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Molecular Biology and Genetics |
Molecular and Population Genetics [K. W-R., I. T.], Human Cytogenetics Laboratory [J. W., D. S.], and Department of Histopathology [R. P., N. W.], Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom; Cancer Genetics, Institute of Cancer Research, Surrey SM2 5NG, United Kingdom [S. B., R. H.]; Queensland Institute of Medical Research, Royal Brisbane Hospital, Brisbane, Herston Q4029 Australia [J. Y., B. L.]; Department of Medical Genetics, Addenbrookes Hospital, Cambridge CB2 2QQ, United Kingdom [I. F.]; Havering Hospitals National Health Service Trust, Essex RM3 OBE, United Kingdom [Y. T.]; Department Clinical Genetics, Guy’s Hospital, London SE1 9RT, United Kingdom [S. H.]; Department of Surgery, University of Ulsan College of Medicine, Asan Institute, Seoul 138-040, Korea [J-C. K.]; Center for Polyposis and Intestinal Disease, Tokyo 113, Japan [T. I.]; and Department of Histopathology, University College London, London WC1E 6JJ, United Kingdom [M. N.]
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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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The epithelium of PJS polyps has a monoclonal origin, as demonstrated by the finding of allelic loss at the LKB1/STK11 locus on chromosome 19p13.3 (2, 3, 4) . PJS-associated cancers also show loss of the wild-type allele at this site, strongly suggesting progression from hamartoma to carcinoma. Hamartomas, adenomas, and carcinomas from patients with CS show LOH at the PTEN/MMAC1 locus on chromosome 10q23.3 (5) , again suggesting that this gene is acting as a tumor suppressor, with loss of the wild-type gene initiating hamartoma growth and leading to subsequent progression to carcinomas in some cases. Basal cell carcinomas from patients with GS have shown allelic loss in the PTCH region of chromosome 9q31, indicating that the PTCH gene is homozygously inactivated in these hereditary tumors (6) ; however, because juvenile polyps occur as an infrequent component of this disease, allele loss has not been investigated in the hamartomas themselves.
Recent work has identified germ-line mutations in the SMAD4 (DPC4) gene on 18q21.1 as a cause of JPS in a subset of families (7 , 8) . The SMAD4 gene encodes a protein that is a cytoplasmic mediator in the transforming growth factor ß signaling pathway. Many of the reported mutations produce SMAD4 proteins that are truncated at the COOH terminus and thus lack normal function. SMAD4 is known to act as a tumor suppressor gene in cancers of the pancreas (9) and the colon (10 , 11) . The high incidence of colorectal cancer in a large JPS kindred with linkage to 18q21.1 and the subsequent observation of mutations in the SMAD4 gene in this kindred led to the reasoning that SMAD4 might also be acting as a tumor suppressor gene in JPS polyps (7 , 8) . However, it has recently been postulated that in cancer predisposition syndromes such as JPS, susceptibility genes work through less direct mechanisms (12) . It was proposed that the neoplastic progression is driven by initial genetic changes within the stromal cells of the hamartoma (classically the clonal component of these lesions) and that an increased risk of cancer is the result of an altered terrain for epithelial cell growth (the "landscaper" effect). In contrast, classic tumor suppressor genes such as APC were said to function as "gatekeepers," preventing runaway growth, and the DNA repair proteins such as MLH1 and MSH2 were thought to be acting as "caretakers" of the genome (13) .
The aim of this study was to investigate whether homozygous inactivation of SMAD4 was present in the polyps of individuals with pure JPS who had a known constitutional SMAD4 mutation. The results have implications not only for the role of SMAD4 as a tumor suppressor gene but also for the clonal origins of JPS polyps and the "landscaper" hypothesis.
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MATERIALS AND METHODS |
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For the assessment of allelic loss, slides from paraffin-embedded
tissue were crudely microdissected using a fine blade or needle to
remove polyp tissue from its stalk and any surrounding normal tissue.
The stromal and epithelial components were not separated. DNA was
extracted using standard methods [for larger polyps, we used the
Qiagen (Hilden, Germany) tissue extraction kit, and for smaller polyps,
we used proteinase K digestion and phenol/chloroform purification].
PCR was performed in duplicate on polyp/normal samples for seven
microsatellite markers (D18S877, D18S851, D18S474, D18S878, ATA7D07,
ATA82B02, and GATA177C03) that lie at approximately 10 cM
intervals along chromosome 18q (Fig. 1)
and one marker on chromosome 18p (D18S542). PCR conditions were as
follows: 25-µl total volume with approximately 12 ng of DNA as a
template with 1x standard PCR buffer, 1.5 mM
Mg2+, 0.25 mM deoxynucleotide
triphosphates, 0.25 unit of Taq polymerase, and 0.5 mM of
each oligonucleotide primer, with the forward primer fluorescence
labeled with HEX, FAM, or TET. Cycling conditions consisted of
an initial denaturation at 94°C for 5 min; 30 cycles each at 94°C
for 30 s, 55°C for 30 s, and 72°C for 30 s; and then
a final extension step at 72°C for 10 min. A portion of each PCR
product (0.2 µl) was combined with 0.2 µl of Tamra350 size standard
(Perkin Elmer Applied Biosystems, Warrington, United Kingdom)
and 3 µl of formamide loading buffer. After denaturation at 94°C
for 5 min, products were electrophoresed on a 4.25% sequencing gel on
an ABI377 semiautomated sequencer for 2 h. Results were analyzed
using Genotyper software, with areas under the peaks (including stutter
bands) compared in all informative (heterozygous) individuals. Allelic
loss was considered present if the relative ratio of normal:tumor peak
areas was less than 0.5 or greater than 2, thereby allowing for
contaminating normal tissue within the microdissected polyp. All PCRs
and Genescan runs were performed in duplicate on separate occasions to
ensure results were consistent.
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-satellite
18-centromere probe (Oncor, Gaithersburg, MD) on the same sections. To
control for the PAC hybridizing efficiency, the experiments were
repeated on normal tissue sections. |
RESULTS |
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: (a) D18S542; (b) D18S877;
(c) D18S851; (d) D18S474; (e) D18S878;
(f) ATA7D07; (g) ATA82B02; and (h)
GATA177C03. The SMAD4 locus reportedly lies between D18S851
and
D18S8785
and is thought to be closest to D18S474 (approximately 0.1 Mb distal).
Fig. 2
shows the results of the allele loss studies.
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), whereas microsatellites proximal and distal to the SMAD4
locus did not show loss. In all cases, it was the wild-type allele that
was lost, as ascertained by genotyping family members. Patient 2 was
informative for six of the eight markers, and in all six juvenile
polyps studied, loss was apparent for one or more microsatellites. The
marker that showed the most frequent loss in juvenile polyps from this
patient was D18S474, the marker closest to SMAD4. In one
polyp (polyp 2.4) from patient 2, only this marker was lost, whereas
the other polyps showed loss over a greater distance. Polyp 2.1 from
this individual shows loss of all informative markers along the long
arm of chromosome 18q, indicating that a major event led to the loss of
the second functional copy of SMAD4; this is interesting to
observe in a benign lesion which for many years was thought to be
nonneoplastic and without malignant potential. It is apparent that
different regions of the chromosome have been lost in different polyps,
indicating that different mechanisms may be involved in inactivation of
the second copy of SMAD4.
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Nine patients (patients 1, 3, 4, 13, 14, 15, 16, 17, and 18) had no detectable germ-line SMAD4 mutation. Of 16 JPS hamartomas from these individuals, 10 showed no allelic loss at any marker, and the remainder showed loss of just one marker. Loss was observed close to SMAD4 in only two polyps (polyps 1.1 and 13.2). One patient had a mixed juvenile/adenomatous polyp (polyp 3.5), and this polyp showed loss around SMAD4. Of eight cancers (from patients 3, 4, 17, and 18), six (polyps 3.2, 3.3, 3.4, 4.2, 17.1, and 18.1) lost alleles at markers around SMAD4. Two cancers (polyps 3.6 and 4.1) showed no loss around SMAD4. Patients 3, 17, and 18 are from the same family. Gastrointestinal cancers and the mixed polyp from this family appear to show loss of a large portion of 18q encompassing SMAD4, although some markers are uninformative. This family has had germ-line mutations in SMAD4 excluded by both linkage analysis and mutation screening. The observed allelic loss in these cancers almost certainly reflects the loss of 18q that is found in approximately 60% of sporadic colorectal cancers (10 , 11) , although no studies of small bowel cancers have been performed.
Single polyps from patients 8 and 11 lost only D18S851, although markers nearer to SMAD4 failed in the PCR; therefore, it is possible that they may also be lost. A single marker (ATA7D07) was lost in the sole polyp from patient 9. No loss was observed in single polyps from patients 10 and 12. None of these four patients has been screened for mutations in SMAD4, and the loss of 18q observed in some of these polyps may reflect a "second hit."
FISH Demonstrates that Individuals with SMAD4
Germ-line Mutations Show Clonal Origin of Epithelial Cells and Some
Stromal Cells in JPS Polyps.
FISH was performed on paraffin blocks from three JPS polyps from
three patients (patients 2, 5, and 19) who have germ-line
SMAD4 mutations. Patient 19 was not included in the allelic
loss study because of a lack of normal material. PAC clone (224 j 22)
was used as a probe for 18q21. To ensure that SMAD4 was the
target for deletion studies, exons 2 and 11 of this gene were amplified
in 100% of clones tested, and the PAC clone was mapped to 18q21
using normal metaphase spreads. Using this probe, we observed only one
signal in epithelial cells of juvenile polyps (90%, 95%, and 82%
from patients 2, 19, and 5, respectively; Fig. 4
, a–c). Infiltrating stromal lymphocytes showed two signals
in over 90% of nuclei from all three polyps (Fig. 4f)
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Intriguingly, the stromal fibroblasts and pericrypt fibroblasts from
each polyp also showed just one signal in between 83% and 90% of
cells, respectively (Fig. 4, d and e)
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. To control for hybridizing efficiency, PAC 224 j 22
was used as a probe against both normal colon and appendix sections
from an unrelated individual. Two signals were observed in the majority
of stromal and epithelial cells (94% and 90% of colon and appendix,
respectively; Fig. 4h
). To ensure that the cells counted
were of the origin indicated by their morphology (stromal, epithelial,
or inflammatory infiltrate), Giemsa staining of the same section used
for FISH was performed, and antibodies particular to these cell types
were hybridized to serial sections of the JPS polyps used for FISH. All
antibodies (AuA1, Mnf116, SmAct, Desmin, CD45, and CD31) and the Giemsa
staining confirmed the cells to be of the expected origin. |
DISCUSSION |
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Allelic loss was observed in individuals (patients 3, 4, 17, and 19) who have had germ-line mutations in SMAD4 excluded as the cause of their JPS, but loss was largely confined to gastrointestinal cancers and a mixed juvenile/adenomatous polyp rather than pure juvenile polyps. Loss around the SMAD4 locus occurs in approximately 60% of colorectal cancers (16) . In about 20% of cases, this loss targets SMAD4 (10 , 11) .
Other patients without detectable SMAD4 mutations showed allelic loss in JPS polyps at occasional microsatellites on 18q. It is possible that these changes, regardless of whether or not they target SMAD4, might be important in the pathogenesis of these JPS polyps. Another possibility to explain these isolated losses is background genetic changes unrelated to tumorigenesis. It is even possible that despite all experiments being performed in duplicate, some of these examples of isolated allelic loss are false positives resulting from variation in allelic amplification efficiency.
Contrary to previous studies (1
, 12
, 17)
, our results
suggest that the juvenile polyp in JPS is not simply a stromal lesion
with an excess of connective tissue. Microscopically, the polyps in
this study were not dissimilar to adenomas, with an abundance of
entrapped epithelium (Fig. 5)
. This was true of juvenile polyps from many different patients, both
with and without SMAD4 mutations. Consistent with these
appearances, we found loss of SMAD4 in epithelial cells from
JPS polyps using FISH. FISH found no loss of SMAD4 in
stromal lymphocytes but did find loss in the less numerous stromal
fibroblasts and pericryptal myofibroblasts. This result suggests that
the epithelium of JPS polyps is clonal and that part of the stroma is
also derived from the same clone, contrary to general histological
theory. Loss of SMAD4 in both epithelium and stroma can also
explain how microsatellite analysis can readily and consistently detect
allele loss at multiple markers on 18q in crudely microdissected
polyps, despite the presence of the considerable inflammatory
infiltrate.
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Previously, after detecting a germ-line deletion of 10q in a patient who had juvenile polyps and multiple congenital abnormalities, Jacoby et al. (17) used allele loss/FISH analysis to show somatic deletion of chromosome 10q22 in juvenile polyps. The putative locus contained within the 10q22 region was termed JP1 and was postulated to account for a large proportion of JPS cases. Loss of chromosome 10q led to the conclusions that JPS may be allelic to CS that had been mapped to the same region and that PTEN may be the "hamartoma" gene. Most of the patients in the study of Jacoby et al. (17) who showed allelic loss were less than 10 years of age, and, given the multiple abnormalities of the patient with the interstitial deletion, it was possible that many of the patients in the study of Jacoby et al. (17) actually had CS. Not all patients could have had CS, however, because Marsh et al. (21) concluded that the minimal region of allele loss in the patients of Jacoby et al. (17) did not include the CS locus. Soon afterward, PTEN mutations were shown to cause CS, but the timing was such that Jacoby et al. (17) were unable to show germ-line PTEN mutation in any of their patients. Therefore, there is no evidence that the 10q22 changes in their polyps were "second hits," and their significance for tumorigenesis was unknown.
Jacoby et al. (17) found that their patients’ polyps harbored 10q deletions in inflammatory lymphocytes in the lamina propria, suggesting that the juvenile polyp may be a lymphomatous neoplasm. This is contrary to our findings. There were potentially important methodological differences between our work and that of Jacoby et al. (17) . It has been demonstrated that when using FISH, sections of 6 µm contain almost no nuclei that are uncut (22) . Therefore, it may not be appropriate to conclude that loss has occurred using the 5-µm sections used by Jacoby et al. (17) , especially if cells comprising the tumor vary in size from normal tissues and/or if stromal and epithelial cells are of different sizes. Jacoby et al. (17) used much lower thresholds than those used in this study to assess allelic loss using microsatellites. Whereas any threshold for the analysis of lesions of uncertain clonal origin must, to some extent, be arbitrary, the lower thresholds of Jacoby et al. (17) are likely to increase sensitivity but run the risk of false positives. For the FISH analysis, Jacoby et al. (17) did not hybridize their 10q probe to control sections to check for differences in probe hybridization efficiency, and they used a chromosome 21 control probe that may itself have undergone changes in polyps or be subject to important differences from the 10q probe.
Our data show that, on the reasonable assumption that loss of the wild-type SMAD4 initiates tumorigenesis, the epithelium of JPS polyps is intimately involved in the formation of the hamartoma and its subsequent progression to carcinoma. In our subset of JPS patients with germ-line SMAD4 mutations, the polyps appear to be composed of an abundance of entrapped epithelium that has become cystically dilated, with the stroma largely containing lymphocytes and other inflammatory cells. We have also found good evidence of loss of wild-type SMAD4 in the epithelium, stromal fibroblasts, and pericryptal myofibroblasts of JPS polyps, a counterintuitive result that may require confirmation by other workers. The causes of differentiation of a single cell of origin into stroma and epithelium within the polyp are, of course, unknown, but it certainly appears that the classical categorization of JPS polyps as simply stromal lesions is incorrect. Thus, the "landscaper" hypothesis, which was developed to explain how epithelial cancers could supposedly arise from the stromal component of a JPS polyp, has no experimental basis and does not apply here, even if it may do so elsewhere. It is therefore not surprising to observe an increase in the incidence of gastrointestinal cancer in patients with JPS. It also becomes much easier to explain how occasional JPS patients can develop cancer very early in life (23) . SMAD4 therefore appears to act as a tumor suppressor gene of the "gatekeeper" type in the epithelium of both JPS polyps and sporadic cancers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Molecular and Population Genetics, Imperial Cancer
Research Fund, London WC2A 3PX, United Kingdom. Phone:
44-0-171-269-3383; Fax: 44-0-171-269-2885; E-mail: woodford{at}icrf.icnet.uk 
2 http://www.ncbi.nlm.nih.gov/omim/.
3 The abbreviations used are: JPS, juvenile
polyposis syndrome; LOH, loss of heterozygosity; FISH, fluorescence
in situ hybridization; PJS, Peutz-Jeghers syndrome; CS,
Cowden syndrome; GS, Gorlin syndrome.
5 http://cedar.genetics.soton.ac.uk/.
Received 12/16/99. Accepted 3/17/00.
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REFERENCES |
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, retention of
heterozygosity; gray circles, uninformative markers;
•, LOH. Crosses, PCR failure. The SMAD4
locus lies between D18s851 and D18s878. *, SMAD4 excluded via
screening of the gene. **, mutations of SMAD4 responsible for JPS.
***, SMAD4 excluded via linkage analysis. ****, SMAD4
mutation status unknown. jp, juvenile polyp (site
unspecified unless indicated). mixed, polyp with
combined juvenile and adenomatous features. p, polyp.
sb ca, carcinoma of the small bowel (site unspecified).
ca, carcinoma.
