Prognostic value of 25-hydroxy vitamin D in extranodal NK/T cell lymphoma

25-hydroxy vitamin D [25-(OH)D] is widely used to determine vitamin D status in clinic. The aim of our study was to evaluate the prognostic value of 25-(OH)D in extranodal NK/T cell lymphoma (ENKTL). Ninety-three (93) ENKTL patients with available serum 25-(OH)D values were enrolled in our study. Vitamin D deficiency is defined as a 25-(OH)D below 50 nmol/L (20 ng/ml). Univariate and multivariate regression analyses were performed to determine independent risk factors for progression-free survival (PFS) and overall survival (OS). Receiver operator characteristic (ROC) curves were plotted, and corresponding areas under the curves (AUC) were calculated to estimate the accuracy of PINK-E (prognostic index of natural killer lymphoma added with Epstein-Barr virus-DNA status) and 25-(OH)D deficiency in ENKTL risk-stratification. Our results suggested that the vitamin D deficiency was an independent inferior prognostic factor for both PFS [hazard ratio (HR), 2.869; 95% confidence interval (CI), 1.540 to 5.346; P = 0.003] and OS (HR, 3.204; 95% CI, 1.559 to 6.583; P = 0.006) in patients with ENKTL. Additionally, we demonstrated that adding 25-(OH)D deficiency to PINK-E score system indeed has a superior prognostic significance than PINK-E alone for PFS [AUC: 0.796 (95% CI: 0.699 to 0.872) vs. 0.759 (95% CI: 0.659 to 0.841), P = 0.020] and OS [AUC: 0.755 (95% CI: 0.655 to 0.838) vs. 0.721 (95% CI: 0.618 to 0.809), P = 0.040]. In conclusion, our study proved that 25-(OH)D deficiency was associated with inferior survival outcome of ENKTL patients.


Introduction
Extranodal natural killer/T-cell lymphoma (ENKTL) is a wellcharacterized subtype of mature T/NK-cell lymphoma. Asparaginase-based or pegaspargase-based chemotherapy regimens or chemoradiation have been evaluated to improve response rates comparing with anthracycline-based regimens [1]. The latest prognostic index of natural killer lymphoma (PINK) added with Epstein-Barr virus (EBV)-DNA status (PINK-E) is a prognostic model based on non-anthracyclinebased chemotherapy, and it consists of five risk factors (age, stage, non-nasal type, distant lymph-node involvement, and EBV-DNA status) [2].
As the active metabolite of vitamin D, 1,25-dihydroxy vitamin D [1,25(OH) 2 D] is produced from primary circulating form of vitamin D, 25-hydroxy vitamin D [25-(OH)D] and the latter one is used to determine vitamin D status [3]. One study has suggested that 25-(OH)D is associated with cancer mortality [4], and there may be a negative correlation with the risk of tumorigenesis [5]. In terms of lymphoma, although studies have suggested that ultraviolet radiation has a protective effect on the development of lymphoma [6,7], but 25-(OH)D has no significant effect on the risk of lymphoma [6][7][8][9][10]. Nonetheless, several studies have shown that 25-(OH)D is Jin Mao and Hua Yin contributed equally to this work.
A previous report on the prognostic role of 25-(OH)D in Tcell lymphoma found that 25-(OH)D had prognostic significance in ENKTL [16]. However, this report did not further explore whether 25-(OH)D could improve the prognostic effect of the recognized prognostic model-PINK-E, which is based on non-anthracycline-based chemotherapy [2]. Our report further analyzed the specific prognostic significance of 25-(OH)D in ENKTL, identified applicable subgroups, and evaluated whether it could improve the prognostic effect of the existing prognostic scoring system.

Ethics
The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. The measurement for serum 25-(OH)D level and other clinical and laboratorial checkups were performed according to the principles of the Declaration of Helsinki. All subjects involved in this study provided Informed consent and permissions.

Data collection
Basic characteristics such as age, gender, B-symptoms, Ann Arbor stage, Eastern Cooperative Oncology Group performance status (ECOG PS), EBV-DNA levels (EBV-DNA copy number in whole blood with the cutoff value of 5000 copies/ ml), primary lesion, PINK-E, and treatment regimens were collected from medical records. Additionally, laboratorial data such as lactate dehydrogenase (LDH) and 25-(OH)D were available within 24 h of first admission from the hospital-based laboratory. The serum 25-(OH)D was measured via electrochemiluminescence immunoassay (Rochee 170, Roche Co., Ltd., Shanghai, China). Vitamin D deficiency is defined as a 25-(OH)D below 50 nmol/L (20 ng/ml) [17].

Statistical analysis
Progression-free survival (PFS) was defined as the period from the date of diagnosis to the date of first-line treatment. Overall survival (OS) was measured from the date of diagnosis to the date of death or the date of final follow-up. Data were analyzed by IBM SPSS statistical software (version 21.0, IBM Inc, NY, USA). The figures of the Kaplan-Meier survival curves were modified using GraphPad Prism (version 6.0, GraphPad Software Inc, CA, USA). Clinicopathologic characteristics of enrolled patients were analyzed using descriptive analysis. Survival time was estimated using Kaplan-Meier survival curves and compared with log-rank tests. Univariate and multivariate regression analyses were performed to determine independent risk factors for PFS and OS. Receiver operator characteristic (ROC) curves were plotted, and corresponding areas under the curves (AUC) were calculated to estimate the accuracy of PINK-E and 25-(OH)D deficiency in ENKTL risk-stratification. P value < 0.05 was considered statistically significant.
The results of univariate and multivariate Cox proportional hazards regression analyses were summarized in Tables 2 and 3. In the univariate analysis, B symptoms, inferior ECOG PS (> 1), elevated LDH, advanced stage (III-IV), extranasal primary lesion, PINK-E score ≥ 3, and 25-(OH)D deficiency were significantly associated with both inferior PFS and OS in the univariate analysis. EBV-DNA positive was only associated with inferior PFS and age > 60 was only associated with inferior OS (Table 2). PINK-E score and 25-(OH)D

Subgroup analysis of 25-(OH)D deficiency in ENKTL
In the subgroup analysis, we separated the patients into two groups according to age, ECOG PS, Ann Arbor stage, and PINK-E score. We found that patients with 25- (   superior prognostic significance than PINK-E alone (P = 0.020 for PFS, P = 0.040 for OS) (Fig. 4).

Discussion
Vitamin D deficiency is an extremely universal phenomenon whether in low, lower-middle, and high-income countries, which is a public health priority [18,19]. The prevalence of serum 25(OH)D < 50 nmol/L is between 24.0-40.4% [20][21][22]. There is no single cause for this condition, low ultraviolet B (UVB) availability and/or exposure, low dietary vitamin D supply, and personal characteristics, such as skin pigmentation, age, and working environment are all of responsibility [18]. Vitamin D deficiency is also common in lymphoma patients. Previous studies have shown that when the threshold was set at 50 nmol/L, the vitamin D deficiency rate ranged from 67 to 88% [11,23], which mainly among B-cell lymphoma. And the prognostic ability of 25-(OH)D in these B-cell tumors such as mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL) has also been demonstrated [11][12][13][14][15]24]. Until now, few studies have reported the relationship between vitamin D deficiency and the prognosis of T cell lymphoma [14,16], and only one study included ENKTL patients, in which 38.6% of the ENKTL patients had below 10 ng/mL (25 nmol/L) of 25-(OH)D at diagnosis [16]. The study demonstrated that 25-(OH)D did have prognostic significance in ENKTL; however, this report did not further explore whether 25-(OH)D could improve the prognostic effect of PINK-E and figure out the applicable subgroups; our report further analyzed the specific prognostic significance of 25-(OH)D in ENKTL. 25-(OH)D is converted to its active form in several body tissues, 1,25(OH) 2 D, which functions as the ligand for vitamin D receptor (VDR). VDR is an essential mediator for the cellular effects of vitamin D; it has been shown to be present in not only the intestine, bone, and kidney, but also the skin, brain, reproductive organs, and certain cells of the immune system. As a nuclear transcription factor, it regulates the  expression of multiple genes, including some responsible for cell cycle regulation, proliferation, differentiation, and apoptosis in cancer cell [3,25,26]. When it comes to lymphoma, different studies have proposed different mechanisms by which vitamin D affects the prognosis of lymphoma patients. For example, vitamin D deficiency leads to inferior outcomes by impairing rituximab-mediated cellular cytotoxicity in patients with DLBCL [27], 1α,25-dihydroxyvitamin enhances the sensitivity of mantle cell lymphoma cells to lenalidomide by pro-apoptotic protein BCL2-interacting killer in mantle cell lymphoma patients [28], and emerging evidence suggests that calcitriol might upregulate the expression of programmed tissue death-ligand 1 [29]. Nevertheless, a recent report suggested that when treated with non-rituximab-containing regimens in FL, vitamin D deficiency is also a significant prognostic indicator [15], and vitamin D deficiency has also been shown to be broadly predictive of inferior outcomes in T cell lymphoma, CLL, or even solid malignancies [3,4,13,16,25]. So vitamin D may affect the prognosis of lymphoma in multiple mechanisms, and further investigation is needed to explore the specific mechanism of how vitamin D affects prognosis in different types of lymphoma.
Considering the realistic effect of vitamin D, several prospective studies have explored whether vitamin D supplementation could improve therapeutic effect and prognosis, and the results show that it does affect tumor cell growth in both Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL) [30][31][32]. No similar studies have been reported in T cell lymphoma. The best schedule for vitamin D replacement in deficient patients remains unclear. One study suggested that vitamin D supplementation should be divided into loading phase (50,000 International Units weekly) and maintenance phase (50,000 International Units monthly), in which 97% of patients met the target value during loading phase [33].
Our results suggested that the vitamin D deficiency was an independent inferior prognostic factor for both PFS and OS in patients with ENKTL. In subgroup analyses, we concluded that the vitamin D deficiency was a significant risk predictor especially in patients with age ≤ 60, ECOG PS ≤1, EBV-DNA positive, stage III-IV, and PINK-E score ≤ 2. Additionally, we demonstrated that adding 25-(OH)D deficiency to PINK-E score system indeed has a superior prognostic significance than PINK-E alone. As a retrospective cohort study, our research has limited number of subjects and many confounding variables that might affect the study indicators are beyond our complete control. In the future, larger scale and multicenter prospective studies are required to clarify the prognostic value of vitamin D deficiency in ENKTL.
In conclusion, our study indicated that 25-(OH)D deficiency was associated with inferior survival outcome of ENKTL patients. PINK-E plus 25-(OH)D deficiency could indeed improve the prognostic effect of the existing prognostic scoring system. Since supplementing vitamin D is a relatively easy task with minimal side effects, we hope that more research will emerge to support this approach in the future.