Systematic Review of Genetic Polymorphisms Associated with Acute Pain Induced by Radiotherapy for Head and Neck Cancers

Highlights • Pain is a complex symptom following radiation therapy for head and neck cancers.• Acute pain control is highly challenging during radiotherapy in head and neck cancers.• Inflammatory, nociceptive and neuropathic pain types are expressed after radiotherapy.• Germline genetic variants are associated with radiation therapy related acute pain.• Mechanisms of variants of radiation related pain are essential for pain management.


Introduction
Head and neck cancers (HNC) are composed of a heterogeneous mix of malignant epithelial cancers that arise from the oral cavity, pharynx, or larynx.Oral cavity and oropharyngeal (OC/OPC) squamous cell carcinoma afflicted more than 54,000 patients in the U. S, in 2022 [1].
Management of head and neck cancer is a stage-dependent multimodality approach that entails surgery, chemotherapy, and radiotherapy (RT).RT is a cornerstone for the management of HNC patients.RT, especially after application of the novel RT techniques, has a favorable impact on the overall survival rates [2].Despite improvement in survival rates, various significant acute and sometimes chronic Abbreviations: HNSCC, Head and neck squamous cell carcinoma; OC/OPC, Oral cavity and oropharyngeal cancer; HPV, Human papilloma virus; RT, Radiotherapy; QOL, Quality of life; SSB, Single strand breaks; DSB, Double strand breaks; ROS, Reactive oxygen species; HNC, Head and neck cancer; NPC, Nasopharyngeal carcinoma; IL, Interleukins; PG, Prostaglandins; TNF, Tumor necrosis factors; Gy, Gray; VEGF, vascular endothelial growth factor signaling; GABA, γ-aminobutyric acid; NGF, Nerve growth factor; ATP, Adenosine triphosphate; SNPs, Single nucleotide polymorphisms; DDR, DNA damage response; EGFR, Epidermal growth factor receptor; OM, Oral mucositis.toxicities are reported during and after RT.RT-attributable toxicities, including mucositis and dermatitis have significant negative impacts on patients' outcome and quality of life (QOL) [3][4][5].
Oral/throat pain, the most common radiation-attributable acute toxicity during and after locoregional RT of HNC, is associated with increase in analgesics use [6].About 68-86% of OC/OPC patients and about one-third of HNC patients present to the emergency department with uncontrolled pain [7][8][9][10][11].The control of RT-induced acute pain is challenging because of the complex nature and multiple mechanisms of pain in these patients.Baseline pain and associated RT toxicities such as mucositis, dermatitis and neuropathy are considered the most common etiologies of RT-induced pain.In addition, pain due to surgery and/or chemotherapy are also observed in these patients [12][13][14].Most clinicians will prescribe opioids to OC/OPC patients during cancer therapy, with 15-40% of patients dependent on opioids for several months post therapy [15][16][17].While trying not to undertreat patients with severe pain, potential overuse of opioids may negatively impact the health status and the QOL of patients.Furthermore, for a subset of patients, the prolonged use of opioids increases the risk for drug abuse and addiction, which may negatively impact the QOL and increase the potential for overdose and death [18].

The mechanism of radiotherapy-induced cell death and normal tissue toxicities
Radiation therapy is a group of ionizing energy beams that induce DNA damage in cells, cancer cells being most susceptible due to their characteristic of rapid cell division.Direct DNA damage is induced by ionizing radiation to DNA, inducing DNA breaks (single-strand breaks (SSBs), double strand breaks (DSBs)) and covalent crosslinking of the complementary DNA strands [19,20].Indirect DNA damage occurs through RT-induced generation of free radicals.For instance, reactive oxygen species (ROS) are produced by the interaction of ionizing radiation with the water molecules [21].After RT, the impairment of DNA repair mechanisms in cancer cells in addition to the accumulation of intracellular ROS induces cell injury and apoptosis, necrosis, and cellular senescence [22][23][24][25].However, normal neighboring healthy tissues will also be exposed to ionizing radiation, inducing normal cell injury and potential cell death despite the lower doses of radiation exposure.Normal tissue damage induces toxicities such as oral mucositis, dermatitis, and neuropathy leading to aggravation of RT-induced acute pain.The severity of RT-induced toxicities and pain is dependent on the total dose, fractions and the duration of radiation delivered to normal tissue in addition to the variation in tissue tolerance to radiation [26,27].

Types and potential mechanisms of radiotherapy induced acute pain in HNC patients
Adequate management of acute pain in NHC patients during RT requires good understanding of the different types and mechanisms of RTinduced pain.Three main types of RT-induced acute pain have been identified in HNC patients receiving RT [28][29][30]  as cytokines and chemokines inducing inflammatory pain.Furthermore, these inflammatory mediators act as noxious stimuli, stimulating nociceptors to induce nociceptive pain.3-Radiotherapy induces direct mechanical and thermal damage in addition to changes in the microenvironment creating an acidic pH, increasing the adenosine triphosphate (ATP), and stimulating release of bradykinin and substance P which act as nociceptors stimuli inducing nociceptive pain.Radiotherapy induces direct nerve injury and inflammation of peripheral nervous system, and fibrosis in the connective tissue can also induce neuropathic pain.
V. Salama et al.

Inflammatory pain
Inflammatory pain, the most common type of RT-induced acute pain in HNC, is caused by activation of inflammatory and immune cells in response to tissue injury and infections induced by RT [21,31].Inflammatory and immune cells in addition to RT-induced damaged cells release inflammatory mediators such as pro-inflammatory cytokines: interleukin (IL)-1β, IL-6, NF-kB, prostaglandins (PG), and tumor necrosis factor-α (TNF-α) [21,32,33].These cytokines promote recruitment of immune and inflammatory cells, inducing more damage of tissues [34].Oral/pharyngeal mucositis and neck dermatitis are the most common inflammatory reactions induced by RT [21,33,35,36].Different signaling pathways involved in RT-induced mucositis and dermatitis have been identified, and they may contribute to RT-induced inflammatory pain, such as: nuclear factor-κB (NF-κB) signaling which upregulates other pathways such as COX2 pathway and downstream tyrosine kinase receptor pathways (e.g., PI3K/AKT signaling and MAPK signaling).Other signaling pathways such as DNA damage checkpoints, cell cycle pathways, WNT/B-catenin and integrin signaling, VEGF signaling, glutamate receptor signaling and IL-6 signaling, have also been identified in correlation with RT-induced mucositis [36,37].

Nociceptive pain
Nociceptive pain is caused by direct damage to non-neural tissue, often from an external injury (i.e., RT-associated) which stimulates nociceptive receptors [30].Noxious stimuli are released from either damaged cancer cells, normal cells or from inflammatory/immune cells recruited after RT [38].Noxious stimuli activate the peripheral sensory neurons through stimulation of afferent sensory neurons which transmit the action potential to neuronal bodies where calcium influx occurs, leading to the release of neurotransmitters (e.g.: substance-P, glutamate, γ-aminobutyric acid (GABA), adenosine triphosphate (ATP), glycine, dopamine, norepinephrine, nitric oxide, and serotonin) which bind to the post-synaptic membrane receptors.The signal then reaches the second order neuron and is transmitted to the somatosensory cortex of the brain where the pain is perceived [39][40][41][42][43].The trigeminal nerves (cranial nerve (CN) V)) and the facial nerves (CN VII) play important roles in pain perception in the head and neck [44].RT-damaged cells and infiltrating immune cells release multiple noxious stimuli.Cytokines and inflammatory mediators released from infiltrating immune cells such as IL-1, TNF-α and IL-6 induce nociceptor sensitization.In addition, the acidic pH of the tumor microenvironment and extracellular ATP act as noxious stimuli to nociceptors at the cancer site [45].

Neuropathic pain
Neuropathic pain is caused by damage to nerves or nervous system [46].RT directly injures the somatosensory nervous system promoting pain signal transmission and peripheral neuropathic pain.RT induces DNA damage which induces apoptosis.Apoptosis is induced through p53 activation which activates the cascade to execute cell death [47,48].Endothelial cell death reduces blood flow to peripheral nerves, including damage and eventual neuronal fibrosis.Furthermore, ionizing radiation and ROS released after RT, cause neuronal cell stress and direct nerve damage.In RT of HNC, the brachial plexus are the neural tissues at high risk of damage.Overexpression of p53 induces apoptosis of neurons after radiation exposure [49].Furthermore, RT induces inflammation and infiltration by immune cells, resulting in immune-mediated peripheral neuropathy.Chronic inflammation in the nervous system microenvironment promotes neuron loss with fibrosis, resulting in chronic neuropathic pain [50].

Genetic variants associated with radiotherapy induced acute pain
Advances in molecular and genetic technologies have motivated researchers and scientists toward exploring the human genome and analyzing genetic variations and their correlations with different diseases and treatment outcomes.
RT-induced acute pain presents a significant morbidity burden on HNC patients receiving RT and drastically reduces patients' quality of life.In an effort to better predict and optimize pain management approaches, various cellular and molecular approaches have been widely explored recently to identify the genomic biomarkers for patients vulnerable to develop RT-induced pain, and examine the influence of these genetic variants on pain modulation and analgesic response [51,52].The advanced technologies in human genome sequencing allowed deep understanding of the genetic variations and mutations related to cancer treatment associated pain.Single nucleotide polymorphisms (SNPs) are the most common DNA sequence variations.These genetic polymorphisms are stable markers and easily and reliably assayed to explore the extent to which genetic variation might prove useful in identifying patients with cancer at high-risk of pain development and their response to pain therapies [53].Likewise, these candidate SNPs could be used in building robust predictive models for pretreatment prediction of acute as well as chronic toxicities for personalized management and precision medicine [54].
Given the multifactorial and complex etiology of RT-induced pain in HNC patients, the challenges in managing RT-associated pain, and the adverse impact of pain on patients QOL, the aim of this literature review is to identify predictive genetic variants and pathways associated with RT-induced pain and related phenotypes in the HNC population.The review will focus on oral mucositis (OM) and dermatitis-related pain which represent common acute, often painful toxicities during HNC radiation treatment.This synthesis of the current literature will provide the basis to develop predictive models of RT-induced acute pain to assist with personalized analgesic therapy.

Search strategies
We conducted a systematic search of databases including Ovid MEDLINE, Ovid Embase, and Clarivate Analytics Web of Science, for publications in English language from the inception of databases to February 28, 2022.The following concepts were searched using subject headings and free text keywords as needed, "radiotherapy", "radiation therapy", "pain", "neuropathy", "analgesics", "acute toxicity", "mucositis", "dermatitis", "single nucleotide polymorphism", "genetic variation", "genetic variability", and "genetic predisposition".The terms were combined using AND/OR Boolean Operators.Animal studies, in vitro studies, and conference abstracts were removed from the search result.Manual searches of journals, publisher databases and reference lists of journal articles were also conducted to supplement the electronic database search.The complete electronic database search strategies were detailed in Supplementary Tables S1-S3.
V. Salama et al.

Inclusion criteria
The included studies met all of the following inclusion criteria: 1) articles published in English, 2) human studies in head and neck cancers, 3) genetic polymorphisms reported to be significantly associated with RT-induced pain in different phenotypes of pain (Inflammatory [oral mucositis, dermatitis], neuropathy, nociceptive pain, or mixed oral/ throat pain).

Exclusion criteria
Articles were excluded if they met any of the following criteria: 1) non-genetic article or no genetic association study, 2) not radiotherapy induced pain or toxicity, 3) meta-analysis, review article or clinical trial, 4) articles written in languages other than English, 5) other cancer types (i.e., non HNC) 6) non-human study, or not blood or buccal DNA or 7) Other unrelated phenotypes.Flow Chart Fig. 2 .

Functional annotation
Manual functional annotation of the identified genes was done using GeneCards database.The function of each gene was assigned according to the most common functional pathway annotated in the GeneCards and the relevant studies published online.[55,56] We divided the collected variants and genes into three groups: 1) variants associated with RT-induced inflammatory pain including; RT-induced acute toxicities; acute mucositis and acute dermatitis (or acute skin reaction), 2) variants associated with radiotherapy induced neuropathic pain, and 3) variants associated with other mixed types of RT-induced acute pain (including post RT-pain, post-RT throat/mouth/neck pain).

Variants associated with radiotherapy induced inflammatory pain
Acute oral/pharyngeal mucositis and acute skin reactions are the most common acute reactions reported following RT.Nineteen articles were identified in our review detected SNPs significantly associated with acute mucositis or dermatitis post-RT.Twenty-three variants in 20 genes were associated with RT-induced inflammatory pain and acute reactions including mucositis or dermatitis.(Table 1).5 (25%) genes are involved in inflammatory pathways and immune systems, 4 (20%) genes are involved in DNA damage and DNA repair pathways, 4 (20%) genes regulate cell death or cell cycle pathways, 2 (10%) genes are involved in metabolisms and microenvironment functional pathways, 5 (25%) genes are functionally involved in other functional pathways (Table 2).
Different grading scales and cut points for toxicity identification were used in the identified studies as well as different covariates and clinical characteristics were considered in data analysis.(Table 3).

Variants associated with radiotherapy induced neuropathic pain
Neuropathic pain is one of the common types of pain developed after receiving RT in HNC.Few studies focused on identification of SNPs associated with radiotherapy-induced neuropathic pain.Reyes-Gibby et al. study was the only literature published to identify SNPs associated with neuropathy and neuropathic pain in HNC.In this study, the authors used the International Classification of Diseases, ninth (ICD-9) and tenth (ICD-10) revisions, for the outcome toxicity identification (i.e., neuropathy/neuropathic pain), they used a binary outcome of neuropathy either yes or no.Age, sex and information of clusters were used as covariates in the statistical analysis.4 variants in 4 genes were identified, associated with radiotherapy induced neuropathic pain.(Table 4).

Variants associated with mixed types of radiotherapy induced acute pain
Our literature search revealed only one study identified SNPs associated with post RT-pain in HNC without classification of the type of pain, we considered this phenotype as mixed types of pain.Liu et al. [77] study did not specify the grading scale used for toxicity identification; however, they divided patients into two groups of with or without toxicity of radiation at three, six and subsequent months.Age, gender, Body mass index (BMI), stage and 131 I dose were used as covariates.5 variants in 4 genes associated with RT-induced throat/neck pain post RT. [77].(Tables 5-6).

Discussion
Acute pain is a significant toxicity during and after RT in HNC, resulting in an increase in disability and morbidity risks and a decrease in QOL of these patients.Understanding the potential pain mechanisms and the host genetic variability can help in pain prediction, prevention and in decision making for personalized pain management.To date, few studies explore the mechanisms of acute pain in HNC patients receiving RT.Moreover, very few variants have been identified associated with RT-induced acute pain in HNC.In this systematic review, we studied the different potential mechanisms for acute pain developed during RT in HNC cancer patients.SNPs to be identified associated with RT-induced acute pain in its different types (i.e., inflammatory/nociceptive, neuropathic, and mixed) in HNC studies were collected.
Our review found that inflammation, DNA damage/repair and cell death induce inflammatory pain following RT.Variants detected in genes involved in DNA damage response or DNA repair are associated with acute toxicities (i.e., mucositis and dermatitis) related to RT.Several studies evaluated the association of variants in DNA damage and DNA repair with RT-induced acute toxicities, 4 studies focused on variants in X-Ray Repair Cross Complementing 1 gene (XRCC1) as a DNA repair gene highly associated with RT toxicity [61,63,73,74].Our review identified other variants in DNA damage/repair genes (ZNF24 [69], NBN [71], and Ku70 [72]).These results match our study of the potential mechanisms of inflammatory pain induced by RT in HNC [22].Several studies identified SNPs in genes functioning in DNA damage and repair in other types of cancers such as; rs3218556 in XRCC2 and rs13181 in XPD were associated with mucositis in non-small-cell lung cancer (NSCLC) [78], rs61915066 in ATM, rs11220184 in CHEK1, rs302877 and rs405684 in RAD51C, and rs60152947, rs10404465, rs1799786 in ERCC2 which were associated with early adverse skin reaction (i.e., dermatitis) post-RT in breast cancer [79] and rs1233255 in PMS1 gene was associated with RT-acute dermatitis in rectal cancer [80].These variants could be explored and validated in HNC in future studies.Additionally, 7 studies identified variants in genes involved in inflammatory pathways and immune systems including; TNF-α [60,77], STING1 [57], HLA-DQB1 [58], TNFRSF1A [64], GHRL [65], and NF-κβ [77], these variants were associated with RT-induced inflammatory pain and mixed throat pain.Studies showed that activation of inflammatory and immune cells after RT induces release of inflammatory cytokines and chemokines promoting more cell damage inducing inflammatory pain in addition to activation of nociceptors causing nociceptive pain [32,33,[81][82][83].Reyes-Gibby et al., studied the role of cytokines in pain activation and sensitization [53,84].Variants in genes regulating release of pro-inflammatory mediators such as cytokines and chemokines are shown here in our review.Other cell signaling pathways including cell cycle and NF-kB pathway (CCND1) [67], autophagy (ATG16L2, ATG10) [62], wnt/β-catenin (APC, GSK3β) [70] and angiogenesis (EDN1) [68] regulating genes were associated with RT related acute mucositis and dermatitis.
Few studies focused on neuropathic pain induced by RT, Reyes-Gibby et al. identified 4 SNPs in 4 genes (SNX8, PCP2, RORA and KNG1) correlated with neuropathy in HNC [76].Although substance-P and other neurotransmitters exaggerate pain response [53,85], no study identified any variant in substance-P or other neurotransmitters correlated with RT-induced pain.More studies are needed to identify the SNPs related to RT-induced neuropathy and neuropathic pain, especially variants in genes involved in neuronal plasticity and neuroinflammatory pathways.
Studies included in this review used either candidate gene approach or genome wide association analysis approach (GWAS), and interestingly through GWAS studies collected, we declared variants in genes involved in new pathways other than the common potential mechanisms, associated with RT-induced acute pain and toxicities in HNC.[58].
Although we did a comprehensive review to identify different potential mechanisms, including molecular pathways and genetic variants associated with RT-induced acute pain in HNC, the limitations in our review include that we did not explore SNPs associated with analgesics response (i.e., pharmacogenomics) or chronic pain.Our search focused on specific phenotypes in HNC population, although few studies were identified focused on HNC.Other variants identified in other cancer types with different phenotypes causing acute pain (e.g., pneumonitis, dysphagia, esophagitis, proctitis or bone pain) could be explored in detail in future studies.Furthermore, most of studies did not specify the type of pain detected, either inflammatory, nociceptive, or neuropathic, while they only mentioned post-RT pain, mucositis, or dermatitis.We involved variants associated with unspecified post RT-pain under the group of other types/mixed pain.These collected studies are just association studies; however, mechanistic studies must be done to test the mechanistic effect of these variants on the phenotype development and alteration of the different pathways associated.The identified candidates need to be studied further for validation of results.
Our future directions include validating these variants and developing predictive algorithms for pain prediction and management using genetic candidates identified in our HNC cohort.

Conclusion
This comprehensive literature review aimed to identify the genetic variants association with RT-induced acute pain phenotypes including; mucositis, dermatitis (Inflammatory pain), neuropathic, nociceptive pain and mixed pain in HNC population.Our review revealed that pain is a complex symptom during and following RT in HNC patients due to its multifactorial origin and diverse phenotype expression.DNA damage/repair, inflammatory pathways, apoptosis, and neuropathy pathways are the most common pathways behind the development of RTrelated acute pain.There is a need for a comprehensive understanding of the genetic profile risk association with acute pain severity, additionally, the clinical findings and treatment plans for HNC patients may help inform development of standardized algorithms for personalized management of acute RT-induced pain to maintain pain relief during RT and improve patients' outcome.

Fig. 1 :Fig. 1 .
Fig. 1.Types and potential mechanisms of radiotherapy induced acute pain.1-Radiotherapy in head and neck cancer induces DNA damage of cancer cells and normal neighboring cells and the release of reactive oxygen species (ROS).DNA damage induces activation of DNA repair mechanisms which is impaired in cancer cells.Impairment of DNA damage repair induces activation of cell death through stimulation of apoptosis processes.Normal tissue death induces mucositis and dermatitis causing inflammatory pain.2-Radiotherapy induces activation of immune responses and inflammatory cells which release inflammatory mediators such as cytokines and chemokines inducing inflammatory pain.Furthermore, these inflammatory mediators act as noxious stimuli, stimulating nociceptors to induce nociceptive pain.3-Radiotherapy induces direct mechanical and thermal damage in addition to changes in the microenvironment creating an acidic pH, increasing the adenosine triphosphate (ATP), and stimulating release of bradykinin and substance P which act as nociceptors stimuli inducing nociceptive pain.Radiotherapy induces direct nerve injury and inflammation of peripheral nervous system, and fibrosis in the connective tissue can also induce neuropathic pain.

V
.Salama et al.

Table 1
Summary of the literature search of variants associated with RT-induced acute inflammatory pain.

Table 2
Pathways and biological functions of genes associated with RT-induced acute inflammatory pain.

Table 3
Grading scale, cut points and covariates in studies of RT-induced inflammatory pain.Radiation Therapy Oncology Group or European Organization for Research and Efficacy of Cancer, CTCAE: The Common toxicity criteria for adverse event, TNM: Tumor, nodal, metastasis staging, BMI: Body mass Index, EBV: Epstein-Barr virus.CCRT: Concurrent chemo-radiotherapy.

Table 4
Summary of literature search of variants associated with RT-induced acute neuropathic pain.

Table 5
Summery of literature search of variants associated with other mixed types of RT-induced acute pain.

Table 6
Pathways and biological functions of genes associated with mixed types of radiotherapy induced acute pain.Sckack et al. conducted GWAS in HNC patients and identified candidate (rs1131769) in DNAJC18 gene regulating a chaperon molecule protecting cellular proteins, significantly associated with OM after RT [57].Yang et al., investigated GWAS of NPC patients and identified variant (rs117157809) in Tankyrase (TNKS) gene regulating telomere capping and maintenance of telomerase activity, is significantly associated with RT-induced OM [59].Li et al., identified GWAS risk loci (rs1265081) in CCHCR1 gene regulating cellular process as keratinocyte proliferation, differentiation, and epidermal growth factor receptor (EGFR) pathways, associated with RT-induced OM in HNC patients