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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References

Background:  Biliary tract cancer is a highly fatal disease with poor prognosis, but the aetiology is poorly understood.

Aim:  We aimed to identify Helicobacter bilis infection in the gallbladder in patients with biliary tract disease.

Methods:  Archival gallbladder specimens from 34 patients (14 males and 20 females) with an average age of 61.4 ± 12.2 years (mean ± SE) were retrieved, consisting of 11 cases of gallbladder cancer, three of bile duct cancer, 16 of cholecystolithiasis and four of pancreatic cancer. DNA was extracted and nested PCR using primers specific for 16S rRNA of H. bilis was performed.

Results : Amplification was observed in 3 of 11 gallbladder cancer cases (27.2%) and one of three cases with biliary duct cancer (33.3%). In total, four of 14 cases with biliary tract cancer were positive for H. bilis (28.6%). In addition, the presence of H. bilis was shown in two of 16 cases (12.5%) with cholecystolithiasis. Notably, although the number of cases examined was small, none of the four cases with pancreatic cancer showed the presence of H. bilis infection in the gallbladder without apparent abnormalities.

Conclusion: H. bilis infection may play a role in biliary tract disease, particularly in biliary tract cancer.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References

Biliary tract cancer is a relatively rare disease worldwide, but it is a highly fatal disease with poor prognosis.1, 2 It occurs relatively more often in some countries, including Chile, India and Japan.1– 4 However, the aetiology and pathogenesis of biliary tract cancer remain unclear.

The relative risk of gallbladder cancer is 2.4–10 times higher in patients with gallstones than in those without.5, 6 However, several other factors are also assumed to play a role in gallbladder cancer development, because about 10–25% of patients with the disease do not have associated cholelithiasis and only a small proportion (1–3%) of patients with gallstones actually develop gallbladder cancer.1 Interestingly, bile from cholecystolithiasis patients in high-risk areas for gallbladder cancer has a higher mutagenic activity.7 Thus, cholecystolithiasis in combination with one or more other factors seems to be causative for gallbladder cancer.

Epidemiologically, the incidence of gallbladder carcinoma varies even within a country. For example, higher incidence rates are observed in northern Japan than in the rest of the country.4 Racial and ethnic background are also involved. In the US, the incidence is 50% higher in Caucasians than in African-Americans.8 In addition, obesity and oestrogens have been reported as risk factors for gallbladder cancer.9 Therefore, a variety of backgrounds, including genetic, geographical and environmental factors, are considered to play a role in gallbladder carcinogenesis.

It is not particularly new to investigate the association of bacteria with malignancy. A few decades ago, for example, typhoid carrier state was reported to be associated with hepatobiliary tract cancer.10 More recently, Helicobacter pylori has been implicated in numerous gastrointestinal diseases, including gastric cancer.11 Detection of Helicobacter sp. in human bile juice has prompted a growing interest as to whether these bacteria colonize the biliary tract of humans and cause hepatobiliary diseases.12, 13 At the same time, however, it is generally known that a limited spectrum of bacteria can survive in bile juice, and doubt has been cast on the existence of pathogenic bacteria in bile juice, with little attention paid to the issue.

It has been reported that some Helicobacter sp. can exist in the gallbladder, liver tissue and bile juice in patients with cholecystolithiasis, primary sclerosing cholangitis and primary biliary cirrhosis.3, 14–17 The involvement of Helicobacter sp. was suggested in hepatobiliary carcinogenesis in a PCR study using bile and hepatobiliary tissue samples.13 In addition, among Helicobacter sp., the existence of H. bilis in bile juice associated with bile duct or gallbladder cancer was also recently suggested by PCR studies.16 However, culture of Helicobacter sp. from the biliary tract has been unsuccessful, and the presence of Helicobacter sp. in this area remains controversial.18,19

Clinically, it is extremely difficult to detect biliary tract cancers at an early stage, with the exception of cases incidentally found at surgery for cholecystolithiasis. However, little is known as to whether or how Helicobacter species are involved in the development of biliary malignancy. Identification of the aetiology of biliary tract cancer, especially the potential causative roles of Helicobacter sp., would therefore be beneficial with regard to prevention.

In this study, we aimed to elucidate the existence of H. bilis in the gallbladder with and without abnormalities in patients with gallbladder cancer, biliary duct cancer, cholecystolithiasis and pancreatic cancer. The existence of H. bilis in the gallbladder was implicated in patients with biliary tract diseases, especially biliary tract cancer.

Specimens

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References

Archival paraffin-embedded gallbladder specimens from 34 cases were retrieved from the pathology laboratory of Osaka Police Hospital. The patients had undergone cholecystectomy for gallbladder cancer, bile duct cancer including common bile duct cancer, cholelithiasis and pancreatic cancer. It was confirmed that there were no apparent macroscopic and pathological abnormalities in gallbladder specimens resected at the time of pancreatoduodenectomy for pancreatic cancer. In all cases, surgery had been elective and potent antibiotic treatment had not been given just before surgery.

DNA collection

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References

Paraffin-embedded specimens were scraped off the slides and put into tubes. They were deparaffinized in xylene and hydrated in a series of graded ethanol. They were then immersed in phosphate-buffered saline, homogenized, and DNA was extracted using a QIAamp DNA blood mini kit (Qiagen, Germany) according to the manufacturer's instructions.

PCR amplification

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References

Nested PCR primer pairs specific for H. bilis were designed to generate 16S rRNA amplicons. The primers were as follows: (A): 5′-ATTTCTCTTTTTAGTGCTTTGTGTTGT T-3′ (sense) and 5′-CTTTCAATAAAAAATTCGTCTTTGGAC-3′ (anti-sense); and (B): 5′-TTGGAATGAGAAATTGATGTTGTGA-3′ (sense) and 5′-ATAAATCGATGAGTTCGCAATTTG-3′ (anti-sense). PCR reactions contained 1 µL of DNA, 1 × PCR buffer, 200 µmol/L of each deoxynucleotide triphosphate, 0.4 µmol/L of each primer and 1 U of Taq DNA polymerase (Qiagen). PCR cycles were: denaturing at 95 °C for 5 min; 40 cycles of denaturing at 95 °C for 15 s and annealing and extension at 58 °C for 1 min; followed by final extension at 72 °C for 7 min. The second PCR was carried out under the same conditions with the different PCR primer set. The PCR products were electrophoresed on 3% agarose gels and visualized by UV light after ethidium bromide staining.

Statistical analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References

To compare mean values, Student's t-test, Welch's t-test and the χ2 test were used. P < 0.05 was considered significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References

The gallbladder archival specimens were obtained from 34 patients with the background diseases as shown in Table 1: gallbladder cancer (11 patients); bile duct cancer (common bile duct cancer, 3; biliary tract cancer, 14 in total); gallstones (16); and pancreatic cancer (4). The mean ± SE age of the patients was 61.4 ± 12.2 years, and there was no significant difference in average age among patients with the four different background diseases. The 34 patients comprised 14 males and 20 females, and gender distribution was not statistically different.

Table 1.  Patient demographics and H. bilis-positive cases
Disease type of cancerDisease (M/F)Age (years) Positive cases
Gallbladder2/964.8 ± 12.63 (0/3)
Bile duct3/061.7 ± 7.81 (1/0)
Biliary tract5/964.1 ± 11.54 (1/3)
Gallstone9/757.0 ± 13.12 (0/2)
Pancreatic0/474.0 ± 5.50 (0/0)

Figure 1 shows a representative result of nested PCR specific for the H. bilis sequence. The PCR product appeared as one 91-bp band on the gel. Amplification was observed in 3/11 cases (27.2%) with gallbladder cancer, and in 1/3 cases with biliary duct cancer (33.3%). In total, 4/14 cases with biliary tract cancer were positive for H. bilis (28.6%).

image

Figure 1. Representative PCR amplification for H. bilis. DNA from a gallbladder with gallbladder cancer was amplified by nested PCR for H. bilis. The cases marked with an asterisk (*) showed positive amplification. M denotes a size marker.

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In gallbladders without cancer in the biliary tract, in contrast, amplification was observed less frequently. The presence of H. bilis was indicated in 2/16 cases (12.5%) with cholecystolithiasis. Notably, although the number of cases was small, none of the four cases with pancreatic cancer showed the presence of H. bilis infection in the gallbladder without apparent morphological or histological abnormalities (Figure 2).

image

Figure 2. Infection rates for H. bilis in biliary tract diseases. H. bilis infection was more marked in cases with biliary tract cancer than in those with cholecystolithiasis and pancreatic cancer. GBCa, gallbladder cancer; BDCa, bile duct cancer; GS, gallstone; and PCa, pancreatic cancer.

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The mean age of the patients with biliary tract cancer who were positive for H. bilis infection was 71.8 years, relatively higher than the average age of all cases with biliary tract cancer, although not statistically significant (P = 0.08) (Table 1). The biliary tract cancer cases comprised five males and nine females, and this malignancy was thus predominant in females. The cholecystolithiasis cases, on the other hand, were composed of nine males and seven females.

Collectively, infection with H. bilis was observed most frequently in gallbladders with biliary tract cancer, with cholecystolithiasis cases showing a lower infection rate. Positive rates were 2–3 times higher in cases with biliary tract cancer than in those without.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References

Since the discovery of H. pylori in the gastrointestinal tract and its association with a variety of diseases, new light has been shed on the involvement of bacterial infection, especially Helicobacter sp. infection, in a variety of diseases.20 Not only H. pylori but also other Helicobacter sp. are known to colonize the gastrointestinal tract of humans and animals.21 Indeed, Helicobacter sp. have been isolated and identified from the stomachs of various mammals, including dogs, cats, ferrets, pigs, monkeys and cheetahs, and an association with gastritis observed in all. Importantly, in an inbred strain of mice, A/JCr, persistent infection with H. hepaticus is linked to the development of hepatic adenomas and adenocarcinomas.22 Identification of Helicobacter sp. in the biliary tract and clarification of its association with biliary tract diseases are therefore highly worthwhile goals.

H. bilis belongs to the enterohepatic Helicobacter sp., and has been isolated from the stomach of dogs and gerbils, and the faeces of gerbils and cats.21 The pathogenicity of H. bilis has been observed in the intestine of various animals infected experimentally and naturally.23–25 Accordingly, it is reasonable to hypothesize the involvement of this bacterium in the pathogenesis of biliary tract cancer.

Fox et al. first reported an association between H. bilis in the gallbladder and cholecystolithiasis in Chilean patients. 3 They identified the existence of the bacteria by Southern blotting, PCR and sequencing in spite of culture failure. Subsequently, conflicting results have been reported.18, 19

Méndez-Sánchez et al. demonstrated a lack of association between Helicobacter sp. colonization and cholecystolithiasis in Mexican populations.18 Rudi et al. demonstrated the absence of Helicobacter sp. in bile from German patients with biliary diseases. 19 When bacterial infection was limited to H. pylori infection, negative colonization of the bacteria was also reported.26 Hence, the colonization of Helicobacter sp. in the biliary tract is considered to be greatly dependent on geographical and ethnic factors.

Recently, Matsukura et al. reported the existence of H. bilis in bile juice in Japanese patients with bile duct cancer and cholecystolithiasis.16 They employed the PCR method in the same way as in the present study. They observed positive amplification in 87% of patients with bile duct or gallbladder cancer, but in only 50% of those with gallstones, cholecystitis or both, and in 29% of Japanese patients with non-biliary diseases. Although positive rates were much lower in our study, the tendency was similar in that biliary tract cancer had a higher positive rate of H. bilis infection than benign and non-biliary disease. We assume that the difference in positive rates derives from the feasibility of PCR amplification: we used DNA extracted from archival specimens, which is usually more difficult to amplify than that collected fresh and stored immediately. Nevertheless, our result is compelling enough to support the idea that H. bilis could exist in gallbladder tissue and might be involved in carcinogenesis in the biliary tract.

Culture of H. bilis from bile juice or biliary tract tissue is probably the best way to prove its involvement in biliary diseases. It is also necessary to elucidate the molecular mechanisms of biliary tract carcinogenesis in relation to bacterial components; for example, CagA, a potential virulence factor from H. pylori, is assumed to play a role in gastrointestinal diseases by disturbing signal transduction in host cells after translocation.27 Further study is warranted to deepen our understanding of chronic bacterial infection, particularly H. bilis, in biliary tract cancer.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Specimens
  6. DNA collection
  7. PCR amplification
  8. Statistical analysis
  9. Results
  10. Discussion
  11. References
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    Méndez-Sánchez N, Pichardo R, González J, et al. Lack of association between Helicobacter sp colonization and gall stone disease. J Clin Gastroenterol 2001; 32: 13841.
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    Rudi J, Rudi A, Maiwald M, et al. Helicobacter sp. are not detectable in bile from German patients with biliary disease. Gastroenterology 1999; 116: 1016.
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    Ward JM, Fox JG, Anver MR, et al. Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J Natl Cancer Inst 1994; 86: 12227.
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    Haines DC, Gorelick PL, Battles JK, et al. Inflammatory large bowel disease in immunodeficient rats naturally and experimentally infected Helicobacter bilis. Vet Pathol 1998; 35: 2028.
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    Shomer NH, Dangler CA, Schrenzel MD, et al. Helicobacter bilis induced inflammatory bowel disease in SCID mice with defined flora. Infect Immun 1997; 65: 485864.
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    Arnaout AH, Abbas SH, Shousha S. Helicobacter pylori is not identified in areas of gastric metaplasia of gallbladder. J Pathol 1990; 160: 3334.
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