Effect of cryotherapy in preventing mucositis associated with the use of 5-fluorouracil

Objective: to evaluate the effect of oral cryotherapy compared to physiological serum on the development of oral mucositis in outpatient cancer patients using the 5-fluorouracil antineoplastic agent. Method: this is a controlled, randomized, double-blind, and multi-center clinical trial, conducted with 60 patients undergoing chemotherapy. The experimental group (n=30) used oral cryotherapy during the infusion of the 5-FU antineoplastic agent, while the control group (n=30) performed mouthwash with physiological serum at their homes. The oral cavity of the participants was assessed at three times: before randomization, and on the 7th and 14th days after using 5-FU. For data analysis, descriptive analyses and the ANOVA, paired t, and McNemar tests were used. Results: there was no statistically significant difference between the experimental and control groups in the assessments regarding the grade of mucositis. However, cryotherapy presented the chance to reduce the presence of intragroup mucositis, between the first and second assessments (p=0.000126). Conclusion: cryotherapy did not obtain statistical significance in relation to oral hygiene with serum, but it proved to be effective intragroup. Record number: RBR-4k7zh3


Introduction
Oral Mucositis (OM) is a complex biological process. The pathogenesis of OM comprises a sequence of biological events possibly influenced by the oral microbiome and by the environment, leading to the positive regulation of pro-inflammatory cytokines, resulting in the thinning of the epithelium through tissue damage and cell death (1) .
People receiving chemotherapy for cancer are at risk of developing mucositis as a side effect (2) , which can occur in 20% to 40% of the patients undergoing conventional chemotherapy regimens (3) .
Among the antineoplastic agents that cause these changes in the oral mucosa is 5-fluorouracil (5-FU) (4) .
5-FU is a drug used in the treatment of solid cancers, such as those of the gastrointestinal tract and breast, and has a short half-life (5) .
OM is a side effect that causes several changes in the patient, such as pain, difficulty in eating, risk of infection and bleeding, and distress, in addition to causing an increase in the treatment cost, both for the patient and for the health system, since it needs medications to control pain and infections and often requires hospitalization for enteral support (1)(2)(3)(4)(5)(6) .
Oral cryotherapy is characterized by the application of ice in the oral cavity or by mouth rinsing with iced water before, during, and after the administration of the chemotherapy drugs (7) . The use of cryotherapy is based on the assumption that ice-induced vasoconstriction will reduce blood flow in the oral mucosa, resulting in lower local concentrations of the chemotherapeutic agents, reducing the chance of OM (1) .
Therefore, the objective of this study was to verify the effect of cryotherapy compared to the oral hygiene protocol with physiological serum in reducing the incidence and severity of oral mucositis in patients using The study population consisted of cancer patients admitted to the aforementioned chemotherapy outpatient clinics. To establish the sample size, it was estimated based on a previous study (8) , where the power calculation to determine the number of participants in each group was performed in relation to the expected change in the grade of mucositis, as assessed by the World Health Organization Scale (9)

Study design
Before the start of collection, the researchers introduced themselves to the nursing staff in the field of study and explained the study, highlighting the criteria for inclusion and exclusion of patients in the research.
Two assistant researchers were trained and instructed not to provide information to any participant about the use of cryotherapy and physiological serum to prevent mucositis, as well as guidelines on oral hygiene, adequate nutrition or others that could interfere with the results of the research.
On the collection days, the nurses signaled the patients considered eligible to the two assistant researchers. In the first moment, the first assistant researcher provided the patient with all the information regarding the study. The patients were informed about the need for new assessments after the intervention and that one of the researchers would contact them by phone to schedule a place and time for the subsequent evaluations, which could take place at the outpatient clinic or at their homes, according to their preference.
After the guidelines, the patients were invited to participate in the research and, if they agreed, the signing of the Free and Informed Consent Form (FICF) and the assessment of the grade of mucositis before the intervention were performed.
In the second moment, the second assistant researcher took out an envelope previously prepared revealing in which group the patient would be included and applied the intervention. The sealed and opaque envelopes, sequenced with the "experimental group" (EG) or "control group" (CG) designation, were www.eerp.usp.br/rlae The person responsible for the statistical analysis was also blinded since, before the data were made available, the CG and EG were coded in G1 and G2 to prevent him from distinguishing the group that received the intervention.
Clinical and sociodemographic data, such as age, schooling, marital status, type of cancer, and chemotherapy treatment protocol were collected from the patient's medical record; information about origin, current occupation, income, religion, smoking and alcoholism habits, use of dental prosthesis, frequency of dental consultations, and use of mouthwashes were collected from interviews with the participant, using an instrument developed by the researchers.
For the participants in the experimental group, the researcher applied ice to the oral cavity, starting 5 minutes before the 5-FU infusion and lasting for 30 minutes of continuous administration, as recommended (10) . It should be noted that there is no determination in the literature about the amount of grams of ice to be applied, only that the ice should be easily moved in the oral cavity, for which ice pieces are recommended. Therefore, the participant was provided with pieces of ice packaged in a plastic cup for individual use and a napkin. The ice was being replaced as it ran out. For the patients with dental prostheses, their removal and packaging in a plastic cup was requested before the intervention.
The participants in the control group were instructed to perform mouthwashes with 10 ml of physiological serum, at room temperature, three times a day, for one minute, and for a period of 14 days after application of the chemotherapy drug. All the instructions for the use of physiological serum were inserted in a label that was attached to each serum bottle provided by the researchers to the patient.
Measurement of mucositis: mucositis was evaluated for both experimental and control groups, according to the World Health Organization (WHO) mucositis assessment scale (9) , a scale considered ideal because it incorporates both clinical and functional factors, and has its validity established in several studies on the theme (8,(11)(12)(13) . On this scale, mucositis is classified into four grades, namely: Grade 0: no changes; Grade 1: erythema; with or without pain; Grade 2: erythema, ulcers, the patient can eat a solid diet; Grade 3: ulcers, extensive erythema, the patient is unable to eat a solid diet; Grade 4: extensive mucositis that does not allow oral feeding. To assess mucositis, personal protective equipment was used, in addition to a pocket flashlight.
The outcome was measured in three moments. The first evaluation was carried out before the administration of 5-FU (T1), the second evaluation 7 days after intervention (T2), and the third, 14 days after (T3).
These evaluation points were established considering that the symptoms of mucositis develop from the 5 th day after the administration of the chemotherapy drug in question, and may last until the 14 th day (3) . For the intervention to show significance of change, the results obtained must be greater than 1, and their respective confidence intervals must not exceed the null value, which is 1. The significance level of the statistical test was set at 5% (ɑ=0.05).
To analyze the intergroup comparison, the ANOVA test was used, with a significance level of 5% (ɑ=0.05).   • Allocation to the Control Group, Center 1 (n=15). • Allocation to the Control Group, Center 2 (n=25).

Follow-up
Experimental Group • Center 1: Loss of follow-up due to impossibility of contact due to absence of telephone network (n=4). • Center 2: Loss of follow-up due to impossibility of contact due to absence of telephone network (n=6).
Control Group • Center 1: Loss of follow-up due to non-adherence to mouthwash with physiological serum (n=3), death (n=2), and impossibility of contact due to absence of telephone network (n=2). • Center 2: Loss of follow-up due to non-adherence to mouthwash with physiological serum (n=1), and impossibility of contact due to absence of telephone network (n=2).

Results
The sample consisted equally of men (n=30) and women (n=30) in the groups, with a mean  (Table 3).
Regarding the reduction of mucositis in the EG (intraclass analysis), it was verified that cryotherapy had a chance of approximately six times (OR: 6.5;   . Cryotherapy is effective in preventing oral mucositis in patients scheduled for chemotherapy with antineoplastic agents with short plasma half-lives, such as bolus doses of 5-fluorouracil (14) . A clinical trial that investigated the effects of oral cryotherapy on chemotherapy-induced oral mucositis in patients undergoing autologous transplantation verified that cryotherapy is more effective than saline mouthwash in reducing the severity of mucositis (6) and, in this sense, a systematic review by Cochrane reported evidence showing that oral cryotherapy can lead to large reductions in the number of adults reporting oral mucositis of all severities after receiving a fluorouracilbased treatment for solid neoplastic malignancies (2) .
Another study, a clinical trial that used oral cryotherapy for 58 patients with esophageal cancer using the DCF (docetaxel, cisplatin and fluorouracil) chemotherapy protocol, verified the reduction of the incidence of oral mucositis of all grades in comparison with the non-cryotherapy group (24.1% x 71.4%, p<0.001) (15) . A meta-analysis that included 14 studies with 1,280 participants also concluded that oral cryotherapy reduced the risk of developing mucositis in patients treated with fluorouracil-based chemotherapy (16) . With regard to mouthwash solutions, the use of chlorhexidine, sodium bicarbonate, benzydamide, and saline solution was identified (5) . A clinical trial aimed at health education on oral hygiene, including mouthwash with salt water with glutamine (17) found no difference between the results regarding OM with the different solutions.
Thus, the scientific evidence points to the effectiveness of interventions such as oral hygiene protocols, which was intended to be tested in comparison to cryotherapy in this study. More than the mouthwash solution itself, the unique component of any oral hygiene protocol for reducing OM is use consistency, which has a positive effect, both in prevention and reduction (17) .
When the effectiveness of intra-group cryotherapy was evaluated, this intervention had the chance to reduce by approximately six times the presence of mucositis between the first and the second evaluations, regardless of the grade, presenting significant statistics. Between the second and third assessments, and between the first and the third, the use of this intervention was able to reduce by approximately seven times the chance of presenting mucositis.
Another relevant fact was that the experimental group did not present higher grades of mucositis in any of the evaluations, differently from the control group. This fact was also verified in other studies ( showing that cryotherapy can, in addition to reducing the onset of mucositis, prevent the occurrence of higher grades of this outcome. A meta-analysis of 29 clinical trials that evaluated, among other interventions, cryotherapy for the prevention of oral mucositis induced by chemotherapy in adult cancer patients, states that it was the most effective intervention in the prevention of OM, with a safety profile similar to the control (18) .
A larger number of studies using cryotherapy in a population of patients with onco-hematological disease was identified than in solid tumors, as in the present study. Onco-hematological patients can be subjected to induction for hematopoietic stem cell transplantation with another drug, melphalan, also a potential etiologic factor for mucositis (7,(19)(20)(21) .
Thus, the measures currently available for the prevention and control of OM, excluding cryotherapy and oral hygiene protocols, are expensive, such as laser therapy, which has a proven indication only for patients undergoing hematopoietic stem cell transplantation and patients with head and neck cancer undergoing chemo or radiation therapy (3) .
In this sense, the cryotherapy intervention is seen as capable of being applied by nurses, as a low It is suggested to conduct research studies that involve more participants, in order to provide more accurate findings and that evaluate oral hygiene protocols with the use of saline solution, still incipient in research.

Conclusion
Although cryotherapy did not obtain statistical significance, when compared to the oral hygiene protocol with saline solution, it proved to be effective intragroup. The inclusion of cryotherapy on an outpatient basis for patients undergoing treatment with the 5-FU chemotherapy drug can be an alternative to reduce the occurrence and severity of mucositis. The results of this study help to clarify evidence that supports the use of oral cryotherapy, which is economical and has few side effects, as a preventive strategy for oral mucositis.