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From the Institute of Molecular Pathology and Immunology of the
University of Porto *
and Departments of
Biophysics
and
Pathology,
Medical Faculty, University of
Porto, Porto, Portugal
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most of the mutations within TGFß RII occur in two microsatellites of the coding region. One of these microsatellites is a poly(A)10 tract, and mutations within this region consist of 1- or 2-base deletions or insertions; the other is a poly(GT)3 microsatellite that was found to have an insertion of an extra GT.13-15 These mutations cause frameshifts of TGFß RII and result in truncation or substitution of conserved residues of the predicted protein product.16 The growth-inhibitory effect of TGF-ß is primarily mediated by a heteromeric complex of two distantly related transmembrane serine/threonine kinases (receptors I and II); the inactivation of either receptor subtype can result in TGF-ß resistance.7,17 Loss of responsiveness to TGF-ß is common in human cancers and is thought to be an important step in tumorigenesis.18,19
The IGFII R gene contains several microsatellites within its coding region, one of which is an 8-deoxyguanosine repeat that is frequently mutated and comprises 1- or 2-bp deletions or insertions, causing frameshifts and premature stop codons.9 IGFII R also inhibits cell proliferation mediated by the IGFII ligand, itself a potent growth stimulant, by internalizing and degrading this protein.20 Thus, IGFII R, by antagonizing the growth-stimulatory effect of IGFII and activating the growth-inhibitory effect of TGF-ß, serves as a growth suppressor gene.9
The human BAX gene contains a tract of eight consecutive deoxyguanosines in the third coding exon; insertions or deletions of one nucleotide are the most frequent mutations in this microsatellite and lead to alterations in the function of the protein.11 BCL2 and BAX proteins are encoded by a family of genes that take part in the maintenance of the balance between cell proliferation and programmed cell death, in multicellular organisms. The BAX gene acts as a promoter of cell death by opposing the death protector effect of the BCL2 gene.21 BAX homodimerizes, as well as heterodimerizes with BCL2, and it was suggested that the ratio of BCL2 to BAX determines survival or death after an apoptotic stimulus.22
We decided to search for microsatellite instability in a series of 152 thoroughly studied cases of sporadic gastric carcinoma and for the presence of mutations in TGFß RII, IGFII R, and BAX genes in a subset of 28 RER+ and 13 RER- carcinomas, with the following aims: 1) to analyze the prognostic meaning of the RER status in a multivariate analysis along with the most important clinicopathological features and 2) to determine the frequency of mutations in each of the three target genes and to find out whether the occurrence of such mutations is related with any clinicopathological feature(s) of RER+ gastric carcinomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients, Tissue Samples, and DNA Extraction
We analyzed the surgical specimens from 152 gastric carcinomas consecutively resected at Hospital of S. João (Porto, Portugal) from 1988 through 1995. Radical extended gastrectomy was performed in the 26 patients with carcinomas involving the cardia. Radical total gastrectomy was performed in the following 60 cases: 36 patients with carcinomas located in the proximal stomach regardless of the histological type, 22 patients with diffuse carcinomas located in the antrum, and 2 patients whose stomachs had been operated on for peptic ulcer. Curative subtotal distal gastrectomy was performed in 66 patients with intestinal or atypical carcinomas located in the antrum.23 In all cases the surgical therapy was performed to achieve a resection without leaving behind macroscopic or microscopic disease. No patient had received preoperative chemo- or radiotherapy. A family history was obtained in every case; none of the patients included in the present study had a family history suggestive of hereditary nonpolyposis colorectal cancer. Follow-up information was obtained in all but five cases. Two cases were excluded from the survival analysis, because they had been in the study for less than 3 years. The range of follow-up was 3 to 9 years (median, 70 months). The six deaths occurring within the 1st month of surgery were considered postoperative deaths.
Hematoxylin and eosin-stained sections were used to classify the tumors according to the classifications of Laurén,23 Ming,24 and Carneiro.25 The pathological staging was achieved using the unified 1987 TNM system for gastric carcinoma. Orcein-stained sections were used for the detection of vascular invasion. Lymphoid infiltration was subjectively scored into absent/minimal and moderate/abundant. Immunohistochemistry was used to classify the lymphocytic infiltration of the tumors, according to the predominance of T or B lymphocytes, using UCHL1 and L26 antibodies (Dako, Glostrup, Denmark), respectively. From each case, tissue fragments from primary tumors and nonneoplastic mucosa were immediately frozen in liquid nitrogen and stored at -70°C until use. High molecular weight DNA was isolated using standard methods26 in a total section of the tumors wherever tumor cells occupied more than 50% of tumor tissue or in microdissected areas where at least 50% of tumor cells were present as evaluated by concurrent cryostat sections.
RER Assays
The 152 gastric carcinomas were studied for microsatellite
instability using a panel of five or six dinucleotide repeat sequences,
as described by Santos et al5
(n =
104), and/or using a primer set localized on intron 5 of the
hMSH2 gene, that amplifies an adenine monomorphic
mononucleotide repeat, BAT 26, as described by Hoang et al1
(n = 152). Fifty of the 152 tumors were
previously reported by Santos et al.5
The polymerase chain
reaction (PCR) products were labeled by [
-32P]dCTP
during amplification reaction, separated by electrophoresis in 6%
denaturing polyacrylamide gels, and visualized through autoradiography.
Cases were considered as having an RER-positive phenotype whenever they
expressed high frequency of microsatellite instability (
40%) using
the dinucleotide repeat markers and/or BAT 26 positivity. In the 104
cases in which we have used both methods, the consistency of the
results was total (100%).
All cases were screened, at least twice, by independent PCRs and independent electrophoretic run. All the scorings were done independently by two observers.
Amplification of the Target Genes (TGFß RII,IGFII R, and BAX) and the "Control Gene" (HPRT)
Detection of mutations in target genes was restricted, in the present study, to the 28 RER+ cases in which constitutional and tumoral DNA was available. We analyzed, furthermore, 13 tumors with an RER- phenotype that were randomly selected from the series of 117 RER- carcinomas.
TGFß RII
Poly(A)10 microsatellite sequence in nucleotides 709 to 718 of the TGFß RII gene was amplified by radioactive PCR using the following set of primers: (RIIU1) 5'-AGA TGC TGC TTC TCC AAA GTG C-3' and (RIID1) 5'-TTG CAC TCA TCA GAG CTA CAG G-3'. These primers amplify a 90-bp target sequence from nucleotides 677 to 766. The primer sequences for the (GT)3 microsatellite sequence in nucleotides 1931 to 1936 were (RIIU2) 5'-ACT GAG TGC TGG GAC CAC G-3' and (RIID2) 5'-AGG AAT CTT CTC CTC CGA GC-3', which amplify the 123-bp target sequence from nucleotides 1887 to 2009.
IGFII R
The 110-bp sequence within IGFII R that contains an 8-deoxyguanosine repeat from nucleotides 4030 to 4140 was amplified by PCR using primer set R4, consisting of (IGFII R1) 5'-GCA GGT CTC CTG ACT CAG AA-3' and (IGFII R2) 5'-GAA GAA GAT GGC TGT GGA GC-3'.
BAX
The 94-bp region encompassing the (G)8 tract of the BAX gene was amplified by PCR with the following primers: (BAX1) 5'-ATC CAG GAT CGA GCA GGG CG-3' and (BAX2) 5'-ACT CGC TCA GCT TCT TGG TG-3', from nucleotides 90 to 184.
HPRT
The 160-bp region within HPRT, which contains a 6-deoxyguanosine repeat in exon 3, was amplified by PCR with the following primers: (HPRT1) 5'-GAC TGA ACG TCT TGC TCG AGA TG-3' and (HPRT2) 5'-AAT CTA CAG TCA TAG GAA TGG A-3'.
The PCR products of the different primer sets, described above, were denaturated in a solution containing 95% formamide for 5 minutes at 94°C and then run in denaturing 6% polyacrylamide sequencing gels. TGFß RII and BAX genes were run in polyacrylamide gels with 32.5% formamide and 6.88 mol/L urea, and IGFII R was run in a gel without formamide and with a lower concentration of urea (6 mol/L). After electrophoresis, the gels were exposed to X-ray film for 4 to 12 hours.
The tumors were considered as having a mutation whenever they showed abnormal bands or shifts in bands in comparison with the respective normal tissue.
Statistical Analysis
The statistical analysis of the results was performed using the
2
test with Yates correction, Fisher's exact test, or
Student's t-test with Statview 4.01 software. Of the 152
patients, 139 were considered for survival analysis; five patients were
lost to follow-up, 2 patients were excluded because they had been on
the study for less than 3 years, and 6 patients died in the
postoperative period. The relationship between RER status and survival
rate of patients was assessed by univariate and multivariate analysis
(BMDP statistical software, Cork, Ireland). The following parameters
were taken into consideration in the survival analysis: age, sex, tumor
site, gross appearance, histological classification, depth of wall
penetration, venous and lymphatic invasion, pathological staging
(pTNM), and RER status. Survival curves were calculated according to
Berkson's actuarial method and compared using the Generalized Savage
(Mantel-Cox) test. The evaluation of the prognostic significance of the
clinicopathological factors was performed by multivariate regression
techniques (Cox's proportional hazards model) in 136 of the 139
patients; three cases were excluded from this analysis because of
missing values on size (n = 2) and gross
appearance (n = 1). A P value of
<0.05 was considered statistically significant, and a P
value of <0.1 was considered suggestively significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows some
examples of BAT 26 analysis with unstable shortened alleles. The
comparison of the clinicopathological features of the cases with RER+
and RER- carcinomas is summarized in Table 1
. Patients with RER+
tumors were significantly (P = 0.03) older
(64.5 ± 12.8 years) than patients with RER- tumors (59.3 ±
12.1 years). Significant associations were also found between RER+
phenotype and distal location of the tumors (P =
0.0003), Laurén's23
intestinal and atypical
histotypes (P = 0.0002),
Carneiro's25
glandular type (P =
0.05), and Ming's24
expansive pattern of growth
(P = 0.0001). The percentage of cases with lymph
node metastases was significantly lower (P =
0.03) in the group of RER+ carcinomas (17.5%) than in the group of
RER- carcinomas (82.5%) (Table 1)
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. The other clinicopathological
features significantly associated with the survival of the patients, in
univariate analysis, were: Carneiro's25
histological
classification (P = 0.008), pTNM stage
(P = 0.012), and vascular invasion
(P = 0.032). The multivariate analysis showed
that pTNM stage was the strongest prognostic factor, followed by wall
invasion, vascular invasion, Carneiro's histological classification,
and RER status (P = 0.048) (Table 2)
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,
whereas 1 of the 13 RER- carcinomas had mutations in IGFII
R and BAX genes (Table 3)
. In the repeat sequence of
HPRT gene, no mutations were found in any of the cases. From
the 23 tumors that presented mutations in the target genes, 11 tumors
(47.8%) displayed mutations in only one gene, and 12 (52.2%) in more
than one gene (Table 3)
. TGFß RII was the most frequently
affected gene. Nineteen of the 28 RER+ tumors (67.9%) presented
alterations within this gene. Eight of the 19 tumors displayed
mutations only in TGFß RII, whereas in the remaining 11
tumors the mutations in TGFß RII were associated with
mutations in other gene(s) (Table 3)
. In the series of the RER+ tumors,
we found that mutations in the TGFß RII gene were
significantly associated (P = 0.02) with the
glandular histotype (Table 4)
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.
A suggestive association (P = 0.06) was found in
the setting of RER+ cases between IGFII R mutations and
lower prevalence of lymph node metastases: RER+ carcinomas with
IGFII R mutations had lower prevalence of lymph node
metastases (11.8%) than RER+ carcinomas without mutations in this gene
(88.2%). BAX mutations were present in 9 of the 28 RER+ tumors (32.1%). Two of the 9 tumors had BAX mutations as a sole alteration; the remaining 7 tumors presented mutations in BAX gene in association with mutations in TGFß RII and/or IGFII R genes. We observed a significantly higher (P = 0.005) percentage of females in the group of cases with BAX mutations (77.8%) than in the group of cases without BAX mutations (31.6%). A suggestive association (P = 0.09) was found between BAX mutations and the degree of wall invasion: 2 of the 9 cases with mutations in BAX were limited to the superficial layers (T1 or T2) of the gastric wall, whereas all 19 carcinomas without BAX mutations invaded the deep layers (T3 or T4) of the stomach.
No other associations were found between the clinicopathological features of the tumors and IGFII R or BAX mutations (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study of 35 RER+ sporadic gastric carcinomas confirms most of the clinicopathological data previously obtained in a series of 12 RER+ carcinomas.5 RER+ gastric carcinomas tend to occur as large and expanding tumors of the distal stomach in relatively old patients; they display usually an intestinal (glandular) or atypical (solid) histotype and often do not give rise to lymph node metastases, regardless of the degree of wall invasion (they usually occur with low pTNM stages). Similar clinicopathological features had been previously pointed out in RER+ carcinomas of other organs.2 The striking differences between RER+ and RER- carcinomas with regard to site and histotype of the tumors (RER+ carcinomas are extremely rare in the cardia and almost never display a pure diffuse (isolated cell) pattern) support the assumption that the etiopathogenesis of cardiac and diffuse carcinomas of the stomach differ from those of antral and intestinal types.29-32
At variance with our previous results,5 we did not find, in the present study, a significantly higher lymphoid infiltration in RER+ carcinomas, despite a trend to more abundant lymphoid cells in this group of tumors. We also did not find any significant difference between the two groups of carcinomas regarding the abundance and relative distribution of B and T lymphocytes (data not shown). It remains therefore questionable whether unrepaired errors of certain genes in the setting of RER phenotype may lead to the appearance of new surface molecules and, in this way, trigger an immune response.5,33
In keeping with most of the on-record studies in gastric and colorectal carcinomas,5,26,34 the RER status was found to be significantly related to survival in univariate analysis. We have shown, moreover, for the first time, to the best of our knowledge, that the same holds true regarding multivariate analysis. The significant association between RER+ phenotype and low prevalence of lymph node metastasization and low pTNM stages indicates that the good outcome of patients with RER+ carcinomas may be ascribed, partly at least, to the close relationship between RER status and staging.
The search for mutations in the three target genes, TGFß RII, IGFII R, and BAX, yielded positive results, in contrast to the absence of mutations in HPRT, a constitutional gene present in all types of cells. This finding supports the assumption that only mutations in target genes that have a direct role in carcinogenesis confer a clonal advantage to the neoplastic cells.16 Mutations in the target genes were almost exclusively detected in RER+ tumors, thus confirming that the involvement of such genes is most likely due to a mismatch repair deficiency.
The higher incidence of mutations in TGFß RII gene suggests that the alterations of the TGFß RII gene occur as an earlier event than those of IGFII R or BAX gene in gastric carcinogenesis. We found just one case with mutations in IGFII R and BAX in the control series composed of RER- tumors. Similar findings were reported by Renault et al,29 Myeroff et al,28 and Akiyama et al.10 As in the series of Ouyang et al,8 the RER- tumor of our series with mutations of IGFII R and BAX genes is an advanced tumor (T3); this finding points to the possibility that such mutations may represent a genetic change occurring during progression, rather than a crucial event in the early steps of tumor development.
In all cases with mutations, we found bands corresponding to the wild-type sequences of the target genes; however, in most of the primary tumors, the band representing the wild-type allele was decreased in intensity, thus suggesting that both alleles were mutant (the residual wild-type signal probably arose from contaminating nonneoplastic cells within the tumor specimens). This assumption was confirmed in xenografts derived from cases 199 and 233 of our series (data not shown), as it had been previously shown in colon cancer.16,28 Alternatively, heterozygous mutations may contribute to tumor progression, as it was described for TGFß RII.35 Rampino et al11 also suggested that reduction of wild-type BAX, because of the inactivation of one allele, facilitates escape from apoptosis by diminishing the BAX-BCL2 ratio.
In the present study, TGFß RII was affected by mutations in 67.9% of the 28 RER+ carcinomas, a percentage that fits within the range of those on record (50 to 92.4%).8,12,13,28,29,35,36 Because the TGFß RII poly(A)10 tract is a mutation target in cells with genetic instability, it is tempting to advance the existence of a carcinogenic pathway in which TGFß RII mutations would confer growth advantage, and would be selected for, in RER+ gastric carcinomas.12,13,37-39 The significant association between TGFß RII mutations and the RER+ phenotype fits with this possibility. Our finding of a close relationship between TGFß RII mutations and the glandular histotype supports, moreover, the role played by TGFß/TGFß RII in the development of glandular-type gastric carcinoma, in a similar way as in colon cancer.12,15,28,40
We found IGFII R mutations in 25% of the RER+ sporadic gastric carcinomas. This percentage is similar to those reported by other groups (range, 23 to 33%).8,9,12,13 We observed, furthermore, a trend toward an association between mutations in IGFII R and low prevalence of lymph node metastases. We still do not know the meaning of this finding, although it fits with the association between RER phenotype and less clinically aggressive tumors. Curiously, the only IGFII R mutated tumor with an RER- phenotype in our series did not display lymph node metastases despite invading the wall of the stomach widely (data not shown). Our results are in accordance with the role on cell motility and metastasization advanced by Minniti et al41 for IGFII R in rhabdomyosarcoma cells. A larger series is necessary to confirm whether or not there is a relationship between IGFII R mutations and decreased nodal metastatic ability of gastric carcinoma.
BAX mutations were detected in 32.1% of the RER+ sporadic gastric carcinomas. This percentage is lower than that observed previously in colon cancer11 and lies in between those reported by Chung et al12 in a series of 6 gastric carcinomas (66%) and by Wu et al13 in a series of 13 gastric carcinomas (15.4%). We found a significantly larger percentage of cases with BAX mutations in women than in men. We do not know the reason behind this finding, which cannot be linked to a particular cancer histotype, because it is known that women tend to have diffuse (isolated cell) carcinoma,25 and this type of gastric carcinoma is extremely rare in the setting of RER+ phenotype in most of the series on record.5,6,13,29,36,42-44 We also found evidence suggesting a putative association (P = 0.09) between the presence of BAX mutations and diminished penetration of the gastric wall by the neoplastic cells; this finding fits with the overall impression on the low clinical aggressiveness of many RER+ carcinomas, but the association is too weak and the series is too small to allow a definitive conclusion on this issue. In conclusion, this retrospective study demonstrates that RER status is a significant prognostic indicator in gastric carcinoma. It shows, moreover, that such prognostic meaning may be mediated by mutations in several "target genes" exhibiting microsatellite instability.
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Footnotes |
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Supported by PRAXIS XXI (project 2/2.1/BIA/250/94).
Accepted for publication July 18, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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