Low serum 25‐hydroxyvitamin D3 levels and late delayed radiation‐induced brain injury in patients with nasopharyngeal carcinoma: A case–control study

Abstract Background and Purpose Inflammatory reaction plays a critical role in the pathogenesis of late delayed radiation‐induced brain injury (RBI). Low vitamin D levels are closely associated with various immuno‐inflammatory diseases, but the relationship with late delayed RBI remains unknown. Here, we aimed to determine the association of serum vitamin D levels with clinical parameters in late delayed RBI patients with nasopharyngeal carcinoma. Methods 25‐Hydroxyvitamin D3 levels and clinical and cerebrospinal fluid parameters were evaluated in 21 patients with RBI and compared with 90 age‐, sex‐, and season‐matched healthy controls. Results 25‐(OH)D3 levels were lower in patients with RBI compared to controls (40.39 ± 22.11 vs. 64.54 ± 19.89 nmol/L, p < .001), especially for aged ≥60 years (vs. <60 years, p = .038), females (vs. males, p = .012), short latency (<5 years) (vs. ≥5 years, p = .015), and severe impairment (LENT/SOMA score ≥3) (vs. LENT/SOMA score <3, p = .010). Serum 25‐(OH)D3 levels were associated with age (r = −.464, p = .015), Latency of RBI (r = .416, p = .031) and LENT/SOMA Scale (r = −.488, p = .010). Conclusions Our data showed that serum 25‐(OH)D3 levels were reduced in late delayed RBI patients with nasopharyngeal carcinoma.

. Late delayed RBI has the highest incidence and the most severe symptoms, can even be life-threatening, and often shows progressive intracranial pressure caused by extensive cerebral edema, cognitive dysfunction, and other neurological deficit (Schultheiss et al., 1995). Immune inflammatory processes play a vital role in the development of RBI, so immunosuppressant such as corticosteroids are commonly used in clinical treatment (Balentova & Adamkov, 2015).
Vitamin D is an organic compound necessary for modulating metabolism of calcium and phosphorus, which is mainly synthesized from 7-dehydrocholesterol in the skin by the action of ultra violet light. Increasing studies have found that vitamin D is a selective immunomodulator in addition to traditional physiological functions (Sassi et al., 2018). When the body is in an immunosuppressive state, vitamin D enhances the function of monocytes and macrophages.
Conversely, when the immune function abnormally increases, vitamin D inhibits the proliferation of activated T and B lymphocytes to maintain immune balance. It is worth noting that vitamin D itself has no physiological function, and only when it is converted into its active form can it become a physiologically active substance. The major active form in circulation is 25-hydroxyvitamin D3 (25-(OH)D3), which can be measured to assess vitamin D status (Holick, 2007).

Recent studies confirmed that low levels of serum 25-(OH)D3
are associated with a variety of immune disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and autoimmune thyroid diseases (Harrison et al., 2020;Islam et al., 2019;Lee et al., 2019). Moreover, vitamin D is independent risk factor for CNS demyelination including multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), and inflammatory spinal cord disease (Mealy et al., 2012;Salzer et al., 2012;Shan et al., 2016). In addition, studies have shown that serum 25-(OH)D3 levels were reduced in patients with anti-N-methyl-d-aspartate receptor encephalitis (Shu et al., 2017). Furthermore, studies have pointed out Vitamin D deficiency is associated with the severity of radiation-induced proctitis in cancer patients (Ghorbanzadeh-Moghaddam et al., 2015).
However, the relationship between low serum 25-(OH)D3 levels and RBI remains unknown. Here, we analyzed 25-(OH)D3 levels in late delayed RBI patients and determined the association of vitamin D levels with clinical parameters in these patients.

| General information
This was a single-center cross-sectional study based on the patients of the First Affiliated Hospital of Sun Yat-sen University, which is approved by the medical ethics committee of the First Affiliated Hospital of Sun Yat-sen University.
From 1 October 2018 to 31 December 2019, we recruited consecutive patients with late delayed RBI (n = 27), and age-, sex-, and season-matched healthy controls (CTLs, n = 80) were selected from the medical Examination Center of First Affiliated Hospital of Sun Yat-sen University. The irradiation dose was 67-75 Gy with a fraction dose of 2 Gy. Season was defined as spring (March to May), summer (June to August), fall (September to November), and winter (December to February) and was also acquired and matched to healthy controls. Inclusion criteria included: (a) age ≥18 years old; (b) Radiation therapy history for histologically confirmed NPC > 6 months prior to study entry; (c) Radiographic evidence to support the diagnosis of RBI without tumor recurrence; (

| MRI scanning
Cranial MRI scanning was used for RBI patients by a GE 1.5T MR scanner (General Electric). The slice thickness of axial scans was 5 mm. Gadopentetate dimeglumine (Gd-DTPA) was administered intravenously for enhanced scanning at a dose of 0.1 mmol/kg.

| Statistical analysis
All the data were analyzed by SPSS 22.0. Measurement data were expressed as mean ± standard deviation (SD) and were compared with Student's t test for normally distributed variables. Non-normal distribution measurement data were described using the median and interquartile range and were compared using Mann-Whitney U test. Categorical data were given as the numbers and percentage (%) and were compared by chi-square test and Fisher's exact test.
Correlations between serum 25-(OH)D3 and age, BMI, LENT/SOMA score, and CSF factors (CSF WBC, TP, Glu, and CL) were analyzed by Spearman's rank test. All analyzes were two-sided, and p < .05 for the difference was statistically significant.

| General characteristics of RBI patients and healthy controls
The details of general characteristic including demographics, clinical features, and laboratory data between RBI patients and healthy controls are summarized in Table 1 spring:summer:fall:winter = 17:10:28:25) were included in our study.
The median LENT/SOMA score in RBI patients was 2 (range, 1-4). All primary tumors of RBI patients are NPC. Of 27 patients with RBI, 5 patients (18.5%) had cranial nerve palsy such as dysarthria and dysphagia; 17 patients (63.0%) had cranial hypertension symptoms such as headache, nausea, and vomiting; 20 patients (74.1%) had cognitive impairment symptoms such as memory deficits and mental disorders; 9 patients (33.3%) had conduction beam damage such as limb weakness and numbness; 6 patients (22.2%) had cerebellar disorder such as vertigo and ataxia; 18 patients (66.7%) had sleep disorder. In addition, as shown in Table 1, there is no statistical difference in the underlying disease rates between the two groups.

| Correlation between serum 25-(OH)D3 levels, clinical characteristics, and CSF parameters in RBI patients
The relationship between serum 25-(OH)D3 levels, clinical characteristics, WBC, and CSF parameters in RBI patients was evaluated (Table 3). It was shown that there was a negative correlation between serum 25-(OH)D3 levels and age (r = −.464, p = .015). Furthermore, there was a positive correlation between serum 25-(OH)D3 levels and latency (r = .416, p = .031). In contrast, there was a negative correlation between serum 25-(OH)D3 levels and LENT/SOMA score.

| D ISCUSS I ON
Emerging evidences have shown that vitamin D not only plays a physiological role in calcium metabolism and bone growth, but also has a strong immune-regulating function. Interestingly, diffuse T-cell infiltration and activated infiltrated macrophages with high expression of proinflammatory factors can be seen in the frozen sections of brain biopsies from RBI patients (Kureshi et al., 1994). Normally, the presence of the blood-brain barrier prevents peripheral immune cells from entering the central nervous system. In our previous studies, it was found that ionizing radiation can induce a decrease in tight junction protein expression (Deng et al., 2017). Increased vascular permeability and disruption of the blood-brain barrier after irradiation can lead peripherally derived macrophages and white blood cells to enter the central nervous system.
Increasing research indicates that vitamin D levels are closely related to radiation-induced inflammatory damage (Yazici et al., 2011 The effect of vitamin D in anti-inflammatory and immunomodulatory may be the link to RBI. Vitamin D has extensive physiological effects in regulation of innate and adaptive immunity, which is mediated by signaling through the vitamin D receptor expressed on immune cells (Delvin et al., 2014). It was reported that vitamin D regulates the proliferation and transformation of monocyte and macrophages (Yuk et al., 2009). In addition, vitamin D controls T-cell antigen receptor signaling and activation of human T cells, while inhibits the proliferation of activated B cells and induced their apoptosis (Chen et al., 2007). In human innate lymphoid cells, Vitamin D downregulates the IL-23 receptor pathway (Konya et al., 2018).

Clinical studies have shown associations of vitamin D deficiency
with various immune diseases such as SLE, RA, and inflammatory bowel disease (IBD) (Agmon- Levin et al., 2013;Bae & Lee, 2018;Kabbani et al., 2016). In central nervous system (CNS), adaptive immunity is the key to the understanding of autoimmune and paraneoplastic inflammatory central nervous system disorders such as MS, NMOSD, and autoimmune encephalitis, which have also been shown to be closely related to vitamin D deficiency (Weissert, 2017).

Disturbance of autoimmune system caused by ionizing radiation
promotes the development of RBI. Consistent with other neurological autoimmune diseases, we found that serum vitamin D levels were significantly reduced in RBI patients in this study. Recently, cumulative evidence links vitamin D and radiation-induced injury.
Therefore, we speculate that low serum vitamin D may be a risk fac- In conclusion, we report that serum 25-(OH)D3 levels are reduced in late delayed RBI patients and are associated with disease disability.

ACK N OWLED G M ENTS
This study was supported by grants from National Natural Science Foundation of China (81971166), including the design of the study, experiment, analysis, and interpretation of data and in writing the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflict of interest.

E TH I C A L A PPROVA L
This study was approved by the medical ethics committee of the First Affiliated Hospital of Sun Yat-sen University.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.1892.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.