Abstract
Helicobacter pylori is a gram-negative bacillus causing benign and malignant gastric diseases such as gastroesophageal reflux (GER). In larynx, H. pylori causes chronic inflammation and mucosal destruction that may lead to malignant changes. Although, H. pylori poses several virulence factors, cagA is probably the main factor in this regard. To evaluate the role of cagA gene in laryngeal squamous cell carcinoma (LSCC), a case–control study was conducted on patients with laryngeal complaints during 2010–2012. Seventy-two patients with LSCC (case group) and 72 patients without malignancy (control group) were included in the study. The H. pylori and cagA factor were assessed in laryngeal specimen of patients with PCR technique. 33 % of patients in case group (24 patients) and 45.8 % (33 patients) of control group were positive for H. pylori. CagA gene was present in 13.8 % (10 patients) of case group specimens and 31.9 % (23 patients) of control group. This difference was statistically significant with Mantel–Haenszel statistical test analyses. The results showed that patients with LSCC have significantly lower incidence of laryngeal H. pylori infection and cagA virulence factor than those without LSCC. Findings from this study support the protective effect of H. pylori infection against laryngeal cancer.
Keywords: Helicobacter pylori, cagA, Laryngeal cancer, SCC
Introduction
Helicobacter pylori (H. pylori) is a gram negative bacillus colonized in humans’ stomach [1] and yet most of cases may remain asymptomatic. H. pylori can cause benign and malignant gastric diseases [2]. Studies have also detected the presence of H. pylori in dental plaque, saliva and adenotonsilar tissue. Different virulence factors such as urease, flagella, adhesion factors, vacuole cytotoxins and cagA have been identified for H. pylori. However, it seems that cagA gene is the main virulence factor of H. pylori [3]. CagA gene encodes a 120–145 kDa protein and has been located on the 40 kb cag pathogenicity island [4]. In vitro experiments on animal models of H. pylori infection have shown that infection with cagA positive H. pylori is more likely to lead to peptic ulcer and malignancy [5]. CagA is not seen in all types of H. pylori but most of East Asian H. pylori isolates are positive for cagA [6]. However, it is used as a marker for pathogenicity [7]. Although, cagA positive H. pylori is mostly reported in gastric tissue, cagA is also detected in adenotonsilar tissue, nasal polyps and laryngeal diseases. Cirak et al. have reported the presence of cagA in 71 % of adenotonsilar tissue samples [8]. It can be hypothesized that H. pylori along with other risk factors such as smoking, alcohol and gastroesophageal reflux (GER) play a role in the pathogenesis of laryngeal squamous cell carcinoma (LSCC).Chronic inflammation of infected tissue with H. pylori followed by epithelial cell proliferation in the laryngeal mucosa might cause laryngeal cancer as in the pathogenesis of H. pylori in gastric cancer [8, 9]. H. pylori exists in oral cavity and stomach independently. H. pylori can be colonized in larynx too [10]. Studies have shown the presence of H. pylori in specimens of benign and malignant laryngeal diseases with serologic, histochemistry and PCR techniques. Most of these studies have utilized the serologic tests of H. pylori with ELISA technique and have evaluated the presence of IgG antibody against H. pylori. Serologic tests measure antibodies (IgA, IgM and IgG) against H. pylori antigens. Although tests for measuring different types of immunoglobulins are available, FDA has only approved the serum IgG-based tests for detection of H. pylori infection. Serologic tests accurately reflect the presence or absence of active H. pylori infection [11]. There are few studies using PCR technique for detection of H. pylori infection, and one study that has used PCR technique suffers from limited number of cases [12]. Laryngeal cancer is one of the most common malignancies of head and neck region.
There are few data available on cagA gene condition of H. pylori variants in Iran. This study aimed to assess the association between cagA positive H. pylori infection and LSCC using a specific PCR technique.
Materials and Methods
Study Population and Specimen Collection
A cross-sectional study was designed and local ethics committee approved the study protocol. Data were collected from patients admitted to otolaryngology-head and neck clinic in Shafa Hospital of Kerman with complaints of laryngeal disease between March 2010 and March 2012. A complete history was obtained and risk factors were assessed for patients with laryngeal complaints. The patients were asked to read, fill out and sign the informed consent form. The exclusion criteria of the study were the followings: rejection to participate in the study, serious gastrointestinal diseases that were confirmed by endoscopy like severe gastritis or gastrointestinal cancers, recent use of drugs against H. pylori activity such as omeprazole, antibiotics (metronidazole, amoxicillin and bismuth), pathologic diagnosis of dysplasia and patients under 30 years of age.
Sample Preparation
In patients with suspicious lesion in indirect laryngoscopy, patients with risk factors for laryngeal malignancy (opium addiction/cigarette smoking) or a history of hoarseness more than 2 weeks, direct laryngoscopy in operation room was done and samples were taken for pathology. From the fresh specimen 3–4 mm2 of tissue was separated, coded, kept at −80 °C and the reminder of the specimen was sent for pathology study with the same coding. If the pathologic diagnosis was in favor of malignancy, the specimen was then placed in case group and if it was in favor of benign lesions (nodule, polyp, cyst or granulation), it was then assigned to control group. If the pathologic diagnosis was in line with dysplasia, the patient was excluded from the study. To prevent any bias in PCR testing of the specimens, the coded samples were sent for PCR technician to blind the lab staff from pathologic diagnosis.
PCR Detection of H. pylori and cagA
The standard strain of H. pylori was provided by Iran Pasteur Institute, Liver and Gastrointestinal department, H. pylori center. The samples were placed in sterile normal saline; DNAs were extracted using High Pure A, DNA Tissue Extract Kit (High Media company). The samples were investigated for existence of H. pylori by performing a real time PCR using Detection Pylori Kit (DNA Technology company, Russia). Positive samples for H. pylori infection were investigated for cagA gene using CagA Kits (Kiagen company, Germany). The Multiple PCR test was carried out on the samples and the results were registered for further investigation.
Statistical Analyses
The sample size with consideration of a relative accuracy of 10 %, type I error (a = 5 %) was computed as 72 persons for each group. SPSS V. 20 and STATA V. 10 were used to calculate odds ratio (OR), relative ratio (RR) and performing Mantel–Haenszel test. Mantel–Haenszel test was performed to consider the confounding effect of opium addiction and cigarette smoking in LSCC and adjust OR for H. pylori and cagA.
Results
In this study 144 men and women with laryngeal complaints aging 30–88 years old were selected. The average age was 52.6. The most common age group in this study was 50–59 followed by 40–49 with frequencies of 22.9 and 28.5 % respectively. Twenty-six patients were female (18.1 %) and 118 were male (81.9 %). Eighty-three percent of patients were opium addicted (inhalation, oral use or both). Of these 83 addicted patients, 75 (90.4 %) were also cigarette smokers. The duration of opium consumption and cigarette smoking ranged from 1 to 50(MEAN ± SD) years and 2 to 50 years (MEAN ± SD), respectively.
Of 144 patients in study, 72 had benign laryngeal lesion (control group) and 72 had LSCC. H. pylori was detected in 57 patients (39.6 %) of which 24 (42.1 %) patient had LSCC and 33 (57.9 %) patient had benign laryngeal lesion (Table 1). In patients with positive samples for H. pylori (57 patients), 33 had positive PCR results for cagA gene (57.9 % in H. pylori positive group and 22.9 % in total patients). CagA was positive in 10 patients with LSCC and 23 patients with benign laryngeal lesions. Comparison of H. pylori frequency between the two groups showed that in control group the presence of H. pylori was more frequent than case group. However, this difference was not statistically significant (P > 0.05). Comparing the age distribution of patients in this study showed no significant difference between the case and control group with the exception of patients aging 60–69 years of old. In this later group, H. pylori was positive in 88.9 % of patients in control group and 42.9 % of patients in case group. This difference was statistically significant (P = 0.027).
Table 1.
Comparing the frequency of H. pylori and CagA in two groups
| Cag A gene existance | ||||||
|---|---|---|---|---|---|---|
| No | Yes | Total | ||||
| Count | Row N % | Count | Row N % | Count | Row N % | |
| Group | ||||||
| Control | ||||||
| Helicobacter pylori existence | ||||||
| No | 38 | 100.0 | 0 | 0.0 | 38 | 100.0 |
| Yes | 10 | 30.3 | 23 | 69.7 | 33 | 100.0 |
| Total | 48 | 67.6 | 23 | 32.4 | 71 | 100.0 |
| Case | ||||||
| Helicobacter pylori existence | ||||||
| No | 49 | 100.0 | 0 | 0.0 | 49 | 100.0 |
| Yes | 14 | 58.3 | 10 | 41.7 | 24 | 100.0 |
| Total | 63 | 86.3 | 10 | 13.7 | 73 | 100.0 |
| Total | ||||||
| Helicobacter pylori existence | ||||||
| No | 87 | 100.0 | 0 | 0.0 | 87 | 100.0 |
| Yes | 24 | 42.1 | 33 | 57.9 | 57 | 100.0 |
| Total | 111 | 77.1 | 33 | 22.9 | 144 | 100.0 |
CagA was less frequent in both control and case group. A comparison between the two groups of the study revealed that the different frequency of cagA positive samples between the control group (23 samples) and case group (10 samples) was statistically significant (P = 0.008). Overall 39.6 % of patients in this study had positive laryngeal PCR results for H. pylori infection. About 45.8 % of patients in control group (33/72) had positive samples for H. pylori in comparison to 33 % (24/72) of samples in case group. Statistical analyses revealed a non-significant (P > 0.09) OR of 0.56. In order to adjust the confounding effect of cigarette smoking and opium addiction, Mantel–Haenszel test was applied (Table 2). This table shows that considering cigarette smoking and opium addiction as confounding factors and using a proper statistical technique for elimination of this confounding effect, the relationship between the two groups and H. pylori remains statistically significant. Table 3 shows the cofounding effect of cigarette smoking and opium consumption on relationship between type of disease and existence of cagA gene. OR less than 1 even after adjustment with Mantel–Haenszel test shows that the presence of cagA gene like H. pylori infection is higher in control group.
Table 2.
Confounding effect of cigarette and opium on correlation between type of disease and H. pylori
| Group/HBP existence | Crude OR | CI 95 % | P value | Adjusted OR* | CI 95 % | P value |
|---|---|---|---|---|---|---|
| No confounding factor | 0.564 | 0.287–1.108 | 0.095 | – | – | – |
| Use of cigarette as confounding factor | – | – | – | 0.383 | 0.166–0.880 | 0.024** |
| Use of opium as confounding factor | – | – | – | 0.257 | 0.104–0.633 | 0.003** |
OR odd ratio, CI 95 % confidence interval 95 %
* Mantel–Haenszel common odd ratio estimate
** Statistically significant
Table 3.
Confounding effect of cigarette and opium on correlation between type of disease and cagA
| Group/CagA Existence | Crude OR | CI95 % | P value | Adjusted OR* | CI 95 % | P value |
|---|---|---|---|---|---|---|
| No confounding factor | 0.331 | 0.144–0.761 | 0.008** | – | – | – |
| Use of cigarette as confounding factor | – | – | – | 0.186 | 0.066–0.522 | 0.001** |
| Use of opium as confounding factor | – | – | – | 0.194 | 0.071–0.530 | 0.001** |
OR odd ratio, CI 95 % confidence interval 95 %
* Mantel–Haenszel common odd ratio estimate
** Statistically significant
Discussion
GER is associated with laryngeal symptoms of which the most commons are posterior glottis erythema and edema. However, hyperkeratosis, thickening (pachyderma laryngitis) and granulation are also reported. Hoarseness, globus sensation and clearing the throat are common. Reflux can lead to contact ulcers in vocal folds, granuloma, leukoplakia and laryngeal malignancy [13]. The relationship between H. pylori and larynx cancer is not fully understood. Some studies have shown reductions in esophagitis and Barrett’s esophagitis in the presence of H. pylori infection. As H. pylori infection reduces gastric acidity and reflux, it might also pose a protective effect against laryngeal cancer [14]. In this study, 144 patients with laryngeal complaints, were assessed by PCR technique for laryngeal infection with H. pylori. The results showed that only 33 % of patients with malignant changes had H. pylori in comparison to 45.8 % of patients with benign lesion. Mantel–Haenszel test was considered to assess the confounding effect of cigarette smoking and opium addiction along with the role of H. pylori infection in laryngeal pathologies. The results suggest that H. pylori may play a role as a protective factor against laryngeal malignancy. However, this finding might be due to other factors like consumption of drugs, nutritional diets (low fat) or individual habits and requires further investigations.
Infection by H. pylori is more common in developing countries, whereas GER is relatively less common in Asia and Africa than North America and Western Europe countries. Reduction of acid secretion after infection with H. pylori can be explained via three mechanisms: severe gastritis of corpus region, increased secretion of gastric bicarbonate and ammonium production by the bacteria itself. After eradication of H. pylori, rising acid secretion and probably reflux can lead to esophagitis, a condition well known to predispose to premalignant conditions like Barrett’s esophagitis [14].In an epidemiologic study by EL-Serag, lower prevalence of H. pylori infection was seen in patients with esophagitis [15]. Wu et al. reported that infection with H. pylori was associated with a decrease in Barrett’s esophagitis [16]. In a study by Majidi et al. in Iran on 45 patients with laryngeal cancer and 42 controls, anti H. pylori antibody titer was significantly higher in controls than cases. It was concluded that infection with H. pylori has an anti secretary effect in stomach which leads to decreased level of gastric acidity. Thus, H. pylori infection can pose a protective effect against diseases associated with GER [17].
In a study by Nurgalieva et al. in USA on 120 patients with larynx cancer and 120 persons in control group, the amount of anti H. pylori IgG antibody was similar between the two groups (32.8 % in patients and 27 % in controls) [18]. In the above study, H. pylori had no association with larynx malignancy and was neither a risk factor nor a protective factor for it. Lower infection rate of H. pylori in both groups of the mentioned study is acceptable due to the geographical distribution of H. pylori infection. Moreover, the presence of strong risk factors of laryngeal cancer such as cigarette smoking and alcohol usage further complicates the finding about the role of H. pylori in laryngeal cancer in this geographical region. In Aygene study in 2001 in Turkey on 26 patients with laryngeal cancer and 32 healthy persons, serum IgG antibodies against H. Pylori antigens were assessed. The results showed that IgG antibody was positive in 73.07 % of cases and 40.62 % of controls. This study supported the role of H. pylori in laryngeal cancer [19]. In 1996, Jenifer et al. conducted a study on 21 cases with laryngeal cancer and 21 controls, serum IgG antibodies against H. pylori antigens were assessed and no significant difference was found [20]. Mokhtari (2003) studied on 30 patients with laryngeal cancer and 30 healthy controls serum IgG antibodies against H. pylori antigens were evaluated. Sixty-three percent of cases and 26 % of controls had positive serologic results [21]. The findings of the study by Mokhtari is in line with Aygenc study but not similar to the findings of the present study [19]. It seems that the main cause of controversial findings in these studies may result from ELIZA technique in detection of serum IgG antibodies against H. pylori that is not specific to H. pylori infection of larynx. An investigation was done in 2004, seeking for H. pylori in specimens of larynx cancer using histopathological and immunohistochemical approaches; however, no correlation was found [7]. In 2009, a study used PCR detection of H. pylori in 29 patients with nasal polyps and laryngeal disease (benign or malignant) and 29 healthy persons. Nevertheless, this study failed to show significant correlation between the disease and H. pylori [12]. However, this study suffered from few numbers of cases. Ozyurt et al. in 2009 identified H. pylori cagA in 78.9 % of the H. pylori positive nasal polyp samples, 89.5 % of normal nasal mucosa samples, and 82.4 % of normal larynx samples [12]. The findings of the study by Ozyurt et al. are not congruent with higher cagA factor in benign lesions in this study.
In this study, the frequency of H. pylori infection and cagA gene was significantly higher in case group than control group. This finding supports the protective effect of H. pylori against laryngeal malignancy. However, this finding merit further investigation with prospective study design, parallel assessment of gastric disease and evaluation of more risk factors of H. pylori infection and laryngeal malignant disease.
Acknowledgement
This study was undertaken under the supervision of Kerman Medical University Research Centre and in cooperation with Kerman Department of Education. We are grateful to Ehsan Mehrabi Kermani for his assistance in English editing of the manuscript and Dr. Sarafi Nejad for his generous help in statistical analyses of the study.
References
- 1.Dunn BE, Cohen H, Blaser MJ. Helicobacter Pylori. Clin Microbiol Rev. 1997;10(4):720–741. doi: 10.1128/cmr.10.4.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Parsonnet J, Hansen S, Rodriguez L, et al. Helicobacter pylori infection and gastric lymphoma. N Engl J Med. 1994;330(18):1267–1271. doi: 10.1056/NEJM199405053301803. [DOI] [PubMed] [Google Scholar]
- 3.Covacci A, Telford JL, DelGuidice G, et al. Helicobacter pylori virulence and genetic geography. Science. 1999;284:1328–1333. doi: 10.1126/science.284.5418.1328. [DOI] [PubMed] [Google Scholar]
- 4.Kikuchi S, Crabtree JE, Forman D, Kurosawa M. Association between infections with CagA-positive or -negative strains of Helicobacter pylori and risk for gastric cancer in young adults. research group on prevention of gastric carcinoma among young adults. Am J Gastroenterol. 1999;94(12):3455–3459. doi: 10.1111/j.1572-0241.1999.01607.x. [DOI] [PubMed] [Google Scholar]
- 5.Blaser MJ, Perez-Perez GI, Kleanthous H, et al. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res. 1995;55(10):2111–2115. [PubMed] [Google Scholar]
- 6.Hatakeyama M, Higashi H. Helicobacter pylori CagA: a new paradigm for bacterial carcinogenesis. Cancer Sci. 2005;96:835–843. doi: 10.1111/j.1349-7006.2005.00130.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Oleastro M, Gerhard M, Lopez Al, et al. H. pylori virulence genotypes in Portuguese children and adults with gastroduodenal pathology. Eur J Clin Microbiol Infect Dis. 2003;22(2):85–91. doi: 10.1007/s10096-002-0865-3. [DOI] [PubMed] [Google Scholar]
- 8.Cirak MY, Ozdek A, Yilmaz D, et al. Detection of Helicobacter pylori and its cagA gen in tonsils and adenoid tissue by PCR. Arch Otolaryngol Head Neck Surg. 2003;129(11):1225–1229. doi: 10.1001/archotol.129.11.1225. [DOI] [PubMed] [Google Scholar]
- 9.Wienholt MG, Erbling MC, Bennetts RW, et al. Detection of antibodies to Helicobacter pylori using oral fluid specimens. Ann N Y Acad Sci. 1993;694:340–342. doi: 10.1111/j.1749-6632.1993.tb18384.x. [DOI] [PubMed] [Google Scholar]
- 10.Akbayir N, Basak T, Seven H, et al. Investigation of Helicobacter pylori colonization in laryngeal neoplasia. Eur Arch Otorhinolaryngol. 2005;262(3):170–172. doi: 10.1007/s00405-004-0794-0. [DOI] [PubMed] [Google Scholar]
- 11.Kosunen TU, Seppala K, Sarna S, et al. Diagnostic value of decreasing IgG, IgA and IgM antibody titers after eradication of Helicobacter pylori. Lancet. 1992;339(8798):893–895. doi: 10.1016/0140-6736(92)90929-W. [DOI] [PubMed] [Google Scholar]
- 12.Ozyrut M, Gunyor A, Ergunanay K, Cekin E, Erkul E, et al. Real time PCR detection of helicobacter pylori and virulence associated cagA in nasal polyps and laryngeal disorders. Otolaryngol Head Neck Surg. 2009;141(1):131–135. doi: 10.1016/j.otohns.2009.04.005. [DOI] [PubMed] [Google Scholar]
- 13.Aviv JE, Savita Collins (2010) Upper aerodigestive manifestations of gastroesophageal reflux disease. In: Flint PW et al (eds) Cummings otolaryngology-head & neck surgery, 5th edn. Mosby, Philadelpia, pp 894
- 14.Feldman M, Freidman LS, Sleisenger MH. Sleisenger and fordtrans gastrointestinal and liver disease. 8. Philadelphia: Saunders; 2006. [Google Scholar]
- 15.Serag HB, Sonnenberg A. Opposing time trends of peptic ulcer and reflux disease. Gut. 1998;43(3):327–333. doi: 10.1136/gut.43.3.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Wu JC, Sung JJ, Chan FK, et al. Helicobacter pylori infection in association with milder gastroesophageal reflux disease. Aliment Pharmacol Ther. 2000;14(4):427–432. doi: 10.1046/j.1365-2036.2000.00714.x. [DOI] [PubMed] [Google Scholar]
- 17.Majidi MR, Rezaei S, Hssanzadeh N, et al. The correlation between helicobacter pylori infection and squamous cell carcinoma of larynx and hypopharynx. Iran J Otorhinolaryngol. 2007;48(19):89–94. [Google Scholar]
- 18.Nurgalieva ZZ, Graham DY, Dahlstrom KR, et al. A pilot study of helicobacter pylori infection and risk of laryngopharyngeal cancer. Head Neck. 2005;27(1):22–27. doi: 10.1002/hed.20108. [DOI] [PubMed] [Google Scholar]
- 19.Aygenc E, Selcuk A, Celikkanat S, et al. The role of helicobacter pylori infection in the cause of squamous cell carcinoma of the larynx. Otolaryngol Head Neck Surg. 2001;125(5):520–521. doi: 10.1067/mhn.2001.119438. [DOI] [PubMed] [Google Scholar]
- 20.Grandis RJ, Perez-Perez GI, Yu VL, et al. Lack of serologic evidence for Helicobacter pylori infection in head and neck cancer. Head Neck. 1997;19(3):216–218. doi: 10.1002/(SICI)1097-0347(199705)19:3<216::AID-HED9>3.0.CO;2-5. [DOI] [PubMed] [Google Scholar]
- 21.Mokhtari N, Khajeh KM, Saadatnia H, Rajati M. Studing the cause effect relation between helicobacter pylori and SCC of larynx and hypopharynx. Iran J otorhinolaryngol. 2004;35:27–32. [Google Scholar]