Serum levels of 25‐hydroxy‐vitamin D in patients with oral squamous cell carcinoma: Making a case for chemoprevention

Abstract Objectives Serum level of vitamin D has been used as a predictor for cancer development. We intend to measure the baseline vitamin D level in patients with oral squamous cell carcinoma (OSCC) and to compare same with non‐cancer controls to determine any association. Materials and methods Patients with OSCC presenting to our clinics were included in this study. Their baseline serum vitamin D levels were measured prior to cancer treatment after obtaining their consents. These patients were then matched with at least 2 cancer‐free subjects to serve as controls and whose serum vitamin D levels were also measured. The serum vitamin D levels obtained for the two groups were then categorized into normal (>35 ng/ml), mild deficiency (25–35 ng/ml), moderate deficiency (12.5–25 ng/ml), and severe deficiency (<12.5 ng/ml). The data were analyzed statistically and the two groups compared. Results A total of 51 patients with OSCC (Male 22 [43%] and female 29 [57%]) and 113 cancer‐free controls (Male 36 [31.86%] and female 77 [68.14%]) were included in the study. The commonest site for OSCC was the tongue, accounting for 45% of the cancer cases. Mean age for cancer patients was 59.33 years ±12.54 and 49.24 years ±15.79 for the control. Among the OSCC patients, 74.51% had moderate to severe vitamin D deficiencies, whereas only 20.35% had a moderate deficiency in the control group with no severe deficiency. Conclusion Logistic regression analysis shows a positive association between vitamin D deficiency and OSCC risk especially in levels below 25 ng/ml. This further corroborates the assertion that vitamin D deficiency may be a useful indicator of OSCC. It may, therefore, be necessary to routinely prescribe vitamin D supplements to subjects with moderate to severe deficiencies in order to decrease the chances of OSCC development.

These extrinsic factors and a host of intrinsic factors lead to complex molecular and cellular changes in a multistep process that eventually evolves to oral cancer (Choudhari, Chaudhary, Gadbail, Sharma, & Tekade, 2014). In this complex process, a study (Sinha, Mukhopadhyay, Das, Panda, & Bhutia, 2013) identified resistance to apoptosis (programmed cell death) by a group of cells known as cancer stem cells as an important factor. This, they claim, confers a "protective autophagy" on these cancer cells and therefore prolong their lifespans. This is believed to be responsible for the cancer metastasis, recurrence, and resistance to both chemotherapy and radiotherapy (Grimm et al., 2015;Sinha et al., 2013). Therefore, treatment options that will promote apoptosis will indeed help in the management of OSCC (Grimm et al., 2015).
The active form of vitamin D (1-alpha, 25-dihydroxycholecalciferol) (VD) has been shown through several studies to affect normal and cancerous cells by enhancing anti-proliferation and pro-apoptotic factors, as well as inhibition of cell-cycle promoters and growth factor signaling pathways (Grimm et al., 2015;Grimm, Alexander, Munz, Hoffmann, & Reinert, 2013;Osafi et al., 2014 andRussel, Rassnick, Erb, Vaughan, &McDonough, 2010). This means that it is pro-apoptotic and is a useful anti-neoplastic agent in the management of several malignancies including those of the head and neck region (Grimm et al., 2015;Osafi et al., 2014 andRussel et al., 2010). Serum level of vitamin D has also been used as a predictor for cancer development (Grimm et al., 2015; Tuohimaa & Lou, 2012).
We, therefore, aim to measure the serum vitamin D levels of OSCC patients visiting our Maxillofacial Centers and comparing it with those in normal healthy OSCC-free patients to determine any associations. This may further elucidate the significance or importance of vitamin D in the etiology and prevention of OSCC.

| MATERIALS AND METHODS
The ethical review committees of King Fahad Medical City (KFMC), Riyadh, and College of Dentistry and King Khalid University (KKU), Abha, approved this study. Consecutive patients attending these hospitals and who were histologically diagnosed with OSCC were included in this study. Demographic data were recorded for these patients and additional information regarding tumor site, duration, and treatment received if any were also documented. Their baseline serum vitamin D levels were measured prior to the cancer treatment after obtaining their consents. These patients were then matched with at least two cancer-free subjects to serve as controls and whose serum vitamin D levels were also measured. Patients who have commenced any form of cancer treatment were excluded from this study. In addition, patients and controls that are on any form of vitamin D supplements were also excluded.
Chi-square test of association was perfumed to assess the association between the two groups. Simple logistics regression analysis was applied to see the association between the levels of Vitamin D in cancer cases and controls. Statistical significance was set at 5% level of significance (p < .05).

| RESULTS
A total of 51 patients with OSCC and 113 cancer-free controls were included in the study. Among the cancer patients, 22 (43%) were male and 29 (57%) female; whereas 36 (31.9%) were male and 77 (68.1%) female among the cancer-free controls. The commonest site for OSCC was the tongue, accounting for 45% of the cancer cases (Table 1).
Mean age for cancer patients was 59.33 years ±12.54, whereas it was 49.24 years ±15.79 for the control. Table 2 gives a full analysis of age group analysis and comparison between the OSCC cases and controls.
Only five patients were below the age of 40 years for the cancer group. Among the OSCC patients, 74.51% had moderate to severe vitamin D deficiencies, whereas only 20.35% had a moderate deficiency in the control group with none severely deficient (Table 3).
Logistic regression analysis for moderate and severe deficiency gave an odds ratio (OR) of 1.65 with a confidence interval (CI) of 0.98-2.77 (Table 4). Furthermore, independent t-test comparing the 2 groups in relation to vitamin D deficiency, revealed a mean of 20.42 ± 12.02 for the cancer group and 34.99 ± 12.38 for the control group with a p-value of .001.

| DISCUSSION
The chemopreventive and therapeutic roles of vitamin D in a wide range of human cancers have been suggested in the literature (Bikle, Oda, & Teichert, 2011;Kamradt et al., 2003). Indeed, many of these malignant lesions including OSCC have been shown to express the receptor for 1,25 Dihydroxyvitamin D 3 ; also known as VDR (vitamin D receptor) (Adisa et al., 2017;Grimm et al., 2013 and2015;Osafi et al., 2014 andRussel et al., 2010). The suggested mechanism of action may be through its effect on p53-mediated DNA damage in cases of skin squamous cell carcinoma (Reichrath & Reichrath, 2013). As for OSCC, Osafi et al. (2014) via an in vitro study opined that the activation of two apoptotic pathways (caspase and bcl:bax) by vitamin D may be responsible for its anti-OSCC effects. The importance of vitamin D in cancer management has been