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Molecular Biology and Genetics |
Departments of Cancer Genetics [G. R. A., W. M. H., J. M. C., M. J. K., N. C., J. D. B., J. K. B., G. P. J., N. J. N., T. B. S.], Surgical Oncology [G. R. A., B. M. B., M. A. R-B., N. J. P.], Cancer Prevention, Epidemiology and Biostatistics [H. S.], Clinical Cytogenetics [S. S.], and Experimental Pathology [D. L. S.], Roswell Park Cancer Institute, Buffalo, New York 14263; Department of Leukemia, M. D. Anderson Cancer Center, Houston, Texas 77030 [J-P. I.]; Department of Surgery, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229 [M. S. K.]; and University of Montreal Hospital Center, Montreal, Quebec, H2W 1T8 Canada [M. B.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genomic instability involved in tumor progression must be a narrowly constrained process. Too great a degree of genomic instability can be expected to give rise to deleterious or lethal mutations at rates exceeding the capacity of cell proliferation and natural selection to overcome, and too low a degree of instability would preclude the timely occurrence of the several essential mutational events necessary to produce malignancy. Although gene targeting of several DNA checkpoint and repair systems in lower organisms is capable of generating genomic instability and in some cases promotes murine tumor development, these same genes have been found to be only rarely associated with human cancers (9 , 10) . Genome safeguards adequate for organisms with short life spans, such as yeast or mice, presumably must be redundantly reinforced in organisms such as humans with much longer life spans; it is these corresponding safeguard mechanisms that must be overcome to generate human cancer.
How might we learn how the genomic instability driving the common human cancers arises? Family cancer genes have provided important starting points, with many of these associated with genomic destabilization (11) . For our studies, we have focused on human sporadic colorectal cancer as an ideal sporadic tumor system because of its relative abundance and the availability of early, premalignant tumor tissues. We have investigated whether intrachromosomal genomic instability is for the most part a single process, potentially controlled by a single gene; is it a single pathway, with multiple potential defect points; or are there several distinct systems and pathways individually and independently involved?
We report here genome-wide scans for LOH, inter-(simple sequence repeat) PCR, and microsatellite instability measurements of genomic instability in a set of 59 sporadic colorectal cancers and 7 premalignant adenomatous polyps. We find these assays detect distinct sets of genomic damage events. Seven principal loci have been selected where LOH is most highly associated with elevated inter-(simple sequence repeat) PCR instability and/or elevated overall rates of LOH (FAL); these loci are flanked by several other loci also associated with elevated genomic instability but to a lesser degree. The seven selected principal loci can be placed in two independent cooperating sets, based on their coordinate loss of heterozygosity patterns.
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MATERIALS AND METHODS |
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LOH Assays.
PCR amplifications were carried out using standard techniques (14
, 15)
. Primers were end labeled with [
-32P]ATP of 3000 Ci/mMol (NEN) and T4-polynucleotide kinase (New England Biolabs). PCR amplification was performed in MJ Research PTC-100 thermocyclers with 30 cycles of 25 s at 95°C denaturation, 30 s at 56°C annealing, and 1 min at 72°C extension. Reaction volumes of 25 µl contained l00 ng of genomic DNA, 200 µM deoxynucleotide triphosphates (Promega), 0.4 µM unlabeled primer, 0.06 µM labeled primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.75 mM MgCl2, and 1.25 units Taq polymerase. After amplification, reactions were mixed with equal volumes of loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol), denatured at 95°C for 5 min, and placed on ice. One µl was directly loaded onto a 0.4-mm-thick 6% acrylamide gel, electrophoresed 2 h at 60 W, and then autoradiographed.
Inter-(Simple Sequence Repeat) PCR.
The inter-SSR PCR method and its reproducibility in multiple independent assays have been described (2
, 12
, 16)
. 32P-end labeled primers homologous to dinucleotide repeats and anchored at the 3' end by two nonrepeat nucleotides were used in a PCR to amplify the sets of genomic sequences present between the repeat elements, where the primer binding sites are inverted and spaced less than 
2 kb apart. PCR was in a volume of 20 µl containing 1 µM primer (1:5 labeled/unlabeled oligonucleotide), 50 ng genomic DNA, and 0.3 unit of Taq polymerase (Life Technologies, Inc., Bethesda, MD) in 1x PCR buffer [10 mM Tris·HCl (pH 9.0), 2% formamide, 50 mM KCl, 0.2 mM deoxynucleotide triphosphates, 1.5 mM MgCl2, 0.01% gelatin, and 0.01% Triton X-100]. Amplification conditions were a 3-min initial denaturation at 94°C, followed by 30 cycles of (30 s at 94°C denaturation, 45 s at 52°C annealing, and 2 min at 72°C elongation), and then a final 7 min at 72°C for extension. The labeled PCR products were analyzed on nondenaturing PAGE at 80 W for 22 min, followed by 50 W for 3400 V-hr for CA-based PCR products and 2800 V-hr for CG-based PCR products. The gels were then dried and autoradiographed. The genomic instability index was computed by dividing the number of altered bands seen in the PCR products amplified from the tumor DNA by the total number of products generated from the corresponding normal tissue DNA.
Screening of BAC Library.
Two 24-bp overlapping oligos (overgos, ctggctcactcttgatTGTGTTGG, agagcccatcctttacCCAACACA), the 3' ends of which have eight base nucleotides complementary to each other (capitalized), were designed within the altered inter-SSR PCR fragment, G2-4-16, missing from tumor 3143. The mix of the two 24-base overgos (10 pmol each) was annealed at 37°C for 10 min, after being heat-denatured for 5 min at 80°C. Both ends of the annealed overgos were filled in at room temperature for 1 h, with 2 units of Klenow, 5 µCi of [
-32P]dATP and [-32P]dCTP each, and 0.1% BSA in OLB buffer [0.24 mM Tris·Cl (pH 8.0), 24 mM MgCl2, 0.1 mM dTTP, 0.1 mM dGTP, 0.35% 2-mercaptoethanol, 1 M HEPES (pH 6.6), 0.9 mM Tris·Cl (pH 7.4), and 0.06 mM EDTA (pH 8.0)] in a total volume of 10 µl. A labeled double-stranded 40-bp probe was thus generated for screening the RPCI-11 BAC library. Four RPCI-11 human library filters were hybridized with the radiolabeled overgos overnight at 60°C in 1 mM EDTA (pH 8.0), 7% SDS, 0.5 M sodium phosphate (pH 7.2). After hybridization, filters were washed in 1.5x SSC, 0.1% SDS at 60°C for 20 min twice and then exposed on a storage phosphor screen and visualized on the Storm 860 phosphorimager.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using a genome-wide set of 348 autosomal markers spaced 10 cM apart, we profiled 59 sporadic colorectal cancers and 7 adenomatous polyps for LOH (Fig. 1)
. The mean LOH rate was 7.3% for this series, with a range of 0 to 45% for each marker. Particularly frequent losses were observed on chromosomes 5, 8, 11, 13, 14, 17, and 18. The FAL rate is defined as the fraction of all alleles informatively assayed for each tumor that exhibited LOH. FAL rates for the set of 59 carcinomas ranged from 0 to 54% with a mean value of 9.5%.
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. If intrachromosomal genomic instability in sporadic colorectal cancer arose from a single mechanism, this result would be difficult to understand. On the other hand, if multiple instability mechanisms exist, and these two assay methods preferentially detect different types of genomic damage, then these data would be consistent with such a model. Alternatively, if the relatively recently developed technique of inter-SSR PCR were somehow detecting PCR artifacts, then we should again fail to see any association with the well-established LOH assay results.
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illustrates the detailed characterization of two additional products, the first of which was seen as intensified in inter-SSR PCR assays of tumor 675 (Fig. 3A)
. Southern blots of tumor and normal DNA showed that in this case an amplification of >2 kb had occurred, which had also independently occurred in tumor 2672 (Fig. 3B)
. The second product characterized was an inter-SSR PCR band missing from tumor 3143 (Fig. 3C)
. The normal specific band was cloned and used in turn to generate overgo oligonucleotides, as described in "Materials and Methods." These were used to screen the BAC library RPCI 11, revealing a single clone 55NO7. When this BAC clone was used for fluorescence in situ hybridization to touch preps of the corresponding tumor 3143 and normal tissue 3144, two spots per nucleus were seen in the normal cells but only one in each tumor cell, revealing that one copy containing at least a majority of the l50-kb BAC clone insert had been deleted in the tumor. (In Fig. 3D
, the two closely spaced spots in what may appear to represent a single tumor cell actually reflects two overlapping cells.)
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. This was a likely possibility, based on the repetitive sequences of the polymorphic microsatellite markers used for these assays (19)
. We cloned and sequenced one such altered product, for marker Rb1.20 near the Rb locus on chromosome 13. This showed a reduction of a segment of 25 (GAAA) repeats to a segment of 24 (GAAA) repeats; such a 4-base change is consistent with a defect in the msh2-msh3 loop recognition system (20)
. Because 348 markers were examined for each colorectal tumor and its corresponding normal colonic mucosa tissue, we were able to generate precise data for the overall extent of MSI instability, termed the FINSTR. Four of the 59 carcinomas had FINSTR values ranging from 0.45 to 0.53, consistent with known rates of MSI in sporadic colorectal cancer, whereas the remaining 55 carcinomas all had FINSTR values of <0.10 (Fig. 5)
. For the 55 tumors that did not show extensive MSI (FINSTR <0.10), consider the one-category and two-category models (21)
. For a one-category model, the damage to microsatellite sequences will build up at a rate of dN/N = kt, and semilog plot of microsatellite damage levels in a set of tumors will produce a straight line. In contrast, if two separate types of processes are represented in the set of tumors, each generating microsatellite damage at significantly different rates in one subset or the other, then damage will be occurring at a rate of dN/N = k1t in one subset, and dN/N = k2t in the other subset. When a semilog plot of MSI is prepared in the second case, a biphasic line would be seen. For all but the four highest FINSTR values which can be regarded as MSI-H, the straight line actually observed on the semilog plot of this data (Fig. 5
, lower panel) is consistent with a stochastic process randomly producing microsatellite alterations in these inherently less stable sequences and is not consistent with separate categories of MSI-L and microsatellite-stable.
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. Although MSI was not correlated with either FAL or inter-SSR PCR (Spearman rank-order correlation procedure for nonnormally distributed values), the graphs show all tumors with elevated MSI had low levels of the other types of instability. The low number of tumors (n = 4) with elevated microsatellite instability precluded conclusively testing a possible inverse association, although recent results using spectral karyotyping would support such an inverse association (22)
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Genomic instability is widely thought to be an essential facilitator of tumor progression, enabling the occurrence of the multiple mutations required for the generation of malignancy, although there might be differences between the types of instability that predominate in facilitating the early and the late progression events. The genomic damage in seven adenomatous polyps detected by inter-SSR PCR had been found to be essentially equal to that of the 59 carcinomas, with mean values of 4.1% of bands altered in adenomas and 3.9% altered in carcinomas. When these same adenomas were assayed for FAL rates, polyps showed levels only one-third that of the carcinomas, with FAL rates of 3.0% for adenomas and 9.5% for carcinomas. Our results should not be taken to mean that the inter-SSR PCR detected instability stops during tumor progression, because laser capture microdissection revealed that late-stage genomic events are obscured when million-cell tissue samples are used for analysis, as was done for these studies.4 Because the same circumstances would similarly affect LOH measurements, our data indicate that instability revealed by inter-SSR PCR typically begins earlier in tumor progression than does that detected as widespread LOH.
Many mechanisms may together contribute to genomic instability in cancer; these can be categorized as three basic possibilities. Dominant mutations may result in processes that somehow actively increase rates of damage to DNA. Recessive mutations in repair or checkpoint systems may, once the second allele is lost, allow normal rates of DNA damage to go unrepaired, enabling damage to accumulate. A third possibility is that an epigenetic event such as global demethylation occurs, simultaneously producing widespread damage throughout the genome. To evaluate this last possibility, we examined the CpG island methylator phenotypes for 19 of our tumor/normal colorectal tissue sets (23
, 24)
. No association of methylation with genomic instability was seen, as defined by inter-SSR PCR genome-wide FAL rates, or MSI (Table 1)
. None of the 19 tumors used for this methylation study showed classical MSI (FINSTR < 0.10). Other investigators have demonstrated that methylation silencing of MLH1 underlies many cases of MSI (25)
, and DNA methylation changes themselves represent one form of epigenetic genomic instability. But methylation evidently does not frequently drive the other intrachromosomal instabilities, which we are measuring at the DNA level.
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); the efficacy of the crypt isolation procedure is evident in photomicrographs of the crypts (Fig. 7b)
. These findings are contrary to what was predicted recently by Rubin (28)
for such experiments. We conclude that the genomic rearrangements seen in cancer cells must arise during the course of the tumor progression process itself.
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. These criteria enabled us to exclude from further analysis those markers less commonly lost and thus less likely to be frequently involved in colorectal tumor progression. Thirteen of the 47 loci, when compared for those tumors with LOH at that locus to those tumors without LOH at that locus, showed relative inter-SSR PCR instabilities >1.5; these were on chromosomes 3 (2 loci), 8, 11, 13 (2)
, 14q (3)
, 17, 18, and 20 (Fig. 8A)
. Several chromosomal regions showing elevated rates of LOH such as 5q, 14p, and most of chromosome 18, showed no association with elevated inter-SSR PCR instability. In contrast, 42 of these 47 loci showed relative genome-wide FAL rates of >1.5; these were on chromosomes 1 (2 loci), 3 (2 loci), 4, 5 (2 loci), 7, 8 (2 loci), 9, 11 (3 loci), 13 (4 loci), 14 (7 loci), 17 (2 loci), 18 (9 loci), 20, 21, and 22 (Fig. 8B)
. All of the loci associated with elevated inter-SSR PCR instability also showed elevated genome-wide FAL rates, although the converse was not the case.
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With 42 loci associated with elevated rates of genome-wide LOH and a subset of 13 also associated with elevated inter-SSR PCR instability, we then selected the most promising for further study. We used the criteria of: (a) LOH of a given marker had to occur at more than twice the background rate of 7.3%, consistent with a frequent overall involvement in malignancy; (b) for inter-SSR PCR, the mean genomic instability index for the set of tumors with LOH at a given locus had to exceed 1.5 times the mean inter-SSR instability rate for the entire set of 59 carcinomas; or (c) for FAL rates, the mean FAL rate for the set with LOH at a given locus had to exceed 1.5 times the mean FAL rate for the entire set. These criteria identified eight separate candidate loci potentially relating to inter-SSR instability and/or FAL, as listed in Table 2
. The significance of the association between LOH at these loci and elevated genomic instability was then tested by using the Mann-Whitney test (two-tailed), and seven of the eight candidate loci were found to have Ps of <0.05. The one locus that failed to meet this criterion, d13s787, had a P of 0.06 and is located relatively close on the p-arm of chromosome 13 to the marker d13s325, which had a P of 0.02. One locus, d8s119, showed a strong association with elevated FAL rate, but no association with inter-SSR instability. Two loci, d18s878 and d20s470, were strongly associated with both types of instability. The other four loci were significantly associated with FAL but less strongly associated with the inter-SSR type instability. If these candidate loci are truly involved in enhancing genomic instability and promoting tumor progression, then we might expect patients whose tumors show LOH at those loci that generate the most rapid progression will reveal earlier onset disease. This was seen for the loci on chromosomes 3, 8, 11, and 13 but not for the loci on 14, 18, or 20; the earlier onset for all was not statistically significant because of the small sample numbers.
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, then LOH of closely associated markers would also be expected to show an association with genomic instability. Consistent with this, LOH of d3s2432, the closest marker to GATA16Y608, which generated substantial informative data (Fig. 8)
, also was associated with elevated genomic instability. For d8s1119, no other close markers were associated with elevated instability, but few LOH events included adjoining markers. For d11s1985, no nearby markers were associated with instability; this was not surprising because none of the LOH events for d11s1985 extended to adjoining markers. For d13s325 and d13s787, LOH of the nearby marker d13s317 was also strongly associated with elevation of both ISSR and FAL instability (Fig. 8)
. For d14s53 on chromosome 14q24, three markers extending to the q-terminus (d14s617, GATA168, and GATA136) were all associated with elevated intrachromosomal instability. For d18s878, LOH of the nearby markers d18s844, d18s851, d18s535, d18s562, and d18s858 were all associated with elevated FAL-type instability but not the ISSR-type instability. For d20s470, no other frequently lost and highly informative markers were seen anywhere else on chromosome 20.
Fine structure mapping around marker d18s878 was carried out to evaluate whether large deletion events were encompassing this region. As shown in Table 3
, for two tumors, LOH events at this position were relatively small, of <5 cM, and for three others the events were >20 cM. Because LOH of d18s878 was strongly associated with intrachromosomal genomic instability of both the FAL and ISSR types, whereas LOH of five other markers on 18q was associated with FAL-type instability alone, there may be multiple genes affecting genomic integrity on 18q.
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. The loci on chromosomes 14 and 18 were found to be coordinately lost, with a P of 0.005. The locus on chromosome 20 was coordinately lost along with any one of the four loci on chromosomes 3, 8, 11, or 13 (P = 0.002).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our approach of identifying those loci where LOH is associated with elevated overall genomic instability at this point establishes only that—an association. The loci we have identified may be where those genes are located, which when lost or rendered defective cause genomic integrity to be rapidly eroded. Alternatively, they may be sites particularly vulnerable to instability processes generating the genomic damage seen in sporadic colorectal cancer. But if this latter possibility were the case, it becomes difficult to envision why these particular loci reveal LOH rates no greater than, or less than, numerous other loci that were not associated with any elevated overall genomic instability. In a more complex model, the loci we have identified might be secondarily involved in pathways affecting the maintenance of integrity, with the true initiating event for genomic instability arising from a means other than gene loss. Testing these possibilities becomes feasible; if the loci we have identified are truly those responsible for genomic integrity and their loss enables tumor progression to occur, then one would expect to see their loss occurring early in progression in premalignant adenomatous polyps (38) . For the first three loci so examined on chromosomes 8, 14, and 18, this has been found to be the case.5
Several interesting genes are known to reside in relatively close proximity to those candidate loci we have identified, which would appear to have functions consistent with roles in colorectal cancer and/or preserving genomic integrity. Beyond MLH3 on chromosome 14, candidates include the VHL, TGFßRII, and XPC genes on chromosome 3, the NBS gene on chromosome 8, the group of FEN-1, DNApol, and DDB-2 on chromosome 11, BRCA2 on chromosome 13, Bcl2, MADH4, and DCC on chromosome l8, PCNA on chromosome 20, as well as p53 on chromosome 17.6
Hall et al. (39) demonstrated in cell fusion studies that amplifiability of the CAD gene in a variety of solid tumor cell lines is a recessive feature, representing at least two complementation groups. It is unknown whether her findings relate to the several candidate loci that have identified in this report. The loci we have identified are associated with intrachromosomal genomic instability in sporadic colorectal cancer, but it is unknown whether they relate to genomic instability in other types of cancers. BRCA2, hMSH2, and hMLH1 all provide examples of genes involved in preserving genomic integrity, yet which have been associated with only very specific tumor types (40 , 41) .
Genome-wide allelotyping may become more useful in elucidating the genetic basis of a number of sporadic tumor phenotypes, in addition to expanding our knowledge of genomic instability itself. The improved resolution offered by array-based approaches, in particular, will greatly assist such studies, although background FAL rates will inevitably complicate such analyses. Nonetheless, it is becoming feasible to identify loci and genes underlying many clinically relevant features of cancer, including those relating to prognosis and treatment response (42 , 43) . Those genes associated with genomic instability and facilitating progression are particularly likely to have prognostic relevance, just as is the case for those colorectal patients with MSI, who have generally better outcomes. Identifying the genes that underlie the several aspects of genomic instability and understanding the mechanisms behind the evolutionary process of tumor progression further offers enormous potential for enhancing the fundamental understanding of cancer. It is from such understanding that will eventually come the truly effective diagnostic tools and therapeutic approaches to this disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grants CA74127 (to G. R. A.) and CA63333 and DC03697 (to T. B. S.) from the NIH; by a grant from the American Cancer Society (to D. L. S.); by a grant from the Charlotte Geyer Foundation (to G. R. A.); and by Roswell Alliance Foundation grants (to N. J. P., M. A. R., T. B. S., D. L. S., and G. R. A.). Services provided through the Roswell Park Biopolymer Facility and the DNA Microarray and Genomics Core Facility were supported by the Institute’s Core Grant P30-CA16056 from the NIH. 
2 To whom requests for reprints should be addressed, at Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263. Phone: (716) 845-4529; Fax: (716) 845-8126; E-mail: garth.anderson{at}roswellpark.org
3 The abbreviations used are: MSI, microsatellite instability; inter-SSR PCR, inter-(simple sequence repeat) PCR; LOH, loss of heterozygosity; FAL, fractional allelic loss; BAC, bacterial artificial chromosome; FINSTR, fractional microsatellite instability rate.
4 G. P. Jahreis, unpublished observations.
5 Bruce Brenner, Nicholas Petrelli, Daniel Stoler, and Garth Anderson, unpublished observations.
6 Online Mendelian Inheritance in Man, OMIM. McKusiik-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library Of Medicine (Bethesda, MD), 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/. A New Gene Map of the Human Genome, Gene Map ’99. National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD) 1999. World Wide Web URL: http:/www.ncbi.nlm.nih.gov/genemap/.
Received 3/ 9/01. Accepted 9/19/01.
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