Keywords
Oral potentially malignant disorders - oral squamous cell carcinoma - saliva - Secretory
leukocyte protease inhibitor - serum
Introduction
Oral cancer is the 15th most prevalent cancer worldwide with a 50% survival rate despite
recent advances in surgery, chemotherapy and radiotherapy.[1] More than 95% of oral cancers are squamous cell carcinomas (SCCs).[2] The high incidence of oral cancer in India is attributed to the habits of tobacco
chewing and smoking. These carcinogens interact with the oral mucosa to cause premalignant
and malignant changes. Most malignancies of the oral cavity arise from preexisting
premalignant lesions.[1] With a view to prevention of oral potentially malignant disorders (OPMD) developing
into oral SCCs (OSCCs), various studies have tried to identify biomarkers in the serum
and saliva.
Secretory leukocyte protease inhibitor (SLPI) was first isolated from secretions of
patients with chronic obstructive pulmonary disease and cystic fibrosis and was thereby
considered a major anti elastase inhibitor. SLPI is produced by neutrophils, macrophages,
beta-cells of pancreatic islets, epithelial cells investing the renal tubules, acinar
cells of parotid and submandibular glands, acinar cells of submucosal glands, and
epithelial cells lining mucous membranes of respiratory and alimentary tracts. SLPI
protects the tissues by inhibiting proteases such as cathepsin G, elastase, and trypsin
from neutrophils; chymotrypsin; and trypsin from pancreatic acinar cells and chymase
and tryptase from mast cells. The main action is inhibition of neutrophil elastase
which protects epithelial surfaces from damage.[3] Initially studied for its role in the prevention of HIV transmission in saliva,
SLPI has recently been recognized as a potential diagnostic and prognostic marker
in the head-and-neck cancer.[4] It is found in most secretions of the body and mainly in saliva.[5] Previous research on SLPI has used brush biopsy specimens in OPMD and OSCC.[6] This report demonstrated a significant decrease in SLPI in participants with oral
precancer and cancer with the levels decreasing progressively from oral precancer
to cancer. A recent study has evaluated archived salivary samples of patients who
went on to develop OSCC and found that high salivary SLPI in smokers was associated
with increased risk of the head-and-neck SCCs (HNSCC).[5] However, studies which have evaluated serum and salivary SLPI in OPMD and OSCC are
lacking.
The present study aims to evaluate the role of serum and salivary SLPI in progression
of OPMD into OSCC.
Materials and Methods
A total of 90 participants reporting to the outpatient department of our institute
were included in the study. They were divided into three groups of 30 participants
each: Group I included participants diagnosed with OPMD, Group II included participants
diagnosed with OSCC, and Group III formed the control group. Participants with any
underlying systemic disease were excluded from the study. Institutional Ethics Committee
Clearance and written informed consent were obtained from each study participant.
Demographic data were acquired from the participants and were followed by an examination
of the oral cavity. Venous blood was withdrawn from the antecubital vein and serum
obtained. Whole saliva was collected by asking the participants to allow saliva to
collect in the mouth and then spit into sterile containers every minute for 5 min.
Saliva and serum were stored at − 20° until analysis.
Serum and salivary SLPI was determined using ELISA (Hycult Laboratories, Netherlands).
The results obtained were subjected to statistical analysis.
Statistical analysis
Kruskal–Wallis test was used to compare the three groups. Intergroup comparison was
done using Mann–Whitney U-test. Correlation analysis was performed using Spearman’s
correlation test.
Results
Of the 30 participants with OPMD (study Group 1), 26 were male and 4 were female.
The third, fourth, and fifth decades all had equal number of cases. Oral submucous
fibrosis (OSMF) was the most common lesion in this group followed by leukoplakia and
erosive lichen planus [Figure 1]. Gutkha chewing was the most common etiologic agent in OSMF, while smoking was associated
with leukoplakia. Study Group II (OSCC) consisted of 24 males and 6 females. Maximum
cases were seen in the 7th decade followed by the 6th decade. The most common site
was the buccal mucosa followed by the tongue and the alveologingival sulcus [Figure 2]. Betel-quid chewing (BLAT) was the most common associated etiological factor [Figure 3].
Figure 1: Graph showing the habits associated with oral potentially malignant disorders.
Gutkha chewing was the most common etiologic agent in oral submucous fibrosis while
smoking was associated with leukoplakia
Figure 2: Graph showing the site distribution of oral squamous cell carcinoma. The
most common site was the buccal mucosa followed by the tongue
Figure 3: Graph showing the habits associated with oral squamous cell carcinoma. Betel-quid
chewing was the most common associated etiological factor
Serum Secretory leukocyte protease inhibitor
The mean level of SLPI in the control, OPMD, and OSCC groups were 26.90, 44.78, and
105.56 pg/ml, respectively. The difference between the groups was statistically nonsignificant
[Figure 4].
Figure 4: Progressive increase in mean levels of serum secretory leukocyte protease
inhibitor in oral potentially malignant disorders and oral squamous cell carcinoma
as compared to controls
Salivary Secretory leukocyte protease inhibitor
The mean level of SLPI in the control, OPMD, and OSCC groups were 80.28, 103.60, and
92.76 pg/ml, respectively. The difference between the groups was statistically nonsignificant
[Figure 5].
Figure 5: Increased mean levels of salivary secretory leukocyte protease inhibitor
in oral potentially malignant disorders and oral squamous cell carcinoma as compared
to controls
Discussion
SLPI is an 11.7 kDa highly basic nonglycosylated protein. SLPI contributes to host
mucosal defense of oral, nasopharyngeal, genital, and respiratory tissues. Salivary
SLPI was initially studied for its anti-HIV activity.[7] Studies have shown a protective function for SLPI against human papillomavirus (HPV)
infection of the oral cavity.[8] It has a major role in protecting tissues against the harmful effects of inflammation.[3] It also has bactericidal and fungicidal properties.[3] Even though the anti-HIV property of SLPI is well established, there appears to
be no role for SLPI in other retroviral infections.[3]
Recently, SLPI is being investigated for its role in a variety of cancers such as
ovarian, breast, colorectal, pancreatic, and oral cancer.[6]
[9]
[10]
[11]
[12]
[13] It has been implicated in cancer progression and metastasis.[13] Cordes et al.[14] demonstrated that lower SLPI was associated with metastatic HNSCC. Devoogdt et al.[15] studied the role of SLPI in lung tumor progression and found that SLPI had definite
promalignant activity. The authors concluded that this property of SLPI was due to
its protease inhibiting activity rather than its property of increasing cell proliferation.[15] Zhang et al.[13] proved that elevated SLPI in pancreatic adenocarcinoma was associated with cell
proliferation and growth of tumor.[13] Decreased SLPI is believed to promote spread of the tumor by allowing degradation
of tissues by proteases produced by tumor cells.[8]
Quabius et al.[16] found that decreased SLPI was associated with HPV-induced OSCC, while smoking-induced
OSCC had higher levels of SLPI and absence of HPV positivity. Quabius et al.[16] proposed that SLPI protects against HPV-induced OSCC in nonsmokers. SLPI increases
in response to smoking and thus high levels are seen in smokers.[8] Pierce Campbell et al. found that salivary SLPI levels were inversely correlated with age.[17]
The present study evaluated the serum and salivary SLPI in participants diagnosed
with OPMD and OSCC to determine its role in the progression of precancer to cancer.
Male preponderance was seen in both OPMD and OSCC groups which is similar to previous
literature reports.[18]
[19]
OPMD were diagnosed from the third to fifth decade of life, whereas OSCC occurred
predominantly in the 7th decade. This is in conformity with previous studies which have stated that OSCC occurs
later due to accumulation of age-related mutagenic and epigenetic effects as also
because most OSCC is believed to arise from pre-existing OPMD.[20]
OPMDs are a group of oral conditions which include leukoplakia, erythroplakia, erosive
lichen planus, and oral submucous fibrosis. The buccal mucosa was the most common
site of OSCC in the present study. Previous studies conducted in India have reported
similar findings.[20] Smoking was the etiological factor in most cases with leukoplakia, while gutkha
chewing was associated with oral submucous fibrosis. Betel quid, a form of chewing
tobacco which is composed of areca nut, lime, tobacco, and betel leaf, was the etiological
agent in most cases of OSCC. According to the World Health Organization, 34% of the
population uses beedis, 31% uses cigarettes, 19% uses smokeless tobacco, 9% uses hookah,
and 7% use other types of tobacco, while the Cancer Patients Aid Association of India
found that cigarettes accounted for 20%, beedis 40%, and chewable tobacco accounted
for 40% of tobacco use in India.[21]
A mass spectrometry-based quantitative shotgun proteomics study of oral brush biopsy
specimens found that SLPI was progressively decreased in patients with OPMD and OSCC
as compared to healthy controls.[6] This decrease was correlated with the development of OPMD and OSCC. They further
suggested that treatment with SLPI could have preventive effects.[6] The present study compared the serum and salivary levels of SLPI in patients with
OPMD, OSCC, and healthy controls.
The present study found that serum levels of SLPI increased progressively from controls
to OPMD patients to OSCC patients. The difference between the groups was not statistically
significant. We were unable to correlate these results with literature reports since
previous studies on serum SLPI levels in OPMD and OSCC are not available.
Salivary levels were higher in OPMD and OSCC as compared to controls. However, the
difference between the groups was not significant. Wen et al.[22] found that SLPI was inversely correlated with tumor invasion in the head-and-neck
OSCC and has implications for prognosis and treatment planning. However, in ovarian
cancers, SLPI was found to be upregulated.[11]
[22] Pierce Campbell et al.[5] evaluated salivary SLPI and found that higher SLPI levels among smokers showed a
higher risk for development of OSCC.
Decreased SLPI levels have been associated previously with a higher risk of tissue
invasion. The present study showed higher levels of SLPI in OPMD and OSCC. These findings
could probably be explained by the anti-inflammatory properties of SLPI which led
to increased levels in saliva and serum. Although the results in the present study
were not statistically significant, there was progressive increase in serum SLPI from
controls to OPMD to OSCC indicating that serum SLPI could have a role in evaluating
the progression of OPMD to OSCC.
The major limitation of the study is the sample size. Our study was more in the nature
of a pilot study, and therefore, studies with larger sample size are required before
definitive conclusions can be drawn.
Conclusion
SLPI is believed to have a role in tumor progression. It also has potential roles
in prognosis and treatment planning in OSCC. Studies on SLPI in different types of
tumors have provided conflicting results. Salivary and serum levels of SLPI have not
been evaluated so far. Our study aimed to determine the role of SLPI as a prognostic
factor in the progression of OPMD to OSCC. The present study showed increase in serum
SLPI in cases with OPMD and OSCC as compared to controls. Salivary levels of SLPI
were also increased in OPMD and OSCC as compared to controls. Our results may reflect
the anti-inflammatory properties of SLPI. These preliminary results appear to indicate
that increasing levels of SLPI in the serum may indicate progression to OSCC. However,
since the sample size of the present study was small, large-scale studies are required
before any definitive results.
