed using Vegan (v.2.5-7, Helsinki, Finland) [52]. Trends in differential abundance of bacterial species over the duration of the study period were explored using a linear mixed model implemented in Maaslin2 [53]. For these approaches, appropriate variance and occurrence filters were implemented (supplementary material and analysis scripts are provided). The tidyverse [54], rstatix [55] and ggpubr [56] packages were used for data cleaning and statistical procedures.

# 3. Results

## 3.1. Patient-Reported Outcomes during the Trial Period

Participants completed symptom-based questionnaires on a weekly basis, assessing typical gastrointestinal symptoms as well as measures of general health and well-being. Additionally, information on the number of bowel movements and their consistency (Bristol Stool Scale, BSS value) was recorded.

Overall perception of health and quality of life of the study participants was consistently within the upper third of the 7-point visual analogue scale used for assessment.
No dynamic was observed along baseline, intervention and washout phase of the trial.
Likewise, no differences between female and male participants were observed within these
parameters (Figure 2A).

<figure><img src="image_107.png" /><figcaption>**Figure 2.** Patient-reported outcomes. (A) Weekly scores for health and quality of life (QoL), (B) results from weekly symptom-based questionnaires including common gastrointestinal symptoms. (C) Number of bowel movements per week in female and male participants. (D) Mean fecal consistency, i.e., the Bristol Stool Scale (BSS) value per week. Data were compared by the Kruskal–Wallis test, no significant differences were observed.</figcaption></figure>
Study subjects were asked for the presence of gastrointestinal symptoms including increase or decrease in appetite, the occurrence of flatulence and diarrhea, nausea, constipation and abdominal discomfort. Of these, only flatulence was a regular complaint, but no clear association with the intervention emerged. Abdominal pain was slightly more severe and more frequent in female participants, although in most instances this complaint coincided with specific events of the menstrual cycle. Overall, no symptom-based signal associated with the intervention was observed (Figure 2B).

The number of bowel openings was recorded and calculated as mean number of bowel movements per week. No changes were observed on a week-to-week basis, particularly not within the intervention period (Table 1, Figure 2C).

<table><caption>**Table 1.** Mean number of bowel movements and mean BSS values. No values for week 3 are available.</caption><thead><tr><th rowspan="2">Week</th><th colspan="2">Mean Number of Bowel Movements/Week</th><th colspan="2">Mean BSS Value/Week</th></tr><tr><th>Mean</th><th>std. Deviation</th><th>Mean</th><th>std. Deviation</th></tr></thead><tbody><tr><td>1</td><td>9.1</td><td>3.2</td><td>3.8</td><td>0.6</td></tr><tr><td>2</td><td>9.1</td><td>4</td><td>3.7</td><td>0.6</td></tr><tr><td>4</td><td>9.1</td><td>3.4</td><td>3.7</td><td>0.6</td></tr><tr><td>5</td><td>9.4</td><td>3</td><td>3.7</td><td>0.6</td></tr><tr><td>6</td><td>9.1</td><td>3.3</td><td>3.7</td><td>0.6</td></tr><tr><td>7</td><td>8.9</td><td>3.5</td><td>3.8</td><td>0.7</td></tr><tr><td>8</td><td>8.7</td><td>2.9</td><td>3.8</td><td>0.7</td></tr><tr><td>9</td><td>9</td><td>3.3</td><td>3.7</td><td>0.6</td></tr></tbody></table>

The Bristol Stool Scale (BSS) is a widely-used and validated tool [57,58] to assess the consistency of a bowel movement. Higher values on the BSS indicate more liquid stool, low values represent very hard consistency. Similar to bowel movements, the mean weekly BSS value (mean of the BSS value of all bowel movements of a single study participant during every week of the trial) was calculated and happened to remain in the middle part of the scale and unchanged during the whole trial period (Table 1, Figure 2D).

## 3.2. Alterations in 16S Amplicon-Based Metagenomic Profiles at the Beginning and after the End of the Prebiotic Intervention

A 16S amplicon based metagenomic approach was applied to investigate temporal alterations in microbiota composition associated with an intervention with ElderCraft®. Weekly fecal samples from 28 participants were analyzed (252 samples overall) and the resulting profiles were compared on a weekly basis to the baseline configuration, i.e., median abundances from weeks 1 to 3.

## 3.3. Changes in Intra-Sample Diversity

In the first week of the intervention period an immediate and strong increase in measures of α-diversity (i.e., intra-sample diversity) was observed (Figure 3A). The number of observed species increased from 623.4 ± 58.0 (mean and standard deviation) during baseline to 925.3 ± 348.2 at the end of week 4. Similarly, the Shannon index increased from 4.1 ± 0.27 to 4.45 ± 0.49 (*p* < 0.001 for both comparisons; pairwise Wilcoxon test). The most prevalent OTUs were mapped to the genus *Bacteroides* (phylum *Bacteroidetes*) and *Faecalibacterium* (phylum *Firmicutes*). Additionally, other genera from the phylum *Firmicutes* were highly abundant both at baseline and throughout the study period (i.e., *Agathobacter*, *Ruminococcus*, *Roseburia*). This was also observed for the genera *Bifidobacterium* (phylum *Actinobacteria*) and *Akkermansia* (phylum *Verrucomicrobia*).

In the later weeks of the intervention period, namely weeks 5 and 6, measures of α-diversity decreased but remained significantly higher in terms of the number of observed species (818.5 ± 356.6, 629.8 ± 43.0). The Shannon index was significantly different to baseline only were not significantly different from baseline apart from week 4 (Figure 3A). After stopping the prebiotic intervention in week 7, again an increase in the number of
observed species was evident (897.9 ± 228.9, *p* < 0.001, pairwise Wilcoxon test). There was no significant difference of Shannon indices.

<figure><img src="image_108.png" /></figure>

**Figure 3.** Microbial ecology measures of diversity. (A) α-diversity—describing intra-group diversity, i.e., the number of observed species—and Shannon indices. (B,C) β-diversity as unweighted Unifrac values by study week (B) and a Principal Coordinate Analysis of unweighted Unifrac distances (C). Statistical comparisons were made using pairwise Wilcoxon tests between the median of the baseline values (weeks 1–3) and the subsequent weeks. *p* values ** < 0.01, *** < 0.001.
### 3.4. *Compositional Differences over Time*

Differences of overall microbial composition between the different study weeks were investigated using the unweighted unifrac index of α-diversity as a measure of difference between groups of samples (Figure 3B,C). Both paired data and principle coordinate analysis (PCoA) revealed changes during, most pronounced in weeks 4 and 5, and in the first week after the prebiotic intervention. These differences met the prespecified significance level. Additionally, a significant effect of sex on microbiome composition was evident (*p* < 0.01 for both timepoint and sex, R-squared 0.07 for timepoint and 0.02 for sex, permutational analysis of variance).

### 3.5. *Alterations on a Species Level Driving Overall Changes in Bacterial Ecology*

Next, we employed linear modelling—taking the longitudinal nature of our data into account—to identify differences in the abundance at the species level responsible for driving the observed microbial ecologic dynamics.

Overall, on the genus level 36 genera and on the species level 71 differently abundant microbial taxa were detected when compared to the baseline abundances (q value < 0.05, Maaslin2 linear random mixed-effects model, Table S1). The taxa identified could be attributed to all major microbial phyla of the human fecal microbiota. Confirming findings from diversity analyses (Figure 3), most species exhibited significantly different abundance in week 4 and week 7 compared to baseline abundances, i.e., at the beginning of the intervention and washout periods, respectively. Most of the detected species showed transient increases such as *Butyricicoccus* spp., Fusobacterium mortiferum, certain species of Ruminococci or decreases including different Roseburia species and Bifidobacterium adolescentis in response to the supply or withdrawal of black elderberry extract. However, some taxa including Akkermannsia were found to have a sustainable expansion throughout the washout period. The most relevant genera and species identified are outlines in Figure 4A,B and a summary statistic for relative abundances as well as information on significance levels derived from Maaslin2 are shown in Table 1.

### 3.6. *Factors Determining Response to the Intervention*

Considerable interest is directed towards inter-individual differences related to differential effects of prebiotic interventions. We therefore associated baseline demographic, behavioral and nutritional covariates with increases in *Akkermannsia* spp. of at least 3-fold during the trial period (i.e., 10 out of 28 participants). Daily intake of foods rich in plant fat was associated with a relevant increase in the abundance of *Akkermansia* spp. in response to the intervention (*p* = 0.03, Fisher's exact test). However, no associations of baseline α- or β-diversity with such an increase were detected. Likewise, we detected no baseline microbial signature predicting a relevant increase in *Akkermansia* abundance.

An increase in the number of observed species or in Shannon index values at week 4 and week 7 of the study compared to baseline was considered a second marker of individual response to the prebiotic. For the number of observed species, this was defined as an increase by at least 50% while for Shannon indices the 3rd quartile value (1.1687 for week 4 and 1.0568) was applied. Thus, based on observed species count, 17 and 8 participants were classified as α-diversity-responders at week 4 and week 7, corresponding to 60.7% and 28.6% of participants. Regarding Shannon index cutoffs, seven individuals were classified as α-diversity-responders at both timepoints (25%). Univariate correlation with baseline nutritional patterns revealed a significant association of α-diversity-response at week 4 with reduced consumption of dairy products such as cheese, eggs and milk (for Shannon index values) and rice (based on observed species counts). Increases in α-diversity at week 7 were associated with less baseline consumption of whole grains (Shannon) and pasta (observed species count). However, there was no association of β-diversity response at weeks 4 and/or 7 with baseline microbiome composition (i.e., Bray–Curtis dissimilarity indices comparing β-diversity responders at baseline) and specific bacterial taxa on the genus and species level (linear mixed effect model Maaslin2; data not shown).
<figure><img src="image_109.png" /><figcaption>**Figure 4.** Analysis for differential abundance of bacterial taxa at the genus (A) and species (B) levels. Blue and red squares depict coefficients of significantly differential abundance for every week of the trial, grey squares represent non-significance (Maaslin2 linear mixed model, *q*-value < 0.25, *p* value < 0.05; detailed results provided in Table S1).</figcaption></figure>

## 4. Discussion

Prebiotic interventions are an attractive and promising platform towards modification of the intestinal microbiome. Their comparatively broad effect on different, albeit rather specific bacterial strains, holds the potential to provide symptom relief yet a potentially mechanistic approach in microbiome-mediated diseases [8,59]. To better understand the interactions of a polyphenol-containing prebiotic based on black a elderberry extract, the ELDERGUT trial has been designed and performed. The design of this 9-week longitudinal trial with a sequence of baseline, intervention and washout periods allowed for assessment of intra-individual dynamics and mitigates the considerable baseline variation that is a common characteristic of studies of the intestinal microbiome [60,61].

Dietary polyphenols have multifaceted and often diverging effects on members of the intestinal microbiota. Extensive metabolization by the microbial enzymatic machinery results in active secondary metabolites [12,15,35]. The aim of the ELDERGUT trial was to investigate the prebiotic properties of a phenol-rich elderberry extract. This highly purified preparation enables a polyphenol content of 18% a dose corresponding to a daily consumption of 30 g raw fruit.

In our population the intake of this concentration of black elderberry extract did not result in any adverse effects. No changes in overall perception of health and quality of life as an array of digestion-associated symptoms was observed. This confirms the good tolerability of the substance which is a prerequisite for further investigation as a prebiotic. Notably, no effects on fecal consistency and the number of bowel openings were observed. These are a typical symptoms induced by many other prebiotics through osmotic and bulk-forming effects [8,62,63].
Although no effects on patient-reported outcomes were detected, the introduction and withdrawal of the study medication at week 4 and week 7 of the trial resulted in changes of measures of microbial α- and β-diversity. As a measure of within-sample variability, indices of α-diversity (observed species count, Shannon index) spiked in these moments of perturbation. Between sample variability, i.e., β-diversity, was quantified using Bray–Curtis dissimilarity. Primary coordinate analysis and PERMANOVA statistical analysis revealed significant, although comparably small, associations with sex and week of the trial, the latter corresponding to the status of supplementation. Overall, 92 bacterial taxa were identified at the species level to be differentially abundant in fecal samples when comparing the weeks during the intervention and/or washout period with the median abundance at baseline. As sudden changes in diversity measures indicate perturbation of microbial community structures this points toward a relevant microbiome-shaping capacity of this prebiotic intervention. Interestingly however, these changes in diversity were only observed immediately after the start and end of the intervention period, respectively. This finding could reflect a quick stabilizing response within the microbial communities. In the context of well-known properties of the intestinal microbiome [7], this observation is likely to reflect the resilience and stability of a healthy microbiome configuration in this very uniform study population of student volunteers. This also underscores that the stability of microbiome compositions might result in obstacles to all variations of microbiome-targeted therapies, underlining the relevance of individual baseline composition and microbiome properties for prebiotic effects.

As the study population in the ELDERGUT trial included only healthy young vol-
unteers, applicability of these findings in the general population and especially in certain
disease groups such as patients suffering from inflammatory bowel disease or irritable
bowel syndrome might be limited. Overall quality of life and subjective perception of
health were already high at baseline in this cohort. Therefore, there is a possibility of a
beneficial effect of black elderberry extract on these measures that could not be detected
in this trial. Furthermore, minor adverse events which could be potentially connected
to the intervention are difficult to validate in the absence of a true placebo control group
and without adequate statistical power to address this issue. Another possible limitation
is, that the study intervention consisted of a natural, although highly purified mixture of
different polyphenols and cannot be considered a chemically well-defined pure substance.
Therefore, attribution of effects to single compounds is not possible.

Statistical methods to assess differential abundance in microbiome studies are notor-
iously non-robust, with different methods resulting in often differing and even contradicting
conclusions. These discrepancies can depend on choices made during processing of the
data and intrinsic properties of the dataset. Furthermore, some methods which have
been used in the field have been reported to suffer from poor specificity and a broadly
accepted 'state of the art' workflow is lacking [64,65]. Nevertheless, recent studies have
made the effort to systematically compare the different methods that are currently in
use [64]. On the basis of these published findings, we chose a method that has been scoring
consistently in these artificial settings and also conceptionally addresses the nature of this
specific dataset [53]. Thus, we are confident, that the taxa that are identified in the present
work represent true hits with biologic significance. In line with earlier reports [66], we
detected a significant increase in the abundance of *Akkermansia* spp. associated with the
study intervention. This bacterial taxon is known for its beneficial effects on inflammation
and metabolism [67,68]. It can influence and strengthen the intestinal barrier despite its
mucolytic properties. Notably, *Akkermansia* has already been reported to bloom under
certain polyphenol-rich diets, although no polyphenol degrading enzymatic capabilities
of this genus could be delineated. It is assumed, that this effect could be due to indirect
interactions between dietary polyphenols and *Akkermansia* [25,28,35], mediated by other
members of the microbiota. Whether this specific effect can be considered truly prebiotic
by the most recent ISAPP definitions is controversial [28]. The bacterial genus *Suterella*
from the phylum *Proteobacteria* was significantly increased immediately in the first week
of supplementation, although it returned to values non-significantly different from the baseline during the intervention period. This taxon has been beneficially associated with glucose metabolism and metabolic changes after Roux-Y-gastric bypass [69]. As members of this genus possess epithelium-adhering and mild pro-inflammatory properties without causing barrier disruption, it has been postulated, that this bacterial group might exert immunomodulatory effects [70]. On the other hand, in studies on fecal microbiota transplantation for Ulcerative Colitis, prevalence of this genus in the fecal transplant has been associated with a lack of remission after the procedure [71]. This detrimental effect on intestinal inflammation was linked to its ability to degrade host IgA [72].

Within the genus Bacteroides (Phylum Bacteroidetes) a more nuanced effect was detected with granular differential effects at the species level. Bacteroides cellulosolyticus increased early during the intervention. This carbohydrate-degrading species [73] has been associated with plant based nutrition [74], better outcomes of anti PD-L1 immuno-oncologic therapy [75] and disease course in COVID-19 [76]. Furthermore, Bacteroides thetaiotaomicron [77] has been implicated in the context of inflammatory bowel diseases [78,79] immune system maturation and obesity [80] and is well adapted to its existence as a gut commensal [81]. The genus Bacteroides seems to emerge as a key target genus of polyphenol supplementation in the setting of this study, probably reflecting the extensive enzymatic repertoire encoded by this bacterial taxon.

Observing the high inter-individual variability of changes in abundance during the intervention, we hypothesized that there might be underlying differences between participants at baseline that could be used to predict probability of taxonomic changes during supplementation. We identified an increase in *Akkermansia* spp. as most relevant to host health and tested for associations of baseline demographic (i.e., sex) and nutritional variables with a 3x increase in *Akkermansia* by week 4. This analysis revealed a significant association of an increase in *Akkermansia* with daily consumption of plant fats. Of note, baseline microbiome diversity indices and taxonomic composition were not significantly associated with this increase in *Akkermansia* abundance. Likewise, we detected associations of changes in α-diversity upon supplementation of black elderberry extract with baseline nutritional features but not with a distinct microbial signature at the baseline. Nevertheless, further analyses could provide better mechanistical insights in the interaction of dietary polyphenol intake, microbial abundance, enzymatic functions within the intestinal microbiota and resulting effects on the host.

**5. Conclusions**

The ELDERGUT trial provided convincing evidence for the prebiotic properties of a polyphenol-rich black elderberry extract in healthy individuals. While intake of this extract was associated with specific and individualized taxonomic changes within the fecal microbiota, no convincing associations with baseline factors emerged in this analysis.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jpm12091479/s1.

**Author Contributions:** Conceptualization, S.R. and S.P.; data curation, S.R.; formal analysis, S.R.; funding acquisition, A.R.M.; investigation, S.R., C.W., J.L., N.P., A.P. and A.Z.; methodology, S.R., A.P. and A.R.M.; project administration, S.R., J.L., U.P. and A.R.M.; resources, H.T., S.P. and A.R.M.; supervision, A.R.M.; visualization, S.R.; writing—original draft, S.R. and S.P.; Writing—review and editing, H.T. and A.R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was planned and conducted within the framework of the VASCage COMET Centre as a non-funded project. Funding was provided by IPRONA AG/SPA. Additionally, this work was supported by the Christian Doppler Research Association (to A.R.M) and we gratefully acknowledge the support by the Austrian Federal Ministry of Science, Research, and Economy and the National Foundation for Research, Technology, and Development.
**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the Medical University Innsbruck (protocol code 1020/2020, 20 February 2020).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in this study.

**Data Availability Statement:** Sequence data are publicly available at EMBL-EBI and scripts detailing the reported statistical analysis and output have been deposited at github (https://github.com/reider-si/ELDERGUT).

**Acknowledgments:** Black elderberry extract was supplied at no additional charge by IPRONA AG/SPA who also provided financial support regarding the analyses in this study. We gratefully acknowledge the support of the VASCAGE trial platform and would like to thank Ulrike Pachmann for invaluable support regarding regulatory approval and project administration. Open Access Funding by the University of Linz.

**Conflicts of Interest:** S.P. is employed at IPRONA AG/SPA. The other authors declare no conflict of interest.

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<figure><img src="image_110.png" /></figure>

Review

# Clinical Application of Ultra-High-Frequency Ultrasound

Anna Russo¹, Alfonso Reginelli ¹ID, Giorgia Viola Lacasella ², Enrico Grassi ³, Michele Ahmed Antonio Karaboue ⁴ID, Tiziana Quarto ⁵, Gian Maria Busetto ⁶ID, Alberto Aliprandi ⁷, Roberta Grassi ⁸,⁹ and Daniela Berritto ⁴,*

¹ Department of Precision Medicine, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy

² Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Sapienza University of Rome, 00185 Rome, Italy

³ Department of Orthopedics, University of Florence, 50121 Florence, Italy

⁴ Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy

⁵ Department of Law, University of Foggia, 71100 Foggia, Italy

⁶ Department of Urology and Renal Transplantation, University of Foggia Policlinico Riuniti of Foggia, 71122 Foggia, Italy

⁷ Department of Radiology, Istituti Clinici Zucchi, 20900 Monza, Italy

⁸ Department of Precision Oncology, University of Campania “Luigi Vanvitelli”, 80138 Naples, Italy

⁹ Italian Society of Medical and Interventional Radiology (SIRM), SIRM Foundation, 20122 Milano, Italy

* Correspondence: berritto.daniela@gmail.com

**Abstract:** Musculoskeletal ultrasound involves the study of many superficial targets, especially in the hands, wrists, and feet. Many of these areas are within the first 3 cm of the skin surface and are ideal targets for ultra-high-frequency ultrasound. The high spatial resolution and the superb image quality achievable allow foreseeing a wider use of this novel technique, which has the potential to bring innovation to diagnostic imaging.

**Keywords:** ultrasound; ultra-high frequency; musculoskeletal; imaging

## 1. Introduction

Ultra-high frequency ultrasound (UHFUS) is a method with the advantage of examining very small structures and providing high-resolution images with a dynamic, real-time, and comparative, where possible, evaluation [1]. UHFUS was established in clinical imaging in the 2000s, with its predominant application in dermatology and angiology [2–4]. In 1979, Alexander and Miller first introduced US as a noninvasive technique to measure normal skin thickness, and, in the 1980s and 1990s, high-resolution ultrasonography (HRUS) was used for the noninvasive assessment of skin nodules and cutaneous diseases [5,6]. The notion of UHFUS is often misunderstood due to a lack of consensus regarding the cutoff of frequencies for the classification of “very high frequencies” and “ultra-high frequencies”. The conventional ultrasonography (CUS) technique refers to the use of probes with frequencies ranging from 10 to 15 MHz. Bhatta et al. [7–10] considered high-frequency US (HFUS) to involve frequencies >10 MHz, while Polanska et al. [7–10] selected 20 MHz as the cutoff for HFUS. Finally, Shung et al. [7–10] defined HFUS for probes with frequencies >30 MHz. UHFUS, with frequencies as high as 50–70 MHz and capable of resolutions as fine as 30 microns, could permit new diagnostic applications in superficial small parts’ examination. A variety of superficial targets within the first 1 cm of the skin surface could be imaged, including for dermatological evaluation, such as the assessment of skin layers, hair follicles, nail units, and vascular, musculoskeletal, intraoral, and small-parts applications [11–14]. Hayaski et al. carried out a description of their experience in the identification and localization of lymphatic vessels in patients with secondary lymphedema. UHFUS provided images with extremely high resolution, demonstrating new characteristics of the lymphatic vessels [15,16]. The introduction of UHFUS in the study of oral lesions is one of the biggest innovations in US imaging of the head and neck region. The available

**Citation:** Russo, A.; Reginelli, A.; Lacasella, G.V.; Grassi, E.; Karaboue, M.A.A.; Quarto, T.; Busetto, G.M.; Aliprandi, A.; Grassi, R.; Berritto, D. Clinical Application of Ultra-High-Frequency Ultrasound. *J. Pers. Med.* **2022**, *12*, 1733. https://doi.org/10.3390/jpm1201733

Academic Editor: Anne-Marie Caminade

Received: 18 August 2022

Accepted: 27 September 2022

Published: 19 October 2022

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<figure><img src="image_111.png" /></figure>

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
frequencies of 48 and 70 MHz can effectively image the superficial layers of the mucosa as well as lesions of the oral cavity [17–21]. In comparison to CUS, UHFUS has a superior spatial resolution, even with the limitation of a low penetration depth, which is within 1–3 cm. In fact, 48 MHz probes have a penetration depth of 23.5 mm, while 70 MHz probes allow the imaging of the first 10.0 mm below the scanning surface.

The study of vascularization can be accomplished with the combination of color Doppler, which allows the observation of the distribution and size of vessels, and the power Doppler, which is usually more sensitive for detecting slow flow.

More than others, the method depends on the operator, as the probes are extremely sensitive to the examiner's hand movements. The literature on UHFUS is still evolving, but this technology seems to be the answer to several diagnostic limitations related to the need for high-resolution investigation for both normal anatomy and disease processes. In this paper, we present the role and potential clinical applications of UHFUS in musculoskeletal imaging based on our experience with a commercially available ultrasound system (Vevo MD; FUJIFILM VisualSonics, Amsterdam, The Netherlands): an ultra-high-frequency clinical ultrasound system equipped with a 48 to 70 MHz linear-array transducer. The system yields an image resolution up to 30 µm, which is equivalent to half a grain of sand, within the first 3 cm from the body surface.

Musculoskeletal evaluation with UHFUS includes, in both adult and pediatric patients, physiological and pathological disorders of superficial joints, tendons, and nerves, as well as presurgical planning and post-treatment follow-up and a guide for non-surgical interventional procedures [22,23].

## 2. Tendons

UHFUS encompasses wide applications, especially in the evaluation of hand anatomy and pathological disorders of the upper arm, including the study of pulleys, fascia, retinacula, and other superficial structures of the soft tissues (Figures 1–3). Particularly concerning the flexor tendon pulleys, the added value of UHFUS becomes crucial due to the subtle thickness of these structures, with a potential role in the identification of traumatic injuries [24–26]. UHFUS can identify pulley ruptures, especially with the dynamic assessment of the tendon-to-bone distance in flexion against resistance. In the evaluation prior to hand surgery, the analysis of hand anatomy can become essential for preoperative planning, postoperative management, and follow-up [27]. Tenosynovitis is defined as hypoechoic or anechoic thickened tissue with or without fluid within the tendon sheath with possible signs of Doppler signals, which are seen in two perpendicular planes [14]. Both tendon disease and tenosynovitis are important features of rheumatoid arthritis (RA), and US represents an ideal tool for their investigation. Tenosynovitis is seen as a combination of synovial thickening within the tendon sheath and tendon sheath effusion [28–36].

<figure><img src="image_112.png" /><figcaption>**Figure 1.** Sagittal view of A1 pulley (white arrows) presenting as a fusiform structure with a hypo-echoic signal contoured by a thin hyperechoic line. The superficial flexor tendon is visible (square parenthesis).</figcaption></figure>
<figure><img src="image_113.png" /><figcaption>**Figure 2.** Extensor tendon of a finger. UHFUS gives a detailed and magnified representation of small structures such as the extensor tendon of the finger, even allowing the visualization of partial lesions. In (a), the sagittal view of the terminal extensor tendon (white arrows) at the level of the distal interphalangeal joint (white asterisk). In (b), the median band of the extensor tendon inserting at the level of the middle phalanx (P2). In (c), the thin sagittal band of the extensor tendon at the level of the metacarpal head.</figcaption></figure>

<figure><img src="image_114.png" /><figcaption>**Figure 3.** Flexor tendon of a finger. Using UHFUS at the flexor tendons, the same spatial resolution can be achieved as in the imaging of the extensor tendons. In (a), the sagittal view of the deep flexor tendon component inserting on the basis of the distal phalanx (P3). In (b), the superficial component of the flexor tendon (white arrows) lying near the deep component (white square parenthesis).</figcaption></figure>

### 3. Small Joints

US is used routinely in rheumatologic and traumatic disorders to evaluate the joints of the wrist, hand, ankle, and foot. The use of UHFUS in the small joints of the hand and foot can demonstrate alterations such as synovial hyperplasia, the presence of calcifications, osteophytes, and an increased vascular signal and can be used to assess the thickness, homogeneity, and sharpness of the articular cartilage of peripheral joints, which is of great help in clinical practice [28–31]. In this field, above all, pediatric patients could benefit from such small transducers with great resolution, and this technology is designed for the smallest of patients with the greatest details and resolution possible (Figures 4 and 5).
<figure><img src="image_115.png" /><figcaption>**Figure 4.** Juvenile idiopathic arthritis (JIA). UHFUS gives clear details of pathological findings in pediatric patients with JIA. In (a), articular effusion (E) at the level of the metacarpal-phalangeal joint (MET-PP). In (b), osseous erosions at the level of the metacarpal head (white arrows).</figcaption></figure>

<figure><img src="image_116.png" /><figcaption>**Figure 5.** Juvenile idiopathic arthritis. UHFUS gives clear details of pathological findings in pediatric patients with JIA. In (a), articular effusion (E) at the level of wrist and enthesophyte (white arrow). Thickened capsule (arrowheads) in (a) presenting with an increased Doppler signal (white square) in (b).</figcaption></figure>

Over the last 10 years, US research in rheumatology has been standardized for early diagnosis and therapy monitoring [32–35].

The term synovitis is used to indicate the presence of synovial hypertrophy with a power Doppler signal and joint effusion, either proliferative or exudative. Its quantification via grayscale US usually uses a semiquantitative scale with three levels of intensity, indicating mild, moderate, or marked synovial changes [28–36]. According to the OMERACT indications, synovial fluid is defined as abnormal hypoechoic or anechoic intra-articular material that is displaceable and compressible and that does not exhibit a Doppler signal.

Erosions appear on US as focal discontinuities in the bone cortex. US assessment can provide detailed imaging of the hyaline cartilage, identifying small cartilage abnormalities in patients affected by RA, especially when dynamic US is performed in flexion of the finger joints and extension. Effusion is defined in US as abnormal hypoechoic or anechoic intra-articular material that is displaceable and compressible, but it does not exhibit a Doppler signal [37]. In the evaluation of crystalline arthropathies such as gouty arthropathy, US allows the evaluation of intra- and para-articular tophaceous deposits as well as the typical
double-contour collateral ligaments. In calcium pyrophosphate deposition disease, US allows the evaluation of crystal deposits as well as hyperechoic dots or an irregular line within the cartilage layer [37–40]. UHFUS with 50 MHz probes is able to accurately identify cartilage thinning, cartilage echogenicity, and subchondral bone.

## *Collateral Digital Ligaments*

UHFUS offers a detailed and magnified representation of collateral ligaments of the hand (Figure 6).

<figure><img src="image_117.png" /><figcaption>**Figure 6.** Collateral ligaments of interphalangeal joints: comparison between 50 MHz probes in (a) and 15 MHz CUS (arrowheads) in (b). In (a), UHFUS gives a more detailed and magnified representation of both deep (white arrows) and superficial (white arrowheads) components of the ligament.</figcaption></figure>

This could become useful in demonstrating even partial injuries of these structures. In particular, injury to the ulnar collateral ligament (UCL) complex of the thumb is a common traumatic lesion that requires prompt imaging evaluation for adequate treatment. UHFUS could have a key role in the illustration of both static and dynamic findings related to UCL injuries with even more details than MRI imaging (Figures 7 and 8).

<figure><img src="image_118.png" /><figcaption>**Figure 7.** Full-thickness UCL tear. In (a), normal appearance of adductor pollicis aponeurosis (white arrows) and below the ulnar collateral ligament (asterisk) of the first metacarpophalangeal joint. In (b), the non-visualization of the ulnar collateral ligament and the presence of a mass-like area (arrowhead) proximal to the joint have high accuracy in depicting a displaced full-thickness ligament tear.</figcaption></figure>
<figure><img src="image_119.png" /><figcaption>**Figure 8.** Same patient as in Figure 7. 1.5 T MRI shows a mass-like area (arrowhead) proximal to the joint representing the injured and displaced ligament in (a). In (b), the contralateral metacarpal phalangeal joint of the thumb. M: metacarpal head.</figcaption></figure>

4. Nerves

Compared to 5–20 MHz probes, UHFUS equipped with 80 MHz probes allows a more detailed evaluation of nerve anatomy in small cutaneous nerve branches, giving a spatial resolution down to 30 microns [6,8,10,41–43] (Figure 9).

<figure><img src="image_120.png" /><figcaption>**Figure 9.** Visualization of small peripheral nerves using UHFUS. Recurrent branch (line) of the median nerve (square parenthesis) at the level of the thenar eminence in (a). Digital branch (dot circle) of the median nerve at the level of the finger in (b).</figcaption></figure>

UHFUS allows the precise evaluation of the normal anatomical appearance as well as the echo-structural alteration of the honeycomb appearance represented by hypoechoic fascicles on a hyperechoic background. Entrapment neuropathies are generally characterized by nerve thickening, the loss of the fascicular pattern, and decreased echogenicity, mainly due to edema (in the acute phase) and fibrosis (in the chronic phase). It is also possible to see the thinning of the nerve at the site of compression. Conversely, in other disorders, such as Charcot-Marie-Tooth and some nerve neoplasms, we can see the enlargement of selected fascicles, an imaging feature recognizable using UHFUS [44–46]. UHFUS allows the determination of the location, size, and type of lesion, nerve swelling, and inflammation or the identification of compressive structures such as calcifications and scar tissue [47,48]. Nerves that are often involved in traumatic and iatrogenic injuries are the superficial cutaneous branches of the median, ulnar, and radial nerves in the hand and wrist and the digital nerves. UHFUS allows the visualization of the small cutaneous branches of peripheral nerves. The advantages of using US in the evaluation of the anatomy and disorders of the median nerve even after surgery are known in the literature: in cases of post-traumatic neoformations, such as a neuroma, both intra- and extra-operative applications are used for the preservation of the nerve after surgical excision. UHFUS could be useful in the study of pediatric patients and in neuromuscular disorders such as chronic inflammatory demyelinating diseases, thanks to the evaluation of the number
of morphologies and the size of the nerve fascicles [49–51]. Benign lesions of the peripheral nerves, such as a lipofibromatous hamartoma and the infiltration of the nerve fascicles, appear as non-homogeneous nerve enlargements, especially in proximal median nerve segments and the brachial plexus [43,52], with variable echogenicity patterns depending on the stage of the disease. UFUS has been used for non-surgical guidance in percutaneous procedures for partial and total wrist denervation. In the treatment of chronic wrist pain due to post-traumatic injuries, degenerative disorders, or arthritis, denervation is the treatment for chronic wrist pain, without impairing motor function, and avoids the need for postoperative immobilization to decrease the risk of stiffness. Under US guidance, the dorsal extensor tendon compartment is visualized, and, using a transverse anatomic projection along the short axis of the tendons, radiofrequency ablation of the posterior and anterior interosseous nerves is performed. The posterior interosseous nerve appears as a thin, hypoechoic, and non-compressible ovoid structure from 1 to 3 mm in diameter (Figure 10) [53–55].

<figure><img src="image_121.png" /><figcaption>**Figure 10.** Percutaneous radiofrequency ablation of the posterior interosseous nerve for chronic wrist pain: In (a), cannula's insertion (white arrowheads) under real-time ultrasound. Guidance for direct visualization of the posterior interosseous nerve (white caliber). The procedure is performed on an awake patient using a noninvasive approach (b).</figcaption></figure>

## 5. Soft-Tissue Masses

UHFUS could represent an advantageous technique for the identification of small and soft-tissue neoformations such as glomus tumors (Figures 11 and 12).

<figure><img src="image_122.png" /><figcaption>**Figure 11.** Glomus tumor. The location corresponds to the pulp of finger IV (a). The patient had the classical triad of symptoms: paroxysmal pain, pinpoint pain, and cold hypersensitivity, lasting for two years. In (b), UHFUS shows a well-delimited nodule in contact with the adjacent phalangeal bone (arrowhead), but no cortical deformity is present. No significant hyperemia on color Doppler was noticed. On CUS (c), the nodule was delineated only thanks to the help of the preliminary UHFUS exam.</figcaption></figure>
<figure><img src="image_123.png" /><figcaption>**Figure 12.** Glomus tumor in the subungual space. No clear alterations were visible during clinical examination at the site of pain (circle in (a)). In (b), UHFUS effectively demonstrates the presence of a hypo-isoechogenic nodule (calibers) in contact with the adjacent phalangeal bone (arrowhead) under the nail plate (arrows). Note the small deformation of the nail plate. Mild vascularization on color Doppler was present (c).</figcaption></figure>

Glomus tumors are rare, benign, vascular neoplasms arising from the glomus body, which is a contractile neuromyoarterial structure found in the reticular dermis. This structure controls blood pressure and temperature by regulating blood flow in the cutaneous vasculature. Hyperplasia in any of these parts can lead to tumor formation, which is extremely painful. Glomus tumors account for 1–5% of soft-tissue tumors of the hand, and 75% of them are subungual in location. Other less commonly involved sites in the hand are the nail matrix, nail bed, and pulp of the finger. The delay in diagnosing these tumors for many years is a significant problem. It is not uncommon that patients are easily misdiagnosed with conditions such as neuropathic complaints, arthritis, or neuralgia and undergo unsuitable treatment. For these reasons, when the clinical examination is equivocal, noninvasive imaging techniques may be needed to aid in the diagnosis and delineate the anatomy preoperatively. Complete surgical excision of the tumor is the only effective treatment. Incomplete excision is considered the main cause of recurrence. US follow-up and/or intraoperative US may be useful for reducing recurrence and ensuring adequate resection, and the UHFUS tool is promising in this field [56].

**6. Conclusions**

Musculoskeletal evaluation with UHFUS includes, in both adult and pediatric patients, physiological and pathological disorders of superficial joints, tendons, and nerve structures, as well as presurgical planning and post-treatment follow-up and a guide for non-surgical interventional procedures. The high spatial resolution and the superb image quality achievable allow foreseeing the wider use of this novel technique, which has the potential to bring innovation to diagnostic imaging.

**Author Contributions:** Conceptualization and data collection, D.B. and A.R. (Anna Russo); writing, R.G. and E.G.; reviewing, T.Q. and A.A.; supervision, A.R. (Alfonso Reginelli) and G.M.B.; English editing, G.V.L. and M.A.A.K.; data curation, A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
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Review

# Non-Oncological Radiotherapy: A Review of Modern Approaches

Valerio Nardone ¹,*,†, Emma D'Ippolito ¹,†,‡, Roberta Grassi ¹, Angelo Sangiovanni ¹, Federico Gagliardi ¹, Giuseppina De Marco ¹, Vittorio Salvatore Menditti ¹, Luca D'Ambrosio ¹, Fabrizio Cioce ¹, Luca Boldrini ²,§, Viola Salvestrini ³, Carlo Greco ⁴, Isacco Desideri ³, Francesca De Felice ⁵, Ida D'Onofrio ⁶, Roberto Grassi ¹, Alfonso Reginelli ¹,‡ and Salvatore Cappabianca ¹,‡

¹ Department of Precision Medicine, University of Campania “L. Vanvitelli”, 80138 Naples, Italy

2 Radiation Oncology, Fondazione Policlinico Universitario A. Gemelli, IRCCS, Largo Agostino Gemelli, 00168 Rome, Italy

3 Radiation Oncology, Azienda Ospedaliero-Universitaria Careggi, Department of Experimental and Clinical Biomedical Sciences, University of Florence, 50134 Florence, Italy

4 Department of Radiation Oncology, Università Campus Bio-Medico di Roma, Fondazione Policlinico Universitario Campus Bio-Medico, Via Alvaro del Portillo, 00128 Rome, Italy

5 Radiation Oncology, Policlinico Umberto I "Sapienza" University of Rome, Viale Regina Elena 326, 00161 Rome, Italy

6 Radiation Oncology, Ospedale del Mare, ASL Napoli 1 Centro, 80147 Naples, Italy

* Correspondence: valerio.nardone@unicampania.it

† These authors contributed equally to this work.

‡ These authors contributed equally to this work.

**Abstract:** Despite being usually delivered in oncological patients, radiotherapy can be used as a successful treatment for several non-malignant disorders. Even though this use of radiotherapy has been scarcely investigated since the 1950s, more recent interest has actually shed the light on this approach. Thus, the aim of this narrative review is to analyze the applications of non-oncological radiotherapy in different disorders. Key references were derived from a PubMed query. Hand searching and clinicaltrials.gov were also used. This review contains a narrative report and a critical discussion of non-oncological radiotherapy approaches. In conclusion, non-oncological radiotherapy is a safe and efficacious approach to treat several disorders that needs to be further investigated and used in clinical practice.

**Keywords:** radiotherapy; non-malignant disorders; non-oncological radiotherapy

## 1. Introduction

Radiation therapy (RT) represents one of the cornerstones of cancer management, together with surgery and systemic therapy. It is reported that almost half of all cancer patients will receive RT during their treatment [1].

In the last decades, RT has undergone several advances driven by the increase in knowledge of radiobiology, use of advanced imaging, and treatment delivery approaches [2–5].

Actually, RT can be delivered with great accuracy, to reach an increasing dose to the targets and at the same time sparing the surrounding organs at risk [2,3].

Non-oncological radiotherapy can be used to treat several disorders and accounts for 20% of all treated patients in Germany [6–8].

In many other countries, including Italy, the use of RT for non-oncological diseases is not very common among the RT centers and often unknown among the other specialists.

The knowledge and the promotion of non-oncological RT, thus, could counterbalance the expected loss of patients or RT fractions foregone in the next future due to the use of hypofractionation.

Herein, we will discuss the different non oncological diseases that can be treated by RT. Following a literature search, we will provide a narrative overview of these topics.

**Citation:** Nardone, V.; D'Ippolito, E.; Grassi, R.; Sangiovanni, A.; Gagliardi, F.; De Marco, G.; Menditti, V.S.; D'Ambrosio, L.; Cioce, F.; Boldrini, L.; et al. Non-Oncological Radiotherapy: A Review of Modern Approaches. *J. Pers. Med.* **2022**, *12*, 1677. https://doi.org/10.3390/jpm12101677

Academic Editor: Anne-Marie Caminade

Received: 22 September 2022

Accepted: 6 October 2022

Published: 9 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_126.png" /></figure>

**Copyright:** © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
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Attribution (CC BY) license (https://
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4.0/).
## 2. Search Strategy

A literature search was conducted to retrieve potential eligible studies using PubMed and the clinicaltrials.gov electronic database.

The literature search was performed in September 2022 (from 1980 to August 2022) using the keywords "Radiotherapy AND non oncological OR non malignant OR benign".

Additionally, we manually searched the reference lists of studies and review papers to identify other relevant studies. No limits were applied to publication type. The results are grouped according to the non-oncological disorders and discussed qualitatively.

## 3. Results

### 3.1. *Heart*

Cardiac arrhythmias affected 8.8 million people in Europe and is an independent risk factor for stroke, [9], heart disease [10] and chronic kidney disease [11]. Recently, RT has been investigated as a potential treatment for recurrent ventricular tachycardia (VT) and atrial fibrillation (AF). The technological improvement of RT techniques might arguably open up to new treatments in this setting. We report the main data in the literature regarding RT treatment of VT and AF. A summary of collected evidence is reported in Table 1.

#### 3.1.1. Ventricular Tachycardia

Stereotactic radiotherapy (SBRT) is under investigation as a treatment option for patients with VT who do not respond to antiarrhythmic drugs and/or catheter ablation. The evidence is scarce, but most studies suggest the dose of 25 Gy in a single session.

Cuculich et al. combined techniques of electrocardiographic imaging to map arrhythmogenic scar regions in patients with refractory VT and non-invasive delivery of precise ablation with SBRT to perform noninvasive cardiac radioablation. SBRT dose was a single fraction of 25 Gy. Treatment efficacy was measured using the number of VT episodes recorded by ICDs [12]. Of the nine evaluated patients, only five underwent SBRT, with no acute high-grade toxicity or complications. Sixty percent of the patients showed mild fatigue and all the patients showed a reduction of VT [12]. Similarly, in the Polish SMART-VT Trial, Kurzelowski et al. used Stereotactic Arrhythmia Radioablation (STAR) for two patients with refractory VT, using the same dose (25 Gy in single fx). After 6 months, ICD showed no VT episodes in one patient and a good response in the other patient [13].

Wight et al., similarly, treated with the same dose 14 patients with refractory VT. In this trial, the clinical target volume was delineated according to the individual patient's characteristics, based on electroanatomic mapping. Two patients died after SBRT, whereas one received a heart transplant, and another patient did not respond. Of the other 10 patients, VT was reduced in 59%, ATP was reduced in 39%, and shocks were reduced in 60% [14].

In the UK multicenter experience, seven patients were treated with the same dose and technique similarly to the previous experience. After 6 months, for the five patients analysed VT burden was reduced by 85%, with no high-grade acute toxicity and three deaths due to heart failure. [15].

The STRA-MI-VT was a phase Ib/II trial that evaluated the feasibility of Cyberknife tracking in treatment planning [16].
<table><caption>Table 1. Summary of evidence regarding ventricular tachycardia (VT) and atrial fibrillation (AF) treatment with stereotactic ablative radiotherapy (SABR).</caption><thead><tr><th>Authors</th><th>Year</th><th>N pts</th><th>Diagnosis</th><th>End-Point</th><th>Dose tot/fx</th><th>Results</th></tr></thead><tbody><tr><td>Cuculich PS [12]</td><td>2017</td><td>5</td><td>VT</td><td>Efficacy and safety of treatment</td><td>25 Gy/1 fx</td><td>No complications during treatment. Fatigue after treatment (three patients), with no acute heart-failure. Marked reduction in the burden of ventricular tachycardia after treatment.</td></tr><tr><td>Kurzelowski R [13]</td><td>2022</td><td>2</td><td>VT</td><td>Efficacy and safety of treatment</td><td>25 Gy/1 fx</td><td>No problem in the first patient. The second one experienced acute side effects with an increase in VT that gradually improved at the end of the follow-up period.</td></tr><tr><td>Wight J [14]</td><td>2022</td><td>14</td><td>VT</td><td>Efficacy and safety of treatment</td><td>25 Gy/1 fx</td><td>VT was reduced in 59%, ATP was reduced in 39%, and shocks were reduced in 60%.</td></tr><tr><td>Lee J [15]</td><td>2021</td><td>7</td><td>VT</td><td>Reduction of VT and safety of treatment</td><td>25 Gy/1 fx</td><td>VT responded in all patients. After 6 months, VT burden was reduced by 85%. No high grade acute toxicity.</td></tr><tr><td>Piccolo C [16]</td><td>2022</td><td>Phantom study</td><td>VT</td><td>Feasibility of Cyberknife on cardiac lesions by tracking as a single marker the lead tip of an implantable cardioverter defibrillator.</td><td>25 Gy/1 fx</td><td>Tracking with a single marker is feasible considering adequate residual planning margins. The volumes could be further reduced by using additional markers.</td></tr><tr><td>Bonaparte I [17]</td><td>2021</td><td>Dosimetric study</td><td>VT</td><td>STAR is efficacy in terms of BDT and MUs.</td><td>25 Gy/1 fx</td><td>Several plans were evaluated for dosimetric considerations.</td></tr><tr><td>Kovacs B [18]</td><td>2021</td><td>57</td><td>VT/FA</td><td>STAR's effectivity and safety for structural VT/VF</td><td>25 Gy/1 fx</td><td>Significant short-term reduction of sustained VT/VF-burden, but recurrences are common.</td></tr><tr><td>Akdag O [19]</td><td>2022</td><td>Phantom study</td><td>VT</td><td>First experimental evidence for real-time cardiorespiratory motion-mitigated MRI-guided STAR on the 1.5 T Unity MRlinac aimed at simultaneously compensating cardiac and respiratory motions.</td><td>25 Gy/1 fx</td><td>Cardiac motion was successfully mitigated using gating, which was demonstrated in the phantom and in-silico experiment.</td></tr><tr><td>Kautzner J [20]</td><td>2021</td><td>3</td><td>VT</td><td>postmortem immunohistochemical was performed early and late after SBRT</td><td>25 Gy/1 fx</td><td>Apoptosis and subsequent fibrosis was shown to be not immediate, thus the antiarrhythmic effects may be delayed after SBRT.</td></tr><tr><td>Di Monaco A [21]</td><td>2022</td><td>5</td><td>AF</td><td>Side effects at 1 month after STAR</td><td>25 Gy/1 fx/25 Gy/1 fx</td><td>No acute treatment-related adverse events (&gt;G1)</td></tr></tbody></table>

Abbreviation: N: number, Pts: patients, RT: radiation therapy, Fx: fractions, VT: Ventricular Tachycardia, AF: Atrial Fibrillation, Gy: Gray, STAR: Stereotactic Arrhythmia Radioablation.
Bonaparte et al. [17] also performed a dosimetric analysis for Linac-based STAR for VT, using different treatment planning approaches. The authors concluded that among the different techniques and energies, the 10 MeV Flattening Filter Free (FFF) approach was the faster but not suitable in patient with cardiac implantable electronic devices [17].

A systematic review including 13 studies and 57 patients confirmed efficacy and safety of STAR for refractory VT/ventricular fibrillation (VF). Thirty-one patients (54%) had ischemic cardiomyopathy and fifty patients (88%) had prior catheter ablation (CA) for VT/VF. A single dose of 25 Gy was delivered to a mean PTV of 64.4 cc (range 3.5–238) with a mean safety margin of 3.3 mm (0–5). Electrical storm was shown in 7% after SABR. VT burden was reduced in all patients, but recurrence affected most of the patients (75%), with several adverse events (81%) and no treatment-related deaths. The authors concluded that STAR preliminary experience appears safe and efficacious despite the recurrence rate and deserve to be further investigated [18]. Despite this, there are still few conflicting data on the follow-up of patients and on the influence of respiratory movement on the cardiac dose. In fact, cardiac motion presents an important challenge because the VT isthmus is subject to both respiratory and cardiac motion. So, Akdag et al. provided first experimental evidence for real-time cardiorespiratory motion-mitigated MRI-guided STAR on the 1.5 T Unity MR-linac. A real-time cardiorespiratory motion-mitigated radiotherapy workflow was developed on the Unity MR-linac. A 15-beam intensity-modulated radiation therapy treatment plan (1 × 25 Gy) was created in Monaco v.5.40.01 (Elekta AB, Stockholm, Sweden) for the Quasar MRI⁴D phantom (ModusQA, Modus Medical Devices, London, Ontario, Canada). Simulations showed that cardiac motion decreased the target’s D98% dose between 0.1 and 1.3 Gy, with gating providing effective mitigation. So, real-time MRI-guided cardiorespiratory motion management greatly reduces motion-induced dosimetric uncertainty and warrants further research and development for potential future use in STAR aimed at simultaneously compensating cardiac and respiratory motions [19].

Kautzner et al. have published case series with the first postmortem immunohistochemical analysis of morphologic changes in the myocardium early and late after SBRT. The authors have found apoptosis followed by fibrosis that could explain the timing of the SBRT efficacy on VT [20].

In conclusion, STAR had reasonable VT suppression in patients where conventional treatment had failed.

### 3.1.2. Atrial Fibrillation

AF affects about 40 million people in the world and increased the risk of stroke and heart failure. Current clinical management include antiarrhythmic therapy and eventually catheter ablation in drug refractory patients. Despite that, a subset of elderly patients are not responsive to systemic therapies and have an high risk of complications following catheter ablation, thus a non-invasive approach as STAR should be investigated.

A systematic review collected available evidence (both preclinical and clinical setting) on the feasibility and efficacy of STAR, including photon RT (XRT) and particle beam therapy (PBT), in the treatment of AF. Twenty-one works (17 for XRT, 3 for PBT, 1 both) published between 2010 and 2021 were included. The main favorable finding consisted in the detection of electrical scar in 4/4 patients undergoing specific evaluation, whereas the minimum dose for efficacy was 25 Gy. No acute complications were observed below this dose and a great heterogeneity was observed among the included studies [22].

Di Monaco et al. performed a phase II trial that reported a preliminary experience of five patients treated for AF. The preliminary results showed no high-grade side effects and a good response in terms of AF control and no further use of antiarrhythmic drugs [21].

Ultimately, STAR represents a safe and effective non-invasive approach in the treatment of drug-refractory arrhythmias, but there is still poor data in the literature regarding long-term efficacy and follow-up. These two aspects, together with the choice of specific patient settings, represent the most important challenge to be investigated with well-defined studies.
## 3.2. Soft Tissue Disorders

Non-malignant, proliferative, soft tissue disorders are a very heterogeneous group of diseases, with a tumor-like phenotype, although they do not have malignant characteristics. These pathologies can reach large dimensions and cause serious organs involved deficits. Abnormal growth of fibroblasts or hypertrophic scar tissue (keloids), or inflammatory factors over-expression, such as bFGF, TGF-β, PDGF, EGF, and CTGF in Dupuytren's disease, can be found at the basis of the onset of these disorders [23,24]. RT may play a role in these diseases control thanks to its anti-inflammatory, anti-proliferative and immunomodulatory effects, as well as has been confirmed by numerous reports, especially when conventional treatments have not achieved sufficient control of symptoms. We report the main data in the literature regarding RT treatment in keloids, Dupuytren's disease, Peyronie's disease, and fibromatosis.

### 3.2.1. Keloids

Keloids are benign skin disorders based on excessive connective tissue proliferation during the normal scarring process. They typically appear after repeated surgeries at the same site, burns, after trauma, and deep dermis injuries. Pathogenetic mechanisms are not fully known, but the fibroblasts present in keloids have different characteristics compared to the fibroblasts present in normal skin [25]. Keloids are common, occurring in 5% to 15% of wounds and affecting both sexes equally. They mainly affect people 10 to 30 years old and are more commonly seen in those with family history of keloids [26]. The treatment of choice for keloids is surgery, which, however, shows 80% local recurrence; therefore, adjuvant RT can lower the risk of recurrence, as well as being the main treatment in cases of scar inoperability. Adjuvant RT can lower the risk of recurrence, as well as being the main treatment in cases of scar inoperability. Brachytherapy is effective to prevent keloid formation: the first session should start the same day as surgery and recommended doses are 5–6 Gy in three fractions or 5 Gy in four fractions [27,28]. Complete response rate of RT (either with electrons or orthovoltage techniques and brachytherapy) range from 50 to 98% according to the literature data (Table 2).

<table><caption>**Table 2.** Soft tissue disorders: included study and radiotherapy parameter.</caption><thead><tr><th>Author</th><th>Year</th><th>N pts</th><th>Diagnosis</th><th>End Point</th><th>Dose</th><th>Results</th></tr></thead><tbody><tr><td>Jiang [29]</td><td>2018</td><td>29</td><td>Keloids</td><td>Control rate</td><td>18 Gy/3 fx</td><td>Response rate 91.9%</td></tr><tr><td>Kim [30]</td><td>2015</td><td>28</td><td>Keloids</td><td>Control rate</td><td>12–15 Gy/3 fx</td><td>Response rate 50%</td></tr><tr><td>Shen [31]</td><td>2015</td><td>568</td><td>Keloids</td><td>Control rate</td><td>18 Gy/3 fx</td><td>Response rate 90.41%</td></tr><tr><td>Emad [32]</td><td>2010</td><td>26</td><td>Keloids</td><td>Control rate</td><td>12 Gy/3 fx</td><td>Response rate 70.4%</td></tr><tr><td>Malaker [33]</td><td>2004</td><td>64</td><td>Keloids</td><td>Control rate</td><td>37.5 Gy/5 fx</td><td>Response rate 97%</td></tr><tr><td>Lo [34]</td><td>1990</td><td>199</td><td>Keloids</td><td>Control rate</td><td>2–20 Gy/1 fx</td><td>Response rate 87% for Dose > 9 Gy, 43% for Dose < 9 Gy.</td></tr><tr><td>Borok [35]</td><td>1988</td><td>250</td><td>Keloids</td><td>Control rate</td><td>4–16 Gy/ various fx</td><td>Response rate 98%</td></tr><tr><td>Van de Kar [36]</td><td>2007</td><td>21</td><td>Keloids</td><td>Control rate</td><td>12 Gy/3–4 fx</td><td>Response rate 71.9%</td></tr><tr><td>Arneja [37]</td><td>2008</td><td>25</td><td>Keloids</td><td>Control rate</td><td>HDR BT 5 Gy/3 fx</td><td>Response rate 92%</td></tr><tr><td>Van Leeuwen [38]</td><td>2014</td><td>67</td><td>Keloids</td><td>Control rate</td><td>HDR BT 6 Gy/2 fx</td><td>Response rate 96.9%</td></tr><tr><td>Jiang [39]</td><td>2016</td><td>32</td><td>Keloids</td><td>Control rate</td><td>HDR BT 6 Gy/3 fx</td><td>Response rate 94%</td></tr><tr><td>Hafkamp [40]</td><td>2017</td><td>29</td><td>Keloids</td><td>Control rate</td><td>HDR BT 13 Gy/1 fx</td><td>Response rate 75.9%</td></tr><tr><td>Kadhum [41]</td><td>2017</td><td>698</td><td>Dupuytren's disease</td><td>Control rate</td><td>21–42 Gy in 3–14 fx</td><td>Good ratio of regressions (6–20% depending on staging), stability (12–81%) and low ratio of progressions (13–65%, depending on staging).</td></tr></tbody></table>
<table><caption>**Table 2.** *Cont.*</caption><thead><tr><th>Author</th><th>Year</th><th>N pts</th><th>Diagnosis</th><th>End Point</th><th>Dose</th><th>Results</th></tr></thead><tbody><tr><td>Seegenschmiedt [7]</td><td>2015</td><td>1762</td><td>Dupuytren's disease</td><td>Control rate</td><td>15-21 Gy in 5-7 fx, 30 Gy split in 2 series of 5fx with a 3 months interval</td><td>Stability of disease in 84% for N stage and 67% for N/I stage</td></tr><tr><td>Betz [42]</td><td>2010</td><td>135</td><td>Dupuytren's disease</td><td>Control rate</td><td>30 Gy split in 2 series of 5 fx separated by a 6- to 8-week interval</td><td>Stability of disease in 59%, 10% improved, and 31% progressed. In stage N 87% and in stage N/I 70% remained stable or regressed</td></tr><tr><td>Seegenschmiedt [8]</td><td>2015</td><td>8732</td><td>Peyronie's disease</td><td>Pain, improvement</td><td>10-20 Gy (2-10 fx)</td><td>Pain regression in 50-90%, Improvement of penile deviation in 30-70%</td></tr><tr><td>Seinen [43]</td><td>2015</td><td>155 RT alone, 815 Surgery + RT</td><td>Fibromatosis</td><td>Local control</td><td>30-74 Gy</td><td>Local control in 78% of the patients treated with surgery and RT versus 85% in patients treated with RT alone</td></tr></tbody></table>

Abbreviation: N: number, Pts: patients, RT: radiation therapy, Fx: fractions, BT: brachytherapy.

### 3.2.2. Dupuytren's Disease

Dupuytren's disease is a fibrotic hyperplasia of connective tissue structures at the level of the finger band and palm. It is a rare condition, with a prevalence of about 2% and with a higher incidence in males (3:1 ratio). The pathogenesis is probably due to factors such as repeated trauma, alcohol and nicotine abuse, and hereditary factors. The diagnosis is purely clinical and seen around the 4th decade of life the presence of fibrotic nodules at the level of the hand that result in digito-palmar contracture leading to severe functional limitations. Dupuytren's disease can be staged by Tubiana classification, which subdivides pathology according to symptomatology. The gold standard treatments for early stages are medical treatment with intralesional applications of xanthine oxidase/dehydrogenase inhibitor, allopurinol, or cytotoxic agents such as vinblastine or colchicine. Surgery is usually reserved for more advanced stages (Stage 3 Tubiana); the role of RT in the treatment of Dupuytren's disease tends to be rather preventive and prophylactic than curative. Thus, the goal is to avoid future functional impairment and a future need for surgery [6]. The efficacy of RT is higher in early stages, inducing a significant reduction in fibroblast proliferation. RT is usually delivered with electron beam at 6MeV energy, while in other centers orthovoltage can be delivered alternatively. The volume treated includes palpable nodules with a safety margin of at least 10mm and the uninvolved structures are protected with lead-based shielding. Among the RT fractionations present in the literature, the most used, with better results both in terms of efficacy and limited toxicity, are the hypofractionations. The main results of the literature are shown in Table 2. Ledderhose disease is a rare type of plantar fibromatosis histologically related to Dupuytren's disease, and it has been also effectively treated with RT [44].

### 3.2.3. Peyronie's Disease

Peyronie's disease (PD) is a benign condition leading to plaque formation at the level of the tunica albuginea of the penis, leading to local pain and a change in curvature during erection. It usually affects men between the 4th and 6th decades of life with an incidence of 0.3-3%. The most likely mechanism is penile trauma causing inflammation of the tunica albuginea and eventually scarring with fibrotic plaque formation. The pathology presents with penile pain, plaque formation, deformity during erection, and subsequent erectile dysfunction. Diagnosis is clinical with identification and measurement of the plaque. Medical treatment involves the use of drugs for oral treatment such as vitamin E, tamoxifen, and colchicine and for intralesional injection verapamil and collagenase. If drug therapy fails, surgery remains the best option, especially in the cases of severe curvature or
angulation and erectile dysfunction [6]. The use of RT is considered indicated in early-stage disease with soft, noncalcified plaques. RT techniques such as x-rays, photons, or low-energy electrons can be used; to have better dose distribution at the surface level, a bolus is also applied over the plaques. The target volume involves the entire plaque with a safety margin of 1 cm, protecting pubic hair, testes, and penile bulb with shielding. Irradiation can be in antero-posterior or latero-lateral projection, the latter with vertical fixation of the penis. The recommended schedule is single dose of 2–3 Gy for a total dose of 10–20 Gy. With this regard, Seegenschmiedt et al. focused their investigation on non-oncological RT publishing numerous case series [7].

### 3.3. Muscle-Skeletal Disorders

Radiation therapy is consolidating over the years its role in the treatment of inflammatory or degenerative skeletal disease. Due to the control of pain, the two most frequent indications for radiotherapy in benign diseases are osteoarthritis and periartthritis. Moreover, RT is a non-invasive approach. Irradiation can be provided by LINAC or orthovoltage. A summary of the collected evidence is reported in Table 3.

<table><caption>**Table 3.** Muscle-skeletal disorders: included study and radiotherapy parameter.</caption><thead><tr><td>Author</td><td>Year</td><td>N pts</td><td>Diagnosis</td><td>End Point</td><td>Dose</td><td>Results</td></tr></thead><tbody><tr><td>Hautmann [45]</td><td>2019</td><td>124</td><td>epicondylitis humeri</td><td>pain relief</td><td>6 Gy(1 Gy)-3 Gy (0.5 Gy)</td><td>complete response 64% at 24 months</td></tr><tr><td>Rogers [46]</td><td>2020</td><td>157</td><td>epicondylitis, plantar fasciitis, and finger osteoarthritis</td><td>pain relief</td><td>4 Gy (0.5 Gy)-8 Gy Orthovoltage</td><td>pain relief at rest and during activity and a corresponding objective improvement in handgrip strength in epicondylitis. Pain relief at rest, during activity and improvement in walking time were demonstrated in plantar fasciitis</td></tr><tr><td>Hautmann [47]</td><td>2020</td><td>86</td><td>Humeral epicondylitis</td><td>pain relief</td><td>3 Gy/2.5 Gy (0.5 Gy/fx); 6 Gy (1 Gy/fx)</td><td></td></tr><tr><td>Micke [48]</td><td>2018</td><td>703</td><td>Calcaneodynia, Achillodynia, Bursitis trochanterica, Shoulder Syndrome, Gonarthrosis</td><td>pain relief</td><td>6 Gy (0.5-1 Gy)</td><td>At follow up, good response: Calcaneodynia 80.7%, Achillodynia 88.9%, Bursitis trochanterica 46.3%, Shoulder Syndrome 60%; only Gonarthrosis 29.2%</td></tr><tr><td>Alvarez [49]</td><td>2019</td><td>108</td><td>OADD</td><td>pain relief</td><td>6 Gy (1 Gy)-12 Gy</td><td>Overall, and with a follow-up of 8 months (range 1-31 months), 91% of patients experienced pain relief. The pain reported according to the VAS scale was 0-3 in 32.6% of the patients, 4-6 in 36.7% and greater or equal to 7 in 20.1% of treated patients.</td></tr><tr><td>Mahler [50]</td><td>2018</td><td>55</td><td>knee osteoarthritis</td><td>pain relief</td><td>6 Gy</td><td>At 3 months follow-up: no substantial beneficial effect on symptoms and inflammatory signs of LDRT in patients knee OA, compared with sham treatment</td></tr><tr><td>Ott [51]</td><td>2015</td><td>112</td><td>Achillodynia</td><td>pain relief</td><td>6 Gy/3 Gy</td><td>Pain control: Early 84% Middle-term 88% Long-term 95%</td></tr><tr><td>Rudat [52]</td><td>2021</td><td>666</td><td>Heel Spur</td><td>pain relief</td><td>3 Gy (Re-irradiation possible)</td><td>Good local control (>75%) and good response to reirradiation</td></tr><tr><td>Hautmann [53]</td><td>2014</td><td>110</td><td>Heel Spur (Re-irradiation)</td><td>pain relief</td><td>3 Gy (Re-irradiation possible)</td><td>73.6% of Pain control after 24 months</td></tr><tr><td>Niewald [54]</td><td>2020</td><td>236</td><td>Kneel and Hand Osteoarthritis</td><td>pain relief</td><td>3 Gy/0.3 Gy</td><td>Good pain control with no difference between the two schemes</td></tr></tbody></table>

Abbreviation: N: number, Pts: patients, RT: radiation therapy, Fx: fractions, OADD: osteoarticular degenerative disorders, LCH: Langerhans cell histiocytosis, LDRT: low dose radiotherapy.
### 3.3.1. Osteoarthritis and Osteoarthrosis

Epicondylitis is commonly divided into two main branches: lateral, also commonly known as tennis elbow, and medial, the golfer's elbow. Epicondylitis has a negative impact on patients quality of life with symptoms such as pain, joint mobility restriction, and even sometimes edema, limiting everyday activities and independence [55].

Hautmann et al. [45] irradiated 138 epicondylitis humeri, with linear accelerator, unlike most studies in the literature that used orthovoltage [46]. Patients had a median NRS (pain numeric rating scale) 7. Total dose was 6 Gy or 3 Gy (every other day). Between elbow treatments, 30% repeated RT for a partial or absent response. The authors reported a complete response of pain, NRS 0, with a follow-up at 24 months and demonstrated a comparable results of linear accelerator RT compared with orthovoltage. The same authors published in 2020 the results of reirradiation at a dose of 3 Gy (0.5 Gy/fx) and 6 Gy (1 Gy/fx), reporting a good control of pain at 24 months of 50.9% and a non-difference statically valid between the two dosages [47]. From the Micke et al. study, in addition, the effectiveness of low-dose RT using LINAC can be inferred compared to RT using orthovoltage. Both techniques manage to obtain an excellent control of the pain, especially in the long term. From the series, only the group of patients with gonarthrosis did not have a good response, probably due to the process of irreversible degeneration typical of this lesion [48].

Alvarez et al. [49] have treated 184 degenerative osteoarticular disorders with a dose of 6 Gy (1 Gy/fr every other day). Patients who did not benefit from the first round of RT repeated treatment, for a total dose of 12 Gy (52% of cases). Median follow-up was 8 months. Although 91% of patients reported improved pain, only 32.6% reported VAS pain 0-3.

The effectiveness of RT is to be found in the anti-inflammatory mechanism of low doses (0.5-1.5 Gy/fx), which determine inhibition of the interactions between leukocytes and endothelial cells, a decrease in the production of adhesion molecules to the endothelium, a decrease of mediators of inflammation, and less expression of pro-inflammatory cytokines. Of note, there is no guideline regarding regimen and dose fractionation, but the strategies have been determined empirically and it has been reported that various doses such as 0.5 Gy performed in six fractions had the same effect of a dose of 1 Gy in six fractions. The choice of fractionation is mostly based on in vitro experiments, which have shown that the anti-inflammatory effect of low doses RT was maximum at 48 h after irradiation, and it was lost after 72 h [56].

In the study of the Netherlands, there were reported negative results for RT, this time in the treatment of the knee osteoarthritis [50]. The authors conducted a randomized, double-blind, controlled study that showed that LDRT (6 Gy) does not lead to a substantial reduction of symptoms in patients with knee osteoarthritis. Note that unlike previous experiences, the follow-up in this study is relatively short and does not allow the long-term benefit to be assessed.

More recently, a prospective trial started in 2020 has been started by Niewald et al. including 236 patients (64 knees and 172 hands) all with a diagnosis of osteoarthritis (OA). The aim of this study was to compare two schemes of treatment (3 Gy vs. 0.3 Gy). The study showed no statistically difference in pain control between the two schemes. Further studies need to be performed because there's no evidence about the effect of low doses such as 0.3 Gy in the literature [54].

Given the increasing interest in benign osteoarticular pathologies in recent years, Alvarez and colleagues have outlined a CT-based contouring atlas for non-malignant skeletal and soft tissue disorders [57]. The aim of the authors is to suggest a correct PTV delineation based on simulation CT, for treatment of painful shoulder syndrome, such as periarthritis humero-scapularis, epicondylitis humeri, finger joint osteoarthritis, trochanteric bursitis, gonarthrosis, plantar fasciitis, and Achilles tendinopathy.
### 3.3.2. Achillodynia

Ott et al. in 2015 evaluated the long-term efficacy of two different RT schemes of 1 Gy/0.5 Gy over 3 weeks, twice per week used for 112 patients with diagnosis of achillodynia. The overall early (right at the end of RT), delayed (6 weeks after RT), and long-term (2 years after RT) response rates for all patients were 84%, 88%, and 95%, respectively. This confirmed the positive role of RT for this kind of treatment [51]. In 2021, a review made by Rudat et al. analysed 666 patients for a total of 864 heels treated between 2009 and 2020. Since 2015, a questionnaire was given to all patients in order to measure the local pain control. For newly treated patients, after 3 months follow-up in case of an unsatisfactory pain control it was offered the possibility of a re-irradiation. Re-irradiation showed an improvement in local pain control of approximately 40%. More than 75% of the patients reported a good pain control, confirming the role of radiotherapy in this field [52]. This confirms the results of another study done by Hautmann in 2014 where 110 heel spur syndrome with lack of pain control has been re-irradiated and results were measured with NRS score system. Re-irradiation confirmed his role because 73.6% of the patients were free from pain 24 months after the treatment [53].

### 3.3.3. Heterotopic Ossification

The role of RT on Heterotopic Ossification (HO) was supported by weak evidence. In 2014, a systematic review made by Ploumis et al. on a total of 27 studies of elbow HO showed that in most cases RT was stopped due to safety reasons. This review confirmed the lack of high-quality findings in the literature about RT in HO syndromes [58]. Thanks to the review made by Galietta et al. in 2022, it was finally confirmed the positive role of RT in the prevention of hip HO. Despite the numerous schemes available, from a single fractionated to several multiple fractionated schemes, no difference has been reported between the different schemes over the surgery alone [59].

### 3.4. Neurological Disorders

Radiation therapy has been widely adopted in the treatment of various neurological disorders, for different aims such as pain relief, control of the symptoms, and obliteration of brain arteriovenous malformations. Moreover, RT is a non-invasive approach that can be safely adopted also in elderly patients. A summary of the collected evidence is reported in Table 4.

<table><caption>**Table 4.** Neurological disorders: included study and radiotherapy parameter.</caption><thead><tr><td>Authors</td><td>Year</td><td>N pts</td><td>Diagnosis</td><td>End Point</td><td>Dose</td><td>Results</td></tr></thead><tbody><tr><td>Rauch [60]</td><td>2012</td><td>11</td><td>Epilepsy</td><td>Tolerability and seizure frequency.</td><td>26.3–58.3 Gy</td><td>Treatment led to an improvement in the frequency of seizures in 63%.</td></tr><tr><td>Liang [61]</td><td>2010</td><td>7</td><td>Epilepsy</td><td>Seizure frequency</td><td>12 Gy</td><td>Reduction of seizure frequency was 50% in two cases, 30% in one case, and 0% in two cases, and seizure frequency increased more than 100% in two cases.</td></tr><tr><td>Bartolomei [62]</td><td>2008</td><td>15</td><td>Epilepsy</td><td>Seizure frequency</td><td>24 Gy</td><td>A total of 60% pts were considered seizure free. All patients who were initially seizure free experienced a relapse of isolated aura (66%) or complex partial seizures (66%) during antiepileptic drug tapering.</td></tr><tr><td>Barbaro [63]</td><td>2009</td><td>28</td><td>Epilepsy</td><td>Seizure frequency</td><td>24 Gy high dose vs. 20 Gy low dose</td><td>At the 36-month follow-up evaluation, 67% of patients were free of seizures for the prior 12 months (high dose: 10/13, 76.9%; low dose 10/17, 58.8%)</td></tr></tbody></table>
<table><caption>**Table 4.** *Cont.*</caption><thead><tr><th>Authors</th><th>Year</th><th>N pts</th><th>Diagnosis</th><th>End Point</th><th>Dose</th><th>Results</th></tr></thead><tbody><tr><td>Smith [64]</td><td>2011</td><td>169</td><td>TN</td><td>Pain relief</td><td>70-85 Gy, 90 Gy</td><td>A total of 79.3% experienced significant relief. A total of 19.0% had recurrent pain. Of 87 patients with idiopathic TN without prior procedures, 79 (90.8%) had initial relief. Among 28 patients treated with 70 Gy, 18 patients (64.3%) had significant relief. Of the patients with 90 Gy at the brainstem, 59 (79.0%) had significant relief.</td></tr><tr><td>Rashid [65]</td><td>2018</td><td>55</td><td>TN</td><td>Pain relief</td><td>90 Gy</td><td>After 30 months median follow-up, 69% of patients were pain free.</td></tr><tr><td>Romanelli [66]</td><td>2019</td><td>387</td><td>TN</td><td>Pain relief</td><td>60 Gy (80% isodose)</td><td>Pain relief rate at 6, 12, 18, 24, 30, and 36 months was, respectively, 92, 87, 87, 82, 78, and 76%.</td></tr><tr><td>Lovo [67]</td><td>2019</td><td>14</td><td>TN</td><td>Pain relief</td><td>140 Gy</td><td>A total of 90% pts reported some form of relief. A total of 60% reached the threshold of 50% pain relief, and for 40% the pain never improved.</td></tr><tr><td>Kundu [68]</td><td>2022</td><td>41</td><td>TN</td><td>Pain relief</td><td>90 Gy</td><td>There has been a significant improvement in the post-radiation pain score in 72% of patients.</td></tr><tr><td>Starke [69]</td><td>2016</td><td>2236</td><td>AVM</td><td>Obliteration rate</td><td>20.5 Gy (mean margin dose)</td><td>Overall obliteration rate was 64.7%.</td></tr><tr><td>Ding [70]</td><td>2017</td><td>232</td><td>AVM</td><td>Obliteration rates, hemorrhage rate</td><td>22.5 Gy</td><td>The actuarial obliteration rates at 5 and 10 years were 72% and 87%, respectively. Annual post-SRS hemorrhage rate was 1.0%</td></tr><tr><td>Patibandla [71]</td><td>2017</td><td>233</td><td>AVM</td><td>Obliteration rates, hemorrhage rate in Grade III-IV AVMs</td><td>Mean dose 17.3 Gy</td><td>The actuarial obliteration rates at 3, 7, 10, and 12 years were 15%, 34%, 37%, and 42%, respectively. The annual post-SRS hemorrhage rate was 3.0%</td></tr><tr><td>Matsuo [72]</td><td>2014</td><td>51</td><td>AVM</td><td>Obliteration rate</td><td>15 Gy (80% isodose)</td><td>The actuarial obliteration rates at 3, 5, 10, and 15 years were 46.9%, 54.0%, 64.4%, and 68.0%, respectively</td></tr><tr><td>Matthiesen [73]</td><td>2012</td><td>211</td><td>GO</td><td>Symptomatic improvement</td><td>20 Gy/10 fx</td><td>A total of 84.2% pts reported a symptomatic improvement</td></tr><tr><td>Kouloulias [74]</td><td>2013</td><td>17</td><td>GO</td><td>Symptomatic improvement and tolerability</td><td>20 Gy/10 fx</td><td>Stabilization of the disease without recurrence was achieved in 12/17 patients. At the end of radiotherapy, the CAS regressed to 4.82 ± 2.24 (p < 0.001, Wilcoxon test). Extraocular motility and pain behind the globe were improved in 14/17 and 16/17 patients, respectively. Five patients developed recurrent signs and symptoms and they underwent surgical decompression</td></tr><tr><td>Li Yim [75]</td><td>2011</td><td>59</td><td>GO</td><td>duration of symptoms, clinical activity score (CAS)</td><td>20 Gy/12 fx (over 2 weeks)</td><td>Response (change in CAS) to orbital radiotherapy was statistically significant from 3.17 ± 1.75 standard deviation (SD) to 0.73 ± 0.92 SD (p < 0.001)</td></tr><tr><td>Kahaly [76]</td><td>2000</td><td>65</td><td>GO</td><td>Symptomatic improvement and toxicity</td><td>A: 20 Gy/20 fx (over 20 weeks) B: 10 Gy/10 fx (over 2 weeks) C: 20 Gy/10 fx (over 2 weeks)</td><td>Response to therapy, defined as a significant amelioration of three objective parameters, was noted in 12 A (67%), 13 B (59%), and 12 C (55%) subjects (C vs. A, p = 0.007). Ophthalmic symptoms and signs regressed most in group A</td></tr><tr><td>Cardoso [77]</td><td>2012</td><td>18</td><td>GO</td><td>Symptomatic improvement and Radiologic response</td><td>10 Gy/10 fx (over 10 weeks)</td><td>Significant decrease in symptoms such as tearing (p < 0.001), diplopia (p = 0.008), and conjunctival hyperemia (p = 0.002). Magnetic resonance imaging showed decrease in ocular muscle thickness and in the intensity of the T2 sequence signal in the majority of patients</td></tr></tbody></table>

Abbreviation: CAS: clinically activity score, SRS: stereotactic radiosurgery, AVM: arteriovenous malformation, GO: Graves ophthalmopathy, TN: Trigeminal neuralgia.
### 3.4.1. Epilepsy

A total of 0.5% of the world's population suffers from epilepsy. About 30–40% of the patients do not benefit from pharmacological therapy and are eligible for surgical treatment. In patients where the pharmacological and surgical alternatives have been exhausted, RT is an option. RT, compared to surgery, has the advantage of not being invasive with low risk of neurological damage to the patient. Rauch C. et al. [60] reported first-time long-term outcome (median 10 years) of fractionated stereotactic radiotherapy (FSRT) in 11 patients with drug-resistant epilepsy. The biologically equivalent dose ranged from 26.3 to 58.3 Gy (α/β = 10). None of the patients developed temporary or permanent neurological deficits. Treatment resulted in improvement of seizure frequency in seven patients: five of them had a decrease in seizure frequency, and two of them were seizure-free at last follow-up.

Liang S et al. [61] reported the long-term outcome of seven patients with temporal lobe epilepsy (TLE) treated with very low-dose LINAC based FSRT, treated with marginal dose of 12 Gy at the 85% isodose line. Reduction of seizure frequency post-FSRT was 50% in two cases, 30% in one case, and 0% in two cases, and seizure frequency increased more than 100% in two cases. No patient was seizure free at the last follow up. Two cases presented transitory complications and two cases showed an obvious drop in IQ, memory decline, and permanent neurologic complications, including partial aphasia and mild hemiplegia in one case, and progressive ataxia and cognition decline in another case. Bartolomei F et al. [62] reported outcome of 15 patients with TLE with median follow-up of 8 years (range 6–10 years) treated with gamma-knife with a marginal dose of 24 Gy. At the last follow-up, 9 of 16 patients (60%) were considered seizure free. A total of 60% of the patients experienced mild headache and were placed on corticosteroid treatment for a short period. All patients who were initially seizure free experienced a relapse of isolated aura (10/15, 66%) or complex partial seizures (10/15, 66%) during antiepileptic drug tapering. Restoration of treatment resulted in good control of seizures. Results are maintained over time with no additional side effects. Long-term results are comparable with conventional surgery.

Barbaro et al. [63] evaluated the effectiveness of radiosurgery, at the level of the amygdala, hippocampus, and parahippocampal gyrus as an alternative to surgery. Thirteen high-dose (24 Gy) and seventeen low-dose (20 Gy) patients were treated. Both groups showed significant reductions in seizures within a year of treatment, without observing major safety issues with high-dose stereotactic radiosurgery (SRS) versus low-dose SRS.

On these bases, radiotherapy has the potential to control the frequency and intensity of seizures in patients with pharmacoresistant epilepsy with mild long-term side effects if administered with proper fractionation, dose prescription, and target volume definition.

### 3.4.2. Trigeminal Neuralgia

Trigeminal neuralgia (TN) is a neurological disease that cause intense facial pain, usually paroxysmal and excruciating, due to an alteration of the V cranial nerve (predominantly the mandibular and maxillary branches). In patients with medications failure/significant adverse events, radiotherapy (SRT, mainly SRS) may be an important therapeutic option. For SRS Gamma-Knife, linear accelerators and Cyber-Knife can be used. The three techniques showed no differences in terms of pain control, whereas the time to recurrence ranged from 6 to 48 months [78].

Smith et al. [64] performed a retrospective study that evaluated cohort of 169 patients treated with LIANC-SRS. The authors investigated different doses and volumes, concluding that increased dose and volume of brainstem irradiation improve clinical outcomes. Similarly, Rashid et al. [65] evaluated 55 patients with TN and treated with LINAC SRS up to a total dose of 90 Gy with 20% isodose line constraint to brainstem. After 30 months median follow-up, 69% of patients were pain free. Another important study has reported the outcomes of using Cyber-Knife SRS administered to 527 patients [66]. Dose prescription was 60–65 Gy to the 80–90% isodose line. A brainstem volume equal to or less than 1 cm³ was exposed at a dose of 10 Gy, with a maximum point dose (0.035 cm³) of 30%
of the prescription dose. The pain relief rates were 87%, 82%, and 76% at 12, 24, and 36 months, respectively.

Lovo EE et al. [67] reported the outcome of 14 patients with TN treated with SRS to the Centromedian and Parafascicular Complex of the contralateral thalamus. They used a gamma angle regularly fixed at 90° and using a 4 mm collimator and the prescribed dose was 140 Gy to Dmax. Almost all patients (90%) reported some form of relief. Six patients (60%) reached the threshold of 50% pain relief. For four patients (40%), the procedure failed because the pain never improved.

Kundu B et al. [68] recently performed a retrospectively evaluation of patients with TN undergoing LINAC-SRS. The patients were evaluated with Barrow Neurological Institute (BNI) pain score and after a median follow-up of 5 months, 72% of the patients showed an improvement in this pain evaluation.

A retrospective study by Fraioli et al. of 45 patients compared SRS to FSRT using a linear accelerator. The authors compared 40 Gy in a single fraction and 72 Gy in six fractions. Patients treated with FSRT showed a higher pain recurrence rate than SRS (27.3% versus 8.3%) [79].

Finally, reirradiation with SRS can still be used in case of recurrence, with a pain relief rate of 50% as showed in two retrospective studies [80,81]. The reirradiation was usually delivered between 15.7 and 26.1 months after the first irradiation, with a dose in the range 70–80 Gy.

### 3.4.3. Brain Arteriovenous Malformations

Brain arteriovenous malformations (AVMs) are the persistence of a direct link between an artery and a vein; the nidus is located where small arteries and veins are connected [82]. Brain AVMs are rare (incidence estimated between 1.12 and 1.42 cases per 100,000 person-years) and mostly occur in young patients [83].

AVMs may be found as an incidental finding. They may be associated with intracranial hemorrhage, seizures, headaches, and/or neurological deficits. CT and MRI angiography are useful for the accurate diagnosis and nidus definition. Treatment approaches for AVMs are neurosurgery, embolization, and intracranial SRS. Stereotactic radiosurgery has a clinical obliteration rate of 60–80% [69–72]. The clinical benefit of SRS induced obliteration appear after 3 years or more. The success rate was increased with smaller volume (up to 30 cm³), lower Spetzler-Martin grade, higher dose, and steeper dose gradient. Embolization performed before SRS provided significantly lower obliteration rates than SRS alone (at 3 years: 41% versus 59%, respectively; *p* < 0.00001) [84,85]. Some AVM locations (functional areas, as thalamus) are related to poorer outcomes and require multimodal management [86]. Fractionated intracranial SBRT is poorly used in patients with AVM, mainly due to the low obliteration and morbidity rates initially reported [87]. Most recent results had an obliteration rate of 50% using fractionated intracranial SBRT delivering equivalent 2 Gy fraction doses higher than 70 Gy [88]. Symptomatic and permanent radiation-induced side effects have been described in 8 to 11% and in 1 to 4% of patients treated with SRS, respectively [71]. Stereotactic radiosurgery can be repeated when no complete obliteration is reported after the initial treatment. This is likely to occur in patients with AVMs larger than 10 cm³ and/or with high Spetzler-Martin grade [71]. Overall, the risks of hemorrhage and radionecrosis have to be considered and the treatment decision needs to be taken in a multidisciplinary setting in which the situation of each patient is assessed individually.

### 3.4.4. Graves Ophthalmopathy

Graves ophthalmopathy (GO) is the most frequent extrathyroidal manifestation of Graves' disease. Although GO is severe in only 3–5% of affected individuals, quality of life is severely impaired even in patients with mild GO. RT is a well-established method of treatment for GO. The main rationale is its anti-inflammatory effect and the high radiosensitivity of T lymphocytes and orbital fibroblasts. Although there are several reports about
the benefits of RT [73–75], optimal initial treatment and its combination with steroids is still controversial. Various RT regimens with different doses and fractionations have been used: 16 or 20 Gy delivered in 8–10 fx (five days/week) is usually considered the standard [89]. A consensus statement from the European Group on Graves’ Orbitopathy does not recommend doses higher than 20 Gy [90]. Recent studies evaluated altered fractionation RT for GO. In a randomized study, Kahaly et al. [76] compared the efficacy and tolerability of three RT regimens of 1 Gy given weekly for 20 weeks for a total dose of 20 Gy; 1 Gy given daily for 2 weeks for a total dose of 10 Gy; and 2 Gy given daily for 2 weeks for a total dose of 20 Gy for patients with moderately severe GO. The authors concluded that whereas all regimens provided similar response rates, the protracted regimen had a better effectiveness and tolerance. Cordoso et al. [77] demonstrated the efficacy of orbital RT with a total dose of 10 Gy, fractionated in 1 Gy once a week over 10 weeks in 18 patients with GO.

Overall, favorable responses have been reported in 60% of cases. The best responses were noted for inflammatory signs and recent onset of extraocular muscle involvement. RT is well tolerated and safe and a careful selection of patients is necessary.

## 4. Discussion

All the above-mentioned disease can benefit from RT with interesting and promising results. At the same time, several pitfalls need to be managed before RT can be considered a safe and efficacious technique to manage non-oncological disorders.

Specifically, more work is needed in the field of radiobiology to correctly quantify the RT damage to organs at risk, especially in the risk of secondary cancers [91–93]. As the development of RT induced secondary malignancies represents one of the most important late side-effects, this topic has significantly influenced treatment decision-making and limited the use of RT for non-oncological disorders. Considering the increased life expectancy and the higher number of older patients, as well as the advanced in treatment delivery, all efforts should be made to prevent the incidence of tumors induced by radiation and to correctly estimate the individual risk.

At the same time, the number of prospective trials investigating the field of non-oncological RT is extremely poor and needs to be improved in the next years (see Table 5).

<table><caption>**Table 5.** Ongoing prospective clinical trial on non-oncological radiotherapy.</caption><thead><tr><td>NCT Number</td><td>Disease</td><td>Design</td><td>Location</td></tr></thead><tbody><tr><td>NCT04722263</td><td>Keloids</td><td>Single arm, interventional pilot study (15 patients). RT: 15 Gy in 3 fractions.</td><td>Montefiore Medical Center, New York, US</td></tr><tr><td>NCT04853433</td><td>Keloids</td><td>Single arm, interventional pilot study (15 patients). The primary endpoint will be toxicity.</td><td>Montefiore Medical Center, New York, US</td></tr><tr><td>NCT04122313</td><td>Dupuytren's Disease</td><td>Prospective, Cohort study. Participants will be treated according to a standard treatment pathway, followed by post-operative radiation. RT: 15 Gy in 5 fx, followed by a 6–8 weeks break then a second identical course. Total dose: 30Gy.</td><td>University of Minnesota, US</td></tr><tr><td>NCT04424628</td><td>Gonarthrosis and Coxarthrosis</td><td>Non-inferiority study in which the investigators compare two low-dose radiotherapy schemes. Arm A will be treated at 3 Gy (0.5 Gy/fraction, 3 fractions/week), and patients in arm B will be treated at 6 Gy (1 Gy/fraction, 3 fractions/week).</td><td>GenesisCare, Malaga, Spain</td></tr><tr><td>NCT02708810</td><td>Trigeminal Neuralgia</td><td>To determine the feasibility of frameless Virtual Cone trigeminal neuralgia radiosurgery at a single institution prior to multi-institutional enrollment.</td><td>Hazelrig-Salter Radiation Oncology Center, Birmingham, Alabama, US</td></tr><tr><td>NCT03995823</td><td>Cerebral Arteriovenous Malformations</td><td>Prospective study including 50 patients with cerebral AVMs treated with GRKS to evaluate the sensitivity for nidus obliteration of MRI.</td><td>Department of Neurosurgery, Medical University of Vienna, Austria</td></tr><tr><td>NCT04843683</td><td>Cardiac Arrhythmias</td><td>Prospective, single-center, phase II trial that will be monitoring the safety and efficacy of using stereotactic ablative radiotherapy (SBRT) to treat arrhythmias.</td><td>University Health Network, Toronto, Canada</td></tr></tbody></table>
<table><caption>**Table 5.** *Cont.*</caption><thead><tr><td>NCT Number</td><td>Disease</td><td>Design</td><td>Location</td></tr></thead><tbody><tr><td>NCT04392193</td><td>Cardiac Arrhythmias</td><td>Proton Particle Therapy for Cardiac Arrhythmia Extracorporeal Energy Source Ablation of Cardiac Tissue: A First Stage Early Feasibility Study</td><td>Mayo Clinic, Rochester, Minnesota, US</td></tr><tr><td>NCT04984265</td><td>Cardiac Arrhythmias (Chagas)</td><td>SBRT in Chagas Disease Ventricular Tachycardia. A single 25 Gy dose will be delivered to the PTV.</td><td>University of Sao Paulo General Hospital, Sao Paulo, Brazil</td></tr><tr><td>NCT04642963</td><td>Cardiac Arrhythmias</td><td>Single arm, aimed at investigate safety requirements for clinical use. A single 25 Gy dose will be delivered to the PTV.</td><td>Medical University of Silesia, Katowice, Poland</td></tr><tr><td>NCT04833712</td><td>Cardiac Arrhythmias</td><td>The study aims to investigate the safety and preliminary efficacy of stereotactic radiotherapy for pulmonary vein isolation to treat refractory atrial fibrillation</td><td>Attikon University Hospital, Chaidari, Greece</td></tr><tr><td>NCT04486339</td><td>Cardiac Arrhythmias</td><td>Pulmonary Vein Isolation Using Stereotactic Radiotherapy System for the Treatment of Refractory Atrial Fibrillation</td><td>Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shangai, China</td></tr><tr><td>NCT03867747</td><td>Cardiac Arrhythmias</td><td>Radiosurgery for the Treatment of Refractory Ventricular Extrasystoles and Tachycardias (RAVENTA)</td><td>University Clinic Mannheim, Mannheim, Baden-Württemberg, Germany</td></tr><tr><td>NCT04162171</td><td>Cardiac Arrhythmias</td><td>Cohort Study—SBRT for VT Radioablation</td><td>Nova Scotia, Canada</td></tr><tr><td>NCT04066517</td><td>Cardiac Arrhythmias</td><td>STRA-MI-VT study is a spontaneous, open-label, not randomized, prospective clinical trial. The objective of the study is to evaluate the safety and efficacy of SBRT in strictly selected patients with refractory VT.</td><td>Istituto Europeo di Oncologia, IRCCS, Milan, Italy</td></tr><tr><td>NCT04612140</td><td>Cardiac Arrhythmias</td><td>Clinical trial: Patients with previously failed conventional RF catheter ablation will be randomized to radiosurgery (active treatment group) or repeated catheter ablation (control treatment group).</td><td>University Hospital Ostrava, Czechia</td></tr></tbody></table>

Abbreviations: SBRT: stereotactic body radiotherapy, RF: radiofrequency, VT: ventricular tachycardia, PTV: planning target volume, GRKS: gamma knife radiosurgery, AVM: Arteriovenous Malformations.

Most of the enrolling trials are investigating the new technique of Heart SBRT for arrhythmia disorders, whereas other diseases that can be managed with RT are not properly investigated. This aspect needs to be properly analyzed to test the role of modern RT in non-oncological fields and to sponsor the results among other specialists that do not know this approach. To this end, especially the scientific societies should undertake to study and spread the use of this type of RT, also involving specialists from other disciplines, following the path of DEGRO German Society.

In this context, it is noteworthy that the spread use of hypofractionation RT schedules is involving several cancer diseases and will decrease the number of fractions of patients at the LINACs [94–96] and so decreasing the reimbursements for RT centers. The knowledge and the use of non-oncological RT, thus, could counterbalance the expected loss of patients and fractions.

Scientific improvements of RT delivery, at the same time, needs to be tested in this particular field of disorders, as several other frontiers could be crossed in the next years that no-one can imagine now, such as the use of heart SBRT.

To the best of our knowledge, the present paper is one of the most recent overviews on RT for non-malignant diseases. Notwithstanding the narrative nature of the review, our results may strongly support the need for further investigation and represent a starting point for future clinical research, also in the context of RT Scientific Societies.

We finally recognize the limits of our review, in that it lacks explicit criteria for article selection and does not evaluate selected articles for validity.

By setting up the ambitious goal of addressing all these challenges, non-oncological RT can become a clinical reality in the next years and a higher number of prospective trials can be designed and conducted in the near future, with the endorsement of RT Scientific Societies.
**Author Contributions:** Conceptualization, V.N., E.D., R.G. (Roberta Grassi), A.S., F.G., G.D.M., V.S.M. and L.D.; methodology, V.N., E.D., R.G. (Roberta Grassi), A.S., F.G., G.D.M., V.S.M. and L.D.; formal analysis, F.C., L.B., V.S., C.G., I.D. (Isacco Desideriand), F.D.F., I.D. (Ida D'Onofrio), R.G. (Roberto Grassi), A.R. and S.C.; investigation V.N., E.D., R.G. (Roberta Grassi), A.S., F.G., G.D.M., V.S.M. and L.D.; resources, F.C., L.B., V.S., C.G., I.D. (Isacco Desideriand), F.D.F., I.D. (Ida D'Onofrio), R.G. (Roberto Grassi), A.R. and S.C.; data curation F.C., L.B., V.S., C.G., I.D. (Isacco Desideriand), F.D.F., I.D. (Ida D'Onofrio), R.G. (Roberto Grassi), A.R. and S.C.; writing—original draft preparation, V.N., E.D., R.G. (Roberta Grassi), A.S., F.G., G.D.M., V.S.M. and L.D.; writing—review and editing, F.C., L.B., V.S., C.G., I.D. (Isacco Desideriand), F.D.F., I.D. (Ida D'Onofrio), R.G. (Roberto Grassi), A.R. and S.C.; visualization, V.N., E.D., R.G. (Roberta Grassi), A.S., F.G., G.D.M., V.S.M., L.D.; supervision, F.C., L.B., V.S., C.G., I.D. (Isacco Desideriand), F.D.F., I.D. (Ida D'Onofrio), R.G. (Roberto Grassi), A.R. and S.C.; project administration, F.C., L.B., V.S., C.G., I.D. (Isacco Desideriand), F.D.F., I.D. (Ida D'Onofrio), R.G. (Roberto Grassi), A.R. and S.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

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