Unveiling Lipid Metabolic Shifts in Different Stages of Oral Submucous Fibrosis: A New Horizon in Predicting Malignant Transformation

Abstract

Background

Oral Submucous Fibrosis (OSMF) is a chronic, progressive condition linked to areca nut consumption, with a high potential for malignant transformation into oral squamous cell carcinoma (OSCC). Changes in lipid metabolism have been implicated in cancer biology, yet the relationship between lipid profiles and OSMF progression remains underexplored. This study investigates the alterations in serum lipid parameters across different clinical stages of OSMF and their association with malignant transformation.

Materials and methods

A cross-sectional study was conducted over 69 OSMF patients, divided into five groups (Stage 1 to 3 and Stage 4a, 4b) based on clinical staging. Serum lipid profiles, including total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and triglycerides (TG), were analyzed. Statistical analysis was performed using ANOVA and Tukey’s post hoc test.

Results

Lipid levels (TC, HDL, LDL, VLDL, TG) showed a significant decline from Stage 1 to Stage 4a (p-0.00), followed by a sharp increase in Stage 4b (p-0.00), coinciding with malignant transformation. Particularly, HDL, VLDL, and TG were significantly elevated in Stage 4b compared to earlier stages.

Conclusion

Alterations in lipid metabolism (hypolipidemia) were observed from Stage 1 to 4a, with a marked shift (hyperlipidemia) during malignant transformation (Stage 4b). Increased levels of HDL, VLDL, and TG in advanced OSMF stages suggest their potential as predictive biomarkers for malignancy. Further research is required to elucidate the mechanisms linking lipid metabolism with OSMF progression and malignancy, paving the way for targeted therapeutic strategies.

Introduction

Oral Submucous Fibrosis (OSMF) is a chronic, debilitating condition of the oral cavity, predominantly seen in Southeast Asia, especially in populations with high areca nut consumption [1]. It is characterized by the progressive fibrosis of the oral mucosa, leading to stiffening of the tissues, reduced mouth opening (trismus), burning sensation, and other complications that severely affect the quality of life. OSMF is considered a potentially malignant disorder (PMD), with a high risk of transforming into oral squamous cell carcinoma (OSCC) [2]. This malignant transformation rate varies, with studies estimating it between 7 and 30%, making it a significant public health concern [3, 4]. Given the irreversible nature of OSMF and its malignant potential, early diagnosis, risk stratification, and continuous monitoring are critical in reducing morbidity and mortality [4].

Lipid metabolism has been extensively studied in cancer biology, as lipids play key roles in maintaining cellular homeostasis, including membrane structure, cell signaling, and energy storage [5–7]. Alterations in lipid profiles, such as variations in cholesterol, triglycerides (TG), and lipoproteins, have been implicated in the pathogenesis of various cancers, including oral cancers [5–8]. The connection between lipid metabolism and carcinogenesis is grounded in the observation that cancer cells exhibit increased lipid synthesis and uptake to support rapid cell division and membrane biogenesis [5, 7]. Moreover, oxidative stress and inflammation, common in OSMF due to continuous irritation from areca nut and other local irritants, can lead to lipid peroxidation, further contributing to the carcinogenic process [6, 8–11].

Several studies have suggested that lipid profile changes may occur in patients with OSMF, possibly reflecting the metabolic alterations linked to the fibrotic process and its progression toward malignancy [9–17]. Dyslipidemia has been observed in several PMDs and malignant conditions, where abnormal lipid levels correlate with disease severity, suggesting that lipid parameters may serve as potential biomarkers for disease progression and malignant transformation [10, 18–21]. However, the exact relationship between serum lipid profiles and the clinical stages of OSMF remains underexplored, and there is limited research investigating whether specific lipid parameters can predict malignant transformation in OSMF.

This study aims to evaluate lipid parameters in different clinical stages of OSMF and explore their association with malignant transformation. By analyzing lipid levels across varying stages of OSMF, this research seeks to uncover potential biomarkers that could help in early diagnosis, risk assessment, and therapeutic monitoring in individuals at risk of developing oral cancer.

Materials and Methods

A cross-sectional prospective study was conducted over 69 enrolled patients aged between 17 and 70 years. The sample size was estimated using mean values of HDL in stage II (38.20 ± 3.12) and stage III (32.73 ± 4.78) OSMF obtained from a previous study conducted by Ajai K et al., [12] the power of a study is 80%, and the level of significance is 5%. Therefore, the sample size estimated was 10 per group. Ethical clearance for the present study was obtained from the institutional ethical committee vide.no. Dean/2023/EC/6961 and written informed consent was also obtained from all participants. The study included patients with clinically proven OSMF cases who will undergo treatment. Patients with systemic diseases, post-operative patients, those treated with chemotherapy and radiotherapy, and individuals with a previous history of cancer or malignancy treatment were excluded. All 69 OSMF patients were divided into four clinical stages according to the classification given by Khanna JN and Andrade NN 1995 [22] (The 4th stage being subdivided into 4a and 4b). The classification is based on functional and clinical features of OSMF:

Stage 1

Burning sensation in the mouth, acute ulceration and recurrent stomatitis, no associated mouth opening limitation, inter-incisal opening of 35 mm and above.

Stage 2

Mottled and marble-like buccal mucosa, dense, pale, depigmented fibrosed areas alternated with pink normal mucosa, occasional red erythroplakic patches, widespread sheets of fibrosis, inter-incisal opening of 26–35 mm.

Stage 3

Pale buccal mucosa firmly attached to the underlying tissues, palpable vertical fibrous bands in the premolar area, unable to blow out cheeks and whistle, in the soft palate, the fibrous bands were seen to radiate from the pterygomandibular raphe or the anterior faucial pillar in a scar-like appearance, the lips may be affected with atrophy of the vermilion border, inter-incisal opening of 15–25 mm.

Stage 4a

Thickened, shortened, and firm fauces, with the tonsils compressed between the fibrosed pillars, small, shrunken, fibrous bud uvula, narrowed isthmus, presence of circular band around entire lip and mouth, restricted tongue movement, diffuse papillary atrophy, atrophy of the vermilion border, inter-incisal opening of 15 mm and below.

Stage 4b

Stage 4a features associated with other premalignant and malignant changes.

The patients were categorized into five groups: Group 1– Stage 1 (16 subjects); Group 2 - Stage 2 (14 subjects); Group 3 - Stage 3 (15 subjects); Group 4 - Stage 4a (11 subjects); and Group 5 - Stage 4b (13 subjects).

Methodology

5 ml of blood was collected under sterile conditions from all the study participants and allowed to clot for 1 h at room temperature. The clotted blood was then centrifuged at 3000 rpm for 5 min, and the serum obtained was stored at 4 degrees celsius for lipid profile estimation [9]. The lipid profile analysis included the assessment of Total Cholesterol (TC), High-Density Lipoprotein (HDL), Low-Density Lipoprotein (LDL), Very Low-Density Lipoprotein (VLDL) and TG analyzed using a semi-automatic analyzer. The data collected was subjected to statistical analysis using SPSS software.

Statistical Analysis

All statistical analyses were performed using a Microsoft Excel worksheet and SPSS Statistics for Windows Version 25.0. As the data were quantitative, the level of significance was set at 5%. To compare the lipid profiles among five groups, one-way ANOVA was used followed by post hoc Tukey test for pairwise comparison.

Results

Out of a total of 69 cases studied, 66 were males and 3 were females, resulting in a male-to-female ratio of 22:1. The mean age of patients was 38.39 ± 12.82 years, with an age range spanning from 14 to 70 years. The mean age of male patients was 38.45 ± 12.62 years, while the mean age for female patients was 37.00 ± 16.51 years. Tobacco consumption, particularly in the forms of gutkha, pan masala, and khaini, emerged as the predominant predisposing factor.

Serum lipid profile parameters, including TC, HDL, LDL, VLDL, and TG were evaluated, and their mean values, standard deviations and p-values were analysed, as summarized in Table 1. Statistical analysis revealed significant differences in the mean values of TC, HDL, LDL, VLDL and TG across the various clinical stages of OSMF (Group 1–5), specifically Stages 1 to 4b (p-0.00). A progressive decline in the mean values of TC, HDL, LDL, VLDL, and TG was observed from Stage 1 (Group 1) to Stage 4a (Group 4), all with considerable statistical significance (p-0.00). Notably, an alarming two- to three-fold significant increase in all these lipid parameters was observed when comparing the values from Stage 4a (Group 4) to Stage 4b (Group 5) indicating malignant transformation (p-0.00).

Table 1.

Association of lipid parameters in different stages of OSMF

S. No	Variables	Group	No. of cases	Mean	Standard deviation	p value
1.	Total Cholesterol	1	16	214.75	17.01	0.000
		2	14	182.29	4.97
		3	15	161.08	9.46
		4	11	117.18	18.36
		5	13	204.65	47.96
2.	HDL	1	16	49.01	9.99	0.000
		2	14	44.91	3.98
		3	15	33.67	3.08
		4	11	28.95	10.82
		5	13	83.40	3.07
3.	LDL	1	16	132.03	20.88	0.000
		2	14	107.64	11.34
		3	15	85.66	9.10
		4	11	55.01	19.76
		5	13	112.50	34.63
4.	VLDL	1	16	33.06	8.87	0.000
		2	14	25.58	12.05
		3	15	30.28	10.30
		4	11	22.42	10.29
		5	13	46.39	19.67
5.	Triglycerides	1	16	156.64	32.41	0.000
		2	14	120.93	41.19
		3	15	124.95	27.82
		4	11	101.32	45.62
		5	13	250.85	121.87

A p-value < 0.05 is statistically Significant

Abbreviations: HDL-High density lipoprotein, LDL-Low density lipoprotein, VLDL-Very low-density lipoprotein, Group 1– stage 1 (16), Group 2 - stage 2 (14), Group 3 - stage 3 (15), Group 4 - stage 4a (11) and Group 5 - stage 4b (13)

Further pairwise comparisons between Group 1 and Group 5 revealed significant elevations in the mean values of HDL (p-0.00), VLDL (p-0.04), and TG (p-0.001) in Group 5, indicating a marked shift in lipid metabolism while progressing towards malignancy (Table 2).

Table 2.

Pair-wise comparison of lipid metrics in different stages of OSMF

S. No	Variables	Pair-wise comparison of Groups		Mean	Standard deviation	p value
1.	Total Cholesterol	1	2	32.46	8.79	0.004
			3	53.67	8.63	0.000
			4	97.57	9.40	0.000
			5	10.10	8.97	0.792
		2	1	-32.46	8.79	0.000
			3	21.21	8.92	0.135
			4	65.10	9.67	0.00
			5	-22.36	9.25	0.124
		3	1	-53.6700	8.63	0.00
			2	-21.205	8.92	0.135
			4	43.90	9.53	0.00
			5	-43.57	9.10	0.00
		4	1	-97.57	9.40	0.000
			2	-65.10	9.67	0.000
			3	-43.90	9.53	0.000
			5	-87.46	9.84	0.000
		5	1	-10.10	8.97	0.792
			2	22.36	9.25	0.124
			3	43.57	9.10	0.000
			4	87.46	9.84	0.000
2.	HDL	1	2	4.09	2.55	0.502
			3	15.34	2.51	0.000
			4	20.06	2.73	0.000
			5	-34.39	2.61	0.000
		2	1	-4.09	2.55	0.502
			3	11.25	2.59	0.000
			4	15.97	2.81	0.000
			5	-38.49	2.69	0.000
		3	1	-15.34	2.51	0.000
			2	-11.25	2.59	0.000
			4	4.72	2.77	0.439
			5	-49.73	2.65	0.000
		4	1	-20.06	2.73	0.000
			2	-15.97	2.81	0.000
			3	-4.72	2.77	0.439
			5	-54.45	2.86	0.000
		5	1	34.39	2.61	0.000
			2	38.49	2.69	0.000
			3	49.73	2.62	0.000
			4	54.45	2.86	0.000
3.	LDL	1	2	24.39	7.64	0.018
			3	46.37	7.51	0.000
			4	77.03	8.18	0.000
			5	19.54	7.80	0.102
		2	1	-24.39	7.64	0.018
			3	21.98	7.76	0.047
			4	52.64	8.41	0.000
			5	-4.85	8.04	0.974
		3	1	-46.37	7.51	0.000
			2	-21.98	7.76	0.047
			4	30.66	8.29	0.004
			5	-26.83	7.91	0.010
		4	1	-77.03	8.18	0.000
			2	-52.64	8.41	0.000
			3	-30.66	8.29	0.004
			5	-57.49	8.55	0.000
		5	1	-19.54	7.80	0.102
			2	4.85	8.04	0.974
			3	26.83	7.91	0.010
			4	57.49	8.55	0.000
4.	VLDL	1	2	7.48	4.63	0.494
			3	2.78	4.55	0.973
			4	10.64	4.96	0.214
			5	-13.33	4.73	0.048
		2	1	-7.48	4.63	0.494
			3	-4.70	4.70	0.854
			4	3.16	5.10	0.972
			5	-20.82	4.88	0.001
		3	1	-2.78	4.55	0.973
			2	4.70	4.70	0.854
			4	7.86	5.03	0.525
			5	-16.11	4.80	0.011
		4	1	-10.64	4.96	0.214
			2	-3.16	5.10	0.972
			3	-7.86	5.03	0.525
			5	-23.97	5.19	0.000
		5	1	13.33	4.73	0.048
			2	20.82	4.88	0.001
			3	16.11	4.80	0.011
			4	23.97	5.19	0.000
5.	Triglycerides	1	2	35.71	22.77	0.523
			3	31.69	22.36	0.619
			4	55.32	24.37	0.168
			5	-94.22	23.23	0.001
		2	1	-35.71	22.77	0.523
			3	-4.02	23.12	1.000
			4	19.61	25.07	0.935
			5	-129.93	23.96	0.000
		3	1	-31.69	22.36	0.619
			2	4.02	23.12	1.000
			4	23.63	24.69	0.873
			5	-125.91	23.57	0.000
		4	1	-55.32	24.37	0.168
			2	-19.61	25.07	0.935
			3	-23.63	24.69	0.873
			5	-149.54	25.49	0.000
		5	1	94.22	23.23	0.001
			2	129.93	23.96	0.000
			3	125.91	23.57	0.000
			4	149.54	25.49	0.000

A p-value < 0.05 is statistically Significant

Abbreviations: HDL-High density lipoprotein, LDL-Low density lipoprotein, VLDL-Very low-density lipoprotein, Group 1– stage 1 (16), Group 2 - stage 2 (14), Group 3 - stage 3 (15), Group 4 - stage 4a (11) and Group 5 - stage 4b (13)

Discussion

OSMF represents a serious public health issue, particularly in regions with high areca nut consumption [9]. Alterations in lipid metabolism and levels are pivotal in the underlying mechanisms of various fibrotic conditions, such as liver fibrosis [23], renal fibrosis [24], and idiopathic pulmonary fibrosis [25] and carcinogenesis including breast cancer [5], colorectal [6], prostate [7] and gastric cancer [8]. Unravelling the precise relationship between altered lipid profiles and the progression of fibrosis and/or malignant transformation of OSMF could pave the way for novel disease-modifying therapies. Thus, the present study attempts to evaluate serum lipid parameters (TC, HDL, LDL, VLDL, TG) across different clinical stages of OSMF to understand the metabolic alterations and their link to malignant transformation. It aims to identify non-invasive biomarkers that can be integrated into routine clinical practice to improve patient outcomes.

A notable male predominance was observed in this study, with a male-to-female ratio of 22:1, consistent with previous research indicating that OSMF is more prevalent among males [26–28]. This is likely due to the higher rates of tobacco and areca nut consumption in men. The mean age (38.39 years) of patients highlights the chronic nature of OSMF, often manifesting after prolonged exposure to environmental risk factors, particularly the habitual use of areca nut products [2, 28].

An extensive search of English literature revealed that the present study is the first one to evaluate the serum lipid profile in OSMF cases with malignant transformation. In the present study, significant differences were found in the mean values of serum lipid parameters (TC, HDL, LDL, VLDL, TG) across different clinical stages of OSMF. The overall trend demonstrated a progressive decline in the mean values of these lipid parameters from early (stage 1) to advanced (stage 4a) OSMF. This is consistent with previous researches conducted by Ajai K et al. (2014), Kanthem RK and Guttikonda VR (2015); Rawson K et al. (2015); Rakheerathnam KK et al. (2018); Pratap M et al. (2018); Sangle VA et al. (2023), who have also reported decreased lipid levels in OSMF patients across various clinical and histopathological stages [12–17]. This reduction in lipid levels may be attributed to chronic inflammation in OSMF that might drive lipid peroxidation, depleting circulating lipids and by greater utilization of lipids for new membrane biogenesis. Also, the accumulation of esterified cholesterol in tissues and subsequent released peroxide radicals damage the cellular structural blocks specifically lipids and malfunction the lipid metabolism. (9–10, 11)

Moreover, the accumulation of lipid facilitates increased endoplasmic reticulum stress and activates fibrogenic signalling pathways by release of profibrotic cytokines such as TGF-beta, VEGF, CTGF, COL1A1, COL3A1, α-SMA leading to increased fibrosis [12–20]. Taken together, all these findings suggest that hypolipidemia is linked to the progression of fibrosis or increased severity of OSMF, reinforcing the idea that lipid parameters could serve as predictive markers for disease progression in OSMF.

The present study findings also revealed a significant upregulation in serum levels of HDL, VLDL and TG in Stage 4b as compared to Stage 1 which is characterized by premalignant/malignant changes. Additionally, while comparing Stage 4a with Stage 4b, it was observed a marked elevation across all lipid parameters including TC, HDL, LDL, VLDL and TG in Stage 4b. These findings are supported by the study conducted by Goyal S et al., 2021, who reported elevated HDL serum lipid levels in patients with OSMF accompanied by leukoplakia compared to those with OSMF alone [27]. However, Goyal’s study also observed a reduction in other lipid metrics such as TC, LDL, VLDL and TG. Few studies reported that lower lipid levels are associated with OSCC cases not arising from OSMF [9–11]. This highlights the need for further investigation into the differences in lipid profiles between OSCC cases not arising from OSMF and OSCC progressed from OSMF and to discriminate the biological distinctiveness between these two lesions.

While, the present study results also align with the earlier studies conducted on different carcinomas such as by Fevrot MC (1984) in haematological carcinoma, Haltom JM (1998) in acute lymphoblastic leukemia, Shah FD et al. (2008) in Breast cancer, Peela, J. & Jarari, A (2012) in Breast cancer, and Seth D et al. (2012) in Endometrial cancer, who found elevated levels of HDL, VLDL and TG in cancer patients [18–20, 29, 30].

The association between lipid metabolism and oral cancer is complex, involving oxidative stress and inflammation, both of which are prevalent in OSMF due to continuous irritation from areca nut and other local irritants [21, 27, 28]. This process can contribute to lipid peroxidation, formation of malondialdehyde which cross-links with DNA, contributes to genomic instability and further exacerbates carcinogenesis [9, 11, 17–19]. In addition, malignant cells exhibit altered lipid metabolism, including increased lipid synthesis and uptake, and decreased lipolysis supporting the need for new cell membranes, energy production, and signalling [12, 17, 19, 29]. This suggests a critical metabolic shift in lipid levels as the disease advances toward malignancy, indicating that elevated lipid levels, especially HDL, VLDL, and TG, could be potential biomarkers for identifying patients at a higher risk of malignancy.

The results of this study have important clinical implications. Monitoring lipid profiles of patients in different stages of OSMF, may provide an additional tool for assessing disease progression and identifying those at risk for malignant transformation. As lipid alterations are relatively easy to measure using routine blood tests, they offer a non-invasive, cost-effective approach to risk stratification. Furthermore, the identification of changes in lipid metabolism could pave the way for novel therapeutic strategies, targeting lipid pathways to prevent or delay the malignant transformation of OSMF.

However, the generalization of the findings of the present study could not be possible due to the small sample size, therefore future studies with larger cohorts are needed to validate these results. Additionally, while this study demonstrates a clear association between lipid profile changes in OSMF progression, the underlying molecular mechanisms driving these alterations remain to be elucidated. Further researches are needed to explore the role of lipid metabolism in the fibrotic and carcinogenic processes of OSMF, including the involvement of specific lipid-related enzymes and pathways such as fatty acid synthase, acetyl-CoA carboxylase, and the mevalonate pathway.

Conclusion

The present study demonstrates a significant decline (hypolipidemia) in lipid parameters across different stages of OSMF (Stage 1 to 4a), with a notable increase in lipid parameters (hyperlipidemia) during the malignant transformation phase (Stage 4b). The findings suggest that lipid profiles, particularly increased HDL, VLDL, and TG could be predictive biomarkers for disease progression and malignant risk in OSMF patients. Future research should explore and validate the molecular basis of lipid metabolism in OSMF and investigate the potential of targeting lipid pathways as a therapeutic strategy to prevent malignant transformation.

Funding

This research received specific grants from the Institute of Eminence Cell, BHU, Varanasi.

Declarations

Conflict of Interest

The author declares that they have no conflict of interest to disclose.

Ethical Approval

The study was approved by the Institute’s scientific & ethical approval committee vide.no. Dean/2023/EC/6961.

Informed Consent

For this type of study informed consent is obtained.