Introduction

Oral submucous fibrosis (OSF) is an oral mucous disease that is mainly associated with betel quid chewing. In 1956, Paymaster described OSF as a precancerous condition and subsequently reported 650 cases with oral squamous cell carcinoma (OSCC), approximately one third of which were associated with OSF [1]. Pindborg et al. [2] then reinforced the hypothesis that oral submucous fibrosis is a precancerous condition or potentially malignant oral mucous disorder [3,4,5,6,7,8]. In 2017, Chaturvedi et al. reported 289 patients with OSCC, and 88 patients of them were associated with OSF. Their study had indicated that the patients with OSCC associated with OSF have better oncologic outcome than those without [9]. Similarly in 2017, Gadbail et al. compared the clinic-pathological features of OSCC in the background of OSF and OSCC. The results had showed that OSCC in the background of OSF was a distinct clinicopathological entity with better prognosis [10].

Although the malignant potential of OSF was first identified in 1956 [1], the molecular mechanisms of malignant transformation is still under investigation. A significant number of studies have been carried out by many researchers. The major carcinogen in the malignant transformation of OSF is the arecoline [11]. It exerts effects by regulating the cell cycle-related proteins [12, 13], inhibiting the expression of suppressor genes [14] and promoting keratinocyte migration and invasion [15]. The increased fibrosis causes the hypoxia and decreased vascularity, which leads to the ischemic atrophy of the epithelium and ultimately leads to cancer [16]. Besides, the epithelial mesenchymal transition is also found to play critical role in the development of invasive carcinoma [17]. As a result, the malignant transformation in the background of OSF appears to be a complex process involving different pathways.

However, these researches are not definitively known whether OSF can transform into squamous cell carcinoma. We, therefore, sought to determine whether OSF is a real oral mucous precancerous lesion or a potentially malignant oral disorder.

Materials and methods

Study design

The design was a multiple-center prospective study, in which 567 patients with OSF were enrolled from 1986 to 2017. This multiple-center trial was approved by the ethics committee and was monitored by the Institutional Data and Safety Monitoring Committee.

Subjects

Five hundred and sixty-seven patients with OSF over aged 18 years were enrolled from September 1986 to August 2017 and followed-up until 2019. The diagnosis and personal information and patients were recorded with the research registration system. All the subjects chewed betel quid before he/she was diagnosed as OSF. All the patients got rid of the habit of chewing betel quid after the diagnosis. There is not any active treatment provided after the diagnosis. However, the pathological fibrosis was not reversed during the follow-up period. Among these subjects, 32 patients were diagnosed with OSCC during the follow-up.

The diagnosis of OSF was indicated according to the criteria of Pindborg [2]:

  1. (1)

    A history of betel quid chewing (more than five slices for every day).

  2. (2)

    When eating spicy food, a burning sensation in the mouth is experienced.

  3. (3)

    The oral mucosa is blanched and white and has fibrous bands.

  4. (4)

    It is difficult for the patient to open their mouth.

The pathological characteristics of OSF tissues were the following:

  1. (1)

    The oral epithelium in the affected areas is atrophic or hypertrophic.

  2. (2)

    In the early stage, the juxta-epithelial area shows hyalinization, and collagen is seen as separate bundles. Blood vessels are dilated and congested. The inflammatory cells, mononuclear lymphocytes, eosinophils, and occasional plasma cells are present.

  3. (3)

    In the moderately advanced stage, collagen is moderately hyalinized, and inflammatory cells are mainly lymphocytes and plasma cells and occasionally eosinophils. Blood vessels are either normal or constricted.

  4. (4)

    In the advanced stage, collagen is completely hyalinized and is seen as a smooth sheet. Inflammatory cells are mainly lymphocytes and plasma cells. Blood vessels are obliterated or narrowed.

The indicated patients were photographed and biopsied with patient consent. Follow-up examination was performed annually to determine the malignant transformation rate and to understand the natural history of OSF.

Patients with TMJ disorders, severe hyperplasia, OSF associated with leukoplakia, or OSF associated with leukoplakia and squamous cell carcinoma in the oral cavity were excluded. Carcinoma in situ and transformation of OSF into squamous cell carcinoma were included in this study. The histopathological diagnostic criteria of dysplasia, carcinoma in situ, and squamous cell carcinoma were as follows:

  1. (1)

    Dysplasia: when hyperchromatic and slightly pleomorphic nuclei are noted in the base and parabasal cell layers of the stratified squamous epithelium, mild epithelial dysplasia is diagnosed. If dysplastic changes extend to the mild point of the epithelium, characterized by nuclear hyperchromatism, pleomorphism, and cellular crowding, moderate epithelial dysplasia is diagnosed. If marked pleomorphism, hyperchromatism, scattered mitotic figures, and atypical cells are present in most of the epithelial thickness, severe epithelial dysplasia is diagnosed [18].

  2. (2)

    Carcinoma in situ: when epithelial cells extend from the basal layer to the surface of the mucosa, carcinoma in situ is defined. The most important feature of carcinoma in situ is that no invasion has occurred, and a focus of invasive squamous cell carcinoma in the adjacent tissue may be present [18].

  3. (3)

    Squamous cell carcinoma: histopathological criteria of squamous cell carcinoma are graded on a 3-point scale as follows.

    1. I.

      Poorly differentiated squamous cell carcinoma: numerous pleomorphic cells within the lamina propria are present.

    2. II.

      Moderately differentiated squamous cell carcinoma: malignant cells are easily recognizable as being of squamous epithelial origin under medium-power and high-power view.

    3. III.

      Well-differentiated squamous cell carcinoma: islands of malignant squamous epithelium invaded into the lamina propria under low-power view. High-power view shows dysplastic epithelial cells with keratin pearl formation [18].

Follow-up

Patients who were diagnosed with OSF were followed-up once every 12 months. When OSF transformation into squamous cell carcinoma in the oral cavity was diagnosed and confirmed by pathological examination, treatment of squamous cell carcinoma was started. After squamous cell carcinoma treatment, patients were followed-up every month for the first year, every 2 months for the second year, and every 3 months thereafter.

Statistical analysis

The results are presented according to the diagnosis of OSF and to the diagnosis and treatment of OSCC. The data cut off point was August 31, 2017. All data were statistically analyzed using the SPSS 20.0 software (International Business Machines, Armonk, New York, USA). The descriptive data were analyzed using the methods of frequencies, percentages, medians, standard deviations, and ranges.

Results

Patients with OSCC

Of the 567 patients with OSF, 32 (5.6%) presented transformation of submucous fibrosis into OSCC (Table 1). Among the 32 patients, 31 were male and 1 was female. Their age distribution was 20–69 years, the average being 52 years (Table 1 and Supplementary Table 1). All 32 patients chewed betel quid. Six percent were aged between 20 and 39 years, 44% were aged between 40 and 49 years, and 50% were aged between 50 and 69 years. Betel quid chewing was most prevalent in patients aged 40–69 years. (Fig. 1a, Table 1).

Table 1 Characteristics of patients with OSF transforming into oral cancer
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Fig. 1
figure 1

Characteristics and betel quid chewing habits of OSF and OSCC patients. (a) Age distribution. (b) Duration of betel quid chewing. (c) Quantity of betel quid chewed. (d) Duration of suffering from OSF. (e) Sites of OSCC transformed from OSF. (f) Degree of OSCC differentiation

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The amount and duration of betel quid chewing in OSCC patients

The OSCC patients had chewed betel quid for different amounts of time: six patients (19%) for 1–9 years, 16 patients (50%) for 10–19 years, 7 patients (22%) for 20–29 years, and 3 patients (9%) for 30–39 years (Fig.1b and Supplementary Table 2).

The amount of betel quid chewing (slices/day) also varied: Five patients (16%) chewed 5–9 slices per day, 19 patients (59%) chewed 10–19 slices per day, 5 patients (16%) chewed 20–29 slices per day, 2 patients (6%) chewed 30–39 slices per day, and 1 patient (3%) chewed ≥ 40 slices per day (Fig.1c and Supplementary Table 3).

When we separated the patients into 4 groups according to the amount of betel quid chewing per day (≤ 5, 6–10, 11–15, and ≥ 16) and compared the duration of suffering from OSF among the groups, the results showed that the more betel quid chewing per day, the less of the duration length required for transformation from OSF to OSCC (P < 0.05) (Fig.2).

Fig. 2
figure 2

Comparison of the duration of suffering from OSF among the groups with different amounts of betel quid chewing per day

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The transformation latency

The transformation rate from OSF to OSCC was 32/567(5.6%), and transformation occurred after 2–24 years with a median time of 8.6 years: transformation occurred in 18 patients (57%) after 1–9 years, in 13 patients (41%) after 10–19 years, and in 1 patient (3%) after 20–29 years (Fig. 3; Fig. 1d).

Fig. 3
figure 3

Duration of follow-up before transformation of OSF to OSCC among patients

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Stages and sites of OSCC transformed from OSF

Among the 32 patients, squamous cell carcinoma was located in left or right buccal regions in 23 patients (72%), in left or right lingual regions in 4 patients (12%), in the right gingival region in 2 patients (6%), and in the right floor of the mouth in 2 patients (6%) (Fig. 1e and Supplementary Table 4).

Well-differentiated squamous cell carcinoma accounted for 23 patients (72%), and moderately differentiated squamous cell carcinoma accounted for 8 patients (25%) (Fig.1f, Fig.4–5, and Supplementary Table 5).

Fig. 4
figure 4

Histopathology of OSF transformation into OSCC. A Histopathological overview of OSF transformation into OSCC. B-B′, Area of OSF transformed into moderately differentiated OSCC; the boxed region is the higher magnification area shown in B′. C-C′, Area of OSF; the boxed region is the higher magnification area shown in C′. C′, Moderate epithelial dysplastic changes

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Fig. 5
figure 5

Differentiation degrees of OSCC transformed from OSF A, A’, Carcinoma in situ. B, B′, Well-differentiated squamous cell carcinoma. C, C′, Moderately differentiated squamous cell carcinoma. Scale bar represents 200 μm in A, B, and C; 100 μm in A’, B′, and C′

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Discussion

OSF is a premalignant and crippling lesion of the oral mucosa that is associated with various chewing habits [19]. In 2006, Ariyawardana et al. showed that betel chewing was the only significantly associated factor in the etiology of OSF. Other investigations then provided further evidence for the role of betel quid chewing in the etiology of OSF [4,5,6,7,8, 20]. Our follow-up study also indicated that betel quid chewing is a significant factor in the etiology of OSF and that there is a strong dose response relationship between the frequency and duration of betel quid chewing and the risk of developing OSF and oral cancer.

OSF was defined as an oral precancerous condition by Paymaster in 1957. In his series, one third of 650 cases with buccal mucous cancer were associated with OSF. In 1984, Pindborg suggested OSF to be a precancerous condition [3] according to the following five criteria: (1) a higher incidence of oral cancer in patients with submucous fibrosis, (2) high occurrence of submucous fibrosis in oral cancer patients, (3) histological diagnosis of oral cancer without any clinical suspicion among submucous fibrosis cases, (4) higher prevalence of leukoplakia among submucous fibrosis cases, and (5) high frequency of epithelial dysplasia. In 2016, Jayasinghe et al. [7] reported five cases of clinically malignant exophytic lesions in a background of OSF. These five patients were all verrucopapillary lesions or verrucous leukoplakia, which were all histologically diagnosed as oral verrucous hyperplasia (OVH) on a background of OSF. Additionally, many other similar studies have been published in the English literature [1,2,3,4,5,6, 8, 20, 21]. However, whether OSF can transform into squamous cell carcinoma remains unclear.

In the present study, among the 567 patients with OSF followed-up over long period, 32 cases had transformed into OSCC. Our study revealed that the average duration of malignant transformation was 8.7 years, longer than that in other studies [5, 6]. Most of the 32 patients with a malignant transformation were presented in the buccal mucosa (72%) and the tongue (12%). Moreover, we found that OSF in the buccal mucosa had a higher malignant transformation potential than that in other regions of the oral cavity. Excepted for the use of betel quid, cigarette and alcohol were identified as risk factors for malignant transformation of OSF in the Mainland China previously. Synergistic effects between betel quid chewing and cigarette or alcohol consumption were revealed [22].

The limitations of the study are that we only observed how OSF in the oral cavity transformed into oral cancer and the patients with both OSF and oral leukoplakia had been excluded in our study. However, the patients who have two or more kind of oral precancerous lesions are gradually increased nowadays. As a result, we need to study the malignant transformation of the patients with both OSF and other precancerous lesions in the future.

Our study indicated that OSF is a really malignant oral disorder. It is, therefore, of great importance to prevent malignant change in patients diagnosed with OSF.