The small intestinal mucosa makes up about 90% of the total surface of the gastrointestinal tract. However, adenocarcinomas arise rarely in this location. To elucidate genetic alterations underlying tumour development in the small intestine we investigated 17 sporadic adenocarcinomas. By comparative genomic hybridization recurrent gains of chromosomal material were found at chromosomes 7, 8, 13q, and 20 (5/17, each), while non-random losses were seen at 8p, 17p (4/17, each), and 18 (8/17 cases). Deletions at 5q, the location of the APC tumour suppressor gene, were seen in three cases. Microsatellite analysis with markers on chromosomal arms 1p, 5q, 8p, 17p, 18q, 19p, and 22q revealed a microsatellite instable phenotype in two cases and a high frequency of loss at 18q21-q22 (80%). Given the high incidence of 18q21-q22 deletions, we performed sequencing analysis of SMAD4, a downstream component of the TGFβ-pathway, located at 18q21. Four tumours displayed mutations in highly conserved domains of the gene indicating disruption of TGFβ-signalling. Our data reveal complex genetic alterations in sporadic small intestinal carcinomas. However, most tumours share deletions of 18q21-q22, which frequently target SMAD4. This indicates that disruption of TGFβ-signalling plays a critical role in small intestinal tumorigenesis.
Carcinomas of the gastrointestinal tract account for approximately 30% of malignancies in adults. But although the small intestinal mucosa makes up about 90% of the surface of the gastrointestine (Ming and Goldman, 1992) adenocarcinomas arise exceedingly rarely in this location. Several reasons are discussed for this disparity. They mainly focus on the reduced exposure to carcinogenic substances due to a rapid transit and a highly diluted nature of the small intestine's nutritional content (Lightdale and Hornsby-Lewis, 1994; Ming and Goldman, 1992).
Small intestinal carcinomas share morphological characteristics and risk factors with other gastrointestinal carcinomas, especially with colorectal carcinomas (Negri et al., 1999; Neugut et al., 1998). Both tumour types may arise from pre-existing adenomas and both are reported to occur more frequently in patients with chronic inflammatory bowel diseases (Pohl et al., 2000; Logan et al., 1989; Lennard-Jones et al., 1977) and in patients with hereditary gastrointestinal cancer prone syndromes like familial adenomatous polyposis coli (FAP), hereditary non polyposis colorectal cancer syndrome (HNPCC), and Peutz-Jeghers syndrome (Neugut et al., 1998; Park et al., 1999; Spigelman et al., 1989).
In contrast to other gastrointestinal carcinomas, only little information is available about the molecular alterations in small intestinal adenocarcinomas. Rashid and Hamilton (1997) have published a study of both sporadic and Crohn's disease associated tumours. By microsatellite analysis they have found low frequencies of deletions at chromosomal arms 5q, the location of the APC gene, and at 18q, where the DCC-, and DPC4/SMAD4 genes are located. The frequency of replication errors, k-ras mutations, and deletions at 17p, the location of the p53 gene, were reported to be similar to the frequency in colorectal carcinomas (Rashid and Hamilton, 1997; Sutter et al., 1996; Arber et al., 2000), while mutational analysis of APC showed infrequent genetic inactivation of this gene in small intestinal carcinomas (Arai et al., 1997).
In order to obtain a more comprehensive picture of molecular alterations in adenocarcinomas of the small intestine we investigated 17 sporadic cases of this tumour type for chromosomal alterations by the use of comparative genomic hybridization (CGH). The study was complemented by microsatellite analysis of 11 loci located in chromosomal areas that are preferentially affected in gastrointestinal carcinomas (chromosomal arms 1p, 5q, 8p, 17p, 18q, 19p, and 22q). Guided by the high frequency of deletions at 18q detected by either CGH and microsatellite analysis we performed sequencing analysis of the SMAD4 gene, a mediator of growth suppression through TGFβ-signalling. SMAD4 is known to be mutated in several tumour types, most frequently in pancreatic carcinoma.
In 14 of the 17 cases CGH analysis revealed chromosomal abnormalities. The distribution of gains and losses is presented in Figure 1. The most common gains of chromosomal material were detected at the following chromosomes: 7 (30%, 5/17 cases: three gains of the entire chromosome, one 7p gain, one 7p and 7qcen-q31 gain); 8 (30%, 5/17 cases: one gain of the entire chromosome, three 8q gains, one terminal 8q gain); 13q (30%, 5/17 cases: three gains of the entire chromosome, one interstitial and one terminal 13q gain); 20q (30%, 5/17 cases: three gains of the entire chromosome, one 20q and one 20p gain), and the X-chromosome (27%, 3/11 cases). Gains of 8q were commonly associated with deletions of 8p indicating the presence of an isochromosome 8q. Other results indicative of isochromosomes were seen for case D04 (6p gain, 6q loss) and case D07 (5p gain, 5q loss).
Gains mostly affected whole chromosomes or chromosomal arms. Only in one case (D27) two interstitial gains at 13q21-q22 and 18qcen-q21 were detected.
Loss of chromosomal material was most commonly detected at chromosome 18 (47%, 8/17 cases). Five cases showed loss of the entire chromosome, in one case the whole 18q arm was affected, and in two other cases deletions were terminal (18q21-ter). Other losses were more frequently seen at chromosomes: 4 (15%, 3/17 cases); 5q (15%, 3/17 cases); 6q (15%, 3/17 cases); 8p (23%, 4/17 cases); 15q (15%, 3/17 cases), and 17p (23%, 4/17 cases). While losses at 8p, 15q, and 17p commonly affected the whole chromosomal arm, an interstitial deletion at 6q14-q22 was seen in one case and a terminal deletion of 6q (6q21-qter) was seen in another case.
For each individual case, CGH-results are given in Table 1 together with the data of microsatellite-analysis and SMAD4-mutational status.
Microsatellite instability at two or more loci was seen for D01 and D30. These cases were classified as MSI-high. Two MSI-low cases were identified (D12 and D27) with one replication error at locus D18S878. In both cases the two wild type alleles were lost.
The highest frequencies of LOH were scored for chromosome 18q (D18S858: nine of 12 informative cases (i.c.), 75%; D18S878: 12 of 15 i.c., 80%) and 17p (D17S520: eight of 31 i.c., 61%). D01 showed LOH at D18S858 and retention of heterozygosity at D18S878. For all other cases with LOH at 18q either both markers showed LOH or one marker showed LOH and the other marker was not interpretable. LOH at chromosome 8p was detected in 31% (D8S1477: five of 16 i.c.), LOH at 5q was detected in 10–31% (D5S820: one of nine i.c., D5S1505; three of 16 i.c.; D5S346: four of 13 i.c.).
LOH at chromosomes 1p, 19p, and 22q was infrequent (D1S1612: one of 14 i.c., D1S2134: one of 13 i.c., D19S591: none of seven i.c., D22S683: three of 13 i.c.). Representative results of the microsatellite analysis are given in Figure 2. The results of microsatellite analysis are presented in Table 2.
SMAD4 mutational analysis
A total of five sequence alterations were detected. Three cases showed missense point mutations with loss of the wild type allele, one case showed a 7 bp deletion with retention of the wild type allele. In D13 a recently described silent polymorphism (Moore et al., 2000) at codon 362 was detected heterozygously in either tumour and surrounding normal tissue (Figure 3). Cases with loss of the wild type allele were: D05: codon 132 CAC → CTC, H132L (Figure 3); D06: codons 350 and 351 GTT GAT → GAT AAT, V350D and D351N (Figure 3); D04: codon 361 CGC → TGC, R361C (Figure 3). D32 showed a deletion of 7 bp within codons 323–325 (TAT TGG TGT TCC ATT) with a retention of the wild type allele (Figure 4). The alteration results in a frame shift with a premature stopcodon 11 codons downstream of the deletion.
Only little information is available on the mutational status of oncogenes and tumor suppressor genes in small intestinal adenocarcinoma. Several authors describe k-ras mutations with a frequency ranging from 36–84% (Sutter et al., 1996; Rashid and Hamilton, 1997; Arber et al., 2000). In a series of 15 small intestinal adenocarcinomas Arai et al. (1997) have found p53 and APC mutations in 27 and 7%, respectively. More recently, Murata et al. (2000) describe a deletion within exon 3 of the β-catenin oncogene in one case. Our study is the first to describe the mutational status of the SMAD4 tumour suppressor gene in small intestinal adenocarcinomas.
Four of 17 investigated small intestinal carcinomas displayed missense mutations of SMAD4. In three cases point mutations with a loss of the wild type allele were found in the DNA-binding N-terminal domain (one case) or the C-terminal homo-oligomerization domain (two cases). The C-terminal SMAD4 mutations at residues 351 and 361 have already been detected in other tumours (Thiagalingam et al., 1996; Schutte et al., 1996) and their functional relevance has been discussed (Shi et al., 1997). The N-terminal mutation at residue 132 is located within the MH1 domain. This domain is considered critical for DNA-binding of SMAD4 (Jones and Kern, 2000). Type and location of all three mutations indicate a complete disruption of the TGFβ-signalling pathway by inactivation of SMAD4.
A protein truncating mutation with a retention of one wild type allele was found in another tumour. The patient affected by the jejunal carcinoma was extraordinarily young (30 years) and except for the SMAD4 mutation and a k-ras mutation (data not shown) only an interstitial deletion at chromosome 6q was detected. The deleted area at 6q overlaps a chromosomal area to which a hereditary mixed polyposis syndrome (HMPS) has recently been mapped (Whitelaw et al., 1997). The somatic SMAD4 mutation in this case consisted of a 7 bp deletion (codons 323–325) and results in a premature stopcodon. The type of this mutation resembles many of the germline SMAD4 mutations associated with juvenile polyposis syndrome (Howe et al., 1998; Roth et al., 1999). Since haploinsufficiency of SMAD4 is considered to trigger tumour development (Xu et al., 2000) a dominant negative effect of the mutation has to be discussed in our case. Given only three alterations found in this tumour, we question if activation of one growth promoting pathway (k-ras) and inactivation of two growth suppressing pathways (SMAD4; 6q tumour suppressor, probably HMPS) were sufficient to initiate the tumour.
In contrast to an LOH frequency of 13% at 18q reported previously for sporadic small intestinal carcinomas (Rashid and Hamilton, 1997) most cases in our study showed LOH at chromosome 18q. Losses affecting specifically 18q were found in nine tumours by either CGH and/or LOH analysis and losses of the entire chromosome 18 were observed in five additional tumours by CGH. A complete loss of chromosome 18 does not necessarily indicate the existence of a targeted tumor suppressor gene at 18q. In one of the cases with loss of the entire chromosome (D06) however, SMAD4 was targeted by mutation of one allele and loss of the wild type allele. There is a discrepancy between LOH frequency at 18q (80%) and frequency of SMAD4 mutations (24%). This indicates that SMAD4 is not the exclusive target of 18q loss in small intestinal adenocarcinomas. Beside the SMAD4 gene, SMAD2 and DCC are potentially inactivated tumour suppressors at 18q.
Losses at 5q (CGH: 17%, LOH: 31%), the location of the APC tumour suppressor, were rare in our study and did not exceed the frequency of losses at several other chromosomes (4q, 6q, 8p, 15q). Thus, confirming previous data (Rashid and Hamilton, 1997, Arai et al., 1997) the presently investigated sporadic carcinomas lack evidence of a significant role of APC inactivation in their development. This is intriguing because the elevated risk of small intestinal carcinomas in patients with familial adenomatous polyposis and the high incidence of small intestinal tumours in mice with germline APC mutations (Moser et al., 1990) suggest a role of APC in the maintenance of regular cell growth in the small intestine. Unlike the situation in the colorectum, however, sporadic and constitutional tumours in the small intestine seem to differ in their pathogenesis.
Losses at 8p are a common finding in gastrointestinal carcinomas (Emi et al., 1992; Yustein et al., 1999) and may target an as yet unidentified tumour suppressor gene (Lerebours et al., 1999). In this study LOH at 8p was found in 31% of cases. It is of note, that our CGH analysis revealed that a loss of 8p was commonly associated with a gain of 8q indicating the presence of an isochromosome 8q. It remains unclear therefore, whether the pathogenic impact of this alteration in small intestinal carcinomas is caused by an inactivation of a tumour suppressor gene at 8p or by unbalanced transcription of growth promoting genes at 8q.
CGH analysis revealed frequent gains at chromosomes 7, 8, 13q, 20, and X. Interstitial gains indicating oncogene amplification were seen in only one case. In the other cases low copy number gains commonly affected whole chromosomes or chromosomal arms. The non-random chromosomal gains detected in small intestinal carcinomas were previously found in a variety of other solid tumours including colorectal, gastric, pancreatic and prostate carcinoma (DeAngelis et al., 1999; Sakakura et al., 1999; Mahlamaki et al., 1997, Visakopi et al., 1995). Given their rather unselective occurrences, particularly gains at chromosomes 7, 8, 20, and X seem to have a general positive impact on the selection of tumour cells, irrespective of their site of origin.
Chromosomal imbalances in the presently investigated tumours were commonly multiple suggesting an inaccurate separation of chromosomes during the mitotic process. Mitotic infidelity is a well known phenomenon in tumours and has been linked to p53 inactivation. It is considered to result from the inability of p53-defective cells to present further progression through the cell cycle in the case of DNA-damage (Tarapore and Fukasawa, 2000). This explanation for complex karyotypic alterations may also apply for small intestinal carcinomas because seven from nine cases in our study with a deletion of chromosomal arm 17p, the location of the p53 gene, showed multiple numerical chromosomal changes. It remains to be determined if p53 was targeted by the deletion.
A microsatellite instability (MSI) high phenotype, indicative of DNA-mismatch repair dysfunction, was detected in only two of our cases. The frequency is similar to the one reported previously for small intestinal carcinomas (Rashid and Hamilton, 1997) and for colorectal carcinomas (Thibodeau et al., 1998). Both MSI high tumours were poorly differentiated and one of the tumours showed excessive mucine production. The latter observation fits within one of the histologic findings in microsatellite instable colorectal carcinomas (Messerini et al., 1997; Kim et al., 1994). Two other cases revealed a replication error at a single locus, which interestingly was located at chromosomal arm 18q and was associated with a deletion of both germline alleles. Yet, although dysfunction of DNA-mismatch repair may play an important role in constitutional small intestinal carcinomas associated with the HNPCC-syndrome (Vasen et al., 1999) it is not implicated in the majority of sporadic small intestinal carcinomas.
In conclusion our data show that although small intestinal carcinomas reveal complex genetic changes a significant number of tumours share karyotypic instability and losses at chromosome 18q21-q22. 18q deletions frequently target the SMAD4 gene and disrupt tumour suppression through TGFβ-signalling.
Materials and methods
During 1988 and 1998, a total of 33 resected specimens of primary, non-periampullary small intestinal adenocarcinomas were registered in the files of the Institute of Pathology, University of Heidelberg, Germany. Specimens from two patients with FAP and from one patient with synchronous rectal carcinoma were excluded. In 13 cases (mostly of the years 1991–1994) the quality of isolated DNA was not sufficient for molecular genetic analysis leaving a total of 17 cases suitable for further analysis. The clinical and morphological data of the cases are summarized in Table 2.
Among the 17 cases, none was associated with chronic inflammatory bowel disease or celiac disease. No patient was suggestive to suffer from a hereditary cancer prone syndrome (HNPCC, FAP). One recurrent small intestinal carcinoma and three cases with metachronous colorectal carcinoma were included. The reason for classifying the latter carcinomas of the small intestine as primary tumours rather than metastasis of the known colorectal tumours was a spread of tumour cells from the mucosal surface into the submucosa with the majority of tumour mass in the luminal orientated layers of the small intestinal wall.
Guided by histology, carcinoma areas with preferential tumour formation (>70%) were sampled from routinely archived paraffin blocks with the help of a bone marrow needle. DNA was extracted from these samples using methods described previously (Isola et al., 1994). Tumour DNA was biotinylated, normal male control DNA was digoxigenin labelled using standard protocols for nick labelling.
After hybridization of the labelled DNA samples on metaphase spreads for 48 h at 37°C the slides were washed and incubated with fluorescence labelled anti-digoxigenin- and anti-biotin-antibodies using standard protocols (Isola et al., 1994). The hybridized metaphase spreads were photographed and analysed using an inverted microscope (Zeiss, Axiovert S 100), a CCD camera and CGH-software as supplied by Metasystems (MetaSystems, Altlussheim, Germany). A minimum of nine metaphases was photographed per case, yielding an average of 13 individual specimens of each chromosome used for comparative fluorescence analysis. Centromeric and telomeric chromosomal areas as well as chromosome 19 were excluded from the investigation. The X-chromosome was only scored in male cases (11 patients) because the control DNA was male.
Fluorescence ratios were determined using both fixed cut off values (0.80 for losses, 1.25 for gains) and cut off values with a twofold standard deviation. Only chromosomal abnormalities that were detected with both evaluation approaches were scored.
Approximately 2 mm2 of areas with preferential tumour tissue of each case and corresponding normal mucosa were microdissected from 5 μM thick sections. DNA was isolated as previously reported (Bläker et al., 1999). PCR analysis was performed in volumes of 10 μl using PCR-primers from the Weber Genome Screening Set (Version 8a, Research Genetics, Huntsville, AL, USA) and additionally synthesized oligonucleotides under standard PCR conditions. Primers from the genome screening set were for loci D1S1612 (1p36), D1S2134 (1p32), D5S820 (5q33), D5S1505 (5q23), D8S1477 (8p21), D18S858 (18q21), D18S878 (18q22), D19S591 (19p13), and D22S683 (22q11-q12). Additionally used primers were for loci D5S346 (5q21) and D17S520 (17p12). Polyacrylamid gelelectrophoresis was performed on 7M Urea, 6–8% polyacrylamid, 0.5×TBE gels at constant voltage of 30 V/cm. The gels were silver stained.
Loss of heterozygosity (LOH) was defined as a reduction of staining intensity of more than 70% of one heterozygous allele in the tumour DNA compared with the staining pattern of the corresponding control DNA alleles. Microsatellite instability (MSI) was scored when longer or shorter alleles were detected in the tumour DNA compared with the control DNA. A MSI-low phenotype was defined as replication errors in less than 40% of the investigated eleven loci; an MSI-high phenotype was defined as replication errors in more than 40% of the investigated eleven loci.
Sequencing analysis of SMAD4
Aliquots of DNA prepared for microsatellite analysis were also used for mutational screening of exons 1, 2, 8, 9, 10, and 11 of the SMAD4 gene. PCR was performed using primers and conditions as described previously (exons 8, 10, 11: Moskaluk et al., 1997; exon 1: Houlston et al., 1998; exon 9: Howe et al., 1998). For exon 2 new primers were designed (S42F: 5′-CTTGCATAATGTGACACATG-3′, S42R: 5′-TGAAACACTATTGAGATCC, annealing temperature 50°C). After purifying, PCR products were subject to direct cycle sequencing using a BigDye Terminator protocol and an ABI Prism 377 DNA sequencer (Applied Biosystems). In case of a mutation, PCR and sequencing were reperformed to rule out artificial base pair substitution by taq-polymerase. In one case with a heterozygous 7 basepair (bp) deletion in exon 8, mutant and wild type allele were separated by polyacrylamid gelelectrophoresis. Bands were cut off separately from the gel, reamplified and sequenced.
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Bläker, H., von Herbay, A., Penzel, R. et al. Genetics of adenocarcinomas of the small intestine: frequent deletions at chromosome 18q and mutations of the SMAD4 gene. Oncogene 21, 158–164 (2002). https://doi.org/10.1038/sj.onc.1205041
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