WORKSHOP
IL CANCRO DELLO STOMACO
BIO-MOLECULAR ASPECTS OF GASTRIC CANCER
Department of Oncology and
Neurosciences, Section of Molecular Pathology
University of Chieti
Alessandro Cama
Messina
15 giugno 2001
Gastrointestinal carcinogenesis is a multistep
process involving the sequential accumulation of alterations in multiple genes
including APC, p53, K-ras and mismatch repair (MMR) genes (1). Genetic
instability in neoplastic cells is required to drive the accumulation of the
large number of mutations that occurs during the tumorigenic process (1). Two
main types of genetic instability have been recognized so far in gastric cancer:
chromosomal instability (CIN) and microsatellite instability (MIN
also known as MSI). Both forms of instability can be investigated by
microsatellite analysis. Therefore, we analyzed microsatellite status in early
gastric cancer (2) and in a series of epidemiologically characterized gastric
cancer patients (3, 4). Furthermore we analyzed the pathway of tumor progression
in gastric cancers with microsatellite instability (5).
In
collaboration with the University of Messina (Proff. Cosimo Inferrera, Rosario
Caruso, Franco Fedele), we analyzed the role of genetic instability in early
gastric carcinogenesis (2). We analyzed the status of 11 microsatellite loci in
paired microdissected samples of tumor and unaffected mucosa from 30 cases of
early gastric carcinoma. Seventeen
tumors (56%) presented one or more microsatellite alterations.
These consisted in contractions/expansions of sequence repeats typical of
MSI and/or loss of microsatellite alleles (loss of heterozygosity = LOH) typical
of CIN. MSI+ typings were
statistically more frequent in intramucosal tumors as compared to tumors with
submucosal spread. The presence of
microsatellite alterations, either LOH alone at the loci analyzed or MSI + LOH
was significantly associated with the absence of lymph node metastasis.
In
a further study conducted in collaboration with the C.S.P.O. of Florence (Dr.
Domenico Palli and collaborators) we analyzed the presence of microsatellite
instability (MSI) in a series of 108 gastric cancers (GCs) previously identified
in an epidemiological study carried out in a high risk area around Florence (3).
To investigate associations between MSI and GC family history 34 cases
(31.5%) who had a GC-affected first-degree
relative were included in the series. A family history positive for colorectal
cancer was reported quite rarely (5.6%). The
analysis of 6 microsatellite loci in DNA from paired normal tissue and tumor
samples, microdissected from paraffin-embedded specimens, revealed varying
degrees of instability: 56 cases
(51.8 %) did not show instability at any of the 6 loci; 19 (17.6 %) showed
instability at one locus; 16 (14.8 %) at two loci; 11 (10.2 %) at three loci; 4
(3.7 %) at four loci; 2 (1.9 %) at five loci.
The replication error positive (RER+) phenotype, defined as the presence of MSI at two loci or more, had a frequency
of 30.6% (33/108) and tended to be positively associated with female sex,
intestinal histologic type, advanced tumor stage, vascular invasion, positive
gastric cancer family history and blood group of A type.
No correlation emerged between age at diagnosis and MSI phenotype, while
a significant association with the MSI+ phenotype was shown by the antral
location. A multivariate analysis
adjusting for a selected group of potential confounding factors confirmed the
strong association of the MSI+ phenotype with the antral location (p=0.001) and
with a positive GC family history (p<0.05). Survival analyses at 5 years
showed improved survival in high-level MSI as compared to MSI-negative patients
and patients with low level MSI combined (p=0.02). By the microdissection
technique, we also used microsatellite allele patterns to investigate
intratumoral heterogeneity and genetic relationships between tumors and adjacent
dysplasia and/or intestinal metaplasia. Areas
of metaplasia and dysplasia demonstrated MSI only in cases with MSI-positive
tumors. In MSI-positive tumors, there was consistent evidence of intratumoral
microsatellite allele heterogeneity, indicating the presence of genetically
divergent tumor cell clones within the same neoplasm.
In collaboration with the
group of Dr. Domenico Palli we also evaluated the role of dietary risk factors
in GC according to MSI status (4). A large series of 382 GC cases and 561
controls were originally identified in a population-based case-control study
carried out in the high-risk area around Florence, Italy; 126 gastric cancer
patients were typed for MSI status. An MSI positive (MSI+) phenotype was
detected in 43/126 cases (34.1%), while 83 cases were classified as MSI-negative
(MSI-). A multinomial logistic regression model was used to compare in the same
analysis the two subgroups of GC classified according to MSI status, with all
the available population controls. A case-case approach was also used. The risk
of MSI+ tumors was positively associated with high consumption of red meat and
meat sauce, and negatively with white meat. A positive association was also seen
with total protein and nitrite intake, while no relation was found with
micronutrient intake. Risk was especially high among subjects reporting both a
positive GC family history and a high consumption of red meat . For MSI- tumors,
a significant protective effect was associated with frequent consumption of
citrus and other fresh fruit, garlic, legumes, vegetables, olive oil and with
high intake of beta carotene and other antioxidants and sugar, while positive
associations were seen with protein and sodium intake. In summary, a specific
dietary pattern emerged for MSI+ gastric tumors suggesting that factors related
to red meat consumption are involved in this pathway, particularly among
individuals with a positive family history. In contrast, the risk of MSI- tumors
was strongly reduced by the frequent consumption of fresh fruit and vegetables.
Clinico-pathological and epidemiological associations of MSI-H GCs (antral
location, GC family history, dietary patterns) observed in our studies suggest
that specific environmental and genetic factors are linked with MMR deficiency.
The analysis of tumor progression pathways has the potential to identify
novel genes that are mutated in gastric carcinogenesis and may lead to the
identification of markers for prognosis and therapeutic response. Therefore, we
analysed MIN 50 gastric carcinomas (GCs) to verify whether mutations at coding
repeats were associated with microsatellite instability (MIN) (5). The tumors
included: ten cases with no MIN, 14 cases with MSI = 1 locus, 13 cases with MIN=
two loci and 13 cases with MIN> or = 3 loci. We investigated coding repeats
within the TGF-beta RII, IGFIIR, BAX, hMSH6, hMSH3 and BRCA2 genes. The TGF-beta
RII, IGFIIR, BAX, hMSH6 and hMSH3 repeats were altered in 11 (22%), five (10%),
four (8%), 16 (32%) and five (10%) cases respectively. Mutations occurred only
in MSI-positive (MIN+) tumors and correlated with increasing MSI levels. No
alterations of the BRCA2 repeat were found. Mutations in genes other than hMSH6
were strongly associated to hMSH6 mutations, suggesting a key role of this gene.
The non-coding BAT-26 and E-Cadherin 3' UTR poly(A)8/(T)15 repeats were analysed
in 44 of the 50 cases. Novel tumor-associated alleles were observed only in
MSI-positive GCs and were in most cases associated with mutations at coding
repeats. Further investigations with BAT-40 confirmed that four cases manifested
mononucleotide repeat alterations restricted to hMSH6 and one case to TGF-beta
RII. A subset of tumors with MSI at two or more dinucleotide loci resulted
negative for mutations at coding and non-coding mononucleotide repeats.
These findings have implications for the molecular mechanisms leading to
the pathogenesis of gastric cancer, for gastric cancer predisposition and for
tumor progression. In fact, a better understanding of molecular pathogenesis and
of tumor progression may lead to: improved cancer prevention, more accurate
prognostic evaluation and more efficient therapy.
In particular, the correlation of MIN phenotype with specific dietary
pattern and family history may be relevant to cancer prevention (3, 4). The
improved survival of patients with MIN+ gastric cancers may help the prognostic
evaluation of patients (3). Moreover the presence of MIN versus CIN phenotype
may be helpful in establishing a tailored chemotherapeutic intervention, as some
chemotherapeutic agents appear to be best suited for tumors with one and not for
tumors with the other form of genetic instability (6). Finally, the specific
pathway of tumor progression and the knowledge of the genes mutated in a
particular tumor may also help to establish a tailored chemotherapeutic
intervention, as mutation in some key genes such as p53 may render the tumor
insensitive to some agents but more sensitive to others (7).
References
1
Lengauer C, Kinzler KW, Vogelstein B Genetic instabilities in human
cancers Nature 1998; 396: 643-649,
2 Battista, P., Palmirotta, R., Vitullo, P., Veŕ, M.C., Colalongo C., Rigoli L., Fedele F., Caruso R., Inferrera C., Romano F., Mariani-Costantini R., Frati F., Cama A. Microsatellite instability in early gastric cancer. Int J Oncol 1997;10:65-70.
3
Ottini L., Palli D., Falchetti M., D'Amico C., Amorosi A., Saieva C.,
Calzolari A., Cimoli F., Tatarelli C., De Marchis L., Masala G.,
Mariani-Costantini R., Cama A. Microsatellite
instability in gastric cancer is associated with tumor location and family
history in a high risk population from Tuscany.
Cancer Res. 1997; 57: 4523-4529.
4
Palli D, Russo A, Ottini L, Masala G, Saieva C, Amorosi A, Cama A,
D’Amico C, Falchetti M, Palmirotta R, Decarli A, Mariani Costantini R, Fraumeni Jr JF. Red
meat, family history and increased risk of gastric cancer with microsatellite
instability. Cancer Res in
press
5
Ottini L, Falchetti M, D'Amico C, Amorosi A, Saieva C, Masala G, Frati L,
Cama A, Palli D, Mariani-Costantini R Mutations
at coding mononucleotide repeats in gastric cancer with the microsatellite
mutator phenotype. Oncogene.
1998;16:2767-2772
6
Fink D, Zheng H, Nebel S, Norris PS, Aebi S, Lin TP, Nehme A, Christen
RD, Haas M, MacLeod CL, Howell SB. In vitro and in vivo resistance to cisplatin
in cells that have lost DNA mismatch repair. Cancer Res 1997;15;57:1841-5.
7 Bunz F, Hwang P M, TorranceC, Waldman T, Zhang Y, Dillehay L, Williams J, Lengauer C, Kinzler KW, Vogelstein B. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin. Invest. 1999; 104:263–269.
A cura di: Unità di Chirurgia Endoscopica - Ospedale Piemonte - Messina
© 
2001
