Affiliations of authors: J. D. Ravenel, E. L. Niemitz, T. M. Jayawardena, H. Uejima, A. P. Feinberg (Institute of Genetic Medicine), E. J. Perlman (Department of Pathology), The Johns Hopkins University School of Medicine, Baltimore, MD; K. W. Broman, Department of Biostatistics, Bloomberg School of Public Health, The Johns Hopkins University; D. W. Bell, D. A. Haber, Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown.
Correspondence to: Andrew P. Feinberg, M.D., M.P.H., Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Ross 1064, 720 Rutland Ave., Baltimore, MD 21205 (e-mail: afeinberg{at}jhu.edu).
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ABSTRACT |
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INTRODUCTION |
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LOI of IGF2 was first described by us and other investigators to occur in Wilms tumor (13), the most common solid tumor of childhood. Subsequently, we and others (424) have found that LOI of IGF2 is one of the most common molecular alterations in cancer, including other embryonal tumors of childhood, such as hepatoblastoma, rhabdomyosarcoma, and Ewing's sarcoma, and major malignancies in adults, such as uterine, cervical, esophageal, prostate, lung, and germ cell tumors. However, despite the prevalence of this alteration, the biologic role of LOI in cancer remains obscure.
Wilms tumor provides a unique opportunity to address the biologic role of LOI in cancer for several reasons. First, it is the first tumor in which LOI was observed (1,2). Second, Wilms tumors are characterized by two distinct subgroups that are distinguishable both by pathology and by the precursor lesions, termed "nephrogenic rests," from which the tumors arise (25). Third, Wilms tumor was the first type of tumor examined by Knudson to formulate his two-hit hypothesis of cancer to explain the bimodal age distribution of children at diagnosis (26). We, therefore, hoped to relate LOI to the known epidemiology of Wilms tumor.
Intralobar nephrogenic rest (ILNR)-like Wilms tumors contain heterologous elements, such as smooth muscle, cartilage, and adipocytes, and are often associated with ILNRs that arise within the interior of the renal lobe early in fetal development. In contrast, perilobar nephrogenic rest (PLNR)-like Wilms tumors lack these heterologous elements and are often associated with PLNRs that arise at the periphery of the renal lobe later in fetal development (25).
We hypothesized that, if LOI of IGF2 is important in Wilms tumorigenesis, LOI might distinguish the two subtypes of Wilms tumors, perhaps providing molecular insight into the pathogenesis of this malignancy. We, therefore, examined the relationship between LOI and the pathologic subtype and stage of Wilms tumor and the patient's age at diagnosis. We also addressed the relationship between LOI in cancer and the abnormal expression of IGF2 and other imprinted genes in the same chromosomal region, including p57KIP2, that might also be affected by LOI, by quantifying and comparing their expression in tumors with LOI with that in tumors with normal imprinting.
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MATERIALS AND METHODS |
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We obtained snap-frozen surgical specimens of 99 Wilms tumors and matched normal kidneys from the same patients from the National Wilms Tumor Study tissue bank (Alberta, Canada) and the Cooperative Human Tissue Network (Columbus, OH). Pathology reports and information on Wilms tumor stage and the patient's age at diagnosis, but no patient identifiers, were provided with the tissue specimens. The Study pathologist (E. J. Perlman) examined all of the tissue samples to verify their identity and homogeneity. PLNR lesions were isolated by microdissecting matched normal kidney tissue.
Homogenized tissue samples were digested overnight at 55 °C with 10 mg/mL proteinase K, and genomic DNA was isolated from them with the use of the Puregene DNA Isolation Kit (Gentra Systems, Research Triangle Park, NC) according to the manufacturer's instructions and resuspended in 10 mM TrisHCl and 1 mM EDTA (pH 8.0). Total RNA was isolated from homogenized tissue samples with the use of the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions and resuspended in 50 µL ribonuclease (RNase)-free water. Twenty-four microliters of RNA was digested with 3 U of Amplification Grade Deoxyribonuclease I (Life Technologies, Inc. [GIBCO BRL], Rockville, MD) in 30 µL according to the manufacturer's instructions. We synthesized complementary DNA (cDNA) in a 40-µL reaction volume that included 5 µL of deoxyribonuclease (DNase) I-digested RNA and 200 ng of random hexamers and either contained or lacked 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) for 2 hours at 37 °C. cDNA was diluted to a final volume of 200 µL with DNase-free and RNase-free water.
Quantitation of Allele-Specific IGF2 RNA Expression to Determine Imprinting Status
We determined the imprinting status of IGF2 by measuring the relative abundance of the two alleles of IGF2 with the use of cDNA derived from Wilms tumor and normal kidney specimens and the Hot-Stop polymerase chain reaction (PCR) method followed by ApaI digestion (27). We amplified 1 µL of each cDNA in a 10-µL PCR reaction that contained 1 pmol each of the unlabeled oligonucleotide primers P3 (5'-CTTGGACTTTGAGTCAAATTGG-3') and P4 (5'-GGTCGTGCCAATTACATTTCA-3'), which are complementary to exon 9 of IGF2, 0.4 µL of the deoxynucleoside triphosphates (2.5 mM each of deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate), 0.6 µL of MgCl2, 1 µL of 10x PCR Buffer (Applied Biosystems, Foster City, CA), and 0.5 U AmpliTaq Polymerase (Applied Biosystems). PCR cycle conditions were as follows: denaturation for 2 minutes at 94 °C and 35 amplification cycles of 94 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds, followed by a final elongation step of 10 minutes at 72 °C. An additional primer extension cycle was performed with the use of 1 pmol of 32P end-labeled oligonucleotide primer P4. We end-labeled 20 pmol of oligonucleotide primer P4 in a 20-µL reaction mixture that contained 4 µL of 5x Forward Reaction Buffer (Invitrogen Corp., Carlsbad, CA), 10 U of T4 polynucleotide kinase (Life Technologies, Inc.), and 25 µCi [-32P]adenosine triphosphate (Amersham Pharmacia, Piscataway, NJ) for 10 minutes at 37 °C and then heat-inactivated the kinase by incubating the reaction mixture for 10 minutes at 70 °C. The PCR products were then digested at 37 °C overnight in a 10-µL volume that contained 1 µL of PCR product, 0.5 µL of 10x ApaI digestion buffer, and 10 U of ApaI (Stratagene, La Jolla, CA). We combined 5 µL of ApaI-digested PCR product with an equal amount of Gel Loading Buffer II (Ambion, Austin, TX), incubated the mixture for 5 minutes at 95 °C, and resolved the DNAs on a 6% acrylamide8M urea gel for 1.5 hours at 75 W. Labeled PCR products were visualized by autoradiography and quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Loss of Heterozygosity Analysis of Wilms Tumors
Loss of heterozygosity (LOH) for 11p was determined by a comparison of tumor and normal DNA at the same ApaI polymorphism used for IGF2 analysis. Samples that were not informative for this marker were examined with the use of the microsatellite marker D11S922 (AFM217yb10, UniSTS). Genomic DNA (100 ng) was PCR amplified including a fluorescently labeled primer. PCR products were separated on an ABI Prism 377 Sequencer and analyzed with Genotyper Software (Applied Biosystems).
Mutational Analysis of the WT1 Gene
The coding region of WT1 that encompasses exons 1 through 10 was amplified in two overlapping fragments by reverse transcription (RT)PCR with the use of 50 ng of cDNA as a template and two sets of oligonucleotide primer pairs. Primers 2F (5'-CCAGTTCACTGGCACAGCCGGAGCCTGTCG-3') and 2R (5'-GTACCCTGTGCTGTGGTTGCTCTGCCCTTC-3') were used to produce WT1 fragment 2, which contains the transactivation domain; primers 3F (5'-GCCACCTTAAAGGGAGTTGCTGCTGGGAGC-3') and 3R (5'-CTTGGAAGTTGGATGAAGAAGATCAACTG-3') were used to produce WT1 fragment 3, which contains the zinc finger domain. The following conditions were used for PCR amplification: 95 °C for 2 minutes, followed by 35 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 1 minute. Because of its high GC content, exon 1 of WT1 was amplified from genomic DNA in a PCR reaction mixture that included 10% dimethyl sulfoxide and oligonucleotide primers 1F (5'-GTGCCTACAGCAGCCAGAGCAGCAGGGA-GTC-3') and 1R (5'-GATTGCGAATAGCGGGCTGGCTCTCGAGG-3'). Amplification conditions were 95 °C for 2 minutes, followed by 35 cycles of 95 °C for 30 seconds, 57 °C for 30 seconds, and 72 °C for 45 seconds.
PCR products were resolved by gel electrophoresis, eluted from the gel, and sequenced with the use of BigDye Terminator Cycle sequencing chemistry (Applied Biosystems) on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Fragment 1 was sequenced with the use of primers 1F and 1R, fragment 2 was sequenced with the use of primers 2F and 2R, and fragment 3 was sequenced with the use of primers 3F, 3R, and 3aR (5'-GTATGAGTCCTG-GTGTGGGTCTTCAGGTGG-3').
Real-Time Quantitative PCR Analysis to Measure Gene Expression
Real-time quantitative PCR was performed in triplicate in 96-well plates; each reaction consisted of 12.5 µL of Taqman Master Mix (Applied Biosystems), 2.5 µL each of 900 nM forward and reverse primers, 2.5 µL of 200 nM Taqman probe, and 5 µL of cDNA. Sequences of the primers and probes that were designed to span and detect intronexon boundaries of IGF2 were as follows: 5'-CCCCTCCGACCGTGCT-3' (forward primer), 5'-TCATATTGGAAGAACTTGCCCA-3' (reverse primer), and VIC-5'-CCGGACAACTTCCCCGAGATACCC-3'-TAMRA (probe). The primers and probe for -actin were purchased from Applied Biosystems. The sequences of the primers and probes used to amplify and detect expression of p57KIP2, WT1, TSSC3, TSSC4, TSSC5, TSSC6, and TAPA1 are provided in supplementary Table 1
at the Journal's Web site (http://jnci.oup-journals.org). Each 96-well real-time quantitative PCR plate included serial dilutions of fetal kidney cDNA, which were used to generate a standard curve for gene expression. The level of IGF2 gene expression was normalized to that of
-actin, after it was demonstrated that there was no statistically significant difference in the expression of IGF2 compared with 10 other housekeeping genes assayed in the multiendogenous control plate (Applied Biosystems) in any of the tissues examined. Expression analysis was performed on an Applied Biosystems Prism 7700 Sequence Detection System. We confirmed reproducibility by comparing the experimental results obtained by two investigators several weeks apart (r = .97).
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Statistical significances of differences were assessed by two-sided Fisher's exact tests (for proportions) or permutation tests with the t statistic (for continuous variables). In the permutation tests [see (28)], we shuffled (randomized) group status relative to the quantitative variable and calculated a two-sample t statistic. This procedure was repeated for a total of 10 000 replicates. The cited P value was the proportion of the replicates for which the absolute value of the t statistic based on randomized data exceeded the absolute value of the observed t statistic. We used permutation tests to avoid assumptions of normality. However, we calculated confidence intervals (CIs) by using the normal assumptions; this inconsistency is not a concern, because the P values obtained by the permutation tests were virtually indistinguishable from those calculated via the t distribution. All statistical tests were two-sided and were performed with the statistical software R (52), version 1.3.1 (see: http://www.R-project.org).
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RESULTS |
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To analyze imprinting of IGF2, we first identified 60 Wilms tumors that could be classified as ILNR-like or PLNR-like. Of these, 36 tumors were heterozygous for a transcribed ApaI polymorphism (1) in IGF2, allowing us to distinguish between the two alleles of the gene and subsequently to determine their imprinting status. Using a quantitative PCR assay (27), we analyzed imprinting of these 36 tumors. Tumors with normal imprinting (n = 21) showed essentially monoallelic expression of IGF2 (i.e., a >5 : 1 ratio of either of the two alleles), whereas tumors with LOI (n = 15) showed equal biallelic expression of the two alleles of IGF2. The tumors also underwent pathologic evaluation by the chief pathologist for the National Wilms Tumor Study (E. J. Perlman). The tumors were scored as objectively as possible, with the use of a double-blinded analysis and conservative criteria (i.e., presence or absence of heterologous elements and presence or absence of nephrogenic rests) to classify them as ILNR-like or PNLR-like. Currently, only approximately 60% of Wilms tumors can be classified into these two groups with the use of conventional histologic approaches; thus, a molecular tool that could improve this rate of classification would be of great value clinically. We found that 25 of the 36 tumors informative for IGF2 imprinting could be classified as being either ILNR-like or PLNR-like. It was remarkable that there was virtually complete segregation of ILNR-like and PLNR-like tumors, depending on the imprinting status, with nine (90%) of 10 PLNR-like tumors showing LOI but only one (6.7%) of 15 ILNR-like tumors showing LOI (Fig. 1). Examples of ILNR-like tumors with normal imprinting and PLNR-like tumors with LOI are shown in Fig. 1
. This difference in the IGF2-imprinting status between these two pathologic subtypes of Wilms tumors was statistically significant (P<.001; Fisher's exact test), indicating a fundamental biologic difference between Wilms tumors with LOI of IGF2 and those with normal imprinting. LOI was not associated with the stage of Wilms tumor and, thus, was not related to tumor progression.
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To determine whether there was overlap between those tumors with abnormal imprinting and those with WT1 mutations, we also examined Wilms tumors directly for the presence of specific inactivating mutations in WT1. We found that only one (6%) of 16 tumors had such a mutation (data not shown). This frequency of mutated WT1 in patients with Wilms tumors is consistent with that reported in a previous study (34). The WT1 mutation that we identified was a tandem duplication of 46 nucleotides inserted within the stop codon, and it resulted in a codon frameshift that would be expected to generate a WT1-like protein containing 83 additional amino acids at the C-terminus. This mutation occurred in an ILNR-like tumor that displayed LOH of 11p. Thus, there was virtually complete segregation between, on the one hand, PLNR-like tumors and LOI and, on the other hand, ILNR-like tumors with normal imprinting, LOH, and WT1 mutations.
Relationship Between LOI and Gene Expression in Wilms Tumors
Despite extensive evidence that LOI occurs in a variety of cancers, it has been difficult to directly link altered expression of specific genes to LOI because of problems related to the quantification of relative levels of specific messenger RNAs (mRNAs). We, therefore, designed real-time quantitative PCR assays to quantify the mRNA expression levels of eight genes that have been directly or indirectly linked to Wilms tumor in samples of Wilms tumor. We assayed the expression of the following genes: IGF2, WT1, p57KIP2, TSSC3, TSSC4, TSSC5, TSSC6, and TAPA1. p57KIP2, which encodes a cyclin-dependent kinase inhibitor, is rarely mutated in BeckwithWiedemann syndrome (35,36), a genetic disorder that predisposes individuals to Wilms tumor. TSSC3, TSSC4, TSSC5, TSSC6, and TAPA1 are genes that lie within a region of chromosome 11, 11p15, that has been shown by microcell-mediated gene transfer to harbor a tumor suppressor gene (37). Previous studies (6,8,38,39) have not demonstrated a statistically significant difference in the expression of any of these genes in Wilms tumor or other cancers in a comparison of tumors with and without LOI of IGF2. However, those studies used techniques, such as conventional semiquantitative RTPCR, northern blots, and RNase protection assays, that lack the sensitivity of real-time quantitative PCR (40) and also failed to link changes in IGF2 expression with imprinting status in Wilms tumors (38,39).
We examined 28 Wilms tumor samples that were informative for the IGF2 ApaI polymorphism, i.e., in which we could determine the imprinting status of IGF2, and found a 2.2-fold (95% confidence interval [CI] = 1.6-fold to 3.1-fold) increase in IGF2 expression in tumors with LOI compared with those with normal imprinting of IGF2 (Table 1). Whereas the magnitude of the difference in IGF2 expression was small, it was both statistically significant (P<.001) and consistent with the activation of the normally silent allele in tumors with LOI. None of the other genes showed a statistically significant difference in expression in tumors with LOI compared with those with normal imprinting of IGF2 (Table 1
), suggesting that the alteration in IGF2 expression is specific for Wilms tumors with LOI and indicating a second fundamental difference in the biology of Wilms tumors with and without LOI.
Relationship Between LOI and Patient's Age at Diagnosis of Wilms Tumor
Patients with Wilms tumor show a bimodal distribution in the age of onset of their tumors, but this has not been fully explained previously. We found a striking difference between the median age at diagnosis of patients who had Wilms tumors with LOI of IGF2 and the median age of patients who had Wilms tumors with normal imprinting. Patients whose tumors showed LOI had a median age at diagnosis (interquartile range [IQR]) of 65 months (4783 months), which was greater than twice that of patients whose tumors showed normal imprinting, who had a median age at diagnosis (IQR) of 24 months (1335 months). This difference in median age of occurrence was statistically significant (P<.001), and it indicates a third fundamental difference in the biology of Wilms tumors in these two groups of patients with and without LOI in their tumors. The relationship of LOI to age was not related to tumor progression, since it was independent of the stage of Wilms tumor (data not shown).
Relationship Between LOI and Nephrogenic Rests
Because LOI appears to distinguish Wilms tumors with PLNR-like pathology from those with ILNR-like pathology, we hypothesized that LOI might be present in the precursor lesions (i.e., the PLNRs themselves). To test this hypothesis, we microdissected normal tissue from a single kidney from a patient with Wilms tumor and identified two PLNRs, both of which were informative for the ApaI polymorphism in IGF2. Both of these rests showed LOI of IGF2 (Fig. 1), confirming that LOI is an early event in Wilms tumorigenesis, in that it occurs in the premalignant lesions themselves.
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DISCUSSION |
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Fig. 2 shows a molecular model for the divergent pathogenesis of Wilms tumor that incorporates the present discovery and results from earlier studies of the molecular biology and pathology of this cancer (41,42). According to this model, WT1 mutations, decreased expression of WT1, or other classic tumor suppressor gene alterations associated with LOH interfere with the maturation of immature nephroblasts at the interior of the developing renal lobe, leading to ILNR-like Wilms tumors. In contrast, LOI in mature nephroblasts leads to the increased expression of IGF2, a failure of proliferation arrest, and the development of PLNR-like tumors at the periphery of the renal lobe. Here, we show for the first time a clear relationship between LOI and IGF2 overexpression. IGF2 is a mitogen that is normally expressed during the proliferative stage of kidney development, in the periphery of the developing renal lobe, where PLNR-like tumors arise. Furthermore, the regions within the kidney that have increased IGF2 expression are those that show increased proliferation in patients with BeckwithWiedemann syndrome and LOI of IGF2 (45).
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These results also support a somewhat provocative view of Breslow and colleagues (4648), who analyzed the two-hit kinetics first proposed by Knudson and Strong (26) in 1972 to explain the bimodal age distribution of patients who develop Wilms tumor.
According to Knudson's model, bilateral Wilms tumors arise in younger individuals than do unilateral tumors because individuals with bilateral tumors have a germline mutation of a tumor suppressor gene and those with unilateral tumors have no such germline mutation (26,29). While that is true, the model does not account for the fact that bilateral Wilms tumors are uncommon [7% incidence overall (46) and only one bilateral case among our study subjects] and it does not explain the observed bimodal age distribution of an unselected group of Wilms tumor patients in the general population, i.e., those with primarily unilateral tumors. Our results support the somewhat provocative study of Breslow and colleagues (46,48) that analyzed Knudson's model. Breslow et al. (47) observed that patients with PLNRs develop tumors at a later age than those with ILNRs. Given that rest-associated tumors constitute a minority of those for which we had imprinting data and that such tumors did not alone account for the bimodal age distribution, we suggest that distinguishing between tumors with or without LOI validates observations by Breslow et al. and provides a biologic foundation for them.
In conclusion, this study demonstrates a striking relationship between LOI and Wilms tumor biology that may distinguish the tumors by their pathology, the premalignant lesions associated with them, and the epidemiology and the genetics of Wilms tumors. Finally, because only approximately 60% of Wilms tumors can presently be classified into ILNR-like or PLNR-like subtypes by histologic examination, this study should provide a valuable molecular tool for Wilms tumor evaluation, similar to that provided by the molecular studies of B-cell lymphoma (49).
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NOTES |
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We thank Hengmi Cui (The Johns Hopkins University, Baltimore, MD) for helpful discussion and Tracy Litzi (The Johns Hopkins University) for technical assistance.
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Manuscript received September 7, 2001; revised October 12, 2001; accepted October 15, 2001.
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