Longitudinal, observational study on associations between postoperative nutritional vitamin D supplementation and clinical outcomes in esophageal cancer patients undergoing esophagectomy

Vitamin D can exert anticancer effect beyond bone and calcium metabolism. We aimed to investigate whether postoperative vitamin D supplementation affects quality of life (QOL) and survival in esophageal cancer (EC) patients. We utilized the widely used EORTC QLQ-C30 and QLQ-OES18 to assess QOL at EC diagnosis and 24 months after surgery. Generalized estimating equations (GEEs) were used to analysis the association of vitamin D supplement use with QOL. Kaplan-Meier method and Cox regression model were used to evaluate the prognostic value of vitamin D supplementation. The notably improved QOL were found among vitamin D supplementation users compared with non-users (p < 0.05). Kaplan-Meier analysis revealed that vitamin D supplement use was significantly associated with improved disease-free survival (DFS) (p = 0.030), but not related to overall survival (OS) (p = 0.303). The multivariable analysis further demonstrated vitamin D supplement use as an independent prognostic factor for DFS (p = 0.040; HR 0.610; 95% CI 0.381–0.978). In conclusion, these results showed that vitamin D supplement use could serve as a promising intervention to enhancing QOL and prolonging DFS in EC.

activities [8][9][10][11][12][13][14] . Observational studies have also revealed significant relationships of vitamin D with breast cancer, colorectal cancer, prostate cancer and pancreatic cancer 6,10,[15][16][17] . Additionally, vitamin D deficiency could increase the risk of developing cancer and lead to a lower QOL 18,19 . Therefore, more and more evidence supported that vitamin D supplement use might enhance QOL and decrease risk of recurrence or mortality among cancer patients. However, no work has been done to address the role of vitamin D supplementation that plays in clinical outcomes in EC patients following surgery.
To investigate the chemopreventive potential and anticancer action of vitamin D, we conducted a longitudinal, observational study of supplementation with vitamin D for improving poor clinical outcomes in EC patients. We primarily hypothesized that vitamin D supplement use could improve QOL during EC treatment and recovery phases. Our secondary hypotheses addressed the relationships between vitamin D supplementation and the risk of recurrence or mortality in cancer patients who underwent esophagectomy.

Association between vitamin D supplement use and QOL.
The prognostic value of vitamin D supplement use. The 3-year OS and 3-year DFS associated with vitamin D supplement use, calculated by Kaplan-Meier method, are shown in Figs 1 and 2. The 3-year OS rates were 49.5% in the non-users group and 55.1% in the users group. The 3-year DFS rates were 40.1% and 53.1% in the non-users group and users group, respectively. Patients who received vitamin D supplementation were more likely to have improved DFS (p = 0.030). However, no positive association of vitamin D supplement use with OS was found in our study (p = 0.303).
Univariate and multivariate analysis. The factors related to OS and DFS on univariate analysis are shown in Table 3. In univariate analysis, physical activity, pathological type, tumor length, T stage, TNM stage, lymph node metastasis, the number of lymph node metastases, positive lymph node ratio and treatment regimen (all p < 0.05) were associated with both OS and DFS. However, vitamin D supplementation was only associated with DFS (p = 0.035; HR 0.611; 95% CI 0.386-0.966) but not related to OS (p = 0.308; HR 0.795; 95% CI 0.511-1.237). The results of multivariate Cox regression analysis of the factors related to OS and DFS are shown in Table 4. It further suggested that vitamin D supplement use was an independent prognostic factor for DFS (p = 0.040; HR 0.610; 95% CI 0.381-0.978). Interestingly, we found physical activity was an independent prognostic factor for both OS (p = 0.004; HR 0.957; 95% CI 0.928-0.986) and DFS (p < 0.001; HR 0.949; 95% CI 0.922-0.978).

Discussion
To the best of our knowledge, this is the first report on relationship between vitamin D supplement use and clinical outcomes of EC. We found a significant improvement in QOL and DFS of EC patients undergoing esophagectomy who received vitamin D supplementation during cancer treatment and recovery phases. Considering imperfect medicare system, relatively low socioeconomic status and weak health conscious in our developing countries, it was not surprising to find that the majority of patients were vitamin D non-users and only 49 patients (16.2%) self-reported to regularly receive vitamin D supplement. The dosage of it was 200-400 IU daily, which was suggested by clinician and also recommended by Chinese Nutrition Society and World Health Organization. In summary, compared to non-users, vitamin D users tended to be younger, attended some college, had a higher income, participated in more physical activity, had lower fat intake, used less tobacco and were more likely to suffer from diabetes in our study.
In view of the limited data supporting positive effect of vitamin D supplement use on QOL for cancer, our results undoubtedly confirmed and extended recently published articles. Although we followed up the patients for 3 years, the sample size of survivors who received vitamin D was too small to sufficient statistical analysis at that time. As a result, the associations between vitamin D supplement use and QOL assessed at 2 years of follow-up were observed. In our study, EC patients who were supplemented with vitamin D had higher scores of physical functioning, social functioning and global health as well as lower scores of fatigue and appetite loss via a 24-month follow-up compared with non-users. It suggested that vitamin D users were more likely to maintain physical function, have more energy and better appetite, feel less faintness as well as enjoy life. These findings were consistent with previous study 20  Continued vitamin D tended to have better QOL. Another cross-sectional study indicated that vitamin D deficiency was quite common as well as associated with fatigue and poor physical and functional well-being in advanced cancer patients 19 . Therefore, it pointed to vitamin D supplementation as a potential therapy to improve cancer patients' QOL. Although vitamin D status is unknown in the current study, vitamin D supplement use did show a significant relationship with QOL.
To date, the association of vitamin D with cancer has been reported by several reviews, 6,9,10,21-25 which proposed it might have potential role as anticancer drug. In addition to the influence of vitamin D on cancer risk [26][27][28] , numerous reports [29][30][31][32] showed that vitamin D sufficiency could improve survival in lung cancer, breast cancer, prostate cancer and follicular lymphoma. However, the studies concerning associations between vitamin D supplementation and DFS or OS were limited and contradictory. In this study, we found that vitamin D supplement use could prolong DFS but not OS of EC patients. Our results were consistent with the findings of Zeichner et al. 33 . They also reported that vitamin D supplementation in patients with nonmetastatic HER2 + breast cancer was associated with improved DFS but not related to OS. While, Lewis et al. 20 studied the correlation of vitamin D supplementation with risk of recurrence or mortality in stage II colorectal cancer patients, no association was observed with DFS or OS. The statistical power for survival analysis may be insufficient or confounding variables could not be completely adjusted to influence the final results or measurement bias, which may in a way explain their null results. In another recent study in women in the UK, Jeffreys et al. 34 found no evidence that pre-diagnostic vitamin D supplementation was associated with survival among women with breast cancer, colorectal cancer, lung cancer and gynaecologic cancer. By contrast, we studied the correlation between post-operative vitamin D supplementation after cancer diagnosis and survival in EC patients, which did show that vitamin D supplement use after surgery could reduce the risk of recurrence to improve DFS. According to numerous existing evidences, the different results might be partially explained that pre-diagnostic vitamin D supplementation was more likely to play a role in reducing cancer risk but little associated with long-term clinical outcomes, while post-diagnostic vitamin D supplement use during cancer treatment and recovery phases might have a crucial effect on long-term survival. Additionally, we interestingly observed physical activity was an independent prognostic factor for both DFS and OS in our study. A number of articles, which have reported the inverse relation of lower physical activity with increased risk of mortality and poor survival, were consistent with our results [35][36][37] . We strongly supported a protective effect of physical activity on prognosis of EC.
In brief, vitamin D can be obtained from foods, sun exposure and supplementation. However, the quantities of vitamin D from dietary are quite small in our country. What's more, cancer patients are more likely to spend relatively large amounts of time indoors because of physical weakness and severe symptoms. It leads to less sun exposure contrasted with healthy people. Therefore, compared to other sources, the crucial role of vitamin D supplementation in cancer prevention cannot be ignored. Furthermore, it is worth mentioning that in addition to the well-known classic metabolic pathway, recently published articles reported that novel pathway of vitamin D metabolism in vivo can be initiated by CYP11A1 and modified by CYP27B1 to generate previously unrecognized vitamin D-hydroxyderivatives, which are different from 25-hydroxyvitamin D [25(OH)D] and 1,25(OH) 2 D [38][39][40] . An amount of studies have revealed that they can not only regulate bone mineralization and calcium homeostasis 41 , but also play an important role in antiproliferative, prodifferentiation and anticancer 11,12,42,43 . Taken together, the in-depth studies about vitamin D as a promising anticancer drug need to be urgently carried out.
At present, the mechanisms, account for the beneficial impacts of vitamin D supplement use on clinical outcomes of cancer, can be summarized as follows. One is that vitamin D are involved in various signaling pathways    Table 3. Univariate analysis of factors associated with OS and DFS. HR, hazard ratio; CI, confidence interval; BMI, body mass index; HBP, high blood pressure; CAD, coronary artery disease; SCC, squamous cell carcinoma; S, surgery; R, radiotherapy; C, chemotherapy; CRT, chemoradiotherapy. *p < 0.05. by its role of anticancer active to affect many important molecular events. As mentioned above, they include inhibition of proliferative, angiogenesis, invasion and metastasis, induction of apoptosis and stimulation of differentiation for malignant cells 6,10,22 . Another recognized mechanism is that vitamin D is also related to immune regulation and inflammation 6,22 , which play an important role in cancer pathogenesis and progression. In addition, the other novel mechanisms will be further studied in future.
There are a few limitations in this study. Firstly, because of a retrospective and single-center study, the collected data is somewhat limited. Secondly, vitamin D concentration in blood is not routinely measured among cancer patients in our clinical practice. As a result, the associations of the level of vitamin D with clinical outcomes could not be observed. Thirdly, pre-operative vitamin D supplement use, which may be related to both post-operative vitamin D supplement use and EC outcomes, is unavailable in our study. Therefore, further studies will be needed to verify that our results are not bias or occasional.
In summary, we initially reported that vitamin D supplement use could enhance QOL and prolong DFS of EC. Our study supported that clinician should recommend the use of vitamin D supplementation as an intervention during cancer treatment and recovery phases to improve poor clinical outcomes. Although our results was strongly supported by previous studies, further prospective, well-designed randomized controlled trials with larger samples are necessary to further verify the impacts of vitamin D supplementation on cancer's clinical outcomes.

Materials and Methods
Patient recruitment. Between January 2012 and December 2012, newly diagnosed and pathologically proven EC patients were recruited from the Department of Thoracic Surgery, Qilu Hospital of Shandong University. All patients enrolled in our study had detailed and authentic clinicopathological data. Patients were excluded from the study: (1) if they were treated with chemotherapy and/or radiotherapy before surgery to reduce the size of the tumor; (2) if they could not clarify whether vitamin D supplementation was used or not after esophagectomy; (3) if they received other supplementations post operative in addition to vitamin D supplement; (4) if they were lost to follow-up. This study was approved by Ethics Committee of Qilu Hospital of Shandong University. Informed consent was obtained from all the patients. All data has been anonymized and de-identified. Moreover, the patient data collection methods were carried out in accordance with the Declaration of Helsinki.
Data collection. Clinicopathological data were obtained from patients' medical records. Metastases to lymph nodes resected at surgery were counted and pathologically examined. Major factors known to influence QOL, recurrence and mortality of EC were extracted from medical records and follow-up, including age at diagnosis, gender, education, income, body mass index (BMI), physical activity, sun exposure, smoking, alcohol, dietary habits, past medical history, tumor characteristics, type of surgery and treatment regimen. All tumors were staged  Vitamin D supplementation assessment. The data of vitamin D supplement use were ascertained through self-report. Each patient or their kin was asked as follows: (1) Did patient take regularly vitamin D supplementation after esophagectomy during EC treatment and recovery phases? (2) If did, what's the frequency and dose of vitamin D supplementation? (3) What's the duration of taking it? (4) What's the brand of vitamin D supplementation? On the basis of these collected data, we finally identified two groups of patients for comparison-those who received vitamin D supplementation after surgery during EC treatment and recovery phases (vitamin D supplementation users group) and those who did not use it ( vitamin D supplementation non-users group).
QOL assessment. EC survivors' QOL were ascertained by using the cancer-specific European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 (EORTC QLQ-C30) and oesophagus-specific module EORTC QLQ-OES18 at diagnosis and 24 months after surgery. Both the two questionnaires are well validated and their utilities among EC patients have been previously described 46,47 . The EORTC QLQ-C30 comprises one global health scale, five function scales (physical, role, emotional, cognitive and social), three symptom scales (fatigue, pain and nausea/vomiting), and six single items (dyspnoea, insomnia, appetite loss, constipation, diarrhea and financial difficulties). The EORTC QLQ-OES18 contains four symptom scales (dysphagia, eating difficulties, reflux and esophageal pain) and six single items (trouble swallowing saliva, choking when swallowing, dry mouth, trouble with taste, trouble with cough and trouble with talking). Each item in both questionnaires, except for the global health scale which has seven responses ranging from "very poor" to "excellent", have four response categories: "not at all", "a little", "quite a bit" and "very much". Patients' responses were converted to a score of 0-100 in accordance with the EORTC scoring manual 46 , with higher scores reflecting better QOL in function scales and global health scale, whereas higher scores in symptom scales and items representing more serious symptoms.
Follow-up and survival assessment. Patients were informed routinely examined in our outpatient clinics every 3 months for the first 2 years post operative and every 6 months interval or until death thereafter. Physical examination, laboratory tests, barium meal fluoroscopy, esophagoscopy, computed tomography scans and other examinations as it fits were included in the follow-up assessments. In brief, the condition of patients' recurrence or death can be timely obtained through a combination of follow-up and medical record review. The follow-up end point was death or January 2016.

Statistical analysis.
Baseline characteristics of patients were presented as mean ± standard deviation or proportion by postoperative vitamin D supplementation status (users versus non-users). The Student's t test or Mann-Whitney U test where appropriate was used to evaluate continuous variables. Categorical variables were assessed by using the Pearson's chi square test or Fisher's exact test. The primary end point of this study was the QOL of EC survivors at 24 months post operative. GEEs were utilized to analysis the association of QOL with vitamin D supplement use. The secondary end points were 3-year DFS and 3-year OS. DFS was calculated from the date of surgery to the first date of tumor recurrence. OS was defined as the time from the date of surgery to the date of death or to last follow-up. Kaplan-Meier curves and log-rank tests were used for survival analyses. The Cox regression model was used in the univariate and multivariate analyses. The variables having statistically significant in the univariate analysis were selected into the multivariable analysis. All p values were two-sided and p < 0.05 indicated statistically significant. All data were performed with the Statistical Package for Social Science program (SPSS for Windows, version 17.0, SPSS Inc., Chicago, IL).