Introduction
Lip and oral cavity cancers represent the 16th overall and the 15th deadliest cancer worldwide with an increasing incidence in the last decades [
1‐
3]. It is estimated that in 2022 the number of lip and oral cavity cancer-related deaths worldwide will be 80,736 [
2]. Similar to other head and neck cancer subtypes, the majority of cancers of the lip and oral cavity are squamous cell carcinomas (SCC) (> 90%) which are characterized by a high degree of local invasion and high rate of metastasis that directly affects the patient prognosis [
4‐
8]. The early diagnosis of lip and oral cavity cancer and premalignant lesions is essential [
9]. In particular, lip squamous cell carcinoma (LSCC) can be prevented either by reducing exposure to solar ultraviolet (UV) radiation, which is its most common cause [
10], or by screening for precancerous lesions [
2]. Precancerous lesions of the lip and the oral cavity represent mucosal lesions, such as oral leukoplakia, oral erythroplakia, proliferative verrucous leukoplakia, oral submucous fibrosis, or actinic cheilitis (AC), while the latter may progress to SCC of the lip (LSCC) [
11] depending on specific prognostic factors such as epithelial dysplasia (ED) [
12‐
14].
AC, also known as actinic cheilosis or solar cheilitis, is the most common precancerous lesion of lip and oral cavity cancers. AC was first described by Ayres in 1923 [
15,
16] as a chronic inflammatory process that affects the lower lip in 95% of cases and is most commonly caused by chronic exposure to sunlight or artificial ultraviolet radiation [
17]. Its clinical presentation includes dryness, erythema, and atrophy at the edge of the vermilion border of the lower lip in predominantly white middle-aged males [
18]. AC can progress into invasive LSCC [
17‐
21] with an estimated incidence ranging from 1.4 to 36% over one to thirty year intervals. Some common risk factors associated with malignant transformation of AC are tobacco smoking, alcohol abuse, HPV, race, family, genetic predisposition, immunosuppressive status, poor diet, and socioeconomic factors [
21]. Histopathological features of AC include hyperplasia, acanthosis or atrophy of the squamous stratified epithelium, hyperkeratosis, and/or different degrees of ED. Also, in subjacent connective tissue, basophilic degeneration of collagen fibers called solar elastosis is usually detected [
22].
According to Dancyger et al., AC pathogenesis is similar to cutaneous actinic keratosis or solar keratosis [
23]. In particular, several genes have been implicated in the progression of AC into LSCC, including tumor suppressor gene
p53, anti-apoptotic
BCL2,
Ki-67, and Murine Double Minute 2 (
MDM2) tumor-suppressor protein [
24]. Although there is a high incidence of UV-specific
p53 mutations in both AC (80%) and SCC (90%) [
25], p53 protein immunoreactivity has not been considered a marker of the malignant transformation of AC into SCC [
26]. On the other hand, the majority of precancerous lesions of the lip, including AC, may not always progress into invasive cancer. Therefore, it is necessary to establish reliable diagnostic histopathologic markers to distinguish precancerous from cancerous lesions and then apply them in clinical practice in order to detect early malignant transformation of the epithelium [
27].
Laminin is the most abundant non-collagenous component of all basement membranes [
28]. Laminins are a family of heterotrimeric glycoproteins composed of three different gene products, α, β, and γ chains. These chains are assembled into a cross-shaped heterotrimer αβγ
3 [
28,
29]. There are at least 16 different laminin isoforms named based on the chain composition [
29]. The α1 chain together with the β1 and γ1 chains forms the prototypic EHS laminin (laminin-1) and is expressed in different basement membranes [
30]. Laminins are involved in several biological processes such as cell differentiation, cell adhesion, migration, proteolytic activity, cell proliferation, and metastatic growth [
29‐
32]. Under the light microscope, laminin presents a continuous expression in the normal oral mucosa or oral hyperplastic lesions [
33,
34]. On the contrary, previous studies suggest a discontinuous distribution of laminin from epithelial hyperplasia to epithelial dysplasia [
35‐
38]. Cytoplasmic laminin expression levels have also been found to be higher in poorly differentiated aggressive oral SCC [
32,
35].
The discontinuous pattern of laminin expression in the basement membrane (BM) as well as the absence of its expression might be useful in the early diagnosis of precancerous lesions of the lips as well as in predicting the biological progression toward malignancy. Therefore, the contribution of laminin to the transformation of AC into LSCC needs further clarification. Here we hypothesized that laminin expression levels are altered during lip carcinogenesis. We also hypothesized that immunohistochemical (IHC) expression of laminin might be a useful diagnostic marker to identify early invasion and distinguish cancerous from precancerous lesions. To investigate our hypothesis, we analyzed laminin expression by IHC analysis in biopsies taken from patients with AC of low-grade and high-grade ED and correlated them with its expression in LSCC biopsies. In order to investigate the possible utility of this marker in the differential diagnosis of precancerous lesions, we examined the integrity of the BM as well as cytoplasmic staining patterns. Understanding the mechanism involved in the progression process of actinic cheilitis into LSCC may provide useful histopathologic markers for its early detection, as well as targets for its therapeutic efficacy to be evaluated by further investigation in larger studies.
Discussion
We present new insights into laminin IHC staining as a diagnostic marker of the progression of AC to invasive LSCC. Specifically, we document the differential staining of laminin in BM between AC with low and high-grade ED or invading LSCC, supporting its use as a histopathologic marker of early neoplastic events and invasion.
The BM is a specialized form of extracellular matrix; its role is to separate the epithelium from underlying connective tissue [
43]. Specifically, the BM consists of a mixture of collagens and glycoproteins, such as laminin, that can bind to each other to make a highly cross-linked extracellular matrix [
44]. It is known that the integrity of the BM changes during inflammation in order to allow the inflammatory cells enter the epithelium. Most importantly, the BM structure undergoes alterations during neoplastic infiltration of cancer cells [
45]. To the best of our knowledge, we present for the first time the differential IHC staining of laminin in AC with ED or LSCC, supporting its role as a useful IHC marker in the differential diagnosis. Specifically, our data showed different patterns of IHC expression of laminin-1 between AC with low-grade ED and AC with high-grade ED/in situ carcinoma and between AC and LSCC developed in AC. According to our findings, diffuse, continuous, and intense laminin staining of the BM is characteristically applied to all biopsies from AC with low-grade ED. Biopsies of AC with high-grade ED/in situ carcinoma also present a continuous expression of laminin, however it may appear less intense, and focal at sites of high-grade/in situ carcinoma than low-grade ED, particularly focally at sites of BM with in
situ carcinoma. Based on our data, the development of invasive carcinoma in AC demonstrates loss of laminin expression in the BM, as documented in all LSCC biopsies. Our findings are consistent with previous studies by Garcia et al. which showed a differential expression of laminin between oral precancerous and cancerous lesions, supporting a loss of continuity of laminin expression in the epithelial BM from the development of oral carcinomas [
35]. Specifically, Garcia et al. noted laminin underexpression along the BM, in 20% of the biopsies with low-grade dysplasia and 57% in high-grade dysplastic lesions, and 70% of oral SCC.
An interesting finding of our study was the cytoplasmic expression of laminin detected in AC with high-grade dysplasia and in LSCC. Specifically, our IHC evaluation for laminin-1, whose expression is largely limited to the epithelial BM, revealed cytoplasmic staining in the lower third of the high-grade ED in AC and in the cytoplasm of infiltrating tumor cells forming focal nests in the dermis. Our findings may support IHC data for the laminin-5 γ2 chain from other studies, which suggested laminin overexpression during the progression of neoplastic disease in invasive oral cancer [
34,
37]. Peixoto da-Silva et al. [
46] reported negative cytoplasmic staining of the laminin-5 γ2 chain in AC cases, and they also found a cytoplasmic accumulation of laminin-5 in invading cancer cells. Cytoplasmic expression of the γ2 chain of laminin has been previously observed in various types of malignancies, and Peixoto da-Silva et al. suggested it may be due to laminin proteolysis products during neoplastic transformation [
46]. Although the exact meaning of this staining remains unknown, the change of laminin expression pattern from linear at the basal cells in low-grade ED to cytoplasmic at the parabasal cells in high-grade ED/in situ carcinoma, not only demonstrates a surrogate biomarker for distinguishing these entities but probably indicates a shift of receptors and cell-cell adhesion molecules from BM to dysplastic epithelial cells, that might contribute to the degradation of the extracellular matrix at the beginning of an invasion. Large-scale studies with different molecules and possibly a combination of methodologies could clarify this phenomenon.
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