CORRESPONDENCE

Re: Helicobacter pylori Seropositivity as a Risk Factor for Pancreatic Cancer

Hans-Olof Nilsson, Unne Stenram, Ingemar Ihse, Torkel Wadström

Affiliations of authors: H.-O. Nilsson, T. Wadström (Department of Medical Microbiology, Dermatology and Infection), U. Stenram, (Department of Pathology), I. Ihse (Department of Surgery), Lund University, Lund, Sweden.

Correspondence to: Torkel Wadström, M.D., Ph.D., Dept. of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 LUND, Sweden (e-mail: Torkel.Wadstrom{at}mmb.lu.se).

In the study of Helicobacter pylori seropositivity as a risk factor for pancreatic cancer, Stolzenberg-Solomon et al. (1) concluded that their findings support a possible role for the presence of H. pylori in the development of pancreatic cancer. We recently reported the identification of Helicobacter species in the livers of patients with hepatocellular carcinoma and cholangiocarcinoma (2) and in those with primary biliary cirrhosis and primary sclerosing cholangitis (3,4). We analyzed pancreatic biopsy specimens from patients undergoing surgery for suspected pancreatic neoplasia for the presence of Helicobacter species and H. pylori by bacterial culture, polymerase chain reaction (PCR), and DNA sequencing. Approval from our institution and the chairman of the ethical committee was obtained.

To isolate Helicobacter species by bacterial culture, portions of pancreatic tissue biopsy specimens obtained during operation were inoculated on Brucella–blood agar plates and incubated in a microaerophilic atmosphere (5% O2, 10% CO2, 85% N2) at 37 °C for 2 weeks. DNA was extracted from other portions of the same pancreatic tissue biopsy specimens by the QiaAmp Tissue DNA protocol (Qiagen, Hilden, Germany), according to the manufacturer's recommendations. For the PCR analysis, Helicobacter genus-specific (corresponding to a sequence that encodes 16S ribosomal DNA [rDNA]) and H. pylori-specific primers (corresponding to a sequence that encodes a 26-kDa alkyl hydroperoxide reductase, AhpC, protein homologue) were used as described (3), with minor modifications, including the addition of 1.25 U rTth DNA polymerase (Applied Biosystems, Foster City, CA) and 5 µL of DNA sample. Reactions were run in 25-µL volumes in a GeneAmp 2400 thermal cycler (Applied Biosystems). DNA sequencing was carried out on the 400-base-pair PCR products obtained after Helicobacter genus-specific PCR amplification, as described (2). Sequences were aligned in the BioEdit Sequence Alignment Editor program (version 5.0.1; North Carolina State University, Raleigh, NC, www.mbio.ncsu.edu/BioEdit/bioedit.html), and a phylogenetic tree was constructed by the neighbor-joining method in CLUSTAL W (www.ebi.ac.uk) (5) and displayed by the TreeView program (version 1.6.1; www.mbio.ncsu.edu/BioEdit/bioedit.html) (6).

Attempts to cultivate Helicobacter species from normal or neoplastic tissue were unsuccessful, even after a prolonged incubation of up to 2 weeks. One endocrine neoplasia type 1 and two normal pancreatic tissue specimens were negative for Helicobacter by PCR with genus-specific primers. Five of six pancreatic ductal carcinomas and one malignant neuroendocrine neoplasia were positive for Helicobacter by PCR with genus-specific primers. All Helicobacter genus-positive samples were negative for H. pylori by PCR with H. pylori-specific primers (Table 1Go). Two 16S rDNA PCR fragments, amplified with the genus-specific primers as described above, were sequenced and compared with the GenBank database by a BLAST search (www.ncbi.nlm.nih.gov). One of the sequences was 98% similar to the 16S rDNA of Helicobacter species liver 3 (accession No. AF142585) and clustered to a phylogenetic group that includes H. pylori. The other 16S rDNA sequence was 99% similar to H. pullorum (accession No. AF047850) and clustered to a phylogenetic group that also contains H. bilis.


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Table 1. Presence of Helicobacter in pancreatic biopsy specimens detected by genus-specific or H. pylori-specific PCR and partial DNA sequencing*
 
Our findings of the presence of Helicobacter DNA in pancreatic cancer tissues and the serologic results of Stolzenberg-Solomon et al. (1) suggest that further studies are needed to analyze the possible role of Helicobacter, and other microaerophilic microbes, in autoimmune pancreatitis and pancreatic carcinoma. A Helicobacter strain, characterized as H. pylori, was recently isolated from a human cirrhotic liver (7), suggesting that Helicobacter and other microorganisms may infect the pancreas, bile tree, and associated tissues by ascending gastric infections or retrograde transfer from the small bowel.

NOTES

Editor's note: Rachael Z. Stolzenberg-Solomon et al. declined to respond to the correspondence by Hans-Olof Nilsson et al.

REFERENCES

1 Stolzenberg-Solomon RZ, Blaser MJ, Limburg PJ, Perez-Perez G, Taylor PR, Virtamo J, et al. Helicobacter pylori seropositivity as a risk factor for pancreatic cancer. J Natl Cancer Inst 2001;93:937–41.[Abstract/Free Full Text]

2 Nilsson HO, Mulchandani R, Tranberg KG, Wadstrom T. Helicobacter species identified in liver from patients with cholangiocarcinoma and hepatocellular carcinoma. Gastroenterology 2001;120:323–4.[Medline]

3 Nilsson HO, Taneera J, Castedal M, Glatz E, Olsson R, Wadstrom T. Identification of Helicobacter pylori and other Helicobacter species by PCR, hybridization, and partial DNA sequencing in human liver samples from patients with primary sclerosing cholangitis or primary biliary cirrhosis. J Clin Microbiol 2000;38:1072–6.[Abstract/Free Full Text]

4 Wadstrom T, Ljungh A, Willen R. Primary biliary cirrhosis and primary sclerosing cholangitis are of infectious origin! Gut 2001;49:454.[Free Full Text]

5 Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–80.[Abstract]

6 Page RD. TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 1996;12:357–8.[Medline]

7 de Magalhaes Queiroz DM, Santos A. Isolation of a Helicobacter strain from the human liver. Gastroenterology 2001;121:1023–4.[Medline]


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