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From the Department of Pathology,*
the Division of
Gastrointestinal/Liver Pathology, and the Department of Internal
Medicine,
Division of Gastroenterology, The
Johns Hopkins University School of Medicine, Baltimore, Maryland; and
the Division of Pathology and Laboratory
Medicine,
University of Texas M. D.
Anderson Cancer Center, Houston, Texas
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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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Histopathologically, FGPs are characterized by cystically dilated and irregularly budded fundic glands in a background of otherwise normal gastric mucosa.18,19 The pathogenesis of FGPs remains uncertain. FGPs have generally been regarded as nonneoplastic lesions, either hamartomatous or hyperplastic/functional in nature.16,19,20 Although some studies have reported no differences in the histology between sporadic FGPs and FGPs in patients with FAP,18-20 others have disputed this. In contrast to sporadic FGPs, FGPs in patients with FAP frequently show foveolar dysplasia,8,21-23 including a 25% prevalence of low-grade dysplasia in FAP-associated FGPs.21 In addition, neoplastic progression of FGPs in FAP patients has occasionally been reported, including the development of a large dysplastic gastric polyp with an activating codon 12 K-ras gene mutation in one patient24 and three cases of infiltrating gastric adenocarcinoma23,25,26 arising in association with fundic gland polyposis.
The molecular pathogenesis of other polypoid lesions in the upper and lower gastrointestinal tract in FAP patients has been well characterized.27-29 Most adenomas of the colorectum, duodenum, and stomach in patients with FAP can be demonstrated to have bi-allelic inactivation of the adenomatous polyposis coli (APC) gene on chromosome 5q21-q22. These neoplasms typically harbor a somatic alteration of one allele of the APC gene locus (typically either truncating mutation or allelic loss) coupled with an inactivating germline APC gene mutation in the other allele. Although the exact cellular functions of the APC protein are not fully known, there is data indicating that it has a role in directing cell migration, particularly in enterocyte migration from the crypt to villus in the small intestine.30
The molecular pathogenesis of foveolar dysplasia in FGPs from FAP patients has not been elucidated. One previous study identified somatic (second-hit) mutations of the APC gene in three (27%) of 11 FAP-associated FGPs, suggesting that at least some FGPs involve the APC pathway typically seen in the adenoma-carcinoma sequence.29 However, the histological features of the FGPs, particularly the presence of foveolar dysplasia, were not reported in that study. To better elucidate the molecular pathogenesis of FGPs, we investigated somatic APC gene alterations in dysplastic and nondysplastic FGPs from FAP patients and in sporadic FGPs. To assess whether somatic alterations in other genes associated with gastrointestinal neoplasia contribute to foveolar dysplasia in FGPs, we additionally evaluated K-ras gene mutations in dysplastic and nondysplastic FAP-associated FGPs.
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Materials and Methods |
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The study population consisted of 17 patients with FAP or
attenuated FAP who had upper gastrointestinal endoscopy with gastric
biopsies at The Johns Hopkins University Hospital between 1991 and
1999. Information concerning germline APC gene mutations in these
individuals, obtained from The Johns Hopkins Hereditary Colorectal
Cancer Registry, is shown in Table 1
. A
total of 41 FGPs from these patients was analyzed. FGPs were separated
into three categories based on examination of hematoxylin and eosin
(H&E)-stained histological sections according to previously published
criteria:21
1) no dysplasia of the foveolar epithelium (15
FGPs), 2) epithelial changes indefinite for dysplasia (six FGPs), and
3) foveolar epithelial dysplasia (20 FGPs: 19 with low-grade dysplasia
and one with high-grade dysplasia). Six gastric body adenomas from
these patients were also included in the study. For comparison, 13
sporadic FGPs from 13 patients without FAP were also analyzed.
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Microdissection of H&E-stained slides for DNA extraction was performed from formalin-fixed, paraffin-embedded specimens. Among the 41 FAP-associated FGPs, separate microdissection of the dilated fundic glands of the polyp and the overlying foveolar epithelium was accomplished in 25 cases. In 11 cases, only the foveolar epithelium could be microdissected because of contamination of the dilated glands by mucous neck cells; in four cases only the dilated glands could be microdissected because of denudation of the foveolar epithelium; and in one case both the foveolar and glandular epithelium of the polyp were included together. In all 13 sporadic FGPs, foveolar epithelium and underlying glands were microdissected and analyzed separately. Genomic DNA was extracted as described previously.31 Corresponding control DNA was extracted from nonneoplastic gastric or duodenal epithelium.
Mutational Analysis of the APC Gene
Two sets of oligonucleotide primers (C1:
5'-GGCATTATAAGCCCCAGTGA-3' and C2: 5'-AAATGGCTCATCGAGGCTCA-3' for
codons 1417 to 1516; D1: 5'-ACTCCAGATGGATTTTCTTG-3' and D2:
5'-GGCTGGCTTTT-TTGCTTTAC-3' for codons 1497 to 1596) were used to
amplify the mutation cluster region of the APC gene for gastroduodenal
polyps.29,32
Polymerase chain reaction (PCR) was performed
under standard conditions in a 50 µl volume using PCR Master
(Boehringer Mannheim, Mannheim, Germany) and 1 µmol/L of both 5' and
3' oligonucleotides with 40 cycles (94°C for 1 minute, 58°C for 1
minute, and 72°C for 2 minutes). PCR products were purified using
shrimp alkaline phosphatase and exonuclease I (Amersham,
Buckinghamshire, United Kingdom). Purified PCR products were sequenced
directly with SequiTherm Excel II DNA Sequencing Kit (Epicentre,
Madison, WI) with the same primers used for DNA amplification.
Oligonucleotides were end-labeled with
[
-32P]-ATP (DuPont-New England Nuclear
Research Products, Boston, MA) using T4 polynucleotide kinase (New
England Biolabs, Beverly, MA). All mutations were verified in both the
sense and anti-sense directions.
Allelic Loss on 5q
Loss of heterozygosity (LOH) on 5q was assessed by microsatellite
assays using PCR amplification of three microsatellite markers (D5S299,
D5S346, and D5S82) as previously described.33
Briefly,
assays were performed in 96-well plates with 38 cycles (95°C for 30
seconds, 55°C for 30 seconds, and 72°C for 1 minute) using PCR
Master in a 10 µl volume, 10 ng of both 5' and 3' oligonucleotides
(200 µmol/L), and 
50 ng of genomic DNA in each well. The 5'
oligonucleotide was end-labeled with
[-32P]-ATP using T4 polynucleotide kinase.
LOH was considered to be present when there was disappearance or at
least a 50% reduction in the intensity of a heterozygous band as
compared with nonneoplastic control mucosa in at least one informative
marker.
Mutational Analysis of the K-ras Gene
Sequencing of the K-ras gene was performed in 39 FAP-associated FGPs and 13 sporadic FGPs. One set of oligonucleotide primers (sense: 5'-GAG-AAT-TCA-TGA-CTG-AAT-ATA-AAC-TTG-T-3' and anti-sense: 5'-TCG-AAT-TCC-TCT-ATT-GTT-GGA-TCA-TAT-TCG-3') was used to amplify a region in exon 1 of K-ras spanning codons 12 and 13. PCR reaction was performed under standard conditions in a 50-µl volume using PCR Master and 1 µmol/L of both 5' and 3' oligonucleotides with 40 cycles (94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute). PCR products were purified using shrimp alkaline phosphatase and exonuclease I. Purified PCR products were sequenced directly with the SequiTherm Excel II DNA Sequencing Kit using an internal primer (5'-ATT-CGT-CCA-CAA-AAT-GAT-3').
Statistical Analysis
Fisher’s exact test was used to compare differences in frequencies of somatic APC gene alterations (mutation or allelic loss) between FAP-associated and sporadic FGPs. The log likelihood ratio test was used to compare somatic APC gene alteration frequencies among FAP-associated FGPs which were negative, indefinite, and positive for foveolar dysplasia. A P value of <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A high frequency of somatic APC alterations (APC gene mutation by
direct sequencing or allelic loss at 5q) was present in FGPs from FAP
patients. Twelve (75%) of 16 FAP patients with FGPs demonstrated an
inactivating somatic APC gene alteration in at least one FGP. Overall,
21 (51%) of 41 FGPs from patients with FAP demonstrated somatic APC
gene alterations. Somatic APC alterations were detected in three (50%)
of six FAP-associated gastric body tubular adenomas, a rate similar to
that detected in FAP-associated FGPs. Somatic APC gene mutations were
of two types: insertion of base A into a 6-base poly(A) tract spanning
codons 1554 to 1556 in 13 FGPs, and CGA
TGA (stop) mutation at codon
1450 in two FGPs and one adenoma. 5q LOH was present in an additional
six FGPs and two adenomas. Notably, these two different pathways for
somatic APC gene inactivation, truncating mutation or allelic loss,
were never demonstrated to occur in the same patient; all patients from
whom multiple polyps were analyzed showed exclusively either LOH or APC
gene mutations in their polyps. (A summary of the specific APC gene
alterations in FGPs and gastric adenomas is presented in Table 1
.)
There were no significant differences between somatic APC alteration
rates among FGPs with dysplasia, indefinite for dysplasia, and no
dysplasia. Specifically, we detected somatic APC gene alterations in
seven (47%) of 15 nondysplastic FGPs, four (67%) of six FGPs
indefinite for dysplasia, and 10 (50%) of 20 dysplastic FGPs
(P = 0.697, log likelihood ratio test) (Figures 1 to 3
and Table 2
). There was high concordance
for genetic alterations in the foveolar epithelium and the underlying
dilated fundic glands of the FGPs. Among the 25 FGPs in which the
foveolar epithelium and underlying fundic glands were microdissected
and analyzed separately, 24 (96%) of 25 showed concordance in APC gene
mutation/5q LOH status. In the one discordant case, a somatic APC gene
mutation (insertion of A in codons 1554 to 1556) was detected in the
foveolar epithelium but not in the underlying glands of an FGP with
low-grade dysplasia (patient 1, Table 1
), possibly secondary to
contamination by inflammatory cells or admixed nonneoplastic fundic
glands. Analysis of control normal DNA from each FAP patient
demonstrated no somatic APC mutation in nonpolypoid mucosa.
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). In this patient, sequencing of control
mucosa demonstrated the mutant and wild-type alleles in equal
intensities, whereas three separate FGPs which had LOH at 5q also
showed a relative decrease in the intensity of the wild-type allele on
sequencing, confirming that the allelic losses were of the wild-type
allele (data not shown). Somatic APC Gene Alterations in Sporadic FGPs
No APC gene mutations were identified in the same sequenced
regions among 13 sporadic FGPs, and LOH on 5q was present in only one
sporadic FGP (8%). In this case, allelic loss was identified with both
the D5S299 and D5S346 markers (D5S82 was noninformative) in the
foveolar epithelium but not in the underlying dilated fundic glands of
the polyp. The frequency of somatic APC alterations in FAP-associated
FGPs was therefore significantly higher than in sporadic FGPs (51%
versus 8%; P = 0.008; Fisher’s exact test)
(Table 2)
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K-ras Gene Mutations in FGPs
A K-ras gene mutation was detected in only one (2.6%)
of 39 FAP-associated FGPs, an FGP showing low-grade foveolar dysplasia
with a GGTGTT mutation in codon 12 (Figure 4)
. No K-ras gene mutations
were detected in any of the sporadic FGPs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results contrast with earlier publications which have regarded both FAP-associated and sporadic FGPs as hyperplastic or hamartomatous lesions with identical morphology. Some investigators have reported no differences between the histological features or mucin histochemistry of FAP-associated and sporadic FGPs, suggesting instead that in both settings FGPs arise from the progressive formation and dilatation of secondary glandular buds, causing glandular dilatation.17,18 Declich et al34 have suggested that FGPs are hyperproliferative polyps based on higher proliferating cell nuclear antigen labeling index in FGPs than in normal fundic mucosa. These observations have led to the opinion by some investigators that dysplastic changes in the foveolar epithelium overlying FAP-associated FGPs are the result simply of superimposed gastric adenomas, which may develop at an increased frequency in association with FGPs because of increased proliferation and increased number of epithelial cells exposed to carcinogenic stimuli.20 Several aspects of the findings presented here suggest that FGPs in patients with FAP are, in fact, neoplasms. First, the molecular pathogenesis of FAP-associated FGPs, attributable to second-hit APC gene alterations, is distinct from that of sporadic FGPs. Second, the same somatic APC gene alterations are consistently found in both the dilated fundic glands comprising the FGP and the overlying foveolar epithelium. Finally, there is no difference in the rate of somatic APC gene alterations detected in dysplastic, indefinite, and nondysplastic FAP-associated FGPs. These findings indicate that somatic APC alterations are intrinsic to the formation of FAP-associated FGPs and are not restricted to superimposed dysplastic, or adenomatous, foveolar epithelium.
The frequent presence of somatic APC gene alterations indicates that FGPs arising in the setting of FAP are neoplastic lesions. FGPs in FAP patients therefore show a similar molecular pathogenesis to that of adenomatous polyps of the colon, duodenum, and stomach, which have been well demonstrated to arise from second-hit APC gene alterations superimposed on the germline APC mutation.27,28 Our detection rates of 51% and 50% for somatic APC alterations in FAP-associated FGPs and gastric body adenomas, respectively, are likely an underestimation of the true frequency of second-hit APC events, because we sequenced only the mutation cluster region for gastroduodenal polyps in this study.29 Although it was not possible in the majority of our cases to prove that the detected somatic APC gene alterations affected the wild-type rather than inherited mutant alleles, in one case (patient 11) we were able to detect the germline 5-bp deletion spanning codons 1544 to 1546 by sequencing of control mucosa. In this case, three FGPs that demonstrated allelic loss at 5q also showed a relative decrease in the intensity of the wild-type allele on sequencing gels, confirming allelic losses on the wild-type APC allele. Interestingly, our data indicate that when multiple gastric polyps from the same patient are analyzed, only one pathway for somatic APC gene inactivation, either mutation or allelic loss, is identified. Recently, Lamlum et al35 have reported that the site of the germline APC mutation strongly influences the nature of the second-hit APC alteration, with some FAP patients showing predominantly allelic loss in their colorectal adenomas and others showing primarily truncating mutations. Confirmation of a similar relationship in the stomach, however, requires analysis of a larger number of gastric polyps.
The findings in this study raise an interesting question about the role
of the APC gene in gastric tumorigenesis. FAP-associated FGPs are the
first polyps in the gastrointestinal tract shown to contain two APC
hits without demonstrating an adenomatous morphology. It is not clear
why, in certain instances, a second hit on the APC gene induces the
formation of FGPs rather than adenomas. This phenomenon cannot be
explained simply by asserting that all FGPs in FAP patients are
secondary to the presence of foveolar dysplasia (morphologically
equivalent to microadenomas in the colon), because only 25% of
FAP-associated FGPs show foveolar dysplasia.21
In fact,
the results of this study indicate that somatic APC events bear
little relationship to the presence or absence of foveolar dyplasia,
because we detected no difference in the rate of somatic APC
alterations in FAP-associated FGPs with and without foveolar dysplasia.
This suggests that additional genetic alterations may account for the
presence of foveolar dysplasia and contribute to neoplastic progression
in a subset of FGPs. In this regard activating K-ras gene
mutations do not seem to play a major role, as we were able to
demonstrate a K-ras gene mutation in only one FAP-associated
FGP with low-grade foveolar dysplasia. Additional genetic events
underlying dysplasia remain to be elucidated.
The role of FGPs in gastric tumorigenesis in patients with FAP remains unclear. In Japanese and Korean populations, FAP has been associated clearly with an increased risk of gastric adenocarcinoma.2,36,37 In Western patients with FAP, this risk seems to be slightly (approximately twofold) increased, but not statistically significantly different from that of the general population in the study reported by Offerhaus et al.38 Neoplastic progression of FGPs in Western patients with FAP has been demonstrated, including reports of a large fundic gland polyp with high-grade dysplasia and K-ras gene mutation, and occasional infiltrating adenocarcinomas arising in association with fundic gland polyposis.23-26 Despite the lack of more exact estimates of the risk of tumor progression in patients with FAP and fundic gland polyposis, molecular evidence indicates that FAP-associated FGPs are neoplastic polyps. Similar to the presence of other neoplastic polyps of the upper gastrointestinal tract in patients with FAP, the presence of fundic gland polyposis may warrant close endoscopic surveillance.
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Acknowledgements |
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Footnotes |
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Supported in part by an award from BioNumerik Pharmaceuticals, Inc.
Accepted for publication June 6, 2000.
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