In this study, we evaluated the potential importance of total, bioavailable, and free 25(OH)D as well as VDBP as pre-treatment prognostic indicators for OS and PFS among patients with NSCLC. We found that all three 25(OH)D fractions had discrimination for long-term survival in NSCLC patients, as high levels of total, bioavailable or free 25(OH)D were associated with better OS and PFS. But we did not find a superior prognostic performance in bioavailable or free 25(OH)D as compared to total 25(OH)D. Moreover, no significant association was found between VDBP and survival in NSCLC.
Vitamin D deficiency is frequently observed in cancer patients, including lung cancer. Herein, we found that 50.1% of the patients had vitamin D deficiency (25(OH)D level <20 ng/mL) and 37.0% had insufficiency (20-30 ng/mL), suggesting a severe vitamin D deficiency status in the current NSCLC cohort, which is consistent with previous studies [11, 21]. In addition, compared with healthy controls, total 25(OH)D was lower in NSCLC patients, among whom survivors had a higher level of total 25(OH)D than non-survivors, indicating a latent impact of vitamin D on the disease prognosis of NSCLC.
Various mechanism may underlie the influence of vitamin D on the progression and prognosis of lung cancer. Vitamin D can inhibit lung cancer tumor growth, migration and proliferation, and promote apoptosis by downregulating Histidine-rich calcium-binding protein (HRC) . As an active metabolite of vitamin D, 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces E-cadherin and decreases EMT-related molecules SNAIL, ZEB1, and vimentin in NSCLC cells, thus preventing NSCLC progression . Besides, vitamin D deficiency may increase inflammation and proliferation of pre-malignant cells thereby enhancing progression of squamous lesions . By contrast, vitamin D treatment in the EGFR mutant NSCLC cells may lead to inhibition of clonogenic growth in a dose-dependent manner . And vitamin D or vitamin D derivatives play a powerful synergic role when used in combination with chemotherapy drugs, promoting the cytotoxic effects of drugs and reducing drug resistance on NSCLC cells compared to using the free single drug [26, 27].
Given the close affinity of vitamin D and NSCLC, the prognostic impact of vitamin D levels on NSCLC has been investigated previously, but the results are controversial. Several cohort studies addressed that higher circulating 25(OH)D showed an association with improved survival of patients with early- or advanced-stage NSCLC [9–11]. In contrast, another two studies demonstrated that there was no effect of 25(OH)D level on survival in advanced NSCLC [12, 13]. In addition, a nested case-control study suggested that patients with higher 25(OH)D was associated with significantly better survival among patients with lung adenocarcinoma but not among those with squamous cell carcinoma .The inconsistent findings indicate that the role of vitamin D in NSCLC prognosis still deserves further exploration.
Recent studies have highlighted bioavailable or free 25(OH)D as a more meaningful marker of vitamin D function [16, 17]. In current study, besides total 25(OH)D, we measured bioavailable and free 25(OH)D levels and investigated their relationships with NSCLC prognosis for the first time. We found that plasma concentrations of bioavailable and free 25(OH)D both were significantly decreased as compared to healthy controls. Additionally, total 25(OH)D was positively with both bioavailable and free 25(OH)D, which may partly explain the decrement of bioavailable and free 25(OH)D. We also found that total, bioavailable, and free 25(OH)D levels were inversely related to both OS and PFS, indicating all three 25(OH)D forms as independent predictors for survival among NSCLC patients. Besides, the AUCs in bioavailable and free 25(OH)D were similar with total 25(OH)D in dynamic trend, thus we could not distinguish if bioavailable and free 25(OH)D were superior to total 25(OH)D in relation to NSCLC prognosis. Our results did not completely correspond with other cancers in previous studies. Chen et al reported that plasma bioavailable 25(OH)D was independently associated with PFS and OS, while total 25(OH)D was independently associated with PFS but not OS in diffuse large B-cell lymphoma . Yang et al showed that higher bioavailable and free 25(OH)D, rather than total 25(OH)D, were associated with better OS in I-III stage colorectal cancer, though only free 25(OH)D was an independent prognostic factor for OS . Similarly, Fang et al suggested that bioavailable 25(OH)D was a preferable biomarker over total 25(OH)D for the prognosis of hepatocellular carcinoma . By contrast, Yuan et al demonstrated that bioavailable 25(OH)D levels were not more strongly associated with risk of advanced and lethal prostate cancer than total 25(OH)D levels . There are several potential reasons for the inconsistent findings. First, the measurements for 25(OH)D fractions varied in these studies. More importantly, there is no uniform standard either measurements or cutoff values for bioavailable and free 25(OH)D at present, which may bias the association between 25(OH)D fractions and cancer prognosis. Second, circulating vitamin D concentration may be influenced by various factors such as liver function, kidney diseases, estrogens and genetic background , thus 25(OH)D fractions only at a time point could not comprehensively represent the status of vitamin D, thereby in part leading to the disagreement of current studies. And finally, total 25(OH)D levels may indeed differ in distinct populations. The mean level of total 25(OH)D in Fang et al study was much higher than Chen et al and our present studies (35.8 ng/mL vs. 16.0-20.8 ng/mL) [29, 31]. It is possible that total 25(OH)D with a small increment provides more benefit for patients with vitamin D deficiency (<20 ng/mL), but no meaningful impact on patients with sufficient vitamin D (>30 ng/mL). Taken together, high level of vitamin D may have a positive association with improved prognosis in various cancers. However, it still should be more prudent about the conclusion that bioavailable or free 25(OH)D could be a better biomarker for the estimation of vitamin D status or the prediction of cancer prognosis.
In addition to different forms of vitamin D, another important biomarker for vitamin D status is VDBP. We found that VDBP levels were significantly higher in NSCLC patients compared with healthy controls. As VDBP is bound to most of vitamin D, the increase of its levels may reduce the free or bioavailable fractions of vitamin D. In other words, the bioavailability of vitamin D may be limited even though circulating vitamin D is sufficient. Previous studies showed an inverse association between VDBP and free or bioavailable 25(OH)D [32, 33], though the association is yet to be found in current study. Furthermore, VDBP per se has multiple biological activities beyond vitamin D transporter, including extracellular actin scavenger and enhancement of neutrophil chemotactic activity . Particularly, the VDBP-derived macrophage activating factor (MAF) demonstrated potent inhibition of both proliferation and migration of tumor cells , and induced tumor cells apoptosis through stimulating macrophages . To date, two study examined VDBP level and lung cancer prognosis. One study including 148 England operated NSCLC patients showed that patients with the highest quartile of VDBP (>430.2 µg/mL) tended to have better prognosis in comparison to those with the lowest quartile of VDBP (<199.4 µg/mL) . The other study conducted in 500 Finland male lung cancer cases demonstrated that circulating concentration of VDBP was not associated with lung cancer specific survival . In this study, no significant association was found between VDBP level and NSCLC survival. Of note, VDBP in the European studies are much higher than that in our study (291-337 µg/mL vs. 204.9 µg/mL), which could be partially attributed to the difference in race and genetic polymorphisms . Whether a sufficiently high level of VDBP (such as >430.2 µg/mL) may have a positive effect on the progression of lung cancer to some extent remains to be further investigated.
Several limitations should be acknowledged in current study. First, the sample size was relatively small, thus the findings of the present study may need to be generalized with caution, and further studies with larger sample sizes are needed to precisely validate these results. Second, the bioavailable 25(OH)D level was calculated based on the total 25(OH)D level, albumin level, and VDBP level in the plasma. Recently, the direct measurement of the bioavailable 25(OH)D level was established , which may more accurately reflect the bioavailable 25(OH)D level in the participants. Finally, other factors affecting vitamin D levels, such as sunlight exposure and vitamin D supplementation, were not evaluated in our study. But season of blood drawing was included as a potential confounder factor, which may reflect sunlight exposure partially. Nevertheless, this study also has several strengths, including the prospective design to minimize the potential for reverse causality. Furthermore, to the best of our knowledge, this is the first study to explore of the relationship between bioavailable and free 25(OH)D and NSCLC survival.