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Molecular Biology and Genetics |
Digestive Diseases [F. Y., R. T. J.] and Genetics and Biochemistry Branches [Y-L. Z., A. M. H., L. J. F.], National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892; Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [I. A. L.]; and Laboratory of Cancer Genetics, University and University Hospital of Tampere, FIN-33101 Tampere, Finland [E. H. M.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because about 25% of gastrinomas arise as a manifestation of the hereditary MEN12 syndrome, a major advance in elucidating the pathogenesis of gastrinomas came with the positional cloning of the MEN1 gene on chromosome 11q13 (4) . Even in nonhereditary sporadic gastrinomas, the MEN1 gene is mutated in 27–37% of cases (5, 6, 7, 8, 9, 10, 11) . Although the function of the MEN1 gene was not evident from its sequence, the protein has been shown to function primarily in the nucleus (12, 13, 14) and to bind to the transcription factor JunD (15) . Other genetic alterations have been shown to occur in gastrinomas, including methylation and deletion of the p16/MTS1 tumor suppressor gene (16) , amplification of the HER-2/neu proto-oncogene (17) , amplification of chromosome 9q (18) , and deletion of chromosomes 1 (19 , 20) or 3p (18 , 21) . Additional, rare defects have been seen in other locations (22) . Aneuploidy has been shown to occur in these tumors, and aneuploidy of the multiple stem line type correlates with a poor prognosis (23) .
In an effort to identify additional loci that might be important in gastrinomas, we have performed an in-depth DNA analysis of these tumors, focusing on sporadic gastrinomas. Four hundred loci distributed evenly throughout the human genome were examined, and the results were confirmed by the complimentary cytogenetic technique of CGH.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. All tumors were frozen in liquid nitrogen at the time of surgery and
stored with paired blood samples at -70°C. One sample (30–100 mg in
size) from a discreet tumor focus was used from each patient. All tumor
samples were examined histologically and judged to consist almost
entirely of tumor cells (>80%), without significant foci of
nonmalignant cells or normal adjacent tissue, and microdissection was
not used to enrich for tumor cells.
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PCR and Product Analysis.
The PCR amplification and analysis were modified from a protocol used
in automated genotyping experiments to robustly amplify microsatellite
markers (24)
. The polymorphic markers used included all
387 markers in the Cooperative Human Linkage Center Screening set
(Weber Version 8), and the fluorescence-labeled primers were purchased
from Research Genetics, Inc. (Huntsville, AL). These markers were
selected because of their even distribution throughout the whole human
genome and because of their high information content with an average
heterozygosity of 76%. Subsequent experiments used 23 markers
distributed throughout chromosome 1 from the ABI PRISM Linkage Mapping
Set (Perkin-Elmer) and 4 markers from the MEN1 region on
chromosome 11q. Two of these markers, D11S1314 and
D11S1357, were from Perkin-Elmer, and the other two,
PPP1CA (25)
and PYGM(26)
, were synthesized.
The 16 DNA samples from the eight patients (blood and tumor DNA from each patient) were individually added to each of the 414 pairs of primers described above using a Biomek 2000 robotic workstation (Beckman Instruments, Inc., Fullerton, CA). Each PCR reaction was conducted in a total volume of 15 µl containing 100 ng of DNA, 0.27 µM each primer, 250 µM each deoxynucleotide triphosphate, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, and 1.25 units of AmpliTaq Gold DNA polymerase (Perkin-Elmer). Reactions were performed in a Perkin-Elmer GeneAmp PCR system 9600 thermal cycler as follows: (a) denaturation at 95°C for 12 min; (b) 10 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 60 s; (c) 20 cycles of denaturation at 89°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 60s; and (d) extension at 72°C for 90 min.
Samples were run on an ABI 377 sequencer (Perkin-Elmer), and because the primers contained one of three different fluorescent labels, and the markers had different size ranges, about eight reactions could be pooled and run in each lane of the gels. Peak heights and areas were quantified, and the data were processed using the Genescan and Genotyper software programs provided with the sequencer.
CGH.
After the full complement of PCR reactions described above were
performed, the nature of the defects detected was confirmed by CGH.
Because the amount of tumor DNA available at this point was limited,
analysis was not attempted on patients 7 and 8. In addition, the tumor
DNA was not used directly for CGH but was amplified using degenerate
oligonucleotide-primed PCR (27
, 28)
. Two steps were used
to amplify the DNA, with the first (preamplification) step done as
described previously (27)
. The second step was conducted
by adding each of the preamplified DNA samples to 50 µl of a solution
that contained 1.5 µM UN1 primer (27)
, 200
µM each deoxynucleotide triphosphate, 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, and 5 units of AmpliTaq DNA polymerase.
Reactions were performed in a PTC-200 thermal cycler (MJ Research,
Inc., Waltham, MA) using 35 cycles of denaturation at 94°C for 1 min,
annealing at 56°C for 1 min, and extension at 72°C for 2 min. A DNA
sample from the MCF-7 breast cancer cell line containing
well-characterized alterations was used as a positive control, and DNA
samples from male and female normal lymphocytes were used as the source
of reference DNA.
The hybridization protocol was performed as described previously (29) . Briefly, the DNA samples were labeled by nick translation using FITC-12-dUTP (DuPont, Boston, MA), and normal reference DNA was labeled using Texas red-6-dUTP (DuPont). Five hundred ng of labeled sample DNA were added to 300 ng of labeled sex-matched reference DNA and 10 µg of Cot-1 DNA (Boehringer Mannheim GmbH, Mannheim, Germany). Hybridization to normal metaphase preparations was done for 48 h, and, after washing, the slides were mounted in an antifade solution containing 4',6-diamidino-2-phenylindole counterstain. The ratios of green:red fluorescence intensities (tumor:normal) were analyzed along each autosome and the X chromosome using a digital image analysis system made up of a BX50 fluorescence microscope (Olympus, Tokyo, Japan) and an Image-Point charge-coupled device camera (Photometrics Ltd, Tuscon, AZ) equipped with IPLab software (Scanalytics, Inc., Fairfax, VA). Images were further analyzed using the Quips CGH software package (Vysis, Inc., Downers Grove, IL).
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RESULTS |
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Patients 2, 3, and 7 had large, aggressive tumors, and the growth of
these tumor was the direct cause of significant morbidity, independent
of the tumors’ production of gastrin. Patient 6 had a large but
slowly growing tumor that was a manifestation of the MEN1 syndrome, and
in the other patients, the tumors were presumably of sporadic origin.
All but one patient had documented metastases to lymph nodes, and three
patients had hepatic metastases.
Nature of the Data and Information Obtained.
The use of fluorescent primers in the PCR reaction and an automated
sequencer facilitated the quantitative analysis of the intensity of
each marker allele. An example of a trace of an individual marker
showing no abnormality is shown in Fig. 1A
. The two alleles from normal blood DNA from a patient are
shown above the trace from the patient’s tumor DNA. Generally, the
amount of PCR product from the normal DNA was slightly less than that
from the tumor, and the larger-sized allele on the right was amplified
slightly less well than the smaller-sized allele on the left;
however, the overall appearance of the traces are very similar. A
simple way to summarize such a result was first used in a genetic
analysis of gastric cancer (30)
. Quantitatively, the ratio
of the intensity of peak a to peak b is very
close to the ratio of peak c to peak d. An
alternative way of expressing this observation is that the value of
(a/b)/(c/d) is close to
unity. The term AI has been used to describe the condition in which
this value is not close to unity.
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shows LOH presumably resulting from a deletion
encompassing one allele in the tumor DNA. A small residual peak remains
from the nonmalignant cells in the sample. Fig. 1C
shows a
more subtle form of AI. Although the result is not as dramatic as LOH,
the degree of AI was more than 2 SDs from the mean, and such results
were often present in multiple adjacent chromosomal markers. This
result is probably due to a duplication of the allele on the right. An
alternative explanation is that the allele on the left was deleted in
about half of the tumor cells, but this is less likely because the DNA
from each tumor was extracted from an individual focus, and the tumor
cells from an individual focus in gastrinomas have been reported to be
genetically clonal in origin (31
, 32)
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Fig. 1D
shows an example of MSI. This was manifested by
extra peaks in the tumor DNA PCR product that were not present in the
normal DNA product. It was seen in 8% of the markers from patient 7,
who had a large and unusually aggressive tumor, and was not seen in any
of the other patients.
In total, just over 6000 PCR reactions were performed using about 400
different markers. Allele homozygosity resulting in a noninformative
result was found in 20% of the reactions, and this value is in
agreement with the heterozygosity reported for the marker sets used.
Insufficient product for a robust quantitative analysis or an
uninterpretable tracing was the result of 18% of the reactions, and
these usually resulted from poor amplification of the normal DNA from
blood. Large defects involving multiple adjacent markers or even entire
chromosomes were found in 10% of the reactions, and these areas are
listed in Table 1
. Of the remaining 52% of the reactions, the mean of
the ratio (a/b)/(c/d) was
1.04 with a SD of 0.18, and this gives an estimate of the precision of
the method when assaying genomic regions free of at least large
rearrangements.
Chromosome 11 Defects.
Thirteen markers from chromosome 11 were used, and the results for all
eight patients are displayed in Fig. 2
. In this figure, the markers are arranged sequentially from the tip of
the short arm of chromosome 11 (top) to the tip of the long
arm (bottom). The result for each marker for each
patient is represented by a bar, and the amount of white in
each bar is proportional to the ratio
(a/b)/(c/d). A typical
example of LOH would have a ratio of 0.2, and in this case, 20% of the
bar would be white, and 80% would be black. For purposes of ease in
using the data processing programs, the lower molecular weight alleles
in blood or tumor DNA were always labeled a or c,
respectively, following the convention shown in Fig. 1A
. A
result of this convention is that half the abnormal ratios were greater
than unity, and half were less than unity, depending on whether the
lower or higher molecular weight allele in the tumor was affected. A
ratio of 0.2, however, would have the same molecular significance as a
ratio of 5, and would be displayed identically in Figs. 2
3
4
by
plotting the reciprocal of the ratios that were greater than unity.
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3
4
), MSI
(MI), or other reasons including insufficient yield from the
PCR reaction (NP). Whereas this is not a significant issue
when there are multiple informative markers showing AI, it is an issue
when evaluating areas such as those in patients 1 and 5 on chromosome
11. In these two patients, there are markers showing LOH on 11q, but an
especially interesting area is the region of the MEN1 gene
at the chromosome band 11q13 (70–80 cM). Whereas markers on the
telomeric side of the MEN1 region show LOH, the markers on
the centromeric side are noninformative or normal. Large deletions of
this region are common in gastrinomas (6
, 9
, 10
, 31
, 32)
,
and when one evaluates for LOH in the immediate vicinity of the
MEN1 gene, deletions are almost universally found
(7)
. Thus, as a validation of the method, finer mapping
around the MEN1 gene was performed in patients 1 and 5 using
four additional markers, and these results confirmed LOH of the
MEN1 gene region (data not shown).
Chromosome 5 Defects and Aneuploidy.
The results from the chromosome 5 markers for all eight patients are
shown in Fig. 3
. Patients 2, 6, and 7 showed AI at every informative marker throughout
the chromosome. Patient 7 had marked AI consistent with LOH, identical
to the results seen in this patient on chromosome 11. Whereas the
tracings in patient 7 showed AI of the magnitude depicted in the
example shown in Fig. 1B
, patients 2 and 6 showed a lesser
extent of AI similar to the result shown in Fig. 1C
. Thus,
it is highly likely that the molecular defect involving chromosome 5 in
patients 2 and 6 is different than the defect found in patient 7. As
discussed above, an attractive and simple explanation is that patients
2 and 6 were trisomic for chromosome 5, and patient 7 was monosomic.
Some additional data to support this explanation were obtained in the
CGH experiments discussed later.
AI involving every informative marker of an entire tumor
chromosome was especially common in patients 6 and 7. Specifically,
patient 6 had AI of nine different chromosomes, and besides the results
shown in Fig. 3
for chromosome 5, this patient’s tumor also showed AI
throughout chromosomes 6, 9, 13, 15, 16, 17, 18, and X. Patient 7, who
had a particularly aggressive tumor, had AI throughout 13 different
chromosomes. The results for chromosomes 11, 5, and 1 are shown in
Figs. 2
3
4
, and similar results were also seen for chromosomes 2, 3, 6,
8, 9, 10, 15, 16, 18, and 22. As shown in Fig. 3
, patient 2 had AI
throughout chromosome 5, but this tumor had no other defects that
involved an entire chromosome. Similarly, patient 3 had AI throughout
the X chromosome alone. As summarized in Table 1
, these four patients
all had large or aggressive tumors, and such defects were not seen in
the four patients with small tumors.
Strictly speaking, the term aneuploidy refers to an abnormal number of chromosomes, but this is usually in the context of abnormal DNA content detected by more direct techniques such as karyotype analysis of metaphase cells or flow cytometry. The PCR-based allelotyping studies used here can detect abnormal DNA content for each chromosome, although the experiments do not directly show whether the content is above or below 2n (n, haploid genome equivalent of DNA). In addition, the results do not directly prove that the abnormality is continuous or corresponds to a cytogenetically distinct structure. For example, LOH at every informative marker on a chromosome could be due to monosomy but does not rule out the possibility that a more complicated deletion exists. Such a scenario might involve deletion of the short arm of one chromosome coexisting with deletion of the long arm of its homologue. Whereas this scenario would probably be functionally identical to monosomy of the chromosome, it would not meet the cytogenetic criteria for monosomy or aneuploidy.
Chromosome 1 Defects.
The largest and most complicated set of defects were seen on chromosome
1. Three patients had abnormalities seen in this chromosome after the
initial screening with 24 markers was done, and these defects were
further investigated by supplementing the screen with an additional 23
markers distributed throughout the chromosome. The results are shown in
Fig. 4
. Patient 7 had LOH at every informative marker, but the results for
patients 2 and 3 were more complicated. Specifically, patient 2 had
normal results at the end of 1p from 0–62 cM, marked AI indicative of
LOH on the rest of 1p from 70–156 cM, and milder AI on 1q from 167 cM
to the end of 1q. Patient 3 had normal results on 1p but had two areas
of LOH on 1q at 167–221 cM and from 251 cM to the end of 1q. The AI
patterns in both patients show a discontinuity in the pericentric
region between 156 and 167 cM, and this area warrants further analysis
as a possible hot spot of rearrangement.
CGH Analysis.
To confirm and extend the allelotyping results, CGH was performed
on patients 1–6 (insufficient DNA remained from patients 7 and 8). The
CGH technique has the potential to supplement AI measurements by
indicating whether any large DNA region is present above or below
2n and by quantifying the copy number. It is also a very
sensitive technique for detecting high-copy number amplifications of
large regions; however, the technique is not consistently accurate in
detecting copy number changes of 1n that are a result of a
deletion, a duplication, monosomy, or trisomy (27
, 29)
.
In contrast to what is usually seen in solid tumors, the tumors in
patients 1–6 had relatively few abnormal areas. In particular, no
areas of high-level amplification were detected. The abnormal areas
that were detected by both CGH and the AI method are shown in Fig. 5
. The chromosome 1 defects in patients 2 and 3 were detected using CGH,
although the magnitude and position of the defects were probably not
determined as precisely as they were with the AI results
discussed above. Nevertheless, the CGH results do support the
interpretation that patient 2 has LOH resulting from a deletion on 1p
and has a duplication of 1q, whereas patient 3 has at least two areas
of LOH resulting from deletions on 1q. Also shown are the results from
chromosome 5 in patient 2, and the CGH results, when combined with the
AI results shown in Fig. 3
, support an interpretation of trisomy for
this chromosome.
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DISCUSSION |
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Whereas this study is somewhat unusual in the large number of loci examined, results using the same basic techniques are available for numerous other types of tumors. Overall, the results in gastrinomas show that large deletions or amplifications are relatively rare and that the genomes of these tumors are generally much more stable than those of other types of malignancies. This is consistent with the clinical observation that sporadic gastrinomas generally grow slowly and have a 10-year survival rate of 94% (1) .
Despite this background of genetic stability, several striking genetic defects were detected. Multiple large defects were found on chromosome 1 in two patients, and both patients had a discontinuity in the pattern of AI in the same pericentromeric region of the chromosome. Whereas this interval is several megabases in size and was not mapped with better resolution, it is possible that this area is a hot spot for chromosome breakage. Interestingly, in a recent compilation of rearrangements common in human neoplasia, the pericentromeric region of chromosome 1 was especially well represented (33) . The results in these two patients, both of whom had hepatic metastases, also confirm and extend a previous observation of chromosome 1 defects in gastrinomas (19) . In that study, 4 of 18 gastrinomas showed LOH on chromosome 1, and this group included all three of the patients who had hepatic metastases. However, the study had limited information on the location of breakpoint regions because only 12 markers were used, and two of the four abnormal gastrinomas had LOH at every informative marker. An additional study (20) found LOH on chromosome 1 in pancreatic tumors, but only two endocrine tumors of unspecified phenotype and two markers on chromosome 1 were included.
The rationale for performing genome mapping studies is that tumor suppressor genes or oncogenes might be present in commonly rearranged regions or at breakpoints. In this regard, the pericentric region of chromosome 1 is known to contain the N-ras and ß subunit of nerve growth factor genes. In colon cancer, mutations of the K-ras gene have been correlated with increased gastrin expression (34) . Whereas the N-ras, K-ras, and H-ras genes have been sequenced in gastrinomas, no mutations have been found (17 , 35 , 36) . It is possible that genetic alterations other than point mutations are important or that mutations are found in only a subset of tumors. Besides the MEN1 gene, no other obvious genes implicated in gastrin expression or regulation are known to be present in the abnormal regions detected in this study. However, the number of components in these pathways is rapidly expanding, and the MEN1 gene product has recently been shown to bind to JunD (15) and to revert the morphology of RAS-transformed NIH3T3 cells (37) .
Although we do not have cytogenetic data, presumptive aneuploidy was detected in several chromosomes that had AI at every informative marker. This result was seen only in the four tumors that were large or aggressive and was not seen in the other four tumors that were indolent and small. This confirms a previous flow cytometry study from this institution (using a different set of tumor specimens) in which aneuploidy (of the multiple stem line type) was found only in aggressive metastatic tumors (23) .
MSI, presumably due to a defect in a mismatch repair pathway, was observed in only one patient who had a large and unusually aggressive tumor. Whereas MSI has been described in multiple different solid tumors including carcinoid tumors of the lung (38) and the gastrointestinal tract (39) , it has not been reported previously in gastrinomas (40) . Given the large number of microsatellite analyses reported previously, especially in the MEN1 gene search literature, MSI should now be considered a possible but very rare event in gastrinomas. In this regard, gastrinomas are almost identical to stromal tumors of the gastrointestinal tract. In a recent report (41) , MSI was found in only 1 of 30 stromal tumor specimens, whereas LOH was a common event. In contrast, leiomyomas have an excellent clinical prognosis, like most gastrinomas, but frequently display MSI (42) .
MSI has emerged as a marker of a mutator phenotype in cancer (43) and has been especially well studied in colon cancer (44) . It has been estimated that each colon cancer cell contains about 10,000 genetic alterations (45) . Whereas relatively few alterations were found in most gastrinomas, they involved large segments of DNA. The one tumor showing 8% MSI, however, is likely an example of the mutator phenotype. There are over 100,000 simple sequence repeats (43) , and this tumor would be expected to show alterations in over 8,000 of them. Even so, this rate is still much lower than that commonly seen in colon cancer, where MSI frequencies can be >40% (44) .
This study was only designed to detect fairly large rearrangements and might have missed previously reported alterations such as amplification of the HER-2/neu proto-oncogene (17) or chromosome 9q (18) , deletion of the p16/MTS1 tumor suppressor gene (16) , or deletion of chromosome 3p (18 , 21) . However, as expected from numerous other studies (reviewed in Ref. 6 ), large deletions on 11q encompassing the MEN1 gene were confirmed in this study. The influence of additional genes is likely on genetic grounds (46) , and the present study implicates chromosome 1 as a likely location.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed. Present address: Division of Gastroenterology and
Hepatology, University of Minnesota School of Medicine, Box 36, 420
Delaware Street SE, Minneapolis, MN 55455. Phone: (612) 626-0922; Fax:
(612) 625-5620; E-mail: ferri015{at}tc.umn.edu 
2 The abbreviations used are: MEN1, multiple
endocrine neoplasia type 1; AI, allelic imbalance; CGH, comparative
genomic hybridization; LOH, loss of heterozygosity; MSI, microsatellite
instability.
Received 2/11/00. Accepted 7/31/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  Y.-J. Chen, A. Vortmeyer, Z. Zhuang, S. Huang, and R. T. Jensen Loss of Heterozygosity of Chromosome 1q in Gastrinomas: Occurrence and Prognostic Significance Cancer Res., February 15, 2003; 63(4): 817 - 823. [Abstract] [Full Text] [PDF]
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