1. EPITHELIAL PROLIFERATION IN H
EPITHELIAL PROLIFERATION IN  H. PYLORI GASTRITIS AND ITS GENETIC REGULATION AFTER BACTERIAL ERADICATION IN INTESTINAL METAPLASIA: ACTUAL FINDINGS AND PERSPECTIVES
E. Ierardi

2. Helicobacter pylori and gastric carcinogenesis
…all long standing inflammations are suitable of neoplastic evolution…

3. Helicobacter pylori and gastric carcinogenesis
WHO: role of H. pylori as gastric carcinogen
June, 1994

4. Helicobacter pylori and gastric carcinogenesis
epidemiology
histopathology
biology

5. EPIDEMIOLOGY
Increased values of incidence and prevalence of the infection in geographic areas with high rates of gastric carcinoma
Forman, 1990 (Cina)
Chen, 1990 (Colombia)
Increased risk of gastric carcinoma in pediatric patients with infection
Sipponen, 1993

6. HISTOPATHOLOGY
Helicobacter pylori  chronic active gastritis  metaplasia intestinale and/or atrophy  dysplasia  carcinoma
Is gastric cancer an infectious disease?
Correa, New Engl J Med, 1991

7. GASTRITIS - CARCINOMA SEQUENCE
intestinal metaplasia
High degree dysplasia and carcinoma

8. E Ierardi, A Pellecchia, P Panza, T Balzano, RA Monno, R Antonacci, A Francavilla.
Epithelial dysplasia arising from intestinal metaplasia in a patient with Helicobacter pylori
Folia Oncologica 1994, 17:

9. BIOLOGY Gastric epithelial turnover Genetic mutations
Interaction with immune system
Bacterial factors
Diet
Animal models

10. EPITHELIAL GASTRIC PROLIFERATION
In normal conditions it sustains mucosal barrier integrity
Proliferating stem cells are confined in the glandular neck and migrate from this site towards foveolar tip undergoing a differentiation
Old and ineffective cells undergo apoptosis
Gastric epithelial turnover:
proliferation/apoptosis ratio
Croitrou, 1993

11. gastritis + gastric epithelial hyperproliferation  eradication
epithelial proliferation
Helicobacter pylori 
gastritis + gastric epithelial
hyperproliferation
 eradication
gastritis healing + normal proliferative pattern
Lynch, 1993
Fraser, 1994
Cahill, 1994
Panella, 1996

12. restore of normal proliferative pattern
epithelial proliferation
Helicobacter pylori 
extension of proliferative site showing a tendency to escalate towards foveolar tip
 eradication
restore of normal proliferative pattern
Testino, 1995
Ierardi, 1996

13. gastritis + intestinal metaplasia + hyperproliferation  eradication
epithelial proliferation
Helicobacter pylori 
gastritis + intestinal metaplasia + hyperproliferation
 eradication
persistence of hyperproliferation in areas of intestinal metaplasia
after 6 months
Ierardi, 1997

14. p53 (“guardian” of genoma integrity) 
genetic mutations
p53 (“guardian” of genoma integrity) 
Its mutation has been observed in H. pylori positive gastritis
frequently, but not always associated to intestinal metaplasia
Tahara.1994
Occhiai, 1996
Nardone, 1999

15. epithelial proliferation)
Genetic mutation
ras p21 (interaction between extra-intracellular environment
aimed to control
epithelial proliferation) 
this mutation is observed in intestinal metaplasia associated to epithelial hyperprolifratio which does not reverse after eradication
Ierardi, 1997

16. Helicobacter pylori and apoptosis:
infection induces an increase of apoptosis
rate
in intestinal metaplasia
apoptosis is normal despite
epithelial hyperproliferation
Moss, 1996
Jones, 1997
Scotiniotis, 1999

17. its mutation may be observed in H. pylori gastritis
genetic mutations
bcl2 (anti-apoptosis) 
its mutation may be observed in H. pylori gastritis
frequently, but not always associated to intestinal metaplasia
Lauwers, 1994
Saegusa, 1995
Nardone, 1999

18. Apoptosis
PARP
TNF alpha
Ierardi, 2002

19. p53 ras p21 Bcl2 apoptosis proliferation
Genes
Inflammation
TNF
p53
ras p21
Bcl2
apoptosis
proliferation
Intestinal metaplasia as precancerosis

20. autoantibodies to parietal cells
Interaction with immune system
Helicobacter pylori
 “antigenic mimicry”
autoantibodies to parietal cells 
glandular atrophy
Negrini,

21. Auto-reaction H. pylori-mediated to parietal cells:
Interaction with immune system
Auto-reaction H. pylori-mediated to parietal cells:
may be observed in pediatric age
associated to epithelial hyperproliferation
extension of proliferative site towards glandular area
Ierardi, 1998

22. Gene CagA (pathogenicity “island”)
Bacterial factors
Gene CagA (pathogenicity “island”) 
“BAD STRAINS”
Major gastric lesions
(ulcers, tumors)

23. Cytotoxic strains: Increase proliferative “response” to infection
Bacterial factors
Cytotoxic strains:
Increase proliferative “response” to infection
induce intestinal metaplasia
are associated to autoimmunity to parietal cells

24. mutagens lack of “protective” factors (anti-oxydants)
Diet
mutagens
(salt, nitrosoderivates)
lack of “protective” factors (anti-oxydants)
Sobala, 1991
Schorach, 1991

25. Ferrets infected with H. mustelae and treated with MNNG
Animal models
Ferrets infected with H. mustelae and treated with MNNG
Development of carcinoma without progressive stages of carcinogenesis
Fox et al, Carcinigenesis, 1993
Mongolian gerbils infected with H. pylori treated with MNU
Observed the progressive stages of carcinogenesis
Not all carcinomas of intstinal type
Sugiyanma et al, Cancer Res, 1998

26. Helicobacter pylori induces gastric cancer in Mongolian Gerbils
Animal models
Helicobacter pylori induces gastric cancer in Mongolian Gerbils
Watanabe et al, Gastroenterology, 1999

27. Animal models
Weeks
Bacterial load (log CFU)
Hystology
chronic active gastritis 100% 100% 100% 100% 100%
ulcer % 100% 100% 40% 59%
epithelial hyperplasia 100% 100% 100% 100% 100%
intestinal metaplasia 0% 60% 80% 100% 85%
hyperplastic polyps 0% 0% 0% 60% 15%
adenocarcinoma 0% 0% 0% 0% 37%
carcinoid 0% 0% 0% 0% 11%

28. Helicobacter pylori and gastric cancer: Koch’ s postulates fullfilled?
Animal models
Helicobacter pylori and gastric cancer: Koch’ s postulates fullfilled?
Wang & Fox, Gastroenterology,1998, 115: 780-3

29. ?
CLINICAL
IMPACT

30. Experimental evidences Evidence-based medicine
CLINICAL IMPACT
Experimental evidences
Evidence-based medicine

31. CLINICAL IMPACT
H. pylori eradication may heal low grade MALT lymphoma (!!!)
H. pylori eradication may prevent the development of gastric carcinoma (???)

32. A 1.8% 10-year cumulative incidence of gastric cancer in IM patients was estimated from the literature.

33. Research project: background
It is conceivable that an array of various genotoxic affections are activated at the time of H. pylori infection
The accumulation of various gene errors with the time may play a role as effector mechanism modulating the course and the final outcome of the disease
NEED OF STUDIES IN THE LONG TERM
(5-10 yrs after eradication)

34. MicroRNAs (miRNA) are a family of small
Genotoxic affections:MicroRNAs
MicroRNAs (miRNA) are a family of small
non-coding RNAs,
powerful post transcriptional regulators of various biological processes from cell fate determination to signaling events
Ambros 2004
Bartel 2004

35. MiRna are involved in:
Tight control of development, differentiation and homeostasis
Regulation of immune innate and adaptative response
Regulation of balance inflammation/autoimmunity
Carcinogenesis
Metabolism

36. MiR-146a
miR-146a reacts to LPS in human monocytes thus inducing expression of NFκB, an important casual link between inflammation and cancer
miR-146a acts as a negative feedback regulator of the innate immune response

37. miR 146 a and H.pylori
Z. Liu 2010
TNF alpha?

38. miR 146 a and H.pylori
H. pylori related proinflammatory cytokines contribute to the induction of miR-146a. NF-қB pathway is required for the induction of miR-146a upon stimulation cytokines
Overexpression of miR-146a reduces H. pylori–induced IL-8, TNF-α and IL-1b
CagA is not necessarily required for miR-146a induction in H. pylori infection
Feed-back?

39. miR146a and cancer
Changes in the expression of miR-146a have been implicated in both the development of multiple cancers and in the negative regulation of inflammation induced via the innate immune response.
miR-146a expression is driven by the transcription factor NF-kappaB, an important causal link between inflammation and cancer. 39

40. Research project: background
microRNAs (mi-R) have emerged as important gene regulators and are recognized as oncogenes or tumor suppressor genes in carcinogenesis
mi-R 146a is upregulated in gastric epithelial cell infected with H. pylori
The same type of micro-RNA is overexpressed in gastric cancer
It is able to interact with cytokines and block apoptosis

41. Research project: summary
in H. pylori carcinogenesis, intestinal metaplasia represents the early pre-cancerous lesion and ras mutation and mi-R 146a are the early genetic changes affecting the alteration in epithelial turnover
there is a lack of evidences about epithelial turnover configuration and associated genetic changes in early stages of H. pylori carcinogenesis, when estimated in the long term (5-10), and of their possible reversibility

42. Methods
Paraffin-embedded samples will be retrieved from H. pylori positive patients showing intestinal metaplasia taken before and after eradication in the course of 5-10 year follow-up
After RNA extraction, reverse transcriptase real time PCR will be performed to detect the mRNA coding for the synthesis of:
Proliferating cell nuclear antigen (PCNA) as marker of epithelial proliferation
Poly ADP ribose polymerase (PARP) as marker of apoptosis
Mutated ras oncogene
Mi-R 146a.

43. PERSPECTIVES
Is it possible a reversibility of early genetic alteration after H. pylori disappearance?
Is it possible a restoration of normal epithelial turnover?
Is it possible a biologic “rest” of intestinal metaplasia?
A decrease in gastric cancer incidence will be obtained by H. pylori eradication