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

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

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

Helicobacter pylori and gastric carcinogenesis epidemiology histopathology biology

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

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

GASTRITIS - CARCINOMA SEQUENCE intestinal metaplasia High degree dysplasia and carcinoma

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: 21-24.

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

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

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

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

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

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

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

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

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

Apoptosis PARP TNF alpha Ierardi, 2002

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

autoantibodies to parietal cells Interaction with immune system Helicobacter pylori  “antigenic mimicry” autoantibodies to parietal cells  glandular atrophy Negrini, 1992-1996

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

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

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

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

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

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

Animal models Weeks 6 26 39 52 62 Bacterial load (log CFU) 5.17 5.33 5.89 4.59 5.40 Hystology chronic active gastritis 100% 100% 100% 100% 100% ulcer 0% 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%

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

? CLINICAL IMPACT

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

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

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

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)

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

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

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

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

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?

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

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

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

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.

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