[page 36↓]

10.  DISCUSSION

Search for LOH has provided a strong tool for gaining insight into the process of cancer neo-genesis and the involvement of a subset of deletions and mutations in the initiation and pro-gression of tumor development. Midgut carcinoids are rare malignant tumors of the small intes-tine. The primary tumor is often inconspicious in size but nevertheless associated with generally larger mesenteric lymph node or liver metastases. The rather indolent malignant behaviour as well as their low incidence has evoked an interest in the genetic events in tumorigenesis of these neoplasms.

Only few midgut carcinoids have been investigated so far (Toliat, Berger et al. 1997; Ghimenti, Lonobile et al. 1999; Jakobovitz, Nass et al. 1996; Debelenko, Emmert-Buck et al. 1997a; Gortz, Roth et al. 1999; Zhao, de Krijger et al. 2000; D´Adda, Pizzi et al. 2002; Kytola, Nord et al. 2002). In contrast, the molecular mechanisms involved in the tumorigenesis of more common highly malignant gastrointestinal carcinomas are better characterized including frequent mutations of the Smad4/DPC4 and DCC genes on chromosome 18q21, APC on 5q21 and p16 on 9p2 .

This genome-wide screening for LOH of eight midgut carcinoid tumors revealed multiple allelic deletions with losses found in all tumors and in most cases encompassing both chromosomal arms. The rate of fractional allellic loss (FAL, in %) varied greatly between the different lesions. Even though the tumor samples still may have contained a certain amount of contaminating fibroblasts after microdissection, the LOH results were striking, especially in tumors 5216 and 5807, presenting with most deleted chromosomes.

Most conspicious were the findings of LOH on chromosome 18 with allelic losses in seven of eight lesions (88%). Only one study, using comparative genomic hybridization, could reveal LOH on chromosome 18 so far, LOH on chromosome 18p in 7/15 and on 18q in 8/15 midgut carcinoids (Zhao, de Krijger et al. 2000). In all but one tumor (2762) the deletions were large and included all informative markers on chromosome 18. LOH on chromosome 18 is a common event in a high proportion of gastroenteropancreatic carcinomas (Fearon, Cho et al. 1990; Schutte, Hruban et al. 1996; Uchida, Nagatake et al. 1996) as well as other tumor types (Papadimitrakopoulou, Oh et al. 1998; Hessman, Lindberg et al. 1999). Moreover, colorectal cancers with LOH on chromosome 18 behave clinically more aggressive than those without LOH (Fearon, Ekstrand et al. 1994). Three candidate tumor suppressor genes have been identified in this region: The DCC (deleted in colorectal cancer), Smad4/DPC4 and Smad2/MADR2/JV18-1 genes located on 18q21 (Fearon, Ekstrand et al. 1994; Eppert, Scherer et al. 1996; Schutte, Hruban et al. 1996). The Smad4/DPC4 gene has been found homozygously mutated in both exocrine and endocrine pancreatic tumors as well as colorectal cancers (Schutte, Hruban et al. 1996; Takagi, Kohmura et al. 1996; Howe, Roth et al. 1998; Bartsch, Hahn et al. 1999; Friedl, Kruse et al. 1999). DCC has also been described to be homozygously deleted in a subset of pancreatic and other cancers (Fearon, Ekstrand et al. 1994; Hilgers, Song et al. 2000) while Smad2/MADR2/JV18-1 alterations have been detected in a limited fraction of colorectal and lung cancers (Eppert, Scherer et al. 1996; Uchida, Nagatake et al. 1996). Although the deletion pattern suggested a tumor suppressor gene telomeric of the gene cluster at 18q21, we chose to analyze the Smad4/DCP4 gene in more detail since exon-specific PCR-primers were available and the described anti-serum for immunohistochemical labeling of Smad4/DPC4 has proven specificity and sensitivity for homozygous gene inactivation of 94% and 91%, respectively (Wilentz, Su et al. 2000). For Smad2/MADR2/JV18-1 and DCC, the correlation of immunohistochemical labeling and gene inactivation is unknown.

In our series of midgut carcinoids we did not identify any mutations in exon 8-11 of the Smad4/DPC4 gene, including the intron-exon boundaries. Although homozygous deletions are difficult to exclude, all exons of all tumors were PCR-amplified with the same efficiency. Immunohistochemical staining of seven tumors, revealed normal expression of the Smad4/DPC4 protein in all investigated lesions. The sequencing results and the distinct staining of Smad4/DPC4 protein strongly suggest the idea that the Smad4/DPC4 gene is unlikely to be in-volved in the development of midgut carcinoids. Tumor 2762, however, displayed a more limited deletion telomeric to D18S858 and the Smad4/DPC4 and DCC loci on 18q, allelic loss of 18q markers D18S541 and D18S844. This same tumor had remained D18S858 on 18q as well as all 18p markers. These findings suggest that this region of LOH might harbour yet another TSG distal to Smad4/DPC4 and DCC loci on 18q unknown to date and that its inactivation is more likely to be involved in the initiation of midgut carcinoid neogenesis.

Previous studies on chromosome 11 have described one somatic missense MEN1 mutation (V531 in [page 37↓]exon 2) in one of 16 midgut carcinoid tumors (Toliat, Berger et al. 1997; Gortz, Roth et al. 1999). Two constitutional putative missense mutations, H50R and G12S on the SDHD (TSG) (succinate-ubiquinone oxidoreductase subunit D) gene locus were found in two midgut carcinoids, both mutations were associated with LOH of the other allele (Kytola, Nord et al. 2002). Microsatellite instability was detected in one of six analyzed midgut carcinoid tumors (Ghimenti, Lonobile et al. 1999). LOH on chromosome 11 was analyzed in 16of 83 midgut carcinoids (Jakobovitz, Nass et al. 1996; Toliat, Berger et al. 1997; Debelenko, Emmert-Buck et al. 1997a; Ghimenti, Lonobile et al. 1999; Gortz, Roth et al. 1999; Zhao, de Krijger et al. 2000; D´Adda, Pizzi et al. 2002; Kytola, Nord et al. 2002. In contrast to these findings, we could not detect LOH on 11q13 in our midgut carcinoids even though both PYGM and INT2 flanking the MEN1 gene were informative in six of eight tumors and for the other two tumors markers D11S2000 at 11q22 was informative. This discrepancy might be due to different patient series and different microsatellites used in the studies. However, the lack of LOH on chromosome 11, especially at 11q13, holds with the fact that sporadic midgut carcinoids are not associated with the MEN1 syndrome and MEN1 deletions only occur in a subset of these tumors. Clearly more tumors have to be analyzed in order to gain a more true insight into involvement of 11q13 in midgut carcinoid neogenesis.

In addition to the frequent LOH on chromosome 18, several other chromosomes were deleted in a subset of tumors. Only one tumor (5807) showed LOH for all informative markers on chromo-some 3, two tumors (5216, 5807) presented with LOH for all informative microsatellites on chromosome 4, 5, 7, 14 and 20, LOH for chromosome 9 was found in two tumors, all chro-mosome 9 microsatellites were deleted in tumor 5216 while tumor 2762 showed LOH for one marker only (GATA 62F03), all chromosome 12 markers were deleted in one tumor (5216), only one chromosome 16 marker (D16S2624) at 16q22.1, the e-cadherin locus, was lost in one tumor (2762). 3p23-3p22 harbours the MLH1 gene causing the familial non-polyposis type of colonic cancer (FCC2) (Panariello, Scarano et al. 1998) as well as the SCLC1 gene being involved in the carcinogenesis of small lung cell cancer (Hibi, Takahashi et al. 1992), the HVBS6 gene maps to 4q32.1 and is rearranged in hepatocellular cancer (Blanquet, Garreau et al. 1988). Mutations of the APC gene located at 5q21 might be important in the evolvement of midgut carcinoid tumors, multiple colonic carcinoid tumors have been reported in one adenomatosis polyposis coli patient (July, Northcott et al. 1999) and APC is known to play a role in the development of a number of colorectal cancers (Kinzler, Nilbert et al. 1991). Down-regulation of the DRA (down-regulated in adenoma) gene on 7q22-7q31.1 is associated with the neoplastic transformation of normal colonic mucosa to polyps to adenocarcinoma (Antalis, Reeder et al. 1998) and p16 at 9p21 is involved in various neogenetic events (Sun, Hildesheim et al. 1995). The SRC gene is located at 20q11.2 and is involved in the procession of advanced colonic cancers (http://www.gdb.org/). The small number of tumors, however, makes it impossible to draw any conclusions of these scattered allelic losses detected in three of eight analyzed carcinoids. They may be caused by random events in a genetic unstable cellular environment.

LOH on chromosomes 4, 5, 7, 9, 14 and 20 were detected in two of eight tumors whereas chromosomes 3, 16 and 19 were affected in only one tumor. Two lesions displayed allelic loss on seven and eight chromosomes respectively. One of the latter, tumor 5807, was the only tumor that had retained chromosome 18 but paradoxically displayed the highest extent of LOH on all chromosomes. In this tumor, all affected chromosomes presented with the same level of allelic retention (40-59%), in the other tumor, 5216, however, we detected markedly lower retention levels of 11-25% for chromosomes 5, 9, and 18 than for chromosomes 4, 7, 12, 14 and 20 (46-60%). This is suggestive of presence of intratumoral heterogeneity in tumor 5216 with chromo-some 5, 9 and 18 deletions in most tumor cells whereas the other chromosomal losses are present in tumor cell subclones only. Tumors displaying LOH are considered to be of monoclonal origin (Guo, Li et al. 2000). The genetic events on chromosomes 5, 9 and 18 might have developped earlier in the neoplastic process than those on the other chromosomes. Another hypothesis is that such deletions might give these mutated cells growth advantage over less altered tumor cells.

Some of the tumor samples contained a rather high amount of of fibroblasts. The normal DNA from these cells will dilute tumor DNA and interfere with the LOH analyses. However, we have been able to detect allelic losses in all lesions and the levels of retention of alleles of all but two tumors have been lower than 50%. One exception (5216) had lower levels of allelic retention in three of eight deleted chromosomes while sample 5807 displayed LOH on several chromosomes with allelic retention levels of approximately 50% for all affected chromosomes. We therefore believe that our figures are a true picture of the deletion patterns of these neoplasms.

Our findings of LOH on chromosome 18 in 88% of the tumors, however, suggests a model wherein the steps required for malignancy in midgut carcinoid tumors commonly involve the loss of genes on chromosome 18 that normally suppress tumorigenesis. Despite the alterations found on chromosome [page 38↓]18, genetic events in midgut carcinoids seem to differ from those found in gastrointestinal carcinomas with regard to the absence of Smad4/DPC4 and K-rasand rare p53 mutations in midgut carcinoid tumors. A more explorative and detailed analysis of loci deleted using a larger number of markers and a larger number of tumor specimens is warranted to clarify the comprehension of the unique neogenetic behaviour of classical midgut carcinoid tumors.


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