Serum levels of single-carbon metabolism vitamins and homocysteine in head-and-neck squamous cell carcinoma: Preliminary report.

Background: Head-and-neck carcinomas are a heterogeneous group of malignancies arising from the upper aerodigestive tract. Tobacco and alcohol are the leading etiological factors; however, bioactive food components, including those that modulate DNA methylation, are being linked to susceptibility. This work assesses the distribution of head-and-neck cancers presenting at a tertiary health institution and determined the serum level of the vitamins and an amino acid involved in the methionine cycle, in view of increasing acceptance of the significant role of DNA methylation in the pathogenesis of cancers. Patients and
Methods: This study involved 30 newly diagnosed cases of head-and-neck squamous cell carcinoma. Thirty apparently healthy volunteers served as controls. The test cases were made up of 19 males and 11 females while controls were made up of 14 males and 16 females. The median ages of the test cases and controls were 59 and 63 years, respectively. Sera obtained from participants' blood were analyzed by high-performance liquid chromatography technique. The study protocol was approved by the joint University of Ibadan/University College Hospital Institution Review Board.
Results: There is a male dominance in the number of cases at male-to-female ratio of 1.7: 1. The oral cavity was the most-affected site. Serum levels of Vitamin B2, B6, B12, and homocysteine were lower in cases compared with controls but not significantly so. However, serum Vitamin A and folic acid levels were significantly lower among the cases ([0.62 vs. 0.71, z = -2.50, P = 0.02], [26.05 vs. 30.82, z = 0.20, P = 0.00]) compared with controls. Only tobacco and alcohol use showed a significant association with head-and-neck cancer, but not family history of cancer or alcohol use alone (P = 0.00).
Conclusion: Significantly low serum Vitamin A and hypofolataemia are associated with head-and-neck squamous cell carcinoma. This is suggestive of a role for these vitamins in the etiopathogenesis of the disease.

INTRODUCTION

Head-and-neck cancers are malignancies affecting the upper aerodigestive tract, the majority of which are head-and-neck squamous cell carcinomas (HNSCC). This disease in the various sites has unique sets of epidemiologic, pathologic, and treatment considerations.[1] Although improved treatment modalities have resulted in improvement in the 5-year survival rate in the last decades, this improved outlook is, however, marginal and dependent on histology and degree of differentiation, clinical staging, tumor site, age of the patient, and comorbidities.[2]

Globally, tobacco and alcohol are the leading etiological factors;[1] others are genetic predisposition, deficiencies in nutritional elements, and involvement of certain viruses.[13] The leading causes of this cancer, however, appear to be less frequently involved in the cases seen in this environment,[345] suggesting the possibility of the involvement of other less globally recognized factor. Results from recent studies also suggest that there could be a low prevalence of human papillomavirus-related HNSCC among the adult population in Nigeria.[6]

Diet in the recent past has been identified as one of the most important modifiable determinants of cancer risk with a number of bioactive components including vitamins and trace elements, protective at different stages of cancer,[7] hence the concept of chemoprevention. Chemoprevention, however, requires careful approach and considerations of such factors as tumor site and type, stage, and type and dose of agent to be employed.[89] In the head-and-neck region, however, high-dose retinoids have been shown to be active against oral premalignant lesions and in the prevention of second primary tumors (SPTs), in the head-and-neck region.[1011]

The current work focuses attention on vitamins involved in the methionine cycle, and homocysteine, a sulfur-containing amino acid, which is an intermediate metabolite of methionine metabolism. This is because of the roles they play in DNA methylation; an epigenetic phenomenon with an increasing relevance in the pathomechanisms of several cancer developments including HNSCC.[12] Furthermore, an increased serum level of homocysteine is associated with malignancies including colorectal cancer[13] and uterine cervical cancer.[14] Elevated homocysteine plasma levels have also been associated with chromosome damage even in the absence of folate deficiency.

PATIENTS AND METHODS

This was a prospective cross-sectional study conducted between 2015 and 2018. It involved 30 newly diagnosed cases of head-and-neck squamous cell carcinoma. The test cases aged between 19 years and 92 years were made up of 19 males and 11 females. Thirty apparently healthy individuals aged between 21 years and 76 years, made up of 14 males and 16 females, served as controls. The median age of the cases and controls were, however, 59 (standard deviation – 19.1) and 63 (standard deviation – 7.5) years, respectively [Table 1]. The study was explained to both cases and controls and informed consent was obtained before the commencement of sample collection. Five milliliter of blood was taken from the antecubital fossa of cases of HNSCC and allowed to clot and retract. Sera were then obtained from the sample. The same was done in the control group. The sera were stored at −20°C until the time of analysis. Analysis was done by high-performance liquid chromatography (HPLC) technique. The study protocol was approved by the UI/UCH joint Institution Review Board.

Table 1

Frequency/Percentages

Characteristics	Cases	Control
Gender
Male	19 (63.3)	20 (66.7)
Female	11 (36.7)	10 (33.3)
Age
<40	9 (30.0)	1 (3.3)
40-49	4 (13.3)	2 (6.7)
50-59	5 (16.7)	3 (10)
59-69	5 (16.7)	16 (53.3)
≥70	7 (23.3)	8 (26.7)
Total	30 (100)	30 (100)

Procedure for the determination of serum homocysteine

Total plasma homocysteine was determined using HPLC according to the method of Cornwell et al.[15]

Procedure for the determination of serum vitamins

Serum levels of Vitamin B2, B6, B12, and folic levels were determined using HPLC method for the determination of B Vitamins by Rada Amidizic et al.[16] The column effluents were monitored at 290 nm for B2, B6, and folic acid and at 550 nm for B12.

Statistical analysis

Data entry was done with Microsoft Excel version 2007 and analysis with STATA version 12. Participants' personal variables were presented as frequency tables. Mann–Whitney U-test, a nonparametric equivalent of the independent t-test, was used to compare values of serum indices between cases and controls. The level of significance was set at P ≤ 0.05.

RESULTS

There is a male dominance in the pattern of presentation at male-to-female ratio of 1.7:1. The oral cavity was the most-affected site (tongue, palate, and floor) with 6 (20%) cases [Table 2]. Serum levels of Vitamin B2, B6, B12, and homocysteine were lower in test cases but not significantly different between the study group and controls [Table 3]. However, median serum Vitamin A and folic acid levels were significantly lower among the cases ([0.62 vs. 0.71, z = −2.50, P = 0.02], [26.05 vs. 30.82, z = 0.20, P = 0.00]). Only tobacco and alcohol use showed significant association with head-and-neck cancer but not family history of cancer or alcohol use alone (P = 0.00) [Table 4].

Table 2

Site and frequency distribution of tumours

Site of tumour	Frequency	Percentage
Antrum	4	13.3
Oral cavity	6	20
(Tongue, Floor of the mouth	1	3.3
Lip, Palate)	5	16.7
Larynx	1	3.3
Mandible	5	16.7
Nasal cavity	1	3.3
Nasopharynx	1	3.3
Orbital mass	5	16.7
Infra-auricular area	1	3.3
Neck (NOS)	30	100
Vocal cord
Total

*NOS: Not otherwise specified (squamous cell carcinoma)

Table 3

Comparisons of Vitamins and Homocysteine between Cases and Control

Micronutrient	Cases	Control	t	P
Vitamin A	0.62	0.71	-2.5	0.02*
Vitamin B2	6.31	6.65	-6	0.49
Vitamin B6	27.42	28.62	-7.5	0.64
Vitamin B12	390.48	420.69	0.75	0.64
Folic acid	26.05	30.82	0.29	0.00*
Homocysteine	7.84	8.44	0.75	0.61

Table 4

Comparisons between Risk Factors in Cases and Control

Variable	Test n (%)	Control n (%)	Total n (%)	t χ2	P
Family history of cancer
None	27 (91.0)	26 (86.7)	53 (88.0)	2.66	0.80
Parents	3 (0.9)	0 (0.0)	3 (4.0)
Sibling	0 (0.0)	2 (6.7)	2 (4.0)
Children	0 (0.0)	2 (6.7)	2 (4.0)
Total n (%)	30(100)	30(100)	60 (100)
Tobacco-alcohol use
Yes	19 (64.0)	2 (6.7)	21 (28.8)		0.00*
No	11 (36.0)	28 (93.3)	39 (72.0)
Total n (%)	30(100)	30(100)	60 (100)
Alcohol intake
Yes	16 (55.0)	18 (60.0)	34 (56.0)	0.24	0.7
No	14 (45.0)	12 (40.0)	26 (44.0)
Total n (%)	30(100)	30(100)	60 (100)

DISCUSSION

DNA methylation is a noncovalent addition of a methyl group to the DNA with consequent alteration in gene expression without an alteration in the base arrangement.[712] Several nutritional components can modulate DNA methylation and cancer susceptibility.[7] The one-carbon metabolism pathway influences the supply of donor methyl groups and consequently the biochemical pathways of methylation processes [Figure 1]. Many of these nutrients have been associated with cancer susceptibility and considered important in the etiopathogenesis of malignancies.[171819] Furthermore, studies on global DNA methylation, nutritional factors, and cancer suggest that patterns of methylation and level of these vitamins may vary depending on the type of cancer.[20] Breast cancer has been reported with global hypomethylation and decreased Vitamin B12 and Vitamin C levels but with elevated folate concentrations.[20] Dietary components, such as Vitamin B12, selenium, and a risk factor (alcohol), may modify the response to inadequate dietary folate; hence, looking at individual nutrients rather than the global picture may be rather simplistic.[720]

Figure 1

DNA methylation role of vitamins

Significantly lower serum folic acid was reported in this study compared with control. This had been previously reported in patients with HNSCC[2122] and associated with an overall increased risk in cancer.[22] Existing results suggest that a role for low serum folate levels as a risk factor for head-and-neck multistep carcinogenesis is plausible, also in consideration of the function of folate in DNA synthesis methylation and repair.[2223] The lower serum folate level in this study supports the rationale for folate as a chemopreventive agent in patients with premalignant lesions and in those with treated HNSCC who are at risk of developing SPTs.[910]

Epidemiologic observations support the concept of a synergistic effect between alcohol consumption and low folate intake in carcinogenesis.[24] While such a relationship is biologically plausible, alcohol alone does not show significant association with HNSCC in this study, the combination of tobacco and alcohol, however, did.

Although a lower level of the B Vitamins has been reported in head-and-neck cancers,[25] the lower serum level when compared with control was not found to be statistically significant in this study. The possible involvement of the B Vitamins in carcinogenesis is largely due to the role played by these vitamins in DNA methylation. Furthermore, noteworthy is the fact that intracellular Vitamin B12 deficiency is reportedly associated with chromosomal damage.[2226]

Vitamin A (retinyl palmitate) deficiency was implicated as a possible cause of head-and-neck cancer when low serum levels were found twice as often in patients with Stages III and IV disease than in healthy individuals.[27] Lower levels of Vitamin A were also found in head-and-neck cancer patients with SPTs compared with those without SPTs, suggesting a possible role in their etiologies[27] and the inclusion in chemopreventive strategies in HNSCC management. The exact mechanism of Vitamin A and its derivatives in potential HNSCC prevention is not fully understood; however, it is thought that these agents restore expression of retinoic acid receptor-beta mRNA, which may promote normal tissue growth and differentiation.[2728] Alcohol intake is associated with an increased risk of a variety of cancers including HNSCC. More specifically, it acts as a competitive inhibitor of Vitamin A oxidation to retinoic acid involving alcohol dehydrogenases and acetaldehyde dehydrogenases.[29] There is also alcohol-induced cytochrome P450 enzyme (CYP), particularly CYP2E1 and the enhanced catabolism of Vitamin A and retinoic acid by alcohol.[29] As a consequence, long-term and excessive alcohol intake results in impaired status of retinoic acid, the most active derivative of Vitamin A. Although the mean serum level of Vitamin A in this study was significantly lower in the test relative to control (P = 0.02), the role of alcohol could not be ascertained as alcohol alone showed no association with HNSCC. A larger cohort is needed for better understanding of the alcohol–retinoid interaction and the mechanisms involved.

CONCLUSION

Statistically significant low levels of Vitamin A and folic acid were found in the study group compared with control. Differences in serum levels of these vitamins might arise from tumor development and consequent metabolic alterations or might precede and promote tumor progression. A follow-up longitudinal study with serial blood sampling and a larger sample size is desirable.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.