Serum levels of folate, homocysteine, and vitamin B12 in head and neck squamous cell carcinoma and in laryngeal leukoplakia

Abstract

BACKGROUND

The authors evaluated serum levels of folate, homocysteine, and vitamin B₁₂ in patients with head and neck squamous cell carcinoma (HNSCC) and in patients with laryngeal leukoplakia, a well known preneoplastic lesion.

METHODS

One hundred forty-four consecutive, untreated patients with HNSCC and 40 consecutive, untreated patients with laryngeal leukoplakia were enrolled in the Department of Otolaryngology at the authors' institution. Data from those patients were compared with data from one control group, which included 90 smokers, and from another control group, which included 120 nonsmokers. Serum levels of homocysteine, folate, and vitamin B₁₂ were measured by an automated immunoassay method based on fluorescence polarization immunoassay technology.

RESULTS

Comparing groups by Student–Newman–Keuls test, serum folate levels were significantly lower in patients with HNSCC and in patients with laryngeal leukoplakia compared with serum folate levels in both the smoker control group and the nonsmoker control group. Serum homocysteine levels in patients with HNSCC were significantly higher compared with homocysteine levels both in the smoker and nonsmoker control groups and in patients with laryngeal leukoplakia. There were no statistically significant differences between groups in serum vitamin B₁₂ levels.

CONCLUSIONS

A role for folate deficiency as a risk factor in head and neck carcinogenesis is plausible. A chemoprevention protocol with folate is both feasible and ethically correct and is in progress at the authors' institution. Homocysteine levels in patients with HNSCC probably are affected largely by the HNSCC phenotype. An accumulation of homocysteine may reveal a genetic defect, which, theoretically, may be a target for pharmacologic therapy, for example, with antifolic drugs. Cancer 2005. © 2004 American Cancer Society.

Head and neck squamous cell carcinoma (HNSCC), as defined herein, includes the common squamous cell carcinomas of the oral cavity, pharynx, and larynx. It is estimated that approximately 42,800 patients were diagnosed with head and neck carcinoma in the United States in 1992, with 11,600 deaths.1–3

The standard therapeutic approach, focused on surgery, irradiation, and chemotherapy, either alone or in combination, has been modified in part over the last 20 years; however, the overall survival of HNSCC patients has not improved substantially. Efforts toward early detection and prevention have not been entirely successful. For patients with early-stage carcinomas, who have a high disease-specific survival rate, second primary tumors represent the first cause of death.1, 4, 5 Conversely, patients with advanced head and neck carcinomas have a high risk of primary treatment failure and death.

To characterize and, thus, to identify high-risk mucosal areas and preclinical tumors, molecular abnormalities in head and neck carcinogenesis have been studied extensively.6–8 Because a genetic predisposition to the development of HNSCC is highly probable, in recent years, a number of genetic polymorphisms have been evaluated in relation to the risk of developing malignancies of the aerodigestive tract.9–11 Metabolic aspects in head and carcinogenesis have been studied less extensively. Nevertheless, we know that metabolic alterations, often aspecific, frequently are associated with carcinoma. These alterations may be secondary, or they may precede tumor development and may favor progression.

In the current work, we focused our attention on compounds involved in the so-called methionine cycle, which leads to the production of S-adenosylmethionine, the body's universal methyl donor. Folate, which is a water-soluble B vitamin that is plentiful in fresh vegetables and fruits, is present under a number of coenzymatic forms, whose main biochemical function in mammalian systems is to mediate the transfer of one-carbon units at different states of oxidation. It is fundamental in the synthesis of serine from glycine, in the synthesis of purine and pirimidine bases, and as a methyl donor to create methylcobalamin, which is used for the remethylation of homocysteine to methionine (Fig. 1).12 Folate deficiency, which almost always is secondary to insufficient dietary intake, has been reported as the most common vitamin deficiency in the United States, affecting up to 10% of the general adult population.13 Homocysteine, which is a sulphur-containing amino acid, is an intermediate metabolite of methionine metabolism that is located at an important metabolic crossroad (Fig. 1), and its serum levels potentially may be affected by a large and nonspecific range of metabolic alterations.12 Folate deficiency often is associated with an elevation of homocysteine, and folate supplementation reduces hyperhomocysteinemia.14 Vitamin B₁₂ (or cobalamin) is a coenzyme of methylmalonil coenzyme A mutase and of methionine synthase (Fig. 1).12

Figure 1. The methionine cycle and its metabolic links with nucleotide synthesis and DNA methylation pathways. THF: tetrahydrofolic acid; MTHFR: methylenetetrahydrofolate reductase; MS: methionine synthase; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine.

Alterations in methionine cycle have been reported in several human malignancies. Maintenance of adequate folate status from dietary sources and/or by synthetic folic acid supplementation has been associated with a protective effect and reduced incidence of a variety of human malignancies.15, 16 In a large, perspective study, it was shown that high dietary folate intake protects heavy smokers from developing lung squamous cell carcinoma.17 Furthermore, it has been reported that folate and vitamin B₁₂ supplementation induced the regression of bronchial squamous metaplasia.18, 19 An inverse correlation between folate intake and the risk of pancreatic and breast carcinoma also was reported.20, 21 A recent animal study evidenced a strong protective effect of folate in beagles that were treated with a gastric carcinogen.22 A role for folate deficiency, as postulated first in the 1960s,23 and for other alterations of methionine cycle metabolites (homocysteine, vitamin B₁₂) as a risk factor for cervical carcinoma never was demonstrated definitively.24–27 Colorectal carcinogenesis was studied extensively in relation to folate metabolism, and several epidemiologic studies showed an increased risk associated to folate deficiency. Patients affected by colorectal carcinoma seem to have lower dietary folate intake,28, 29 higher homocysteine levels, and lower folate serum levels.30 A role of folate deficiency in colorectal carcinogenesis also was confirmed in animal studies.31, 32 Folate currently is considered one of the most promising chemopreventive agents for colorectal carcinogenesis.33 Plasma homocysteine levels also are increased in patients with hematologic tumors and, in particular, in children with acute lymphoblastic leukemia.34 In patients with ovarian carcinoma, increased homocysteine ascitic and cystic concentrations may derive from a biochemical defect of the methionine cycle in tumor cells,35 as determined, for example, by an alteration in the 5,10-methylenetetrahydrofolate reductase gene.36

On a molecular level, several cellular effects have been described that may account for the involvement of these metabolites in carcinogenesis. Methionine cycle disruptions, by reducing intracellular S-adenosylmethionine (SAM), can alter cytosine methylation in DNA (Fig. 1), leading to inappropriate activation of protooncogenes, repression of tumor suppressor genes, and induction of malignant transformation. Alterations in DNA methylation and, in particular, a global hypomethylation and a regional hypermethylation, especially of promoters of tumor suppressor genes, have been described in human tumors.37 In head and neck carcinoma, promoter hypermethylation of key genes in critical pathways, such as INK4A, is common and also has been described recently.38 Alternatively, abnormal DNA metabolism and a variety of cytogenetic lesions have been associated with folate deficiency in laboratory models as well as in human folate deficiency.39, 40 Normal levels of the precursor nucleotides (2′deoxynucleotide-S′ triphosphates [dNTPs] and nucleotide-S′ triphosphates [NTPs]) for DNA/RNA synthesis are dependent directly on intracellular folate availability,41 and it seems that dNTP pool imbalance is of great relevance for the carcinogenic effects of folate deficiency.42 A specific and important biochemical alteration, secondary to a defect in deoxythymidilate (dNTP) synthesis (Fig. 1), is uracil misincorporation in DNA, which may be sufficient to determine double strand breaks, mutations, and chromosomal aberrations.43, 44 Overall, dNTP pool imbalance reduces the efficiency both of DNA-synthesizing enzymes, with an increase in the background mutation rate, and of DNA-repairing enzymes, with an enhancement of mutagen-induced carcinogenesis.15, 16, 45 Convincing evidence from clinical studies indicates that moderate folate deficiency independently would not be mutagenic in vivo but probably interacts with other risk factors in promoting tumor progression. In fact, studies about lung and cervical carcinogenesis suggest that folate deficiency enhances an underlying predisposition due to environmental factors, as heavy cigarette smoking and human Papillomavirus infection.16, 46, 47 Conversely, hypofolatemia also can interact with genetic factors. Homozygous mutant individuals for a common methylenetetrahydrofolate reductase (MTHFR) gene polymorphism (677 C→T; Ala→Val) reportedly have a reduced risk of colorectal carcinoma49 and of adult acute lymphocytic leukemia,50 probably because the normal, more efficient enzyme reduces the amount of folate available for pathways involved in DNA synthesis and repair (see Fig. 1). Notably, in patients with low systemic folate status, the protective effect for colon carcinogenesis of the mutant TT genotype is lost anyway.

In head and neck carcinogenesis, nutrition may play an important role, and an inverse association between the consumption of fruits and vegetables and the incidence of HNSCC has been reported.51 In a preliminary study, we previously evaluated the concentrations of folate and homocysteine in the serum of patients with HNSCC, with statistically significant findings.52 In the current study, we evaluated folate, homocysteine, and vitamin B₁₂ serum levels in a larger series of patients with HNSCC in relation to disease site, local extension, and the neck status. We also collected data about these methionine cycle metabolites in patients with laryngeal leukoplakia, a well known preneoplastic lesion, to assess whether such alterations are early or should be considered a late consequence of tumor progression.

MATERIALS AND METHODS

Patients

One hundred forty-four consecutive, untreated patients suffering from primary HNSCC (Table 1) and 40 consecutive, untreated patients with laryngeal leukoplakia were enrolled in our Department of Otolaryngology after obtaining their informed consent. Because most of the patients with HNSCC (89.5%) and most patients with leukoplakia (92.5%) were smokers, and because cigarette smoking can determine alterations in homocysteine, vitamin B₁₂, and folate status and may be a confounding factor,53 we compared the results from patients with HNSCC and patients with laryngeal leukoplakia with the results from an age-matched and gender-matched control group of 90 smokers and with the results from another age-matched and gender-matched control group of 120 nonsmokers (Table 2). All participants in our smoker group were current smokers, and all participants in our non-smoker group did not ever smoke; among our patient groups, we also considered smokers who had quit smoking not more than 5 years before their diagnosis of malignancy. Patients with HNSCC were divided according to disease progression in patients with early-stage disease (T1–T2N0; n = 74 patients), locally advanced disease (T3–T4N0; n = 40 patients), regionally advanced disease (n+; n = 30 patients) (Table 1). Control participants were from the same geographic area as the patients. Both control participants and patients were enrolled after obtaining informed consent to the use of part of their blood samples for an experimental study. We excluded from our study all individuals who had an estimated habitual alcohol consumption > 35 g of alcohol, or > 4 glasses of alcoholic beverages, per day, because it is well known and it has been described clearly that heavy drinking can alter folate absorption and metabolism and, at the same time, is a risk factor for head and neck carcinoma and, thus, may have been a relevant confounding factor in our study. Conversely, there is evidence that low-to-moderate alcohol consumption does not determine any change in serum levels of folate or homocysteine.54 No participants who were included in the study had received folate or vitamin B₁₂ supplements in the last 6 months before the study. In addition, both the patients and the controls had normal renal function. Overall nutritional status may be the primary determinant of folate and vitamin B₁₂ levels. We excluded from our study any participant with clinically evident nutritional deficiencies. Characteristics of the patients and controls are shown in Table 2.

Table 1. Patients with Head and Neck Squamous Cell Carcinoma: Disease Site and Stage

Disease site	No. of patients
	Early-stage disease (T1–T2,N0)	Locally advanced disease (T3–T4,N0)	Regionally advanced disease (any T,N+)	Total
Oral	20	9	9	38
Laryngeal, glottic	46	5	1	52
Laryngeal, supraglottic	5	2	3	10
Laryngeal, transglottic	—	15	4	19
Pharyngolaryngeal	—	4	5	9
Oropharyngeal	2	3	4	9
Nasopharyngeal	1	2	4	7
Total	74	40	30	144

Table 2. Characteristics of the Patient and Control Groups

Characteristic	No. of patients (%)		No. of controls (%)
	HNSCC	Laryngeal leucoplakia	Nonsmokers	Smokers
Total no.	144	40	120	90
Age (yrs)
Mean	64	56	55	58
Range	37–93	39–77	35–70	30–78
Gender (female/male)	7/29	1/8	1/3	11/34
Smokers	129 (89.5)	37 (92.5)	—	90 (100.0)
10–20 Cigarettes/day	38 (29.5)	8 (20.0)	—	25 (28.0)
> 20 Cigarettes/day	91 (60.0)	29 (72.5)	—	65 (72.0)
Smoking duration
< 20 Yrs	41 (32.0)	19 (47.5)	—	28 (31.0)
> 20 Yrs	88 (57.5)	18 (45.0)	—	62 (69.0)
Drinkers	115 (80.0)	27 (67.5)	40 (33.0)	63 (70.0)
≤ Glasses/day	65 (45.0)	17 (42.5%)	30 (25.0)	28 (31.0)
2–4 Glasses/day	50 (35.0)	10 (25.0)	10 (8.0)	35 (39.0)

Methods

Homocysteine was measured by a fully automated AxSYM method (Abbott, USA) according to the manufacturer's recommendations. The automatic method is based on the determination of S-adenosyl-L-homocysteine (SAH) obtained from the enzymatic conversion of homocysteine, previously reduced with dithiothreitol, to SAH by bovine SAH hydrolase. The Abbott AxSYM immunoassay is based on fluorescence polarization immunoassay technology. After the addition of mouse monoclonal SAH antibody to the sample, S-adenosyl-L-cysteine fluorescein tracer, which competes with SAH for antibody binding sites, is added. Homocysteine concentration is then quantified by the intensity of polarized fluorescent light. Serum B₁₂ was measured by microparticle enzyme immunoassay, and serum folate was measured by ion capture assay on an AxSYM Analyzer (Abbott Diagnostics, Abbott Park, IL).

Statistical Analysis

The α level was fixed at 0.05. Statistical analysis was performed using STATA 6.0 with an analysis of variance and the Student–Newman–Keuls test.

RESULTS

Results are reported in detail in Tables 3 and 4 and are represented schematically by box plots in Figure 2. In an analysis of variance using an F test for serum folate levels, the results between groups differed significantly (F = 120; P < 0.001). Comparing groups by Student–Newman–Keuls test, statistically significant differences resulted between patients with HNSCC and the smoker control group (q = 19.048; P < 0.05), between patients with HNSCC and the nonsmoker control group (q = 23.644; P < 0.05), between patients with laryngeal leukoplakia and the smoker control group (q = 11.59; P < 0.05), and between patients with laryngeal leukoplakia and the nonsmoker control group (q = 14.052; P < 0.05).

Table 3. Serum Levels of Folate, Homocysteine, and Vitamin B₁₂ in the Control Groups, in Patients with Head and Neck Squamous Cell Carcinoma, and in Patients with Laryngeal Leucoplakia

Group	Folate			Homocysteine			Vitamin B12
	Level (ng/mL)a	Q score	P value	Level (μmol/L)a	O score	P value	Level (pg/mL)a	Q score	P value
HNSCC: head and neck squamous cell carcinoma; NS: not significant. a Values shown are the mean ± standard deviation levels.
HNSCC group	4.87 ± 2.26	23.644	< 0.05	13.4 ± 10.2	7.681	< 0.05	429 ± 281	—	NS
Nonsmoker controls	9.7 ± 2.2			8.7 ± 3.9			480 ± 256
HNSCC group	4.87 ± 2.26	19.048	< 0.05	13.4 ± 10.2	6.464	< 0.05	429 ± 281	—	NS
Smoker controls	9.1 ± 2.7			9.1 ± 5.0			472 ± 225
Leucoplakia group	5.46 ± 2.12	14.052	< 0.05	8.45 ± 2.29	—	NS	373 ± 152	3.3	> 0.05 (NS)
Nonsmoker controls	9.7 ± 2.2			8.7 ± 3.9			480 ± 256
Leucoplakia group	5.46 ± 2.12	11.59	< 0.05	8.45 ± 2.29	0.691	> 0.05 (NS)	373 ± 152	—	NS
Smoker controls	9.1 ± 2.7			9.1 ± 5.0			472 ± 225
HNSCC group	4.87 ± 2.26	0.691	> 0.05 (NS)	13.4 ± 10.2	5.594	< 0.05	429 ± 281	—	NS
Leucoplakia group	5.46 ± 2.12			8.45 ± 2.29			373 ± 152

Table 4. Serum Levels of Folate, Homocysteine, and Vitamin B₁₂ in Patients with Head and Neck Squamous Cell Carcinoma According to Disease Stage

Disease stage	Folate			Homocysteine			Vitamin B12
	Level (ng/mL)a	Q score	P value	Level (μmol/L)a	Q score	P value	Level (pg/mL)a	Q score	P value
HNSCC: head and neck squamous cell carcinoma; NS: not significant. a Values shown are the mean ± standard deviation levels.
Early stage	4.96 ± 2.21	0.634	> 0.05 (NS)	12.9 ± 11.43	1.405	> 0.05 (NS)	479 ± 390	2.94	> 0.05 (NS)
Locally advanced	4.76 ± 2.44			15.33 ± 7.6			351 ± 195
Early stage	4.96 ± 2.21	—	NS	12.9 ± 11.43	—	NS	479 ± 390	—	NS
Regionally advanced	4.78 ± 2.2			12.9 ± 9.6			408 ± 211
Locally advanced	4.76 ± 2.44	—	NS	15.33 ± 7.6	—	NS	351 ± 195	—	NS
Regionally advanced	4.78 ± 2.2			12.9 ± 9.6			408 ± 211

Figure 2. In these box plots, serum levels of the 3 metabolites (folate, homocysteine, and vitamin B₁₂) in the patient groups and in the control groups are represented. (A) For serum folate levels, an analysis of variance (F test) showed that differences between groups were highly probable (F = 120; P < 0.001). (B) For serum homocysteine levels, an analysis of variance (F test) showed significant differences between groups (F = 13.22; P < 0.001). (C) For serum vitamin B₁₂ levels, an analysis of variance (F test) showed that differences between the groups were not significant (F = 2.4; P = 0.068).

In an analysis of variance using an F test for serum homocysteine, the results between groups also differed significantly (F = 13.22; P < 0.001). Comparing groups by Student–Newman–Keuls test, statistically significant differences resulted between patients with HNSCC and the smoker control group (q = 6.464; P < 0.05), between patients with HNSCC and the nonsmoker control group (q = 7.681; P < 0.05), and between patients with HNSCC and patients with laryngeal leukoplakia (q = 5.594; P < 0.05).

In an analysis of variance using an F test for serum levels of vitamin B₁₂, the results between groups did not differ significantly (F = 2.4; P = 0.068). Comparing groups by Student–Newman–Keuls test for vitamin B₁₂ serum levels, no statistically significant differences resulted between groups (Table 3).

We divided our patients according to disease progression in patients with early-stage disease (T1–T2N0), locally advanced disease (T3–T4N0), and regionally advanced disease (n+), evaluating and comparing serum levels of metabolites in each group (Table 4). In an analysis of variance with an F test for serum folate levels, the results between groups did not differ significantly (F = 0.13; P = 0.880), and no statistically significant differences were found between groups in a comparison by Student–Newman–Keuls test (Table 4). In addition, no differences were found when comparing serum B₁₂ and homocysteine levels among these groups of patients (Table 4). Serum levels of the three metabolites also did not differ among patients with SCC at various sites in the head and neck (Table 1) (data not shown).

DISCUSSION

Lower serum folate levels and higher serum homocysteine levels were observed in patients with HNSCC compared with the smoker and nonsmoker control groups, thus confirming our previous results.52 Patients with head and neck carcinomas in various sites at different stages substantially had the same folate levels (Table 4), which, thus, were not a marker of disease progression. Serum folate levels are low both in patients with leukoplakia and in patients with HNSCC, without a statistically significant difference (Table 3); thus, the serum folate level cannot be considered a diagnostic marker. Nevertheless, the current 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 and repair. Like what was postulated for other malignancies, in head and neck carcinogenesis, hypofolatemia probably does not have an independent role as an initiating factor. Instead, presumably, hypofolatemia acts synergistically with other genetic and environmental factors, such as tobacco carcinogens (in fact, almost all of our patients were smokers), making cells more susceptible to mutagens and increasing the rate of tumor progression (see above).

Homocysteine serum levels were higher in patients with HNSCC than in patients with leukoplakia, who did not differ significantly from the control groups; furthermore, serum homocysteine levels did not differ significantly among patients with HNSCC in different sites and at different stages (Table 4). These data exclude a role for a high homocysteine serum level both as a risk marker, because they are not altered in presence of preneoplastic lesions, and as a marker of neoplastic progression. Nevertheless, the evaluation of serum homocysteine does not seem useful for diagnostic purposes, because hyperhomocysteinemia is a quite frequent and nonspecific finding and because it would not be sensitive for detecting HNSCC. In fact, in patients with HNSCC, values of homocysteine in the serum are extremely heterogeneous, as also documented by the high standard deviation (10.2 μmol/L) compared with the other groups (Table 3). This suggests that, in patients with HNSCC, homocysteine levels do not depend exclusively on folate levels but probably are affected largely by the phenotype of HNSCCs, which are a very heterogeneous subset of tumors from a molecular point of view. An accumulation of homocysteine may reveal a genetic defect of the methionine cycle in tumor cells, as described previously in ovarian carcinoma.35 Such a metabolic defect, theoretically, offers a target for pharmacologic therapy, for example, with antifolic drugs, in a defined subset of patients with HNSCC.

The hypothesis of an involvement of vitamin B₁₂ in carcinogenesis has a scientific basis, as discussed above, and intracellular vitamin B¹² deficiency reportedly has been associated with chromosomal damage to buccal mucosal cells in smokers.55 Nevertheless, in the current study focused on head and neck carcinoma, differences in vitamin B₁₂ serum levels lacked significance.

Epidemiologic data suggest an inverse association between the consumption of fruits and vegetables and the incidence of head and neck carcinoma,51 and we previously reported lower folate levels in patients with HNSCC.52 The current data confirm that, for patients with head and neck carcinoma, like in other malignancies, the protective effect of dietary fruits and vegetables may be due, at least in part, to the presence of folic acid. Hypofolatemia may be a common risk factor for head and neck and colon carcinoma that may explain in part the high incidence of colon second primary tumors (SPTs) in patients with HNSCC.4

A diet rich in folate, thus, may be a simple and low-cost preventive measure for the population. An increase in recommended dietary allowances for folate and for other micronutrients involved in DNA metabolism has been proposed.56 In fact, a folate intake in the recommended range often is insufficient to reach in the cells the optimal levels of the precursor nucleotides (dNTPs and NTPs) for DNA/RNA synthesis and repair.39, 41, 56

The definition of a role for hypofolatemia as a risk factor for HNSCC opens intriguing perspectives for chemoprevention, that are particularly relevant from a clinical viewpoint, considering the peculiar behavior of such tumors. In fact, SPTs, which develop at an annual rate of 3–7%, are the leading cause of disease-related mortality in patients with HNSCC.4, 5 Furthermore, precursor lesions (i.e., leukoplakia, erythroplakia), which can be identified directly by clinical examination and often precede the development of malignancy, supply a definite target for secondary prevention and an immediate experimental verification during clinical trials, because the response to treatment can be assessed very simply. Retinoids, even with a noninnocuous toxicity profile, were proposed initially as chemopreventive agents; however, in the EUROSCAN trial (the largest scale clinical trial to date), a 2-year supplementation of retinyl palmitate and/or N-acetylcysteine resulted in no benefit—in terms of survival, event-free survival, or second primary tumors—to patients with HNSCC or lung carcinoma.57 Folate supplementation has no known toxic effects and was reported as effective in reducing the incidence of various malignancies and in inducing regression of precancerous lesions. Therefore, a chemoprevention protocol with folate in patients with leukoplakia of the oral cavity, oropharynx, and larynx, under strict histologic and clinical follow up, currently is rational and ethically correct and already is in progress at our institution with encouraging preliminary results.