Effect of smoking on failure of H. pylori therapy and gastric histology in a high gastric cancer risk area of Colombia

Summary

It has been proposed that eradication of Helicobacter pylori infection is a sound strategy for gastric cancer prevention. Several factors including smoking have been associated to treatment failure rates. This study aimed to evaluate the smoking effect on the efficacy of H. pylori therapy, as well as on the histological parameters in the gastric mucosa from subjects from a high gastric cancer risk area. Two-hundred-sixty-four Colombian subjects with gastric precancerous lesions who participated in a chemoprevention trial, received anti-H. pylori treatment at baseline and had data recorded on cigarette use, were included in this study. A detailed histopathological assessment of the gastric mucosa was performed in biopsies taken before any intervention. H. pylori eradication was assessed in gastric biopsies at 36 months post-treatment. The overall eradication rate was 52.3%; rates of 41.3% and 57.1% were observed for active-smokers and non-smokers, respectively. Multivariate logistic regression analysis showed that smokers had a 2-fold higher probability of failure in Helicobacter pylori eradication than non-smokers (OR: 2.0; 95% CI: 1.01–3.95). At baseline, active-smokers had a higher score of intestinal metaplasia compared to non-smokers. In the corpus mucosa, active- smokers showed lower scores of H. pylori density, total inflammation, neutrophil infiltration, and mucus depletion than non-smokers. In the antrum, no significant differences were observed between active-smokers and non-smokers. In summary, in patients who smoked, H. pylori treatment was less effective. Smoking cessation may benefit H. pylori eradication rates.

Helicobacter pylori (H. pylori) infection is a well-recognized cause of gastric cancer.¹ The potential role of H. pylori eradication in preventing gastric adenocarcinoma is a focus of great public health interest. Although major improvements have been made in the efficacy of treatment regimes, virtually all of them result in some failures to eradicate the bacterial infection.² Several factors including smoking have been reported to influence this adverse outcome.^3–7 A recent meta-analysis evaluating mainly studies from Europe, Asia, and North America showed that smoking increases the treatment failure rate for H. pylori eradication.⁶ Because limited and contrasting data from developing countries were available for the meta- analysis (one report from Brazil),³ we believe that information from a high gastric cancer risk population from Latin America will complement the published findings. Additionally, the effects of smoking on gastric histopathology are of interest.

Both tobacco and tobacco smoke contain thousands of compounds, including a variety of carcinogens, ⁸ among which N-nitroso compounds have been associated to gastric carcinogenesis.⁹ Tobacco smoking is a long-term known risk factor for development of gastric preneoplastic lesions and gastric cancer.^8,10–15 Furthermore, in subjects infected with H. pylori, smoking increases the risk of intestinal metaplasia¹⁶ and gastric cancer.¹⁷

In the present study, including subjects from a high gastric cancer risk area in Colombia, we evaluated the smoking effect on the efficacy of H. pylori therapy and on the histological parameters in the gastric mucosa.

Methods

Study subjects, H. pylori treatment, and follow-up

Detailed description of characteristics of the subjects, interventions, follow-up, and results of this chemoprevention trial were reported previously.^18,19 Briefly, adults from two towns from a high risk gastric cancer area in Colombia were screened in 1991. The subjects underwent upper gastrointestinal endoscopy and gastric mucosa biopsies from antrum, incisura angularis and corpus. Individuals with gastric preneoplastic lesions were randomly assigned to receive anti-H. pylori treatment with or without vitamin supplementation (ascorbic acid and beta-carotene), and/or placebo in a 2 × 2 × 2 factorial design for 6 years.¹⁸ Anti-H. pylori treatment consisted of amoxicillin (500 mg three times per day), metronidazole (375 mg three times per day), and bismuth subsalicylate (262 mg three times per day) for 14 days. As assessed by interviews and recorded pill counts every three months, the patients´ compliance with therapy protocol was satisfactory. The subjects included in this report represent patients who received anti-H. pylori treatment at entry, had a follow- up endoscopy at 36 months and provided information about cigarette use at enrollment. According to their smoking habit, subjects were classified into "non-smokers" (those who never smoked), "active-smokers" (smokers at the date of the recruitment, who had been smoking for at least one year), and "ex-smokers". The latter group was excluded from this analysis. The prevalence of smoking was 30.3%. Most of them were light smokers (median: 3 cigarettes per day); therefore, no dose-response was evaluated.

Histopathology

Gastric biopsy specimens taken at baseline and 36 months of follow-up were fixed in formalin and embedded in paraffin. Four-micron-thick sections were stained with haematoxylin and eosin for regular histology, with Alcian blue-periodic acid Schiff to detect intestinal metaplasia, and with the modified Steiner silver technique to detect H. pylori.²⁰ Histological diagnostic categories were: multifocal atrophic gastritis without metaplasia (MAG) and multifocal atrophic gastritis with intestinal metaplasia (IM). MAG, defined as loss of glands, was graded as mild, moderate, or marked.²¹ IM was defined as replacement of the gastric epithelium by intestinal-type epithelium. It was further subclassified as complete (small intestine-type), defined by the presence of absorptive enterocytes with brush border alternating with goblet cells, or incomplete (colonic type), defined by the presence of columnar cells with foamy cytoplasm, lacking brush border. Based on the global diagnostic categories (3=MAG and 4=IM), a previously used histopathology score ¹⁹ that reflects the grade of atrophy and the type and extent of IM, was applied. Briefly, the MAG score was augmented according to the degree of atrophy: 0.25=indefinite for atrophy, 0.50=mild, 0.75=moderate, and 1.0=severe. The IM score was modified according to the type and extension. IM type was classified into four categories in an ordinal scale: 0.1=complete type, 0.2=mixed predominant complete type, 0.3=mixed predominant incomplete type, and 0.4=incomplete type. The average extension of the IM was grouped by tertiles. Each tertile was given a value: 0.2, 0.4, or 0.6, respectively. In order to obtain a total score of IM, values for type and extension were added to the original score for IM.

Infiltration of mononuclear (MN) cells, polymorphonuclear neutrophils (PMN), intraepithelial lymphocytes, mucus depletion, regenerative activity, and density of H. pylori colonization were graded in a semiquantitative scale 0–3 (absent, mild, moderate, and marked). MN, PMN, and H. pylori density were evaluated according to the updated Sydney system.²¹ The number of lymphoid follicles observed in each biopsy sample was registered.

Statistical analysis

Average values of every histological parameter were estimated considering the total number of biopsies and separately by gastric subsite (corpus and antrum). Data obtained from transitional mucosa were combined with those from antrum. Total inflammation score was the average of the scores for PMN and MN inflammation.

H. pylori status at 36 months was used as the proxy variable for eradication (presence of infection was considered as failure, and lack of infection as success). Student’s t tests were used to test associations between histological parameters and smoking status. Multivariate logistic regression models were used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) to assess the risk of eradication failure. A linear regression model was used to assess the relationship of IM score and smoking status, known risk factors of IM, such as age and sex were considered. All analyses were performed using Stata 9.0 software (Stata Corporation, College Station, TX).

Results

Results of the analysis of H. pylori eradication by smoking status and baseline diagnosis are presented in table 1. The overall eradication rate was 52.3%. Eradication rates in active-smokers and non-smokers were 41.3% and 57.1%, respectively. These rates were not significantly influenced by age and sex. The adjusted OR for eradication failure for active-smokers was 2.0 (95% CI: 1.01–3.95), indicating a 2-fold higher probability of failure in H. pylori eradication. Subjects with IM had a decreased risk of eradication failure compared with those with MAG (OR: 0.48; 95% CI: 0.26–0.87).

Table 1.

Odds ratios and 95% confidence intervals of eradication failure by smoking status and baseline histological diagnosis.

	H. pylori eradicationa
	Success n=138	Failure n=126	Adjusted OR (95% CI)
Smoking status, n(%)
Non-smoker	105(57.1)	79(42.9)	1.0
Active-smoker	33(41.3)	47(58.7)	2.0(1.01 – 3.95)b
Histopathological diagnosis, n(%)
Multifocal atrophic gastritis	23(37.7)	38(62.3)	1.0
Intestinal metaplasia	115(56.6)	88(43.4)	0.48(0.26 – 0.87)c

Abbreviations: OR, Odds ratio; CI, confidence interval

^aStatus based on H. pylori status at 36 months of follow-up

^bAdjusted by age, sex, town, intervention (consumption of ascorbic acid and beta-carotene), and baseline histological diagnosis

^cAdjusted by age, sex, town, intervention (consumption of ascorbic acid and beta-carotene), and smoking status

Histopathological parameters in the gastric mucosa at baseline according to smoking status are shown in table 2. Although there was not a difference between active- smokers and non-smokers regarding the global histopathological diagnosis, the unadjusted mean of the IM score (which considers the type and extension) was significantly higher among active-smokers than among non-smokers (~13 percent difference; p=0.042). The difference remained statistically significant after adjustment for age, sex, and town (p=0.016). Stratified analysis by gastric subsite showed significantly lower scores of H. pylori density, PMN infiltrate, total inflammation, and mucus depletion in the corpus of active-smokers compared to non-smokers. There were no differences in any of the scores in the antrum between the two groups.

Table 2.

Histological parameters by smoking status at baseline

	Smoking status at baseline
Histological parameters at baseline	Non- smoker n=184	Active- smoker n=80	Two-sided p-value
Histopathological diagnosis, n(%)
Multifocal atrophic gastritis	43(23.4)	18(22.5)	0.878
Intestinal metaplasia	141(76.6)	62(77.5)
Histopathological score, mean (SD)	4.26(0.43)	4.33(0.44)	0.251
Total score of Intestinal metaplasia, mean (SD)	0.48(0.19)	0.54(0.22)	0.042
Density of H. pylori colonization, mean (SD)
Total	1.69(0.74)	1.75(0.67)	0.516
Antrum	1.58(0.78)	1.71(0.76)	0.186
Corpus	2.10(0.77)	1.88(0.72)	0.050
Polymorphonuclears, mean (SD)
Total	1.91(0.66)	1.97(0.58)	0.484
Antrum	1.87(0.71)	2.00(0.63)	0.165
Corpus	2.08(0.74)	1.78(0.87)	0.011
Mononuclears, mean (SD)
Total	1.93(0.26)	1.92(0.22)	0.740
Antrum	1.94(0.29)	1.93(0.23)	0.764
Corpus	1.93(0.35)	1.86(0.42)	0.230
Total inflammation, mean (SD)
Total	1.92(0.44)	1.95(0.37)	0.662
Antrum	1.91(0.47)	1.97(0.40)	0.334
Corpus	2.00(0.50)	1.83(0.59)	0.020
Intraepithelial lymphocytes, mean (SD)
Total	0.31(0.32)	0.37(0.37)	0.195
Antrum	0.36(0.39)	0.45(0.43)	0.112
Corpus	0.16(0.36)	0.10(0.28)	0.255
Mucus depletion, mean (SD)
Total	2.11(0.97)	2.13(0.92)	0.917
Antrum	2.11(1.02)	2.20(0.98)	0.511
Corpus	2.19(1.09)	1.87(1.14)	0.050
Lymphoid follicles, mean (SD)
Total	0.33(0.30)	0.33(0.31)	0.937
Antrum	0.37(0.36)	0.34(0.35)	0.598
Corpus	0.22(0.47)	0.22(0.43)	0.963
Regenerative activity, mean (SD)
Total	0.60(0.52)	0.66(0.53)	0.405
Antrum	0.68(0.59)	0.75(0.62)	0.358
Corpus	0.43(0.63)	0.32(0.61)	0.204

Abbreviation: SD: Standard deviation

Interestingly, subjects who did not eradicate the bacterium had a significantly higher score of H. pylori density at baseline than those with a successful eradication (1.82 versus 1.61, p-value for mean difference=0.019).

Discussion

Increasing evidence supports that H. pylori eradication prevents the development of gastric adenocarcinoma. A prospective, randomized study in China found that eradication of H. pylori significantly decreased the incidence of gastric cancer in subjects without precancerous lesions.¹⁴ In a retrospective, large-scale study in Japan, a significantly lower incidence of gastric cancer was observed in subjects with eradication compared to those in whom the infection persisted.²² In addition, studies considering preneoplastic lesions as an end-point also support the benefits of H. pylori therapy.^19,23,24

Our results indicate that smoking was associated with anti-H. pylori treatment failure. Although this association has been described previously and addressed in a recent meta-analysis,⁶ limited and conflicting evidence from studies conducted in developing countries exist.³

Regarding the possible mechanisms involved in treatment failure, it is known that smoking decreases gastric mucosal blood flow and mucus secretion, ^25,26 which may reduce the delivery of antibiotics to the gastric mucosa. In addition, smoking increases acid gastric secretion, which lowers the efficacy of some acid labile antibiotics, such as amoxicillin and clarithromycin.²⁷ These two effects seem to be supported by a recent large study that reports increase in the cure rate in smokers (but not in nonsmokers) who received increased doses of clarithromycin. ²⁸

Besides smoking, multiple other factors have been associated with H. pylori eradication failure: poor compliance,²⁹ antibiotic resistance,⁵ low grades of inflammation in antrum and activity in the fundus,⁴ H2-receptor antagonist pre-treatment,³⁰ genetic polymorphisms of CYP2C19 (with different effects on the metabolism of PPIs),³¹ excessive bacterial burden,⁷ and infection with low-virulence H. pylori strains.^3,32,33

Previous studies in Colombian patients reported higher prevalence of more virulent H. pylori strains (vacAs1m1, cagA-positive, and cagE-positive) in subjects with gastric cancer and IM in comparison with subjects with gastritis (atrophic and non-atrophic). ^34,35 Assuming that in our study more advanced lesions (IM) may have been caused by strains with higher virulence, it is not surprising that the failure rate was higher in patients with MAG than in those with IM (table 1). It has been suggested that treatment may be more effective in eliminating more virulent strains, resulting in only partial and/or selective eradication of H. pylori in mixed infections. ³²

In the United States, the current recommended primary therapies for H. pylori infection include: a clarithromicyn-based triple therapy (a proton pump inhibitor [PPI], clarithromycin, and amoxicillin or metronidazole) for 14 days, or a bismuth quadruple therapy (a PPI or H₂-receptor antagonist, bismuth, metronidazole, and tetracycline) for 10–14 days.² Eradication rates are in the range of 70–90%.² Subjects in our chemoprevention trial were treated (in 1991) with bismuth subsalicylate, amoxicillin, and metronidazole for 2 weeks, reaching a cure rate of 52.3% at 36 months of follow-up. This relatively low rate may have been caused by the treatment protocol, which did not include clarithromycin or an antisecretory drug. It has been suggested that the eradication rates correlate positively with the degree of inhibition of gastric acid secretion.³⁶ H. pylori resistance against metronidazole may have also played a role in treatment failure. The prevalence of H. pylori resistance to nitroimidazoles has been reported to be about 80% in tropical regions, where these drugs are frequently used for diarrhea treatment.³⁷ In our chemoprevention study, subjects who tested positive for H. pylori at 36 months were treated again for 14 days with amoxicillin, clarithromycin, and a PPI. The eradication rate was 74% at 72 months of follow-up.¹⁸ Finally, as previously reported, ⁷ we observed that excessive bacterial burden may have been also involved in failure to the initial treatment.

Smoking is a risk factor for the development of gastric preneoplastic and neoplastic lesions ^8,10–16 and H. pylori infection seems to increase this risk.^16,17 We did not find association between the histological diagnosis and the smoking status. It is likely that the presence of precancerous lesions in all the individuals influenced this result. However, active-smokers had significantly higher IM score (including extension and type) than non-smokers, indicating the presence of more advanced lesions.

Besides the carcinogenic effects of the N-nitroso compounds generated from the tobacco smoke components,⁹ it is known that smoking impairs cell renewal process by several mechanisms, including reduced levels of epidermal growth factor, reduction of ornithine decarboxilase activity, and inhibition of constitutive nitric oxide synthesis.^38,39 In addition, smoking increases bile salt reflux rate and gastric bile salt concentration.^38,40 Several experimental studies have demonstrated that duodenogastric reflux (bile and/or pancreaticoduodenal secretions) induces gastric adenocarcinoma.^41–43 In vitro, bile acid exposure has shown to promote intestinal differentiation in esophageal cell lines,⁴⁴ thereby starting the metaplasia-dysplasia-carcinoma sequence.

Regarding the histological parameters associated with inflammation and mucus depletion, we did not find differences in the antral mucosa between nonsmokers and active-smokers. It is likely that the effects of the H. pylori infection overlap those of the smoking, influencing the results. The lower scores in H. pylori density, total inflammation, and PMNs observed in the corpus mucosa of active-smokers compared to those of non-smokers may have a physiological explanation. Cigarette smoking increases gastric acid secretion by the parietal cells, located in the fundus and corpus of the stomach. The lower pH resultant may prevent H. pylori infection (which is predominantly antral) to disseminate to corpus and fundus, and therefore, lower scores of inflammation would be expected in these areas. The minor differences observed in histological parameters may be related to the low doses of tobacco smoke exposure (median: 3 cigarettes/day). Increased acid secretion has been observed in subjects who smoked at least 3 cigarettes per day compared to non-smokers.⁴⁵

Despite its harmful effects on the gastric mucosa, cigarette smoking is consistently associated with lower incidence of ulcerative colitis and a lower relapse rate of the disease.^46,47 Studies have suggested that the beneficial effects of nicotine in ulcerative colitis are due to increased mucus secretion, and decreased production of proinflammatory cytokines and nitric oxide.⁴⁷ Future studies are needed to investigate these effects in the gastric mucosa.

A limitation of the present study was the assessment time of H. pylori eradication. The annual reinfection/ recrudescence rate of the cohort from which this sample was taken, was 5.4%.¹⁹ Urea breath tests were performed on all subjects in the H. pylori treatment arm between the initial and the 3 year biopsies. Since the discrepancy in the infection status between the biopsy results and the breath tests was small, and because the breath tests had a significant variability in time, we favored the use of the biopsy results.

Considering that eradication of H. pylori infection may be a promising strategy to control gastric cancer, the observed adverse effect of smoking on eradication has an important public health implication. Particularly because while smoking rates have been decreasing for several decades in the United States and other industrialized countries, those in developing countries are increasing at around 3% per year.^48–50 While anti-smoking efforts are showing favorable results in developed countries, those efforts are far less effective in developing countries, where transnational tobacco companies have been increasingly promoting their products. This trend may result in poorer treatment outcomes in developing countries where H. pylori infection is highly prevalent.

In conclusion, on the basis of the findings of the published meta-analysis, and the additional information provided by our study, smoking increases the risk of H. pylori treatment failure. Smoking cessation may benefit H. pylori eradication rates. Therefore, infected smoker patients should be encouraged to quit their habit. Smoking also influences the gastric histopathologic parameters, decreasing the severity of the lesions in the corpus.

Abbreviations

CI

confidence interval

H. pylori

Helicobacter pylori

IM

intestinal metaplasia

MAG

multifocal atrophic gastritis without metaplasia

MN

mononuclear cells

PMN

polymorphonuclear neutrophils

OR

odds ratio