Journal of Investigative and Clinical Dentistry (2014), 5, 1–6
ORIGINAL ARTICLE Oral Pathology
Analysis of salivary secretor status in patients with oral submucous fibrosis: a case-control study Kaveri Hallikeri1, Ravichandra Udupa1, Krutika Guttal2 & Venktesh Naikmasur2 1 Department of Oral and Maxillofacial Pathology, Sri Dharamasthala Manjunatheshwara College of Dental Sciences & Hospital, Dharwad, Karnataka, India 2 Department of Oral Diagnosis and Radiology, Sri Dharamasthala Manjunatheshwara College of Dental Sciences & Hospital, Dharwad, Karnataka, India
Keywords blood group, carcinoma, oral submucous fibrosis, saliva, secretor status. Correspondence Dr Kaveri Hallikeri, Department of Oral and Maxillofacial Pathology, S.D.M. College of Dental Sciences and Hospital, Sattur Dharwad, Karnataka 580009, India. Tel: +91-0836-2468142 Fax: +91-0836-2467612 Email: [email protected] Received 11 October 2013; accepted 4 March 2014. doi: 10.1111/jicd.12100
Abstract Aim: Many individuals have various tobacco-related habits, yet only some develop clinical manifestation of lesions. This raises the question of whether there any inherent or host risk factors involved in the pathogenesis which need to be further investigated. The aim of the present study was to analyze the ABO antigen, secretor status, and blood groups of patients. Methods: The study consisted of 99 participants, with 33 patients allocated to three groups: (a) patients with a tobacco-related habit and oral submucous fibrosis (OSF); (b) patients with a tobacco-related habit, but no lesions; and (c) healthy controls. A total of 1 mL unstimulated saliva was collected in a sterile test tube, and the Wiener agglutination test was performed to analyze the ABO antigen in all three groups. Results: All of the OSF patients were non-secretors, whereas 84.8% were nonsecretors in the group of individuals with habits as compared to 15.2% in the healthy group. A statistically-significant difference was observed between the OSF and healthy groups. The patients in the OSF group were predominantly blood-group A, followed by groups O, B, and AB. Conclusion: There is a correlation between salivary secretor status and the development of OSF. Thus, non-secretors are at greater risk of and more prone to the development of oral lesions. Blood-groups A and O predominate over the B and AB blood groups.
Introduction Oral submucous fibrosis (OSF) is a high-risk precancerous condition related to chewing areca nut alone or as a component of betel quid. Approximately 600 000 000 people worldwide chew raw areca nut or in any processed form.1 In India, there is increasing addiction tendency among young people due to easy access of tobacco products, effective price changes, and marketing strategies. There are numerous commercially-prepared areca nut preparations, and gutkha is the most popular. Recent epidemiological data indicate that the number of OSF cases has increased rapidly in India from an estimated 250 000 cases in 1980 to 2 000 000 cases in 1993.2 This disease ª 2014 Wiley Publishing Asia Pty Ltd
has become a major public health issue in India, owing to its prevalence among 20–30 year olds, and its association with malignant transformation. OSF is an irreversible condition; once developed there is no effective treatment. The malignant transformation rate associated with OSF ranges from 7% to 13%.3 More than 2400 new cases of oral cancer arising from OSF are diagnosed every year in Taiwan due to the prevalent use of betel quid.4 Jian et al.5 analyzed the clinical symptoms and signs in 29 cases of OSF cases, which transformed into squamous cell carcinoma. Andakumar et al. and Ko et al.6,7 reported a malignant transformation rate of 4–30.4% in India and 12.28% in Taiwan, respectively. Recent data show that the potential of malignant transformation 1
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in OSF is much higher compared to other premalignancies.8 Therefore, early diagnosis of this potentially-malignant oral lesion is essential for better prognosis. Although biopsy is the gold standard to observe the earliest changes in the epithelium of premalignant lesions, repeat biopsies might not be feasible. To overcome these problems, the salivary secretor status would be useful. It has been reported that non-secretor individuals are more prone to develop lesions, and certain types of cancers are more prevalent in particular blood-group types. Various authors have shown the relationship between the pathogenesis of disease and secretor status, which is a possible factor influencing disease status.9,10 A higher intensity of oral disease is found among non-secretors in terms of precancerous and cancerous changes to tissues of the oral cavity. Oral disease susceptibility is reflected in the occurrence of epithelial dysplasia, which was found almost exclusively in the non-secretor group. The presence of the epithelial antigen has a protective role in infections by interfering with the binding sites either on the surface of microorganisms or to epithelial cells. Studies have shown that infection is more common in non-secretors.11,12 It has also been reported that individuals with blood-group A have a predisposition for oral cancer. The functional loss of the A enzyme in dysplasia and carcinoma has been reported, whereas in normal epithelium it is strongly positive. This is due to the downregulation of glycosyltransferase, which is involved in the biosynthesis of A and B antigens, and is associated with tumor development.13 Based on this, the present study was undertaken in order to determine secretor status and blood group in OSF patients and those with a tobacco-related habit without lesions, and healthy controls. Materials and methods Salivary secretor status was studied in 99 individuals; each group consisted of 33 patients: (a) patients with a tobacco-related habit and OSF (group I); (b) patients with a tobacco-related habit, but no lesions (group II); and (c) healthy controls (group III). Establishing secretor status in saliva The methodology followed for assessment was adapted from Vidas et al.14 A total of 1 mL non-stimulated saliva was collected from each patient and stored in a sterile glass vial. The saliva was then poured into a sterile test tube and closed with a lid. The test tube was then placed in boiling water bath for approximately 10 min to destroy enzymes. The supernatant fluid was then extracted by a centrifugal force of 1700 turns in 10 min. The Wiener agglutination test was used to establish secretor status. 2
The test serum was diluted in a salted physiological solution of 1:10 dilution. The following antiserum was then placed into test tubes marked I–IV: (a) I, one drop of saliva + one drop of antiB serum; (b) II, one drop of saliva + one drop of antiA serum; (c) one drop of physiological solution + one drop of antiB serum; and (d) one drop of physiological solution + one drop of antiA serum. After 10 min at room temperature, one drop of 2–3% of suspension A erythrocytes was added into sterile tubes II and IV, and one drop of suspension B erythrocytes into tubes I and III. All the test tubes were agitated, and left at room temperature. Readings were available after 1 h. Test tubes III and IV (controls) showed agglutination. Agglutination in tube I was as a result of the presence of substance A2 in saliva, that is, of secretor A, while the agglutination in tube II was proof of secretor B. The absence of agglutination in tubes I and II designated AB secretor, and at the same time, agglutination in tubes I and II confirmed that the person was a non-secretor. Results The type and distribution of tobacco-related habits and the detailed percentages among the study groups are outlined in Table 1. The percentage of secretor status among patients with a tobacco-related habit and OSF (group I), patients with a tobacco-related habit, but no lesions (group II), and healthy controls (group III) is shown in Figure 1. All of the patients in group I were non-secretors, whereas in group II, 84.8% were non-secretors compared to 15.2% in group III. A statistically-significant difference was observed between groups I and III. The correlation of duration and intensity of habits with secretor status could not be done due to the large difference in secretor status among the individuals in group II (correlation requires at least equal distribution of secretor status among the study group). Details of the association between study groups and blood-group type is shown in Table 2. It was also observed that OSF was more prevalent among those with blood-group A (36.36%) compared to groups O (33.33%), B (27.27%), and AB (3.03%). Table 1. Type of tobacco-related habit and distribution percentage among the study groups Habits
OSMF group
%
Habit group
%
Total
Areca nut Gutkha Pan masala Combination Total
4 20 2 7 33
12.12 60.61 6.06 21.21 100
5 6 3 19 33
15.15 18.18 9.09 57.58 100
9 26 5 26 66
OSMF, oral submucous fibrosis.
ª 2014 Wiley Publishing Asia Pty Ltd
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assess the cause of lesion development in individuals with tobacco-related habits. Based on this, in the present study, we determined the secretor status in the above-mentioned groups. A secretor is defined as a person who secretes their blood-type antigens into body fluids, such as saliva in the mouth and mucous in the digestive tract and respiratory cavity. A non-secretor puts little or none of their blood type into these same fluids.14 The blood-group antigens are genetically regulated by A, B, and H levels and secretor genes, and subsequently, blood-group antigens are expressed on erythrocytes and oral epithelial cells. These cell surface carbohydrates are involved in cell-to-cell and cell-to-substrate interactions, and changes in cell surface carbohydrates are reflected in cellular behavior.13 The non-secretor status of a patient could influence the development of systemic and oral manifestations. The ability to secrete blood-group antigens plays an important role in the natural resistance of organism to infection. Increased susceptibility to microbial infection and a higher occurrence of urinary tract infection have been reported in non-secretor individuals.17–19 Clark et al.18 reported that non-secretors of the ABO group antigen have an increased risk of developing peptic ulcers, increased susceptibility to microbial infection, and a higher occurrence of urinary tract infections. The ability to secrete blood-group antigens plays an important role in the natural resistance of organisms to infection. In a study of 100 cases of leukoplakia, most of the non-secretors were in the diseased group. Oral candidiasis among non-secretors is also frequently. The non-secretor status facilitates the prolonged candidal adhesion to mucosal sites.20 In the present study, all the individuals in group I were non-secretors; 84.8% of the individuals in groups II were non-secretors and 15.2% were secretors. In contrast, 84.8% of the individuals in group III were secretors, and only 15.2% were non-secretors. We hypothesize that non-secretors might have an added effect in the pathogenesis OSF, as all of the OSF patients were non-secretors, and non-secretor individuals with a tobacco-related habit are more likely to be prone
Figure 1. Secretor status among the study groups. Group I, patients with a tobacco-related habit and oral submucous fibrosis; group II, patients with a tobacco-related habit, but no lesions; and group II, healthy controls. ( ) Non Secretor; ( ) Secretor.
Discussion The prevalence rate of OSF in India ranges from 0.2% to 1.2%. Individuals between 20 and 40 years of age are most commonly affected, but individuals aged between 2 and 89 years have also been reported to be affected.2 In the present study, OSF was more prevalent in the second decade of life, and was found to be strongly associated with a gutkha habit (20/33) of 1–10 years. Gutkha chewing is a popular habit, with potential links to the occurrence of oral cancer.15 Other tobacco-related habits included betel nut chewing (4/33), pan masala (2/33), and mixed habits (7/33). The components of gutkha, such as areca nut extract, are known to induce mutagenic and genotoxic effects, in addition to inducing preneoplastic, as well as neoplastic lesions of the oral cavity. Reactive oxygen species produced during auto-oxidation of areca nut polyphenols are crucial in the initiation and promotion of oral cancer.16 The development of disease in patients with tobaccorelated habits is unpredictable. Inherent or host risk factors are most likely responsible for the development of OSF. A detailed analysis of these factors is required to
Table 2. Blood groups among the study groups Blood
OSMF group
%
Habit group
%
Control group
%
Total
%
A B AB O Total
12 9 1 11 33
36.36 27.27 3.03 33.33 100.0
14 8 2 9 33
42.42 24.24 6.06 27.27 100
17 10 4 2 33
51.52 30.30 12.12 6.06 100
27 43 7 22 99
27.27 43.43 7.07 22.22 100.00
OSMF, oral submucous fibrosis.
ª 2014 Wiley Publishing Asia Pty Ltd
3
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to developing oral lesions. These patients would require regular follow up to diagnose the disease as early as possible to provide the best treatment. To the best of our knowledge, there have been no published studies comparing secretor status in individuals with tobacco-related habits; thus, ours is the first to estimate secretor status in these individuals. Previous studies have correlated secretor status and the severity of the disease, as in leukoplakia.21 Secretor status is well documented in oral cancer and premalignant lesions, such as like leukoplakia, and fungal infections, such as candidiasis.11,22 Vidas et al.14 studied the secretor status in patients with pathological changes of the oral mucosa in lichen ruber planus, erosivos, and leukoplakia. Of the 61 patients, only 15 subjects were non-secretors. The patients with a non-secretor status had severe degree of the disease, whereas a mild degree of the disease was recorded for the secretors. Secretor status has been linked to the potentially-malignant oral lesions of candidal leukoplakia; the non-secretor status facilitates the prolonged adhesion of Candida to the mucosa, and in addition, epithelial dysplastic changes seen exclusively in non-secretors.23 The prevalence of Candida in OSF has been reported to range from 40% to 63.6%, with Candida albicans the predominant species isolated. These carry significant risks of malignant transformation. The non-secretor saliva not only fails to prevent attachment of Candida, but also promotes the binding of Candida to tissue. With potentially-malignant disease, the superadded candidal infection needs special attention, as it is postulated that nitrosamine compounds produced by Candida species might directly, or together with other chemical carcinogens, activate specific proto-oncogenes, and thus facilitate the development of malignant lesion.24–26 Cervoic et al. studied the presence of the ABO antigen in the saliva of patients with oral cancer, and found that 12 of 57 patients were non-secretors of blood-group antigens in saliva. There was no significant difference between the experimental and control groups.23 The interesting molecular basis is that epithelial cells in non-secretors do not have an active flucosyl-transferase, the activity of which is associated with the expression of the ABO blood-group oligosaccharides. Human carcinoma colon cell line has shown the loss of AB glycosyltransferse, which can enhance the malignancy of the cell lines. Molecular studies of bladder cancer have shown that the loss of expression of A-transferase is related to the loss of chromosome at 9q34, which is a locus for the ABO gene.27 The distribution of ABO blood groups in and around Bangalore has been reported to be 9.81% for the O group, 29.95% for the B group, 23.85% for the A group, and 6.37% for the AB group.28 The South Indian study by Das et al.29 showed that group O is the most predom4
inant, followed by groups B and A. Another South Indian study, conducted in the Chittoor district of Andhra Pradesh, India, also showed a similar pattern of blood-group distribution.30 In the present study, it was also observed that OSF was prevalent in patients with blood-group A (36.36%), followed by groups O (33.33%), B (27.27%), and AB (3.03%); however, these correlations were not statistically significant. There are no data available regarding the blood group and incidence of OSF occurrence in these individuals. The present study is a pilot study and the first of its kind to correlate secretor status in individuals with OSF. However, future studies with larger sample sizes are required to confirm the present findings. The deletion of blood-group antigen A from premalignant oral mucosal lesions is regarded as an example of impaired synthesis of cell surface carbohydrates associated with malignant change. This is due to the accumulation of precursors or carbohydrates, that is, a H antigen in premalignant lesions. The loss of the A antigen with the accumulation of the H antigen suggests that the activity of enzymes, which converts the H antigen to A, decreases in premalignant lesions. In an immunohistochemistry study, it was revealed that the H antigen accumulates in the cell membrane of premalignant lesions with dysplasia, and is irregularly distributed in oral squamous cell carcinoma. This difference in distribution can be beneficial for the early detection of cancer.21,31 Bryne et al.32 reported that the loss of H-antigen expression at the invasive front was well correlated with tumor development and the histologic grade of tumor malignancy, rather than H-antigen staining in the entire tumor. In a prospective study by Lamey et al.,33 which included 100 Sri Lankan patients with oral cancer and controls, secretor status appeared to be an associated risk marker for the development of lesions. In a hospitalbased study by Jaleel et al., blood groups were recorded in oral cancer patients. It was concluded that people with blood-group A were 1.46 times at a greater risk of developing oral carcinoma, followed by those with blood-groups B, AB, and O.12 There are various studies reporting that individuals with blood-group A are prone to developing carcinomas. In their studies, Raghavan et al. and Toto and Nadimi34,35 also reported the increased susceptibility of blood-group A to oral cancer. Conclusion Non-secretor status in an individual with a tobaccorelated habit indicates that there is alteration in the epithelial surface carbohydrates, which marks the changes in the epithelium and makes oral mucosa prone to disease development. The present study demonstrated that ª 2014 Wiley Publishing Asia Pty Ltd
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non-secretor individuals with a tobacco-related habit developed oral lesions, thus patients with a history of betel quid or areca nut habits in any form can be considered to be at risk. Secretor status can be part of routine investigations to assess the disease status and whether an individual’s susceptibility manifests the disease. There is a need to analyze which particular groups of individuals are more prone to develop OSF using larger sample sizes.
References 1 Gupta PC, Warnakulasuriya S. Global epidemiology of areca nut usage. Addict Biol 2002; 7: 77–83. 2 Ranganathan K, Umadevi M, Joshua E, Kirankumar K, Saraswathi TR. Oral submucous fibrosis: a case control study in Chennai South India. J Oral Pathol Med 2004; 33: 274–7. 3 Tilkarartne WM, Klinikowski MF, Saku T, Peters TJ, Warnakulasuriya S. Oral submucous fibrosis: review on aetiology and pathogenesis. Oral Oncol 2006; 42: 561–8. 4 Li N, Jian X, Hu Y, Xu C, Yao Z, Zhong X. Discovery of novel biomarkers in oral submucous fibrosis by microarray analysis. Cancer Epidemiol Biomarkers Prev 2008; 17: 2249–59. 5 Jian XC, Shen ZH, Liu SF. Oral submucous fibrosis-case reports. J Clinical Stomatol 1985; 1: 12–3. 6 Andakumar A, Thimmasethy KT, Sreeramareddy NM. A populationbased case-control investigation on cancers of the oral cavity in Bangalore, India. Br J Cancer 1990; 62: 847– 51. 7 Ko YC, Huang YL, Lee CH. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995; 24: 450–3. 8 Gupta MK, Mhaske S, Ragavendra R, Imtiyaz. Oral submucous fibrosis —current concepts in etiopathogenesis. People’s J Sci Res 2008; 1: 39–44. 9 Sheinfield J, Shaeffer AJ, CordonCordo C, Rogatko A, Fair WR. Association of Lewis blood group phenotype with recurrent urinary tract infections in women. N Engl J Med 1989; 320: 773–7.
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Acknowledgments The authors would like to thank Mrs Ambika Patil (Sri Dharamasthala Manjunatheshwara College of Dental Sciences & Hospital, Dharwad. Karnataka, India) for her assistance in examining the salivary secretary status, and Dr Arvind M. Yeri (Sri Dharamasthala Manjunatheshwara College of Dental Sciences & Hospital, Dharwad. Karnataka, India) for permitting us to utilize the laboratory facilities.
10 Lomberg H, Hollstrom J, Jodal U, Svanborg-Eden C.. Secretor status and renal scarring in girls with recurrent pyelonephritis. Microbiol Immunol 1991; 35: 371–4. 11 Lamey PJ, Darwazeh AM, Muirhead J, Rennie JS, Samaranayake LP, MacFarlane TW. Chronic hyperplastic candidosis and secretor status. J Oral Pathol Med 1991; 20: 64–7. 12 Jaleel BF, Nagrajappa R. Relationship between ABO blood groups and oral cancer. Indian J Dent Res 2012; 23: 7–10. 13 Dabelsten E, Gao S. ABO blood group antigens in oral cancer. J Dent Res 2004; 84: 21–8. 14 Vidas I, Delajlija M, Temmer-Vuksan B, Stipetic-Mravak M, Cindric N, Maricıc D. Examining the secretor status in the saliva of patients with oral pre-cancerous lesions. J Oral Rehabil 1999; 26: 177–82. 15 Javed F, Chotai M, Mehmood A, Almas K. Oral mucosal disorders associated with habitual gutka usage: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 857–64. 16 Jeng JH, Chang MC, Hahn LJ. Role of areca nut in betel quid associated chemical carcinogenesis: current awareness and future perspectives. Oral Oncol 2001; 37: 477–92. 17 Kinane DF, Blackwell CC, Brettle RP, Weir DM, Winstanley FP, Elton RA. ABO blood group secretor state and susceptibility to recurrent urinary tract infection in women. Br Med J 1982; 285: 423–4. 18 Clark CA, Price-Evans DA, MacConnel DA, Sheppard PM. Secretion of blood group antigen and peptic ulcer. Br Med J 1959; 1: 603–7.
19 Lamey PJ, Darwazeh AMG, Muirhead J, Rennie JS, Samaranayake LP, MacFarlane TW. Chronic hyperplastic candidosis and secretory status. J Oral Pathol Med 1991; 20: 67–70. 20 Pourazar A, Joshi SR, Sathe MS, Advani SH, Bhatia HM. Antigens in leukoplakia. Indian J Med Res 1986; 89: 83–8. 21 Dabelsteen E, Vedtotte P, Hakomori S, Young WW Jr. Accumulation of blood group antigen precursor in oral premalignant lesions. Cancer Res 1983; 43: 1451–4. 22 Dabelsteen E, Clausen H, Mandel U. Aberrant glycosylation in oral malignant and premalignant lesions. J Oral Pathol Med 1991; 20: 361–8. 23 Cerovic R, Juretic M, Balen S, Belusic M, Caser L, Rogic M. Examining the presence of ABO(H) antigens of blood types in the saliva of patients with oral cancer. Coll Antropol 2008; 32: 509–12. 24 Ariyawardana GA, Panagoda J, Fernando HN, Ellepola AN, Tilakaratne WM, Samaranayake LP. Oral submucous fibrosis and oral yeast carriage – a case control study in Sri Lankan patients. Mycoses 2005; 50: 116–20. 25 Koneru A, Hallikeri K, Shubhda C, Naikmasur VG, Kulkarni RD. Comparative study of Candida in oral submucous fibrosis and healthy in individuals. Rev Odonto Cienc 2011; 26: 71–6. 26 Fiueld EA, Field JK, Martin MV. Does Candida have a role in oral epithelial neoplasia? J Med Vet Mycol 1989; 27: 277–94. 27 Orlow I, Lacombe L, Pellicer I. Phenotypic and genotypic characterization of the histoblood group ABO
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(H) in primary bladder tumors. Int J Cancer 1998; 75: 819–24. 28 Periyavan S, Sangeetha SK, Marimuthu P, Manjunath BK, Seema DM. Distribution of ABO and Rhesus-D blood groups in and around Bangalore. Asian J Transfus Sci 2010; 4: 41–2. 29 Das PK, Nair SC, Harris VK et al. Distribution of ABO and Rh-D blood groups among blood donors in a tertiary care centre in South India. Trop Doct 2001; 31: 47–8. 30 Reddy KS, Sudha G. ABO and Rh(D) blood groups among the Desuri Red-
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dis of Chittur District, Andhra Pradesh. Anthrapol 2009; 11: 237–8. 31 Biondi C, Campi C, Escovich L, Garcia B, Racca A, Cotorruelo C. Loss of A, B, and H antigens in oral cancer. Immunologica 2008; 27: 127–31. 32 Bryne M, Thrane PS, Dabelsteen E. Loss of expression of blood group antigen H is associated with cellular invasion and spread of oral squamous cell carcinoma. Cancer 1991; 67: 613– 8. 33 Lamey PJ, Douglas PS, Napier SS. Secretor status and oral cancer. Br J Oral Maxillofac Surg 1994; 32: 214–7.
34 Raghavan VM, Bailoor DN, Jhansirani P. Incidence of ABO blood groups in oral cancer in South Kanara district. J Indian Dent Assoc 1986; 58: 305–8. 35 Toto PD, Nadimi H. Co-expression of cytokeratins, involucrin and blood group antigens in oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1990; 70: 75–80.
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ORIGINAL ARTICLE Oral Pathology
Analysis of salivary secretor status in patients with oral submucous fibrosis: a case-control study Kaveri Hallikeri1, Ravichandra Udupa1, Krutika Guttal2 & Venktesh Naikmasur2 1 Department of Oral and Maxillofacial Pathology, Sri Dharamasthala Manjunatheshwara College of Dental Sciences & Hospital, Dharwad, Karnataka, India 2 Department of Oral Diagnosis and Radiology, Sri Dharamasthala Manjunatheshwara College of Dental Sciences & Hospital, Dharwad, Karnataka, India
Keywords blood group, carcinoma, oral submucous fibrosis, saliva, secretor status. Correspondence Dr Kaveri Hallikeri, Department of Oral and Maxillofacial Pathology, S.D.M. College of Dental Sciences and Hospital, Sattur Dharwad, Karnataka 580009, India. Tel: +91-0836-2468142 Fax: +91-0836-2467612 Email: [email protected] Received 11 October 2013; accepted 4 March 2014. doi: 10.1111/jicd.12100
Abstract Aim: Many individuals have various tobacco-related habits, yet only some develop clinical manifestation of lesions. This raises the question of whether there any inherent or host risk factors involved in the pathogenesis which need to be further investigated. The aim of the present study was to analyze the ABO antigen, secretor status, and blood groups of patients. Methods: The study consisted of 99 participants, with 33 patients allocated to three groups: (a) patients with a tobacco-related habit and oral submucous fibrosis (OSF); (b) patients with a tobacco-related habit, but no lesions; and (c) healthy controls. A total of 1 mL unstimulated saliva was collected in a sterile test tube, and the Wiener agglutination test was performed to analyze the ABO antigen in all three groups. Results: All of the OSF patients were non-secretors, whereas 84.8% were nonsecretors in the group of individuals with habits as compared to 15.2% in the healthy group. A statistically-significant difference was observed between the OSF and healthy groups. The patients in the OSF group were predominantly blood-group A, followed by groups O, B, and AB. Conclusion: There is a correlation between salivary secretor status and the development of OSF. Thus, non-secretors are at greater risk of and more prone to the development of oral lesions. Blood-groups A and O predominate over the B and AB blood groups.
Introduction Oral submucous fibrosis (OSF) is a high-risk precancerous condition related to chewing areca nut alone or as a component of betel quid. Approximately 600 000 000 people worldwide chew raw areca nut or in any processed form.1 In India, there is increasing addiction tendency among young people due to easy access of tobacco products, effective price changes, and marketing strategies. There are numerous commercially-prepared areca nut preparations, and gutkha is the most popular. Recent epidemiological data indicate that the number of OSF cases has increased rapidly in India from an estimated 250 000 cases in 1980 to 2 000 000 cases in 1993.2 This disease ª 2014 Wiley Publishing Asia Pty Ltd
has become a major public health issue in India, owing to its prevalence among 20–30 year olds, and its association with malignant transformation. OSF is an irreversible condition; once developed there is no effective treatment. The malignant transformation rate associated with OSF ranges from 7% to 13%.3 More than 2400 new cases of oral cancer arising from OSF are diagnosed every year in Taiwan due to the prevalent use of betel quid.4 Jian et al.5 analyzed the clinical symptoms and signs in 29 cases of OSF cases, which transformed into squamous cell carcinoma. Andakumar et al. and Ko et al.6,7 reported a malignant transformation rate of 4–30.4% in India and 12.28% in Taiwan, respectively. Recent data show that the potential of malignant transformation 1
K. Hallikeri et al.
Salivary secretory status
in OSF is much higher compared to other premalignancies.8 Therefore, early diagnosis of this potentially-malignant oral lesion is essential for better prognosis. Although biopsy is the gold standard to observe the earliest changes in the epithelium of premalignant lesions, repeat biopsies might not be feasible. To overcome these problems, the salivary secretor status would be useful. It has been reported that non-secretor individuals are more prone to develop lesions, and certain types of cancers are more prevalent in particular blood-group types. Various authors have shown the relationship between the pathogenesis of disease and secretor status, which is a possible factor influencing disease status.9,10 A higher intensity of oral disease is found among non-secretors in terms of precancerous and cancerous changes to tissues of the oral cavity. Oral disease susceptibility is reflected in the occurrence of epithelial dysplasia, which was found almost exclusively in the non-secretor group. The presence of the epithelial antigen has a protective role in infections by interfering with the binding sites either on the surface of microorganisms or to epithelial cells. Studies have shown that infection is more common in non-secretors.11,12 It has also been reported that individuals with blood-group A have a predisposition for oral cancer. The functional loss of the A enzyme in dysplasia and carcinoma has been reported, whereas in normal epithelium it is strongly positive. This is due to the downregulation of glycosyltransferase, which is involved in the biosynthesis of A and B antigens, and is associated with tumor development.13 Based on this, the present study was undertaken in order to determine secretor status and blood group in OSF patients and those with a tobacco-related habit without lesions, and healthy controls. Materials and methods Salivary secretor status was studied in 99 individuals; each group consisted of 33 patients: (a) patients with a tobacco-related habit and OSF (group I); (b) patients with a tobacco-related habit, but no lesions (group II); and (c) healthy controls (group III). Establishing secretor status in saliva The methodology followed for assessment was adapted from Vidas et al.14 A total of 1 mL non-stimulated saliva was collected from each patient and stored in a sterile glass vial. The saliva was then poured into a sterile test tube and closed with a lid. The test tube was then placed in boiling water bath for approximately 10 min to destroy enzymes. The supernatant fluid was then extracted by a centrifugal force of 1700 turns in 10 min. The Wiener agglutination test was used to establish secretor status. 2
The test serum was diluted in a salted physiological solution of 1:10 dilution. The following antiserum was then placed into test tubes marked I–IV: (a) I, one drop of saliva + one drop of antiB serum; (b) II, one drop of saliva + one drop of antiA serum; (c) one drop of physiological solution + one drop of antiB serum; and (d) one drop of physiological solution + one drop of antiA serum. After 10 min at room temperature, one drop of 2–3% of suspension A erythrocytes was added into sterile tubes II and IV, and one drop of suspension B erythrocytes into tubes I and III. All the test tubes were agitated, and left at room temperature. Readings were available after 1 h. Test tubes III and IV (controls) showed agglutination. Agglutination in tube I was as a result of the presence of substance A2 in saliva, that is, of secretor A, while the agglutination in tube II was proof of secretor B. The absence of agglutination in tubes I and II designated AB secretor, and at the same time, agglutination in tubes I and II confirmed that the person was a non-secretor. Results The type and distribution of tobacco-related habits and the detailed percentages among the study groups are outlined in Table 1. The percentage of secretor status among patients with a tobacco-related habit and OSF (group I), patients with a tobacco-related habit, but no lesions (group II), and healthy controls (group III) is shown in Figure 1. All of the patients in group I were non-secretors, whereas in group II, 84.8% were non-secretors compared to 15.2% in group III. A statistically-significant difference was observed between groups I and III. The correlation of duration and intensity of habits with secretor status could not be done due to the large difference in secretor status among the individuals in group II (correlation requires at least equal distribution of secretor status among the study group). Details of the association between study groups and blood-group type is shown in Table 2. It was also observed that OSF was more prevalent among those with blood-group A (36.36%) compared to groups O (33.33%), B (27.27%), and AB (3.03%). Table 1. Type of tobacco-related habit and distribution percentage among the study groups Habits
OSMF group
%
Habit group
%
Total
Areca nut Gutkha Pan masala Combination Total
4 20 2 7 33
12.12 60.61 6.06 21.21 100
5 6 3 19 33
15.15 18.18 9.09 57.58 100
9 26 5 26 66
OSMF, oral submucous fibrosis.
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assess the cause of lesion development in individuals with tobacco-related habits. Based on this, in the present study, we determined the secretor status in the above-mentioned groups. A secretor is defined as a person who secretes their blood-type antigens into body fluids, such as saliva in the mouth and mucous in the digestive tract and respiratory cavity. A non-secretor puts little or none of their blood type into these same fluids.14 The blood-group antigens are genetically regulated by A, B, and H levels and secretor genes, and subsequently, blood-group antigens are expressed on erythrocytes and oral epithelial cells. These cell surface carbohydrates are involved in cell-to-cell and cell-to-substrate interactions, and changes in cell surface carbohydrates are reflected in cellular behavior.13 The non-secretor status of a patient could influence the development of systemic and oral manifestations. The ability to secrete blood-group antigens plays an important role in the natural resistance of organism to infection. Increased susceptibility to microbial infection and a higher occurrence of urinary tract infection have been reported in non-secretor individuals.17–19 Clark et al.18 reported that non-secretors of the ABO group antigen have an increased risk of developing peptic ulcers, increased susceptibility to microbial infection, and a higher occurrence of urinary tract infections. The ability to secrete blood-group antigens plays an important role in the natural resistance of organisms to infection. In a study of 100 cases of leukoplakia, most of the non-secretors were in the diseased group. Oral candidiasis among non-secretors is also frequently. The non-secretor status facilitates the prolonged candidal adhesion to mucosal sites.20 In the present study, all the individuals in group I were non-secretors; 84.8% of the individuals in groups II were non-secretors and 15.2% were secretors. In contrast, 84.8% of the individuals in group III were secretors, and only 15.2% were non-secretors. We hypothesize that non-secretors might have an added effect in the pathogenesis OSF, as all of the OSF patients were non-secretors, and non-secretor individuals with a tobacco-related habit are more likely to be prone
Figure 1. Secretor status among the study groups. Group I, patients with a tobacco-related habit and oral submucous fibrosis; group II, patients with a tobacco-related habit, but no lesions; and group II, healthy controls. ( ) Non Secretor; ( ) Secretor.
Discussion The prevalence rate of OSF in India ranges from 0.2% to 1.2%. Individuals between 20 and 40 years of age are most commonly affected, but individuals aged between 2 and 89 years have also been reported to be affected.2 In the present study, OSF was more prevalent in the second decade of life, and was found to be strongly associated with a gutkha habit (20/33) of 1–10 years. Gutkha chewing is a popular habit, with potential links to the occurrence of oral cancer.15 Other tobacco-related habits included betel nut chewing (4/33), pan masala (2/33), and mixed habits (7/33). The components of gutkha, such as areca nut extract, are known to induce mutagenic and genotoxic effects, in addition to inducing preneoplastic, as well as neoplastic lesions of the oral cavity. Reactive oxygen species produced during auto-oxidation of areca nut polyphenols are crucial in the initiation and promotion of oral cancer.16 The development of disease in patients with tobaccorelated habits is unpredictable. Inherent or host risk factors are most likely responsible for the development of OSF. A detailed analysis of these factors is required to
Table 2. Blood groups among the study groups Blood
OSMF group
%
Habit group
%
Control group
%
Total
%
A B AB O Total
12 9 1 11 33
36.36 27.27 3.03 33.33 100.0
14 8 2 9 33
42.42 24.24 6.06 27.27 100
17 10 4 2 33
51.52 30.30 12.12 6.06 100
27 43 7 22 99
27.27 43.43 7.07 22.22 100.00
OSMF, oral submucous fibrosis.
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to developing oral lesions. These patients would require regular follow up to diagnose the disease as early as possible to provide the best treatment. To the best of our knowledge, there have been no published studies comparing secretor status in individuals with tobacco-related habits; thus, ours is the first to estimate secretor status in these individuals. Previous studies have correlated secretor status and the severity of the disease, as in leukoplakia.21 Secretor status is well documented in oral cancer and premalignant lesions, such as like leukoplakia, and fungal infections, such as candidiasis.11,22 Vidas et al.14 studied the secretor status in patients with pathological changes of the oral mucosa in lichen ruber planus, erosivos, and leukoplakia. Of the 61 patients, only 15 subjects were non-secretors. The patients with a non-secretor status had severe degree of the disease, whereas a mild degree of the disease was recorded for the secretors. Secretor status has been linked to the potentially-malignant oral lesions of candidal leukoplakia; the non-secretor status facilitates the prolonged adhesion of Candida to the mucosa, and in addition, epithelial dysplastic changes seen exclusively in non-secretors.23 The prevalence of Candida in OSF has been reported to range from 40% to 63.6%, with Candida albicans the predominant species isolated. These carry significant risks of malignant transformation. The non-secretor saliva not only fails to prevent attachment of Candida, but also promotes the binding of Candida to tissue. With potentially-malignant disease, the superadded candidal infection needs special attention, as it is postulated that nitrosamine compounds produced by Candida species might directly, or together with other chemical carcinogens, activate specific proto-oncogenes, and thus facilitate the development of malignant lesion.24–26 Cervoic et al. studied the presence of the ABO antigen in the saliva of patients with oral cancer, and found that 12 of 57 patients were non-secretors of blood-group antigens in saliva. There was no significant difference between the experimental and control groups.23 The interesting molecular basis is that epithelial cells in non-secretors do not have an active flucosyl-transferase, the activity of which is associated with the expression of the ABO blood-group oligosaccharides. Human carcinoma colon cell line has shown the loss of AB glycosyltransferse, which can enhance the malignancy of the cell lines. Molecular studies of bladder cancer have shown that the loss of expression of A-transferase is related to the loss of chromosome at 9q34, which is a locus for the ABO gene.27 The distribution of ABO blood groups in and around Bangalore has been reported to be 9.81% for the O group, 29.95% for the B group, 23.85% for the A group, and 6.37% for the AB group.28 The South Indian study by Das et al.29 showed that group O is the most predom4
inant, followed by groups B and A. Another South Indian study, conducted in the Chittoor district of Andhra Pradesh, India, also showed a similar pattern of blood-group distribution.30 In the present study, it was also observed that OSF was prevalent in patients with blood-group A (36.36%), followed by groups O (33.33%), B (27.27%), and AB (3.03%); however, these correlations were not statistically significant. There are no data available regarding the blood group and incidence of OSF occurrence in these individuals. The present study is a pilot study and the first of its kind to correlate secretor status in individuals with OSF. However, future studies with larger sample sizes are required to confirm the present findings. The deletion of blood-group antigen A from premalignant oral mucosal lesions is regarded as an example of impaired synthesis of cell surface carbohydrates associated with malignant change. This is due to the accumulation of precursors or carbohydrates, that is, a H antigen in premalignant lesions. The loss of the A antigen with the accumulation of the H antigen suggests that the activity of enzymes, which converts the H antigen to A, decreases in premalignant lesions. In an immunohistochemistry study, it was revealed that the H antigen accumulates in the cell membrane of premalignant lesions with dysplasia, and is irregularly distributed in oral squamous cell carcinoma. This difference in distribution can be beneficial for the early detection of cancer.21,31 Bryne et al.32 reported that the loss of H-antigen expression at the invasive front was well correlated with tumor development and the histologic grade of tumor malignancy, rather than H-antigen staining in the entire tumor. In a prospective study by Lamey et al.,33 which included 100 Sri Lankan patients with oral cancer and controls, secretor status appeared to be an associated risk marker for the development of lesions. In a hospitalbased study by Jaleel et al., blood groups were recorded in oral cancer patients. It was concluded that people with blood-group A were 1.46 times at a greater risk of developing oral carcinoma, followed by those with blood-groups B, AB, and O.12 There are various studies reporting that individuals with blood-group A are prone to developing carcinomas. In their studies, Raghavan et al. and Toto and Nadimi34,35 also reported the increased susceptibility of blood-group A to oral cancer. Conclusion Non-secretor status in an individual with a tobaccorelated habit indicates that there is alteration in the epithelial surface carbohydrates, which marks the changes in the epithelium and makes oral mucosa prone to disease development. The present study demonstrated that ª 2014 Wiley Publishing Asia Pty Ltd
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non-secretor individuals with a tobacco-related habit developed oral lesions, thus patients with a history of betel quid or areca nut habits in any form can be considered to be at risk. Secretor status can be part of routine investigations to assess the disease status and whether an individual’s susceptibility manifests the disease. There is a need to analyze which particular groups of individuals are more prone to develop OSF using larger sample sizes.
References 1 Gupta PC, Warnakulasuriya S. Global epidemiology of areca nut usage. Addict Biol 2002; 7: 77–83. 2 Ranganathan K, Umadevi M, Joshua E, Kirankumar K, Saraswathi TR. Oral submucous fibrosis: a case control study in Chennai South India. J Oral Pathol Med 2004; 33: 274–7. 3 Tilkarartne WM, Klinikowski MF, Saku T, Peters TJ, Warnakulasuriya S. Oral submucous fibrosis: review on aetiology and pathogenesis. Oral Oncol 2006; 42: 561–8. 4 Li N, Jian X, Hu Y, Xu C, Yao Z, Zhong X. Discovery of novel biomarkers in oral submucous fibrosis by microarray analysis. Cancer Epidemiol Biomarkers Prev 2008; 17: 2249–59. 5 Jian XC, Shen ZH, Liu SF. Oral submucous fibrosis-case reports. J Clinical Stomatol 1985; 1: 12–3. 6 Andakumar A, Thimmasethy KT, Sreeramareddy NM. A populationbased case-control investigation on cancers of the oral cavity in Bangalore, India. Br J Cancer 1990; 62: 847– 51. 7 Ko YC, Huang YL, Lee CH. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995; 24: 450–3. 8 Gupta MK, Mhaske S, Ragavendra R, Imtiyaz. Oral submucous fibrosis —current concepts in etiopathogenesis. People’s J Sci Res 2008; 1: 39–44. 9 Sheinfield J, Shaeffer AJ, CordonCordo C, Rogatko A, Fair WR. Association of Lewis blood group phenotype with recurrent urinary tract infections in women. N Engl J Med 1989; 320: 773–7.
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Acknowledgments The authors would like to thank Mrs Ambika Patil (Sri Dharamasthala Manjunatheshwara College of Dental Sciences & Hospital, Dharwad. Karnataka, India) for her assistance in examining the salivary secretary status, and Dr Arvind M. Yeri (Sri Dharamasthala Manjunatheshwara College of Dental Sciences & Hospital, Dharwad. Karnataka, India) for permitting us to utilize the laboratory facilities.
10 Lomberg H, Hollstrom J, Jodal U, Svanborg-Eden C.. Secretor status and renal scarring in girls with recurrent pyelonephritis. Microbiol Immunol 1991; 35: 371–4. 11 Lamey PJ, Darwazeh AM, Muirhead J, Rennie JS, Samaranayake LP, MacFarlane TW. Chronic hyperplastic candidosis and secretor status. J Oral Pathol Med 1991; 20: 64–7. 12 Jaleel BF, Nagrajappa R. Relationship between ABO blood groups and oral cancer. Indian J Dent Res 2012; 23: 7–10. 13 Dabelsten E, Gao S. ABO blood group antigens in oral cancer. J Dent Res 2004; 84: 21–8. 14 Vidas I, Delajlija M, Temmer-Vuksan B, Stipetic-Mravak M, Cindric N, Maricıc D. Examining the secretor status in the saliva of patients with oral pre-cancerous lesions. J Oral Rehabil 1999; 26: 177–82. 15 Javed F, Chotai M, Mehmood A, Almas K. Oral mucosal disorders associated with habitual gutka usage: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 857–64. 16 Jeng JH, Chang MC, Hahn LJ. Role of areca nut in betel quid associated chemical carcinogenesis: current awareness and future perspectives. Oral Oncol 2001; 37: 477–92. 17 Kinane DF, Blackwell CC, Brettle RP, Weir DM, Winstanley FP, Elton RA. ABO blood group secretor state and susceptibility to recurrent urinary tract infection in women. Br Med J 1982; 285: 423–4. 18 Clark CA, Price-Evans DA, MacConnel DA, Sheppard PM. Secretion of blood group antigen and peptic ulcer. Br Med J 1959; 1: 603–7.
19 Lamey PJ, Darwazeh AMG, Muirhead J, Rennie JS, Samaranayake LP, MacFarlane TW. Chronic hyperplastic candidosis and secretory status. J Oral Pathol Med 1991; 20: 67–70. 20 Pourazar A, Joshi SR, Sathe MS, Advani SH, Bhatia HM. Antigens in leukoplakia. Indian J Med Res 1986; 89: 83–8. 21 Dabelsteen E, Vedtotte P, Hakomori S, Young WW Jr. Accumulation of blood group antigen precursor in oral premalignant lesions. Cancer Res 1983; 43: 1451–4. 22 Dabelsteen E, Clausen H, Mandel U. Aberrant glycosylation in oral malignant and premalignant lesions. J Oral Pathol Med 1991; 20: 361–8. 23 Cerovic R, Juretic M, Balen S, Belusic M, Caser L, Rogic M. Examining the presence of ABO(H) antigens of blood types in the saliva of patients with oral cancer. Coll Antropol 2008; 32: 509–12. 24 Ariyawardana GA, Panagoda J, Fernando HN, Ellepola AN, Tilakaratne WM, Samaranayake LP. Oral submucous fibrosis and oral yeast carriage – a case control study in Sri Lankan patients. Mycoses 2005; 50: 116–20. 25 Koneru A, Hallikeri K, Shubhda C, Naikmasur VG, Kulkarni RD. Comparative study of Candida in oral submucous fibrosis and healthy in individuals. Rev Odonto Cienc 2011; 26: 71–6. 26 Fiueld EA, Field JK, Martin MV. Does Candida have a role in oral epithelial neoplasia? J Med Vet Mycol 1989; 27: 277–94. 27 Orlow I, Lacombe L, Pellicer I. Phenotypic and genotypic characterization of the histoblood group ABO
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(H) in primary bladder tumors. Int J Cancer 1998; 75: 819–24. 28 Periyavan S, Sangeetha SK, Marimuthu P, Manjunath BK, Seema DM. Distribution of ABO and Rhesus-D blood groups in and around Bangalore. Asian J Transfus Sci 2010; 4: 41–2. 29 Das PK, Nair SC, Harris VK et al. Distribution of ABO and Rh-D blood groups among blood donors in a tertiary care centre in South India. Trop Doct 2001; 31: 47–8. 30 Reddy KS, Sudha G. ABO and Rh(D) blood groups among the Desuri Red-
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dis of Chittur District, Andhra Pradesh. Anthrapol 2009; 11: 237–8. 31 Biondi C, Campi C, Escovich L, Garcia B, Racca A, Cotorruelo C. Loss of A, B, and H antigens in oral cancer. Immunologica 2008; 27: 127–31. 32 Bryne M, Thrane PS, Dabelsteen E. Loss of expression of blood group antigen H is associated with cellular invasion and spread of oral squamous cell carcinoma. Cancer 1991; 67: 613– 8. 33 Lamey PJ, Douglas PS, Napier SS. Secretor status and oral cancer. Br J Oral Maxillofac Surg 1994; 32: 214–7.
34 Raghavan VM, Bailoor DN, Jhansirani P. Incidence of ABO blood groups in oral cancer in South Kanara district. J Indian Dent Assoc 1986; 58: 305–8. 35 Toto PD, Nadimi H. Co-expression of cytokeratins, involucrin and blood group antigens in oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1990; 70: 75–80.
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