Tissue-Specific Quantification of Radiation-Induced Cervical Fibrosis and Correlation with Cervical Range of Motion

Background Cervical fibrosis (CF) as a late consequence in patients after radiotherapy significantly impacts the long-term symptoms, functionality, and quality of life of these cancer patients due to a hardening process of different histological tissues. Modern Shear Wave Ultrasound Elastography now enables a differentiated analysis of the changes in various tissue types. In this study, tissue-specific changes in CF induced by radiation therapy in head and neck (ENT) cancer patients were quantified and correlated with cervical range of motion (CROM). Materials and Methods 16 patients after radiation of the cervical lymphatic drainage were selected as the observation group (OG). Further, 16 people without radiation in the head and neck region were matched by gender, age, and BMI as the control group (CG). Stiffness measurements in kilopascal (kPa; 1 Pa = 1 N m−2) were performed using shear wave elastography (SWE) to assess the elasticity of muscle, fascia, and subcutaneous tissue within and surrounding the sternocleidomastoid muscle (SCM). Specific parameters of the OG were compared to the CG and correlated with functional parameters and quality of life (QoL). Results The OG exhibited significantly higher stiffness values (Emean, Emax, Emin) across all tissue types than the CG, suggesting a tangible effect of radiation therapy on tissue stiffness. Muscle compartment analysis revealed the most significant stiffness differences. Thickness measurements indicated changes in the muscle and skin but not in the subcutaneous tissue. CROM measurements within the OG fell within normal ranges, suggesting a possible homogenizing effect of radiation treatment on CROM variability. Strong correlations were observed between age and specific stiffness measures, particularly in the OG group, indicating a broader impact of aging or radiation therapy on physiological measures. Significant correlations between tissue stiffness and CROM were found. Conclusion CF after radiotherapy occurs primarily in the muscle tissue and its fascia, with the hardening being about twice as pronounced as in the average population and becoming more pronounced with increasing age and correlates with CROM.


Materials and Methods
16 patients after radiation of the cervical lymphatic drainage were selected as the observation group (OG).Further, 16 people without radiation in the head and neck region were matched by gender, age, and BMI as the control group (CG).Stiffness measurements in kilopascal (kPa; 1 Pa = 1 N m − 2 ) were performed using shear wave elastography (SWE) to assess the elasticity of muscle, fascia, and subcutaneous tissue within and surrounding the sternocleidomastoid muscle (SCM).Speci c parameters of the OG were compared to the CG and correlated with functional parameters and quality of life (QoL).

Results
The OG exhibited signi cantly higher stiffness values (Emean, Emax, Emin) across all tissue types than the CG, suggesting a tangible effect of radiation therapy on tissue stiffness.Muscle compartment analysis revealed the most signi cant stiffness differences.Thickness measurements indicated changes in the muscle and skin but not in the subcutaneous tissue.CROM measurements within the OG fell within normal ranges, suggesting a possible homogenizing effect of radiation treatment on CROM variability.Strong correlations were observed between age and speci c stiffness measures, particularly in the OG group, indicating a broader impact of aging or radiation therapy on physiological measures.Signi cant correlations between tissue stiffness and CROM were found.

Conclusion
CF after radiotherapy occurs primarily in the muscle tissue and its fascia, with the hardening being about twice as pronounced as in the average population and becoming more pronounced with increasing age and correlates with CROM. 1 Background Fibrosis is a descriptive term that characterizes a dynamic pathological process involving damage to parenchymal cells, stromal tissue remodeling, and the affected tissue's contraction Straub et al (2015); Stubble eld (2011Stubble eld ( , 2017)); Cox et al (1995).Fibrosis is a late effect of radiotherapy that rarely improves over time and persists as a chronic condition affecting a patient's functioning and quality of life (QoL) Cox et al (1995); Gupta et al (2021); Andreassen et al (2006).Essentially, brosis results from a misdirected wound healing process Mann et al (2011).A crucial component of brosis is the hardening of the affected tissue.Blood and lymph vessels get displaced, and fat reservoirs get damaged, which leads to edema, ischemia, and increased vulnerability to injury Straub et al (2015); Koerdt et al (2015); Kosmacek and Oberley-Deegan (2020).As a result, the tissue atrophies and loses its functionality Straub et al (2015); Noel et al (2021).Currently, there is no uni ed and universally accepted de nition of radiation-induced brosis.The pathophysiological changes and the resulting symptoms are referred to as " brosis", depending on the context Straub et al (2015); Stubble eld (2011).Therefore, " brosis" is often used as an umbrella term for chronic radiogenic changes in various tissues and organs and the resulting symptoms Cox et al (1995).A key reason for this is that it is still largely unclear which speci c pathophysiological changes of diverse tissues lead to which symptoms and functional de cits and to what extent these, in turn, affect the QoL.This illustrates the need for more speci c consideration of tissue changes and their in uence on other clinically relevant consequences, such as functionalities and QoL.One group that is particularly affected by chronic soft tissue changes after radiotherapy is patients with head and neck (ENT) tumors.Due to the close anatomical relationship between various types of healthy tissue and the irradiated tumor, radiation induced cervical brosis (RICF) has particular importance for ENT tumor patients.The process of RICF impairs the QoL due to a range of complications, such as cervical dystonia, muscle contracture, dysphagia, and neck muscle atrophy Stubble eld (2011); Kim et al (2015); Nachalon et al (2020); Lindblom et al (2014).With modern intensity modulated radiotherapy (IMRT) Hodapp (2012), signi cantly lower levels of symptomatic brosis can already be achieved compared to conventional 3D radiotherapy.However, approximately 30% of patients still exhibit symptomatic brosis of grade 2 or higher one year after radiation Gupta et al (2021).The diagnostic methods for detecting RICF are manifold Stubble eld (2017); Liu et al (2015); Davis et al (2003); Moloney et al (2015).This can be attributed to brosis being subject to complex and multiple pathophysiological processes for which no diagnostic standard has yet been de ned.The diagnosis generally comprises either the detection of hardening of the tissue or image morphological changes such as induration of the tissue.Historically, however, all these methods have signi cant limitations.Manual palpation of radiation-induced stiffness in the neck muscles can be classi ed with a palpation score, but it is subjective Marinus et al (2000).Stiffness as Young's modulus of elasticity (YM) expressed in kPa through compression with a known force was obtained to validate the palpatory ndings.This approach measured the overall stiffness as YM, correlating with the palpation score Leung et al (2002).
Another objective approach is indentometry, which measures the overall stiffness in Newton per mm (N/mm) Koch and Wilke (2022).Still, these methods only assess overall stiffness but do not provide any differentiated conclusions about which tissue -subcutaneous tissue, fascia, or muscle -contributes to the overall stiffness.Shear wave elastography (SWE) was rst described in 1995 Sarvazyan et al (1995) as a quantitative method for stiffness measurement Liu et al (2015).Shear waves are generated by an acoustic radiation force (Push Beam), causing a displacement of tissue particles, resulting in an orthogonal shear wave generation Bercoff et al (2004); Nightingale et al (2002) (Fig. 1).The ultrasound probe emits the push beam and records the propagated shear waves immediately afterward 1.The propagation speed Cs of the shear wave, measured in m/sec, is higher in stiff and lower in soft tissue.
The ultrasound machines can give out both the propagation speed or, through a calculation, the Shear Modulus G (in kPa) or Young's modulus of elasticity E (in kPa).The expression in kPa is more widely used since clinicians are usually more familiar with the expression of stiffness in kPa.The validity of SWE was examined in a histology-controlled study in a mouse model evaluating skeletal muscle brosis, which strongly correlates with histology Martins-Bach et al (2021).Several studies about SWE of muscle tissue have been published for the lower extremities, showing excellent interrater reliability Wu et al (2020).Because the relationships between the extent of connective tissue changes in different tissue types and their clinical relevance of RICF have not yet been su ciently investigated, we used modern SWE to quantify tissue-speci c stiffness in ENT cancer patients without previous surgery in the head and neck area.We compared the results with those of a CG and correlated them with the CROM.

Patient selection
From 2020 to 2022, 16 ENT cancer patients were selected as the observation group (OG), and they were treated between 2016 and 2021 with de nitive radiochemotherapy, including bilateral, regional lymphatic drainage pathways (at least level IIa to IVb.For inclusion in the study, the completion of the oncological treatment had to be at least 16 weeks previously.The exclusion criteria were operations or functional restrictions in the head-neck region regarding the musculoskeletal system (such as chronic pain and restricted shoulder and neck movement) before radiation.Furthermore, no recurrence and no oncological treatment other than de nitive radiochemotherapy had been performed or was pending.
Further, 16 patients were included in the study as a CG, and they had not received any radiotherapy in the head and neck region or the shoulder and had never received systemic therapy due to oncological disease.These patients were supposed not to have previous chronic complaints in the head and neck region.The patients in the CG were matched in gender, age, and body mass index.

Clinical baseline assessment
In addition to patient-speci c data collection, tumor characteristics, and treatment speci cations were collected for all patients.Occurrences of lymphedema and musculoskeletal pain (Numerical rating scale 0-10) were recorded as chronic symptoms.Furthermore, the validated and recognized questionnaires on the general oncological quality of life EORTC QLQ-C30 and the head and neck-speci c quality of life of all ENT patients completed HN35.The thickness of the skin, the subcutaneous tissue, and the muscle were measured with the electronic caliper in the o ine mode using ultrasound.Additionally, CROM was measured in both groups with the CROM Instrument (Performance Attainment Associates, Roseville, Minnesota, US) with atwo-degree grading (Appendix Figure A1).A compass goniometer measured the cervical rotation Audette et al (2010) (Appendix Figure A2).The gravity goniometer measured the cervical lateral exion.The CROM-Device showed a good inter-and intra-rater-reliability Williams et al (2012).

Ultrasound Examination -Shear Wave Elastography
The stiffness measurements were done with a Resona 7 (Mindray China) using a linear transducer 11 − 3 MHz with a width of 43 mm.The subjects were supine with the head resting on a pillow, avoiding stretching on the SCM to avoid a stiffness increase.The entire muscle length was examined left and right in three sections (proximal, middle, and distal).The elastograms were stored for o ine analysis.
Measurements were obtained using Young's Modulus E with mean, maximum, and minimum values.The de nitive stiffness calculations were done o ine using a tracing with an ellipsoid covering the largest possible for the subcutaneous tissue and the fascia and manual tracing for the muscle (Fig. 2).The three measurements of each tissue compartment were averaged to gather information on the entire muscle.The SCM's transverse diameter was measured by selecting the thickest part of the muscle mass above the bifurcation of the SCM's sternal and clavicular part.

Statistical Analysis
The analysis was conducted using the Pandas data analysis toolkit.To compare groups, the dependent and independent t-tests were employed for parametric distributions, while the Mann-Whitney U Test was used for non-parametric distributions.Correlation analyses were performed with the Spearman rank correlation coe cient for non-parametric distributions and the Pearson correlation coe cient for parametric distributions.

Baseline characteristics
A total of 32 patients were included in the study.Of the 16 patients in the OG who received radiochemotherapy for ENT cancer, there were twelve men and four women.The median age was 65; the median BMI was 22,7 kg/m 2 .Most patients had oropharyngeal cancer (9), whereas 4 had hypopharyngeal, two laryngeal and one nasopharyngeal cancer.The median time from the end of treatment to study inclusion was 26 months (range 6-60).13 of 16 patients had at least one macroscopically suspicious cervical lymph node (cN+) at primary diagnosis.The radiation technique was IMRT for all patients, which was applied by volumetric arc therapy (Varian Medical Systems, Palo Alto,CA, USA) or Tomotherapy (Tomotherapy Hi-Art, Accuray, Sunnyvale, USA).All patients received at least 50 Gy (single dose 2.0 Gy) on elective lymphatic drainage (at least Level II -IV on both sides) and a boost up to 70 Gy (single dose 2.0 Gy) on macroscopic tumor disease.Radiation planning was based on CT and MRI for all patients.Image guided radiation therapy (IGRT) was performed generally and regularly during irradiation.Furthermore, all patients received 4-6 cycles of cisplatin 40mg/m 2 /KOF weekly during radiation therapy.At the time of study inclusion, all patients had a subjectively determined mild degree of brosis, which was determined by palpation.9 out of 16 patients had submental lymph edema, and all nine were currently treated with lymphatic drainage.In the entire course since the end of therapy, 12 patients have ever received lymphatic drainage.Only two patients reported mild musculoskeletal pain in the neck (NRS 2-3/10).40% of the patients (7/16) stated that they felt tension in the neck and shoulder muscles.The median general score for global health (EORTC QLQ-C30) was 83,3 (mean 75,5; range 41,7-100; SD 15).For the head and neck-speci c QoL, EORTC QLQ-HN35, only the answers for pain, swallowing, teeth, opening mouth, dry mouth, sticky saliva, and sense problems were fully completed by all ENT cancer patients.Symptom scales for pain and swallowing were relatively low (median 0 and 8,3) with a small standard deviation (SD 10,0 and 16,7).Half of the patients had no problems with their teeth, while eight patients had values ranging from 33 to 100 (SD of all patients 33.8).Most patients (10) had no problem with mouth opening.However, when it did occur, the extent was pronounced (median 0, SD 42,9).Only ve patients had issues with dry mouth (median 0, mean 14,8).The most common limitations were sticky saliva and sensory problems.The most signi cant restrictions occurred regarding sticky saliva.
Here, 13 out of 16 patients were affected, with a median value of 66.6.Twelve patients (75%) had sensory problems.Twelve patients (75%) had sensory issues, with a mean score of 26.5.

Stiffness -Shear Wave Elastography
The OG exhibited signi cantly increased stiffness E measured in kPa for the mean, maximum, and minimum measurements in the left and right sternocleidomastoid muscles, fascia, and subcutaneous tissues.This indicates a rise in stiffness across all examined tissue types within the OG, with calculated median ratios from 1.2 to 2.4.Overall, the examination results were very similar on both sides of the neck, which underlines the consistency of the examination method.The hardening of all tissue types in the CG was about half as great as in the OG.Only the median values of the mean and max values in the subcutaneous adipose tissue were only slightly increased compared to the other tissues (ratio 1,2 − 1,6).In summary of the OG, the mean stiffness values in the muscle and muscle fascia were broadly similar.In contrast, the stiffness in the subcutaneous fatty tissue was slightly lower (Table 1).

Cervical Range of Motion
CROM showed no differences between the OG and the CG, with the measurements in both groups falling within the normal ranges.The means and medians of the CROM are presented in Table 3.When considering the scope of movement, an age-dependent differentiation is necessary.The OG showed higher and lower values in speci c age and movement categories and a lower incidence of deviations.
The OG showed negative correlations with all CROM parameters, indicating a decreased range of motion with age accentuated by RT (Table 4).

Correlations of Stiffness with Function and QoL
Mean Stiffness values of various tissues with the general score for global health (EORTC QLQ-C30), as well as pain, swallowing, opening mouth, and senses problems (EORTC QLQ-HN35), showed very weak or weak mainly negative correlations for all function scores (range: -0,322-0,266).No clear correlations were found for the occurrence of lymphedema either.The values here were between − 0.256 and 0.024.

Discussion
To our knowledge, this is the rst study to quantify RICF in terms of stiffness in a tissue-speci c manner.
There is a signi cant hardening of all tissue types compared to the average population, with an approximate doubling of the mean hardening in the muscle and muscle fascia.In contrast, the hardening effects are less pronounced in the subcutaneous fatty tissue.The values can be regarded as very reliable due to the meticulous measurement method and the consistent measurement results on both sides of the neck, which underlines the suitability of SWE for quantifying RICF in soft tissue.From this, it can be assumed that CF follows a relatively reliable inter-individual dynamic.In a previous study by Liu et al, which also investigated muscle stiffness in ENT cancer patients after radiotherapy, the stiffness values E had a normal distribution, whereas, in this study, only E values of the subcutaneous tissue of the right SCM showed a normal distribution Liu et al (2015).The stiffness ratios for the muscle tissue were approximately 3 and 4 for the subcutaneous tissue, which is signi cantly higher than the ratios for muscle, subcutaneous tissue, and fascia of 2 in our present study.The degree of dispersion is higher in the study by Liu et  subcutaneous tissue, the relatively pronounced changes in the muscle and fascia can be explained by increased myo broblast activity and, thus, the increased incorporation of collagen.On the other hand, a general not fully understood "densi cation" of the tissue could be responsible, which is indicated by the signi cant decrease in the tissue thickness of muscle and fascia in contrast to the subcutaneous tissue compared to the CG.Overall, symptoms such as restricted movement or dystonia, pain, or lymphoedema were relatively low in our OG and were essentially evenly distributed.The relationship between radiation dose and volume in the development of radiation-induced brosis has already been demonstrated Borger et al (1994).The level of the irradiation dose to the muscle, e.g., correlates with clinical symptoms, as has been shown with the occurrence of trismus in relation to the dose to the pterygoid muscles Goldstein et al (1999); Teguh et al (2008).Both the relatively homogeneous changes in stiffness of all tissues between the respective patients, as well as the moderate musculoskeletal side effects and lymphedema in our OG, can be explained by a very homogeneous dose prescription (50 Gy) of the elective lymphatic drainage pathways with strict adherence to the plan quality using IMRT Hodapp (2012).The essentially good preservation of the mobility of the cervical spine in the irradiated group can also be explained by the widespread strict protection of the myelon and the associated lower dose to the cervical vertebral joints, which is made possible by volumetric arc therapy.Muscle function appears to be largely preserved, despite a general increase in density.Overall, the symptoms in our OG were relatively mild.Only 2 out of 16 patients reported musculoskeletal pain, and the overall quality of life was good.For this reason and because the hardening within the OG was relatively homogeneous and constantly increased, few effects were shown in the correlation analyses with functional parameters and QoL.However, the negative correlations between age and ROM parameters, particularly in the OG, suggest an additive effect of RT and aging on reducing cervical exibility.This observation is crucial for developing age-speci c rehabilitation strategies that account for the compounded impact of RT and natural aging on cervical motion.As described, due to uncertainty and lack of clarity, various radiationinduced effects on different tissue types are grouped as " brosis" Straub et al (2015).There is no consensus for a concrete de nition or a clear distinction between whether brosis is a pathophysiological effect, clinical syndrome, or both.Therefore, the pathophysiological mechanisms of radiation-induced brosis must be understood in greater detail, and the explicit causal relationship between these mechanisms and clearly de ned clinically relevant symptoms must be elucidated.This is necessary to establish clear dose limits to prevent symptoms and develop targeted potential therapeutic approaches.We have relatively robust dose-volume constraints for many organ functions Teguh et al (2008).However, for musculoskeletal complaints or late effects in soft tissue (such as lymphedema, etc.), there are few correlations between dose exposure and soft tissue complications (lymphedema, tension, pain, restricted movement, etc.).Furthermore, given the complex pathophysiological interplay of musculoskeletal functional impairments after radiotherapy, we still do not know enough about which structures we need to look at precisely when protecting the patient.Dose-volume-related Normal Tissue Complication Probability (NTCP) calculations are necessary for large patient cohorts experiencing higher-grade symptoms to establish meaningful soft tissue constraints regarding higher-grade endpoints.Due to its tissue-speci c and reliable, reproducible brosis quanti cation, SWE holds excellent potential for conducting corresponding analyses.
Limitations.The OG was relatively small.Additionally, the patients were recruited from regular follow-up care, representing a positive selection bias, as typically mainly healed and generally tter patients attend follow-up appointments.Both factors contribute to observing mostly mild long-term radiation-induced effects in our OG.There needs to be more than this number of patients to show clear correlations between the various degrees of hardening and quality of life parameters, particularly given the signi cant inter-individual differences in QoL.

Conclusion
Cervical brosis after radiotherapy occurs primarily in the muscle tissue and its fascia, with the hardening being about twice as pronounced as in the average population and becoming more pronounced with increasing age and correlates with CROM.Shear-wave elastography holds excellent potential for quantifying brosis reliably, speci cally in various tissue types.

CF
Principle of shear wave elastography: The ultrasound probe emits a push beam, displacing tissue particles.This generates shear waves propagating orthogonally, which are subsequently recorded by the same ultrasound probe.

Table 1
Stiffness in kPa of the Muscle, Fascia, and Subcutaneous tissue

Table 3
Rodemann and Bamberg (1995)ctor (CTGF) or TGF-β leads to stimulation and migration of myo broblasts and, nally, to increased production of collagen and bronectin.The connective tissue becomes more rigid and denser, ultimately perceived as hardening Stubble eld (2017); Burger et al (1998); Flechsig et al (2012); Frangogiannis (2020);Rodemann and Bamberg (1995).The complexity of the pathophysiological relationships and their consequences are not fully under-stood.For example, the consequences of radiotherapy regarding the function of the smallest nerve branches have not yet been su ciently clari ed.Peripheral nervous system dysfunction arises from either external compression of soft tissues due to brosis, ischemia resulting from brosis, or a combination of both factors Stubble eld (2011).We have shown that the resulting hardening effects on the muscle and muscle fascia are relatively stronger than in the subcutaneous fatty tissue.The broadly comparable changes in the muscle and muscle fascia can be attributed to relatively new insights, indicating that fascia tissues are not merely individual supportive structures of a muscle involved in power transmission and proprioception.Instead, they constitute a coherent, multi-layered, and interacting system within the body.They possess complex mechanosensory functions and are even capable of contraction independently Schleip et al (2012); Klingler et al (2014); Schleip et al (2014).The matrix is a dynamic structure with viscoelastic properties and dynamic variabilities.This viscoelasticity can adapt actively to the viscoelasticity of the adjacent tissue by mechanoreceptors Stecco et al (2013).Compared to al. than in the present study when comparing the SD to the MAD multiplied by the factor 1.4826.The differences can be explained by the region of interest (ROI) size, which was signi cantly larger in our study, resulting in more homogeneous values.Radiation primarily harms tissues by triggering apoptosis or clonogenic cell death through DNA damage caused by free radicals Hauer-Jensen et al (2004).These cause an in ammatory reaction.Increased formation of mediators such as