Correlated factors of acute oral mucosal and skin reaction induced by radiotherapy in hypopharyngeal carcinoma

Background This present retrospective single center study was intended to investigate the factors Methods From May 2012 to December 2018, previously untreated 93 patients with hypopharyngeal squamous cell carcinoma received radiotherapy in Affiliated Cancer Hospital of Zhengzhou University were enrolled. Radiation Therapy Oncology Group (RTOG) scoring criteria were used for assessing the severity of toxicities. Patients are therefore classified into “mild reaction group” (G0~G1) and “acute reaction group” (G2~G4). Single variate was applied to screen out factors with significant difference between mild and acute reaction groups, multivariate analysis was used to detect independent risk factors from those related. A total of 16 medical and laboratory indexes were included to examine, i.e., gender, age, smoking history, primary site, history of hypotension and diabetes, treatment modalities, dose, T (tumor) staging, N (reginal lymph node) staging, as well as hemoglobin value (1 week before radiotherapy). Relevant data used for the study were collected from clinical records. Results Total of 93 subjects completed radiotherapy. Acute mucositis occurred in 75 patients, and 27 cases developed acute radiation dermatitis. Smoking history, post-operative radiotherapy, concurrent chemotherapy, T staging, N staging, total dose (Gy) for GTV, single fraction dose (Gy) for GTV, and hemoglobin value (1 week before radiotherapy) showed significant differences between G0~G1 and G2~G4 groups of oral mucosa reaction; significant differences between mild and acute dermatitis reaction groups were found in diabetes history, hemoglobin value, age, total dose (Gy). Multivariate analysis showed that higher hemoglobin value ( OR = 1.120, P = 0.031), smoking history ( OR = 0.070, P = 0.031) were independent risk factor of acute OM; significant relationships for acute skin reaction were found with hemoglobin value ( OR = 1.059, P = 0.034) and older age ( OR = 1.068,

cases developed acute radiation dermatitis. Smoking history, post-operative radiotherapy, concurrent chemotherapy, T staging, N staging, total dose (Gy) for GTV, single fraction dose (Gy) for GTV, and hemoglobin value (1 week before radiotherapy) showed significant differences between G0~G1 and G2~G4 groups of oral mucosa reaction; significant differences between mild and acute dermatitis reaction groups were found in diabetes history, hemoglobin value, age, total dose (Gy). Multivariate analysis showed that higher hemoglobin value ( OR = 1.120, P = 0.031), smoking history ( OR = 0.070, P = 0.031) were independent risk factor of acute OM; significant relationships for acute skin reaction were found with hemoglobin value ( OR = 1.059, P = 0.034) and older age ( OR = 1.068, P = 0.036).
Conclusion Multivariate analysis showed higher hemoglobin value and smoking history to be the most relevant factors independently predicting grades 2 or higher OM; higher hemoglobin value and older age were found to be significantly associated with acute skin reaction. 3

Background
Patients with hypopharyngeal squamous cell carcinoma (HSCC) are often diagnosed at advanced stages, and accounts for 3 ~ 5% of head and neck cancer suffers [1] . Radiotherapy has well been acknowledged as a major or indispensable measure in the curative treatment of head and neck cancer. However, even with the benefits of conformal IMRT( intensity modulated radiation therapy) technique, up to 90% of patients are inclined to experience different toxicities of various degrees [2] , especially the radiation oral mucositis and dermatitis, which are the most common complications. Therefore, great number of patients are expected to experience oral mucosa and skin reaction during RT treatment. Patient's ability to eat and drink can be temporarily or permanently impaired, as the result, the quality of patient's life was lowered heavily [3] . Even worse, in severe conditions, radiation oral mucositis and dermatitis can lead to super infection and interrupted treatment. The effectiveness of treatment was consequently interfered [4] . Many clinic characteristics or concurrent diseases history have been suggested as potential risk factors for acute radiation oral mucosa and skin reaction of head and neck cancer, such as smoking history, diabetes history, higher hemoglobin value, dosage, as well as sex, age et al. However, there is still no consensus on the predictors of adverse effects due to the limited sample size and controversial results. In addition, there are few reports on the predictors of adverse effects of hypopharyngeal cancer radiotherapy. The purposes of this study were intended to investigate the risk factors that contribute to increased severity of radiation skin and mucosal reaction and to provide theoretical basis and important considerations for clinical practice.

Patient and the study setting:
From May 2012 to December 2018, a total of 93 patients with hypopharyngeal cancer who were undergoing radiotherapy were identified. They were staged IIB-IVA (8th edition AJCC Hypopharyngeal cancer TNM staging standard was referenced for staging). Patients inclusion criteria: previously untreated with hypopharyngeal cancer, staging IIB-IVA; completed radiotherapy and the records of acute toxicities were available; pathologically diagnosed hypopharyngeal cancer squamous cell carcinoma; there were no distant organ metastasis proved by MRI/CT examination; KPS (pretreatment Karnofsky score) ≥70; blood tests, kidney and liver function were in normal range. Patients exclusion criteria: patients have history of drug allergy; patients with others tumor. Clinical characteristics and relative data of patients were collected from the records. All subjects in present study had signed informed consent before they accepted treatment. This study was approved by the Ethical Committee of Affiliated Cancer Hospital of Zhengzhou University.

Treatment:
Radiotherapy Treatment: Patients were all accepted for conformal intensity modulated radiation therapy using 6MV x-ray medical linear accelerator (Varian company) as standard treatment modality.
The prescription dose for gross tumor volume, including prime site of tumor and involved lymph nodes (GTVnx / GTVnd), ranges from 60 to 70Gy, with daily fraction dose of 2.0 -2.2 5Gy, and 5 fractions per week. Prescription dose of clinical target volume (CTV) ranges from 50.4Gy to 60Gy.
Normal tissues were managed with tolerable RTOG0615 and RTOG0225 normal tissue limit dosage [5] .

Management of radiation induced oral toxicities
Oral care was delivered to subjects with OM (once a day); compound VitminB12 solution was compressed and used for atomization inhalation; recombinant fibroblast growth factor 1(Acidic, FGF1) 5 local spraying and antibiotic were used for patients of G3 OM. Antibiotics usage was based on throat swab culture results. Oxycodone hydrochloride prolonged-release tablets was administered when pain score of patients was up to 4. In severe situations, fentanyl would be applied for patients, and patients would be suggested to discontinue RT for 2 to 3 days (no more than 5 days) if even worse.

Radiation reaction and criteria
RTOG standard for acute radiation mucositis and dermatitis were used for grading [6,7] (Table 1); G0 G1was defined as mild adverse reaction; G2 ~ G4 was defined as acute adverse reaction ( there is no one with grade 4 in this study).

Statistical analysis
Statistical analysis were carried out using SPSS 25 (IBM, Armonk, NY). Each variable was analyzed by univariate Cox regression, Student T-Test and Chi square test (or Fisher's exact test) for continuous quantitative data and categorical variables respectively. The variables were considered correlated with oral mucositis when p < 0.05, and these variables were put into the multivariable Cox regression model to identify independent risk factors. Two-tailed test p < 0.05 were considered statistically significant.

Results
Patient's characteristics and clinical indexes were shown in Table 2  respectively. 36 cases had no skin reaction. There is no one with grades 4 of oral mucositis or dermatitis.
Univariate analysis showed that smoking history, post-operative radiotherapy, fraction dose (Gy), total prescription dose (Gy), T staging, and N staging, were significant characteristics between "mild" and "acute" OM; hemoglobin value, fraction dose (Gy) and age were significant factors between the two groups of skin reaction. (as shown the Table 2). These significant different characteristics between the two groups were put into the multivariable cox regression model to identify independent risk factors. The result showed that hemoglobin value and CRT were independent factors of OM ( Table   3), and that hemoglobin value, age, total dose have influence on grades of acute radiation dermatitis independently ( Table 4).

Discussions
Post-operative radiotherapy, radiotherapy alone or in conjunction with concurrent chemotherapy have well been acknowledged as standard curative treatment for patient with head and neck cancer [8] . OM and acute skin reaction are the most common complications, which always posed great challenge to clinical physician. Previous studies indicated that the two adverse effects showed dose and individual dependent. We retrospectively analyzed 93 patients and identified that higher hemoglobin value and smoking history are independent risk factors of OM; higher hemoglobin value, elder age and higher total dose are significantly associated with the severity of radiation skin reaction.
Smoking are shown to be positively correlated with OM in present study. It's always been known that tobacco comprised of aldehydic, phenolic and other organics. These compounds of toxic would be released during combustion and do damage to the oral mucosa, and the ability of proliferation of epithelia cell would be affected as a result. Besides, these substances could reduce the level of epidermal growth factor in saliva as well. Therefore, the epithelial cell of oral mucosa proliferation and repair was delayed, and the healing process was impeded directly. Moreover, nicotine could shrink skin capillary [9] , which contributes to increased severity of OM also.
Diabetic patients' skin is often in poor nutritional status due to microvascular and peripheral nerve disease. Peripheral vascular suffered from injury and atherosclerosis within radiated area may induce severe mucositis and radiation skin reaction. The body is in a state of stress when patient was exposed to radiotherapy, in which more glucagon, adrenal corticosteroid, growth hormones were released to blood. Moreover, the stress condition can reduce the sensitivity of tissue cell to insulin, resulting in aggravated disorder of glucose metabolism [10] . Previously reports indicated that higher blood glucose is significantly associated with acute skin reaction and OM. In present study diabetes is not a risk factor of acute skin reaction. It can be explained by well controlled blood glucose level during radiotherapy treatment.
Oxygenation plays crucial role in radiotherapy of tumor. It is well been accepted that the reduction of blood hemoglobin concentration can induce impaired tumor oxygenation and increased X-ray resistance. Therefore, the effectiveness of radiotherapy was impeded. Increased blood hemoglobin level can increase radio-sensitivity of tumor and the normal tissue. Cheng H et al reported that patients with higher value of blood hemoglobin at 1 week before radiotherapy were more inclined to develop acute skin reaction and OM, in consistent with this study [11] .
Chemotherapy and radiotherapy can complement each other and exert synergistic action on treatment of tumor because RT is significant on local control while chemotherapy is particularly important to systemic metastasis [12,13] . However, the normal tissues couldn't be spared totally when chemo-therapeutic drugs destroying tumor cells. DNA synthesis and cell regeneration can be inhibited by chemotherapy drugs and x-ray, resulting in oral ulcers and damaged normal tissue and organs. It has been well documented that some chemo-therapeutic drugs can enhance the biological effects of ionizing radiation [14][15][16] . In addition, chemotherapy can lead to bone marrow suppression 13 (such as thrombocytopenia, neutropenia) and decreased immune-defense ability, indirectly impairing normal tissue healing. It has been reported by Bonomo P [17] that cisplatin-based regimen were potentially more susceptible to the development of acute toxicity and concurrent chemotherapy could increase the incidence of OM or acute skin reaction. Although not identified as independent risk factors in this study, significant higher (39/40, 40 patients received CCRT, 39 patients developed G2 or above OM) oral mucositis were observed in patients receiving CCRT, which may due to the smaller samples size.
Radiation of high energy particles can cause DNA damage directly or indirectly. Total GTV dose and single fraction dose have been well-recognized as risk factors to develop severe OM. A previous report indicated that a radiation dose of 30 Gy is a related factor of severe OM [18] . Doctor Vera-Lionch M [19] reported that risk increased when dose reached 50 Gy. Dose-volume parameters can also successfully predict acute skin reactions [20] . The current study demonstrates that total dose and fraction dose are associate with Grade 2 or above OM and acute skin reaction.
Patients with higher N staging were administered with a larger radiation field and more dosage, and therefore were more likely to develop G2 or worse radiation mucositis. This has previously been reported [6,21] . One of the limitations of present study was that the sample size is small, partially due to that the incidence of hypopharyngeal cancer was low. In the current study, nearly all (13/14) patients with T4 staging developed G2 or above OM. There are few researches about it and the extensive efforts are needed for further investigation.
There is still no consent on how age impacts on radiation dermatitis. The aging of cell lines leads to thinning of the epidermis, losing of collagen and decreasing of capillary network [16] . In addition, older patients often have poor nutrition status, which increases the difficulty in healing. That may explain that aging significantly increases the possibility of acute skin reaction., This result was also demonstrated in the paper of Dr Porcked [22] .

Conclusions 14
The current study indicates that risk factors of OM and dermatitis should be taken into consideration in clinical practice by physician when making decisions on therapeutic time, fraction dose and biological effects. Declarations

Ethics declarations
The current study was approved by the Ethical Committee of Affiliated Cancer Hospital of Zhengzhou University.

Ethics approval and consent to participate
The current study was approved by the Ethical Committee of Affiliated Cancer Hospital of Zhengzhou University.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author upon request.