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Single-Nucleotide Polymorphism in WT1 Gene in a Hyperplastic Intralobar Nephrogenic Rest With Botryoid Protrusion
Neoplasms Single-Nucleotide Polymorphism in WT1 Gene in a Hyperplastic Intralobar Nephrogenic Rest With Botryoid Protrusion Kentaro Mizuno, Yutaro Hayashi, Keiichi Tozawa, Shoichiro Iwatsuki, Yoshiyuki Kojima, and Kenjiro Kohri Nephrogenic rests are nodular collections of undifferentiated renal blastema cells in the postnatal kidney that are recognized as putative precursor lesions of Wilms tumor. In this study, we report the case of a 3-year-old boy who was diagnosed with a hyperplastic intralobar nephrogenic rest extending through the renal pelvis and ureter. After radical nephrectomy, adjuvant chemotherapy was performed as done in stage II Wilms tumor. We also investigated the mutation of the WT1 gene and identified a related single-nucleotide polymorphism (rs16754). We consider that not only various genetic mutations but also single-nucleotide polymorphisms or other epigenetic factors might be involved in the development of Wilms tumor. UROLOGY 76: 149 –152, 2010. © 2010 Elsevier Inc.
W
ilms tumor (WT) is the most common renal tumor of childhood; it is an embryonal tumor that develops from the remnants of the immature kidney. A nephrogenic rest (NR) is a collection of persistent undifferentiated renal blastema cells in the postnatal kidney that is considered as a precursor lesion of WT.1 We report here a rare case of hyperplastic NR extending through the renal pelvis and ureter, and attempt to investigate the genetic characterization of this lesion according to the mutation analysis of the Wilms tumor suppressor (WT1) gene. To our knowledge, there are no reports that describe the relationship between the development of WT or NR and single-nucleotide polymorphisms (SNPs) in the WT1 gene.
CASE REPORT A 3-year-old boy presented with gross hematuria to the pediatric outpatient clinic in our hospital. He had no abdominal pain or painful micturition. Physical examination revealed a left abdominal mass. No ophthalmic or genitourinary abnormality or psychomotor retardation was observed. Blood and chromosomal analysis showed no abnormalities except for mild anemia. Abdominal ultrasonography revealed a 7-cm mass of mixed echogenicity occupying the left renal pelvis. Computed tomography and magnetic resonance imaging revealed a heterogeneous, contrast-enhanced mass in the dilated collecting system (Fig. 1). There was no
From the Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan Reprint requests: Yutaro Hayashi, M.D., Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467– 8601, Aichi, Japan. E-mail: [email protected] Submitted: June 24, 2009, accepted (with revisions): September 5, 2009
© 2010 Elsevier Inc. All Rights Reserved
evidence of metastasis. WT with protrusion into the renal pelvis was suspected, and radical left nephrectomy was performed. The surface of the left kidney appeared smooth, with no tumor invasion into the surrounding tissues. The left renal pelvis was dilated and filled with an elastic mass. The ureter was divided at the position of the lower pole of the kidney. After the left kidney was removed, a light yellow, polypoid, glistening mass occupying a large part of renal pelvis and extending into the proximal ureter was observed. The tumor was pedunculated and attached to the upper part of the renal pelvic mucosa. No parenchymal mass or renal venous invasion was present (Fig. 2A). The detailed histopathologic examination was performed by individual pathologists. The pedicle of the tumor was attached to the renal parenchyma near the pelvic wall, and the tumor protruded into the renal pelvis (Fig. 2B). The tumor had typical features of WT with epithelial, stromal, and blastermal components. However, the tumor was not covered by a pseudocapsule and no anaplasia was observed (Fig. 2C). There was no evidence that tumor cells existed in the renal parenchyma or the distal ureteral stump. The final pathologic diagnosis was hyperplastic intralobar NR; however, a part of the tumor was not distinguishable from the histology of WT. Therefore, we clinically diagnosed it as stage II WT, and administered adjuvant chemotherapy with actinomycin-D and vincristine according to the regimen of the National Wilms’ Tumor Study Group-5. The patient showed no local recurrences or metastasis, and is making satisfactory progress as was noted in the follow-up on August 2009, that is, 14 months after the surgery. DNA was extracted from leukocytes isolated from the patient’s peripheral blood and tumor tissue using 0090-4295/10/$34.00 doi:10.1016/j.urology.2009.09.012
149
Figure 1. (A) CT (noncontrast, corticomedullary, and nephrographic phase, respectively) and (B) MRI (T2-weighted, T1-weighted, and Gd-enhanced images, respectively) show a heterogeneous, subtly enhancing mass extending through the renal pelvis (arrowhead).
Figure 2. Macroscopic appearance of the tumor (A), and permanent slide preparation (B,C) in hematoxylin-eosin staining. (A) The tumor was pedunculated and attached to the renal pelvis; (B) the pedicle of the tumor was attached to the renal parenchyma near the pelvic wall (arrowhead), and the tumor protruded into the renal pelvis, ⫻1; (C) anaplasia was not observed in most of the tumor, and the final pathologic diagnosis was hyperplastic nephrogenic rest, ⫻200.
the nucleic acid purification system, MagExtractor MFX-2100 (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Amplification of exons 1-10 was performed by polymerase chain reaction (PCR) using specific primers.2-4 After the purification of PCR products using MinElute PCR purification kit (Qiagen, Tokyo, Japan), direct sequencing was performed with ABI PRISM 310 genetic Analyzer (Applied Biosystems, Foster City, CA). DNA sequence analyses indicated a 1252A-G substitution in exon 7, 150
the zinc-finger domain, without an amino acid change (Arg 352) (Fig. 3).
COMMENT We encountered an uncommon case of a hyperplastic intralobar NR with botryoid protrusion and could detect the SNP rs16754 of the WT1 gene in the tumor tissue. Microscopically, NRs are often observed as islet clusters of embryonal cells in the developing kidney; moreover, they are classified as perilobar or intralobar on the basis of UROLOGY 76 (1), 2010
Figure 3. DNA substitution in exon 7 of the WT1 gene (1252A-G). This is the SNP rs16754.
their position within the renal lobe. Morphologic features that reflect growth or regressive changes further characterize both perilobar and intralobar NRs, and hyperplastic NRs can produce a renal mass that can easily be mistaken for a small WT.1 Infrequently, the polypoid expansion of WT into the renal collecting system resembling botryoid sarcoma may occur.5 In the present case, the final pathologic diagnosis was hyperplastic intralobar NR based on the small number of dyskaryotic blastemal cells and the lack of the pseudocapsule; however, a part of the tumor was not distinguishable from the histologic features of WT. The etiology of ureteral extension of WT is still unknown, but intralobar NRs have been noted in nearly 50% of such children;5 this may signify a relationship between intralobar NRs and the development of WT involving the collecting system and the ureter. Since Beckwith et al6 have provided us with a new concept of NR as a precursor lesion of WT, it is recognized that the biological pathways leading to the development of most WTs are complex and probably involve several genetic loci, for example, WT1,7,8 CTNNB1,7,8 and p53.9 The WT1 gene is located on chromosome 11p13, and encodes a transcriptional factor containing a domain of 4 zinc-finger motifs and is thought to function as a classic tumor suppressor gene.10,11 The normal functioning of the WT1 gene is essential to genitourinary development. Recently, genetic and epigenetic alterations of the WT1 gene have been found to be involved in the development of WT,12 and the specific WT1 mutation that correlates with the histology of WT has also been reported.2 However, molecular analysis of WTs revealed that intragenic microdeletions/insertions and point mutations of 1 allele within the WT1 gene are found in only 10%-15% of WTs, suggesting that other alterations must contribute to Wilms’ tumorigenesis.3 In the present case, to clarify the genetic characteristics of UROLOGY 76 (1), 2010
this lesion, we investigated the mutation of the WT1 gene. Consequently, we could detect a 1252A-G substitution in exon 7, that is, SNP rs16754. Because SNP rs16754 is considered a cis-acting putative regulatory SNP,13 it is likely that this SNP affected the regulation of WT1 gene expression. In fact, Orloff et al14 have reported that some SNPs in WT1 genes are significantly associated with focal segmental glomerulosclerosis. Because it is feasible that the abnormal expression of transcriptional factors such as the WT1 gene cause the development of sporadic WT or NR, it is important to clarify not only the mutation but also SNPs of the candidate genes to accurately assess pathology. Recently, Safford et al15 have reported that decreased E-cadherin expression correlates with higher stage of WT, and 11 of 15 high-stage tumors contained intronic SNPs. In future, it is likely that these genetic or epigenetic information influence the policies of treatment or follow-up in patients with WTs. Further studies in a large number of cases of WT are needed to gain more knowledge about the correlation of specific SNPs and tumorigenesis or the styles of tumor expansion.
CONCLUSIONS We report a rare case of hyperplastic NR extending through the renal pelvis and ureter. A part of the tumor was not distinguishable from the histology of WT, and an SNP (rs16754) in the WT1 gene was recognized. NRs are considered as putative precursor lesions of WT, and various genetic mutations might be involved in their development to WT. Further investigations are needed to clarify the relationship between specific SNPs and tumorigenesis. Acknowledgment. The 242nd JUA Tokai Divisional Meeting Best Presentation Award was given for the summary of this case report.
References 1. Beckwith JB. Nephrogenic rests and the pathogenesis of Wilms tumor: developmental and clinical considerations. Am J Med Genet. 1998;79:268-273. 2. Shibata R, Hashiguchi A, Sakamoto J, et al. Correlation between a specific Wilms tumour suppressor gene (WT1) mutation and the histological findings in Wilms tumour (WT). J Med Genet. 2002; 39:e83-e87. 3. Schumacher V, Schneider S, Figge A, et al. Correlation of germline mutations and two-hit inactivation of the WT1 gene with Wilms tumors of stromal-predominant histology. Proc Natl Acad Sci USA. 1997;94:3972-3977. 4. Virappane P, Gale R, Hills R, et al. Mutation of the Wilms’ tumor 1 gene is a poor prognostic factor associated with chemotherapy resistance in normal karyotype acute myeloid leukemia: the United Kingdom Medical Research Council Adult Leukaemia Working party. J Clin Oncol. 2008;26:5429-5435. 5. Ritchey M, Daley S, Shamberger RC, et al. Ureteral extension in Wilms’ tumor: a report from the National Wilms’ Tumor Study Group (NWTSG). J Pediatr Surg. 2008;43:1625-1629. 6. Beckwith JB, Kiviat NB, Bonadio JF. Nephrogenic rests, nephroblastomatosis, and the pathogenesis of Wilms’ tumor. Pediatr Pathol. 1990;10:1-36.
151
7. Royer-Pokora B, Weirich A, Schumacher V, et al. Clinical relevance of mutations in the Wilms tumor suppressor 1 gene WT1 and the cadherin-associated protein beta1 gene CTNNB1 for patients with Wilms tumors: results of long-term surveillance of 71 patients from International Society of Pediatric Oncology Study 9/Society for Pediatric Oncology. Cancer. 2008;113:10801089. 8. Fukuzawa R, Anaka MR, Heathcott RW, et al. Wilms tumour histology is determined by distinct types of precursor lesions and not epigenetic changes. J Pathol. 2008;215:377-387. 9. Malkin D, Sexsmith E, Yeger H, et al. Mutations of the p53 tumor suppressor gene occur infrequently in Wilms’ tumor. Cancer Res. 1994;54:2077-2079. 10. Gessler M, Poustka A, Cavenee W, et al. Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature. 1990;343:774-778.
152
11. Call KM, Glaser T, Ito CY, et al. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell. 1990;60:509-520. 12. Satoh Y, Nakadate H, Nakagawachi T, et al. Genetic and epigenetic alterations on the short arm of chromosome 11 are involved in a majority of sporadic Wilms’ tumours. Br J Cancer. 2006;95: 541-547. 13. Milani L, Gupta M, Andersen M, et al. Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells. Nucleic Acids Res. 2007;35:e34-e43. 14. Orloff MS, Iyengar SK, Winkler CA, et al. Variants in the Wilms’ tumor gene are associated with focal segmental glomerulosclerosis in the African American population. Physiol Genom. 2005;21:212-221. 15. Safford SD, Freemerman AJ, Langdon S, et al. Decreased E-cadherin expression correlates with higher stage of Wilms’ tumors. J Pediatr Surg. 2005;40:341-348.
UROLOGY 76 (1), 2010
W
ilms tumor (WT) is the most common renal tumor of childhood; it is an embryonal tumor that develops from the remnants of the immature kidney. A nephrogenic rest (NR) is a collection of persistent undifferentiated renal blastema cells in the postnatal kidney that is considered as a precursor lesion of WT.1 We report here a rare case of hyperplastic NR extending through the renal pelvis and ureter, and attempt to investigate the genetic characterization of this lesion according to the mutation analysis of the Wilms tumor suppressor (WT1) gene. To our knowledge, there are no reports that describe the relationship between the development of WT or NR and single-nucleotide polymorphisms (SNPs) in the WT1 gene.
CASE REPORT A 3-year-old boy presented with gross hematuria to the pediatric outpatient clinic in our hospital. He had no abdominal pain or painful micturition. Physical examination revealed a left abdominal mass. No ophthalmic or genitourinary abnormality or psychomotor retardation was observed. Blood and chromosomal analysis showed no abnormalities except for mild anemia. Abdominal ultrasonography revealed a 7-cm mass of mixed echogenicity occupying the left renal pelvis. Computed tomography and magnetic resonance imaging revealed a heterogeneous, contrast-enhanced mass in the dilated collecting system (Fig. 1). There was no
From the Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan Reprint requests: Yutaro Hayashi, M.D., Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467– 8601, Aichi, Japan. E-mail: [email protected] Submitted: June 24, 2009, accepted (with revisions): September 5, 2009
© 2010 Elsevier Inc. All Rights Reserved
evidence of metastasis. WT with protrusion into the renal pelvis was suspected, and radical left nephrectomy was performed. The surface of the left kidney appeared smooth, with no tumor invasion into the surrounding tissues. The left renal pelvis was dilated and filled with an elastic mass. The ureter was divided at the position of the lower pole of the kidney. After the left kidney was removed, a light yellow, polypoid, glistening mass occupying a large part of renal pelvis and extending into the proximal ureter was observed. The tumor was pedunculated and attached to the upper part of the renal pelvic mucosa. No parenchymal mass or renal venous invasion was present (Fig. 2A). The detailed histopathologic examination was performed by individual pathologists. The pedicle of the tumor was attached to the renal parenchyma near the pelvic wall, and the tumor protruded into the renal pelvis (Fig. 2B). The tumor had typical features of WT with epithelial, stromal, and blastermal components. However, the tumor was not covered by a pseudocapsule and no anaplasia was observed (Fig. 2C). There was no evidence that tumor cells existed in the renal parenchyma or the distal ureteral stump. The final pathologic diagnosis was hyperplastic intralobar NR; however, a part of the tumor was not distinguishable from the histology of WT. Therefore, we clinically diagnosed it as stage II WT, and administered adjuvant chemotherapy with actinomycin-D and vincristine according to the regimen of the National Wilms’ Tumor Study Group-5. The patient showed no local recurrences or metastasis, and is making satisfactory progress as was noted in the follow-up on August 2009, that is, 14 months after the surgery. DNA was extracted from leukocytes isolated from the patient’s peripheral blood and tumor tissue using 0090-4295/10/$34.00 doi:10.1016/j.urology.2009.09.012
149
Figure 1. (A) CT (noncontrast, corticomedullary, and nephrographic phase, respectively) and (B) MRI (T2-weighted, T1-weighted, and Gd-enhanced images, respectively) show a heterogeneous, subtly enhancing mass extending through the renal pelvis (arrowhead).
Figure 2. Macroscopic appearance of the tumor (A), and permanent slide preparation (B,C) in hematoxylin-eosin staining. (A) The tumor was pedunculated and attached to the renal pelvis; (B) the pedicle of the tumor was attached to the renal parenchyma near the pelvic wall (arrowhead), and the tumor protruded into the renal pelvis, ⫻1; (C) anaplasia was not observed in most of the tumor, and the final pathologic diagnosis was hyperplastic nephrogenic rest, ⫻200.
the nucleic acid purification system, MagExtractor MFX-2100 (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Amplification of exons 1-10 was performed by polymerase chain reaction (PCR) using specific primers.2-4 After the purification of PCR products using MinElute PCR purification kit (Qiagen, Tokyo, Japan), direct sequencing was performed with ABI PRISM 310 genetic Analyzer (Applied Biosystems, Foster City, CA). DNA sequence analyses indicated a 1252A-G substitution in exon 7, 150
the zinc-finger domain, without an amino acid change (Arg 352) (Fig. 3).
COMMENT We encountered an uncommon case of a hyperplastic intralobar NR with botryoid protrusion and could detect the SNP rs16754 of the WT1 gene in the tumor tissue. Microscopically, NRs are often observed as islet clusters of embryonal cells in the developing kidney; moreover, they are classified as perilobar or intralobar on the basis of UROLOGY 76 (1), 2010
Figure 3. DNA substitution in exon 7 of the WT1 gene (1252A-G). This is the SNP rs16754.
their position within the renal lobe. Morphologic features that reflect growth or regressive changes further characterize both perilobar and intralobar NRs, and hyperplastic NRs can produce a renal mass that can easily be mistaken for a small WT.1 Infrequently, the polypoid expansion of WT into the renal collecting system resembling botryoid sarcoma may occur.5 In the present case, the final pathologic diagnosis was hyperplastic intralobar NR based on the small number of dyskaryotic blastemal cells and the lack of the pseudocapsule; however, a part of the tumor was not distinguishable from the histologic features of WT. The etiology of ureteral extension of WT is still unknown, but intralobar NRs have been noted in nearly 50% of such children;5 this may signify a relationship between intralobar NRs and the development of WT involving the collecting system and the ureter. Since Beckwith et al6 have provided us with a new concept of NR as a precursor lesion of WT, it is recognized that the biological pathways leading to the development of most WTs are complex and probably involve several genetic loci, for example, WT1,7,8 CTNNB1,7,8 and p53.9 The WT1 gene is located on chromosome 11p13, and encodes a transcriptional factor containing a domain of 4 zinc-finger motifs and is thought to function as a classic tumor suppressor gene.10,11 The normal functioning of the WT1 gene is essential to genitourinary development. Recently, genetic and epigenetic alterations of the WT1 gene have been found to be involved in the development of WT,12 and the specific WT1 mutation that correlates with the histology of WT has also been reported.2 However, molecular analysis of WTs revealed that intragenic microdeletions/insertions and point mutations of 1 allele within the WT1 gene are found in only 10%-15% of WTs, suggesting that other alterations must contribute to Wilms’ tumorigenesis.3 In the present case, to clarify the genetic characteristics of UROLOGY 76 (1), 2010
this lesion, we investigated the mutation of the WT1 gene. Consequently, we could detect a 1252A-G substitution in exon 7, that is, SNP rs16754. Because SNP rs16754 is considered a cis-acting putative regulatory SNP,13 it is likely that this SNP affected the regulation of WT1 gene expression. In fact, Orloff et al14 have reported that some SNPs in WT1 genes are significantly associated with focal segmental glomerulosclerosis. Because it is feasible that the abnormal expression of transcriptional factors such as the WT1 gene cause the development of sporadic WT or NR, it is important to clarify not only the mutation but also SNPs of the candidate genes to accurately assess pathology. Recently, Safford et al15 have reported that decreased E-cadherin expression correlates with higher stage of WT, and 11 of 15 high-stage tumors contained intronic SNPs. In future, it is likely that these genetic or epigenetic information influence the policies of treatment or follow-up in patients with WTs. Further studies in a large number of cases of WT are needed to gain more knowledge about the correlation of specific SNPs and tumorigenesis or the styles of tumor expansion.
CONCLUSIONS We report a rare case of hyperplastic NR extending through the renal pelvis and ureter. A part of the tumor was not distinguishable from the histology of WT, and an SNP (rs16754) in the WT1 gene was recognized. NRs are considered as putative precursor lesions of WT, and various genetic mutations might be involved in their development to WT. Further investigations are needed to clarify the relationship between specific SNPs and tumorigenesis. Acknowledgment. The 242nd JUA Tokai Divisional Meeting Best Presentation Award was given for the summary of this case report.
References 1. Beckwith JB. Nephrogenic rests and the pathogenesis of Wilms tumor: developmental and clinical considerations. Am J Med Genet. 1998;79:268-273. 2. Shibata R, Hashiguchi A, Sakamoto J, et al. Correlation between a specific Wilms tumour suppressor gene (WT1) mutation and the histological findings in Wilms tumour (WT). J Med Genet. 2002; 39:e83-e87. 3. Schumacher V, Schneider S, Figge A, et al. Correlation of germline mutations and two-hit inactivation of the WT1 gene with Wilms tumors of stromal-predominant histology. Proc Natl Acad Sci USA. 1997;94:3972-3977. 4. Virappane P, Gale R, Hills R, et al. Mutation of the Wilms’ tumor 1 gene is a poor prognostic factor associated with chemotherapy resistance in normal karyotype acute myeloid leukemia: the United Kingdom Medical Research Council Adult Leukaemia Working party. J Clin Oncol. 2008;26:5429-5435. 5. Ritchey M, Daley S, Shamberger RC, et al. Ureteral extension in Wilms’ tumor: a report from the National Wilms’ Tumor Study Group (NWTSG). J Pediatr Surg. 2008;43:1625-1629. 6. Beckwith JB, Kiviat NB, Bonadio JF. Nephrogenic rests, nephroblastomatosis, and the pathogenesis of Wilms’ tumor. Pediatr Pathol. 1990;10:1-36.
151
7. Royer-Pokora B, Weirich A, Schumacher V, et al. Clinical relevance of mutations in the Wilms tumor suppressor 1 gene WT1 and the cadherin-associated protein beta1 gene CTNNB1 for patients with Wilms tumors: results of long-term surveillance of 71 patients from International Society of Pediatric Oncology Study 9/Society for Pediatric Oncology. Cancer. 2008;113:10801089. 8. Fukuzawa R, Anaka MR, Heathcott RW, et al. Wilms tumour histology is determined by distinct types of precursor lesions and not epigenetic changes. J Pathol. 2008;215:377-387. 9. Malkin D, Sexsmith E, Yeger H, et al. Mutations of the p53 tumor suppressor gene occur infrequently in Wilms’ tumor. Cancer Res. 1994;54:2077-2079. 10. Gessler M, Poustka A, Cavenee W, et al. Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature. 1990;343:774-778.
152
11. Call KM, Glaser T, Ito CY, et al. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell. 1990;60:509-520. 12. Satoh Y, Nakadate H, Nakagawachi T, et al. Genetic and epigenetic alterations on the short arm of chromosome 11 are involved in a majority of sporadic Wilms’ tumours. Br J Cancer. 2006;95: 541-547. 13. Milani L, Gupta M, Andersen M, et al. Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells. Nucleic Acids Res. 2007;35:e34-e43. 14. Orloff MS, Iyengar SK, Winkler CA, et al. Variants in the Wilms’ tumor gene are associated with focal segmental glomerulosclerosis in the African American population. Physiol Genom. 2005;21:212-221. 15. Safford SD, Freemerman AJ, Langdon S, et al. Decreased E-cadherin expression correlates with higher stage of Wilms’ tumors. J Pediatr Surg. 2005;40:341-348.
UROLOGY 76 (1), 2010