Advertisement

Molecular Medicine

, Volume 20, Issue 1, pp 57–71 | Cite as

Differential Proteomics of Helicobacter pylori Associated with Autoimmune Atrophic Gastritis

  • Ombretta Repetto
  • Stefania Zanussi
  • Mariateresa Casarotto
  • Vincenzo Canzonieri
  • Paolo De Paoli
  • Renato Cannizzaro
  • Valli De Re
Research Article

Abstract

Atrophic autoimmune gastritis (AAG) is a condition of chronic inflammation and atrophy of stomach mucosa, for which development can be partially triggered by the bacterial pathogen Helicobacter pylori (HP). HP can cause a variety of gastric diseases, such as duodenal ulcer (DU) or gastric cancer (GC). In this study, a comparative proteomic approach was used by two-dimensional fluorescence difference gel electrophoresis (DIGE) to identify differentially expressed proteins of HP strains isolated from patients with AAG, to identify markers of HP strain associated with AAG. Proteome profiles of HP isolated from GC or DU were used as a reference to compare proteomic levels. Proteomics analyses revealed 27 differentially expressed spots in AAG-associated HP in comparison with GC, whereas only 9 differential spots were found in AAG-associated HP profiles compared with DU. Proteins were identified after matrix-assisted laser desorption ionization (MALDI)-TOF and peptide mass fingerprinting. Some AAG-HP differential proteins were common between DU- and GC-HP (peroxiredoxin, heat shock protein 70 (HSP70), adenosine 5′-triphosphate (ATP) synthase subunit a, flagellin A). Our results presented here may suggest that comparative proteomes of HP isolated from AAG and DU share more common protein expression than GC and provide subsets of putative AAG-specific upregulated or downregulated proteins that could be proposed as putative markers of AAG-associated HP Other comparative studies by two-dimensional maps integrated with functional genomics of candidate proteins will undoubtedly contribute to better decipher the biology of AAG-associated HP strains.

Notes

Acknowledgments

This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) grant #10266 (to V De Re); AIRC grant #12214 (to R Cannizzaro); and the Bio-Proteomics Core Facility, CRO Scientific Direction. We thank Bruno Bacher for the use of DeCyder.

Supplementary material

10020_2014_2001057_MOESM1_ESM.pdf (97 kb)
Supplementary material, approximately 96 KB.

References

  1. 1.
    Bettington M, Brown I. (2013) Autoimmune gastritis: novel clues to histological diagnosis. Pathology. 45:145–49.CrossRefPubMedGoogle Scholar
  2. 2.
    Miceli E, et al. (2012) Common features of patients with autoimmune atrophic gastritis. Clin. Gastroenterol. Hepatol. 10:812–14.CrossRefPubMedGoogle Scholar
  3. 3.
    Erdoðan A, Yilmaz U. (2011) Is there a relationship between Helicobacter pylori and gastric autoimmunity? Turk. J. Gastroenterol. 22:134–8.CrossRefGoogle Scholar
  4. 4.
    Bergman MP, et al. (2005) The story so far: Helicobacter pylori and gastric autoimmunity. Int. Rev. Immunol. 24:63–91.CrossRefPubMedGoogle Scholar
  5. 5.
    Correa P. (1996) Helicobacter pylori and gastric cancer: state of the art. Cancer Epidemiol. Biomarkers Prev. 5:477–81.PubMedGoogle Scholar
  6. 6.
    Presotto F, et al. (2003) Helicobacter pylori infection and gastric autoimmune diseases: is there a link? Helicobacter. 8:578–84.CrossRefPubMedGoogle Scholar
  7. 7.
    Ferreira AC, et al. Helicobacter and gastric malignancies. Helicobacter. 1:28–34.Google Scholar
  8. 8.
    Blaser MJ, Atherton JC. (2004) Helicobacter pylori: persistence: biology and disease. J. Clin. Invest. 113:321–33.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ricci V, Romano M, Boquet P. (2011) Molecular cross-talk between Helicobacter pylori and human gastric mucosa. World J. Gastroenterol. 17:1383–99.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Costa AC, Figueiredo C, Touati E. (2009) Pathogenesis of Helicobacter pylori infection. Helicobacter. 14 (Suppl. 1):15–20.CrossRefPubMedGoogle Scholar
  11. 11.
    Wen S, Moss SF. (2009) Helicobacter pylori virulence factors in gastric carcinogenesis. Cancer Lett. 282:1–8.CrossRefPubMedGoogle Scholar
  12. 12.
    Rieder G, Fischer W, Haas R. (2005) Interaction of Helicobacter pylori with host cells: function of secreted and translocated molecules. Curr. Opin. Microbiol. 8:67–73.CrossRefPubMedGoogle Scholar
  13. 13.
    Enroth H, Akerlund T, Sillén A, Engstrand L. (2000) Clustering of clinical strains of Helicobacter pylori analyzed by two-dimensional gel electrophoresis. Clin. Diagn. Lab. Immuno. 7:301–6.Google Scholar
  14. 14.
    Proença-Modena JL, Acrani GO, Brocchi M. (2009) Helicobacter pylori: phenotypes, genotypes and virulence genes. Future Microbiol. 4:223–40.CrossRefPubMedGoogle Scholar
  15. 15.
    Duncan SS, et al. (2013) Comparative genomic analysis of east Asian and non-Asian Helicobacter pylori strains identifies rapidly evolving genes. PLoS One. 8:e55120.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Dong QJ, et al. (2012) Relatedness of Helicobacter pylori populations to gastric carcinogenesis. World J. Gastroenterol. 18:6571–6.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Dong QJ, Wang Q, Xin YN, Li N, Xuan SY. (2009) Comparative genomics of Helicobacter pylori. World J. Gastroenterol. 15:3984–91.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    McClain MS, Shaffer CL, Israel DA, Peek RM Jr, Cover TL. (2009) Genome sequence analysis of Helicobacter pylori strains associated with gastric ulceration and gastric cancer. BMC Genomics. 10:3.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Tomb JF, et al. (1997) The complete genome sequence of the gastric pathogen. Helicobacter pylori. Nature. 388:539–47.CrossRefPubMedGoogle Scholar
  20. 20.
    Alm RA, et al. (1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature. 397:176–80.CrossRefPubMedGoogle Scholar
  21. 21.
    Gomceli I, Demiriz B, Tez M. (2012) Gastric carcinogenesis. World J. Gastroenterol. 18:5164–70.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Gashi Z, Zekaj S, Haziri A, Bakalli A. (2011) The influence of the type of ulcers in the degree of atrophic gastritis. Medicinski Arhiv. 65:20–2.PubMedGoogle Scholar
  23. 23.
    Zhang Z. (2007) The risk of gastric cancer in patients with duodenal and gastric ulcer: research progresses and clinical implications. J. Gastrointest. Cancer. 38:38–45.CrossRefPubMedGoogle Scholar
  24. 24.
    Mini R, et al. (2006) Comparative proteomics and immunoproteomics of Helicobacter pylori related to different gastric pathologies. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 833:63–79.CrossRefPubMedGoogle Scholar
  25. 25.
    Oleastro M, Monteiro L, Lehours P, Megraud F, Menard A. (2006) Identification of markers dell’Helicobacter pylori strains isolated from children with peptic ulcer disease by suppressive subtractive hybridization. Infect. Immun. 74:4064–74.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Schmidt HM, et al. (2010) The cag PAI is intact and functional but HP0521 varies significantly in Helicobacter pylori isolates from Malaysia and Singapore. Eur. J. Clin. Microbiol. Infect. Dis. 29:439–51.CrossRefPubMedGoogle Scholar
  27. 27.
    Tomasini ML, et al. (2003) Heterogeneity of cag genotypes in Helicobacter pylori isolates from human biopsy specimens. J. Clin. Microbiol. 41:976–80.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Karnes WE Jr, et al. (1991) Positive serum antibody and negative tissue staining for Helicobacter pylori in subjects with atrophic body gastritis. Gastroenterology. 101:167–74.CrossRefPubMedGoogle Scholar
  29. 29.
    Kokkola A, et al. (2003) Spontaneous disappearance of Helicobacter pylori antibodies in patients with advanced atrophic corpus gastritis. APMIS. 111:619–24.CrossRefPubMedGoogle Scholar
  30. 30.
    Simula MP, et al. (2010) PPAR signaling pathway and cancer-related proteins are involved in celiac disease-associated tissue damage. Mol. Med. 16:199–209.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Franco AT, et al. (2009) Delineation of a carcinogenic Helicobacter pylori proteome. Mol. Cell. Proteomics. 8:1947–58.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Momynaliev KT, et al. (2010) Functional divergence of Helicobacter pylori related to early gastric cancer. J. Proteome Res. 9:254–67.CrossRefPubMedGoogle Scholar
  33. 33.
    Oh JD, Kling-Bäckhed H, Giannakis M, Engstrand LG, Gordon JI. (2006) Interactions between gastric epithelial stem cells and Helicobacter pylori in the setting of chronic atrophic gastritis. Curr. Opin. Microbiol. 9:21–7.CrossRefPubMedGoogle Scholar
  34. 34.
    Ohata H, et al. (2004) Progression of chronic atrophic gastritis associated with Helicobacter pylori infection increases risk of gastric cancer. Int. J. Cancer. 109:138–43.CrossRefPubMedGoogle Scholar
  35. 35.
    McNamara LE, Dalby MJ, Riehle MO, Burchmore R. (2010) Fluorescence two-dimensional difference gel electrophoresis for biomaterial applications. J R Soc. Interface. 6:S107–18.CrossRefGoogle Scholar
  36. 36.
    O’Toole PW, Logan SM, Kostrzynska M, Wadström T, Trust TJ. (1991) Isolation and biochemical and molecular analyses of a species-specific protein antigen from the gastric pathogen Helicobacter pylori. J. Bacteriol. 173:505–13.PubMedGoogle Scholar
  37. 37.
    Kern R, Malki A, Holmgren A, Richarme G. (2003) Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase. Biochem. J. 371:965–72.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. (2007) Chronic inflammation and oxidative stress in human carcinogenesis. Int. J. Cancer. 121:2381–6.CrossRefPubMedGoogle Scholar
  39. 39.
    Stent A, Every AL, Sutton P. (2012) Helicobacter pylori defense against oxidative attack. Am. J. Physiol. Gastr. Liver Physiol. 302:G579–87.CrossRefGoogle Scholar
  40. 40.
    Wang G, Alamuri P, Maier RJ. (2006) The diverse antioxidant systems of Helicobacter pylori. Mol. Microbiol. 61:847–60.PubMedGoogle Scholar
  41. 41.
    Backert S, et al. (2005) Subproteomes of soluble and structure-bound HP proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry. Proteomics. 5:1331–45.CrossRefPubMedGoogle Scholar
  42. 42.
    Lock RA, Cordwell SJ, Coombs GW, Walsh BJ, Forbes GM. (2001) Proteome analysis of Helicobacter pylori: major proteins of type strain NCTC 11637. Pathology. 33:365–74.CrossRefPubMedGoogle Scholar
  43. 43.
    Jungblut PR, et al. (2000) Comparative proteome analysis of H. pylori. Mol Microbiol. 36:710–25.CrossRefPubMedGoogle Scholar
  44. 44.
    Cao P, McClain MS, Forsyth MH, Cover TL. (1998) Extracellular release of antigenic proteins by Helicobacter pylori. Infect. Immun. 66:2984–6.PubMedGoogle Scholar
  45. 45.
    McAtee CP, et al. (1998) Identification of potential diagnostic and vaccine candidates of Helicobacter pylori by two-dimensional gel electrophoresis, sequence analysis, and serum profiling. Clin. Diagn. Lab. Immunol. 5:537–42.PubMedPubMedCentralGoogle Scholar
  46. 46.
    el Yaagoubi A, Kohiyama M, Richarme G. (1994) Localization of DnaK (chaperone 70) from Escherichia coli in an osmotic-shock-sensitive compartment of the cytoplasm. J. Bacteriol. 176:7074–8.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Hoffman PS, Garduno RA. (1999) Surface-associated heat shock proteins of Legionella pneumophila and Helicobacter pylori: roles in pathogenesis and immunity. Infect. Dis Obstet. Gynecol. 7:58–63.PubMedPubMedCentralGoogle Scholar
  48. 48.
    Osawa H, et al. (2001) Comparative analysis of colonization of Helicobacter pylori and glycolipids receptor density in Mongolian gerbils and mice. Dig. Dis. Sci. 46:69–74.CrossRefPubMedGoogle Scholar
  49. 49.
    Hamma T, Adrian R, Ferré-D’Amaré AR. (2006) Pseudouridine synthases. Chem. Biol. 13:1125–35.CrossRefPubMedGoogle Scholar
  50. 50.
    Ge J, Yu YT. (2013) RNA pseudouridylation: new insights into an old modification. Trends Biochem. Sci. 338:210–8.CrossRefGoogle Scholar
  51. 51.
    Shajani Z, Sykes MT, Williamson JR. (2011) Assembly of bacterial ribosomes. Annu. Rev. Biochem. 80:501–26.CrossRefPubMedGoogle Scholar
  52. 52.
    Dunn BE, et al. (1997) Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies. Infect. Immun. 65:1181–8.PubMedPubMedCentralGoogle Scholar
  53. 53.
    Pyndiah S, et al. (2007) Two-dimensional blue native/SDS gel electrophoresis of multiprotein complexes from Helicobacter pylori. Mol Cell Proteomics. 6:193–206.CrossRefPubMedGoogle Scholar
  54. 54.
    Kobayashi F, et al. (2011) Production of autoantibodies by murine B-1a cells stimulated with Helicobacter pylori urease through toll-like receptor 2 signaling. Infect. Immun. 79:4791–801.CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Huang CH, Chiou SH. (2011) Proteomic analysis of upregulated proteins in Helicobacter pylori under oxidative stress induced by hydrogen peroxide. Kaohsiung J. Med. Sci. 27:544–53.CrossRefPubMedGoogle Scholar
  56. 56.
    Ni J, Mei M, Sun L. (2012) Oxidative DNA damage and repair in chronic atrophic gastritis and gastric cancer. Hepatogastroenterology. 59:671–5.PubMedGoogle Scholar
  57. 57.
    Yonekura K, Maki-Yonekura S, Namba K. (2002) Growth mechanism of the bacterial flagellar filament. Res. Microbiol. 153:191–7.CrossRefPubMedGoogle Scholar
  58. 58.
    Godlewska R, Wiśniewska K, Pietras Z, Jagusztyn-Krynicka EK. (2009) Peptidoglycan-associated lipoprotein (Pal) of gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis. FEMS Microbiol. Lett. 298:1–11.CrossRefPubMedGoogle Scholar
  59. 59.
    Govorun VM, et al. (2003) Comparative analysis of proteome maps of Helicobacter pylori clinical isolates. Biochemistry. 68:42–9.PubMedGoogle Scholar
  60. 60.
    Lee HW, Choe YH, Kim DK, Jung SY, Lee NG. (2004) Proteomic analysis of a ferric uptake regulator mutant of Helicobacter pylori: regulation of Helicobacter pylori gene expression by ferric uptake regulator and iron. Proteomics. 4:2014–27.CrossRefPubMedGoogle Scholar
  61. 61.
    Hopf PS, et al. (2011) Protein glycosylation in Helicobacter pylori: beyond the flagellins? PLoS One. 6:e25722.CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Park JW, et al. (2006) Quantitative analysis of representative proteome components and clustering of Helicobacter pylori clinical strains. Helicobacter. 11:533–43.CrossRefPubMedGoogle Scholar
  63. 63.
    Meyer-Roseberg K, Scott DR, Melchers K, Sachs G. (1996) The effect of environmental pH on the proton motive force of H. pylori. Gastroenterology. 111:886–900.CrossRefGoogle Scholar
  64. 64.
    Dixon MF. (2001) Pathology of gastritis and peptic ulceration. In: Helicobacter pylori: Physiology and Genetics. Mobley HLT, Mendz GL, Hazell SL, Eds. Washington, DC: ASM Press.Google Scholar
  65. 65.
    Ferrero RL, Lee A. (1991) The importance of urease in acid protection for the gastric-colonising bacteria H. pylori and H. felis sp. nov. Microb. Ecol. Health. Dis. 4:121–34.CrossRefGoogle Scholar
  66. 66.
    Kansau I, Labigne A. (1996) Heat shock proteins of Helicobacter pylori. Aliment Pharm. Ther. 1:51–6.CrossRefGoogle Scholar
  67. 67.
    Yamaguchi H, et al. (1999) Induction of secretion of interleukin 8 from human gastric epithelial cells by heat-shock protein 60 homologue of Helicobacter pylori. J. Med. Microbiol. 48:927–33.CrossRefPubMedGoogle Scholar
  68. 68.
    Yamaoka Y, et al. (2001) Relation between cytokines and Helicobacter pylori in gastric cancer. Helicobacter. 6:116–24.CrossRefPubMedGoogle Scholar
  69. 69.
    Kitadai Y, et al. (1999) Transfection of interleukin-8 increases angiogenesis and tumorigenesis of human gastric carcinoma cells in nude mice. Br. J. Cancer. 81:647–53.CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Kavaliauskas D, Nissen P, Knudsen CR. (2012) The busiest of all ribosomal assistants: elongation factor Tu. Biochemistry. 51:2642–51.CrossRefPubMedGoogle Scholar
  71. 71.
    Backert S, et al. (2005) Gene expression and protein profiling of AGS gastric epithelial cells upon infection with Helicobacter pylori. Proteomics. 5:3902–18.PubMedGoogle Scholar
  72. 72.
    Janosi L, Shimizu I, Kaji A. (1994) Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth. Proc. Natl. Acad. Sci. U. S. A. 91:4249–53.CrossRefPubMedPubMedCentralGoogle Scholar
  73. 73.
    Liu LN, et al. (2011) A comparison of proteomic analysis of Helicobacter pylori in patients with gastritis and gastric cancer between areas of high and low incidence of gastric cancer. Beijing Da Xue Xue Bao. 43:827–32.PubMedGoogle Scholar
  74. 74.
    Jung SW, Sugimoto M, Graham DY, Yamaoka Y. (2009) homB status of Helicobacter pylori as a novel marker to distinguish gastric cancer from duodenal ulcer. J. Clin. Microbiol. 47:3241–5.CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Park JW, et al. (2008) Proteomic analysis of Helicobacter pylori cellular proteins fractionated by ammonium sulfate precipitation. Electrophoresis. 29:2891–903.CrossRefPubMedGoogle Scholar
  76. 76.
    Baik SC, et al. (2004) Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Helicobacter pylori strain 26695. J. Bacteriol. 186:949–55.CrossRefPubMedPubMedCentralGoogle Scholar
  77. 77.
    Cho MJ, et al. (2002) Identifying the major proteome components of Helicobacter pylori strain 26695. Electrophoresis. 23:1161–73.CrossRefPubMedGoogle Scholar
  78. 78.
    Sabarth N, et al. (2002) Identification of surface proteins of Helicobacter pylori by selective biotinylation, affinity purification, and two-dimensional gel electrophoresis. J. Biol. Chem. 277:27896–902.CrossRefPubMedGoogle Scholar
  79. 79.
    Bumann D, et al. (2002) Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori. Infect. Immun. 70:3396–403.CrossRefPubMedGoogle Scholar
  80. 80.
    Krah A, et al. (2003) Analysis of automatically generated peptide mass fingerprints of cellular proteins and antigens from Helicobacter pylori 26695 separated by two-dimensional electrophoresis. Mol Cell Proteomics. 2:1271–83.CrossRefPubMedGoogle Scholar
  81. 81.
    Yonezawa H, et al. (2011) Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori. Anaerobe. 117:388–90.CrossRefGoogle Scholar
  82. 82.
    Jungblut PR, et al. (2010) Helicobacter pylori proteomics by 2-DE/MS, 1-DE-LC/MS and functional data mining. Proteomics. 10:182–93.CrossRefPubMedGoogle Scholar
  83. 83.
    Hynes SO, McGuire J, Wadström T. (2003) Potential for proteomic profiling of Helicobacter pylori and other Helicobacter spp. using a ProteinChip array. FEMS Immunol. Med. Microbiol. 36:151–8.CrossRefPubMedGoogle Scholar
  84. 84.
    Nilsson CL, Larsson T, Gustafsson E, Karlsson KA, Davidsson P. (2000) Identification of protein vaccine candidates from Helicobacter pylori using a preparative two-dimensional electrophoretic procedure and mass spectrometry. Anal. Chem. 72:2148–53.CrossRefPubMedGoogle Scholar
  85. 85.
    Zhang YN, et al. (2011) Comparative proteome analysis of Helicobacter pylori clinical strains by two-dimensional gel electrophoresis. J. Zhejiang Univ. Sci. B. 12:820–27.CrossRefPubMedPubMedCentralGoogle Scholar
  86. 86.
    Khoder G, Yamaoka Y, Fauchère JL, Burucoa C, Atanassov C. (2009) Proteomic Helicobacter pylori biomarkers discriminating between duodenal ulcer and gastric cancer. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 877:1193–9.CrossRefPubMedPubMedCentralGoogle Scholar
  87. 87.
    Yamaoka Y, et al. (2006) Helicobacter pylori outer membrane proteins and gastroduodenal disease. Gut. 55:775–81.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Author(s) 2014

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (https://doi.org/creativecommons.org/licenses/by-nc-nd/4.0/)

Authors and Affiliations

  • Ombretta Repetto
    • 1
  • Stefania Zanussi
    • 2
  • Mariateresa Casarotto
    • 2
  • Vincenzo Canzonieri
    • 3
  • Paolo De Paoli
    • 1
  • Renato Cannizzaro
    • 4
  • Valli De Re
    • 1
  1. 1.Facility of Bio-ProteomicsCentro di Riferimento Oncologico (CRO), Aviano National Cancer InstituteAvianoItaly
  2. 2.Microbiology-Immunology and VirologyCentro di Riferimento Oncologico (CRO), Aviano National Cancer InstituteAvianoItaly
  3. 3.Pathology UnitCentro di Riferimento Oncologico (CRO), Aviano National Cancer InstituteAvianoItaly
  4. 4.Gastroenterology UnitCentro di Riferimento Oncologico (CRO), Aviano National Cancer InstituteAvianoItaly

Personalised recommendations