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Keywords:

  • urinary bladder;
  • neoplasms;
  • urothelial carcinoma;
  • laser-capture microdissection;
  • carcinogenesis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

Urothelial carcinoma commonly manifested loss of heterozygosity (LOH) at different regions of chromosomes 17p, 3p, and 9q. Recent studies suggested that bladder stromal cells may be implicated in the growth and progression of urothelial carcinoma. To better understand the genetic alterations in the stromal cells in patients with bladder carcinoma, the authors evaluated the prevalence of allelic loss at three microsatellite polymorphic markers on chromosomes 17p13 (TP53), 3p25-26 (D3S3050), and 9q32-33 (D9S177). In addition, the pattern of X-chromosome inactivation of the stromal cells was evaluated by analyzing the DNA methylation pattern at a polymorphic site on the androgen receptor gene.

METHODS

The authors studied 18 female patients who underwent either transurethral resection (n = 2) or radical cystectomy (n = 16) for high-grade muscle-invasive urothelial carcinoma of the urinary bladder. Genomic DNA samples from the stromal cells immediately adjacent to the tumor and the tumor itself were prepared from formalin-fixed, paraffin-processed tissues using laser-assisted microdissection and LOH was determined.

RESULTS

The stromal cells showed a high frequency of LOH on chromosomes 17p13 (29%), 3p25-26 (61%), and 9q32-33 (47%) with no clear concordance with the adjacent tumor cells. Fourteen specimens (78%) showed LOH in the stroma in at least 1 of 3 markers examined. Nonrandom X-chromosome inactivation was frequent in the stromal cells (50% of informative specimens).

CONCLUSIONS

The current study revealed that some of the genetic changes that commonly occur with invasive urothelial carcinoma were frequently found in the adjacent stroma and suggested that the stroma of urothelial carcinoma may play an important role in bladder carcinogenesis. Cancer 2003. © 2003 American Cancer Society.

Human bladder carcinoma is believed to arise from a field change that affects the entire urothelium.1, 2 Recent studies investigating the genetic basis of bladder carcinoma have focused on the occurrence of loss of heterozygosity (LOH) at key loci.3–6 The extent of allelic loss has been found to vary with the different grades and stages of cancer, supporting the notion that tumor suppressor genes are inactivated during bladder carcinogenesis and stepwise cancer progression.3–5 It is important to note that recent reports suggested that these genetic changes may not be isolated to the epithelium of various carcinomas, but may also occur in the adjacent stromal cells.7–13 Epithelial–stromal interactions are known to be central to the development, growth, and cellular differentiation of the urinary bladder.14 Indeed, most of the intercellular substances and extracellular matrix molecules that are required for tumor growth and progression are produced by the stromal cells.15 Thus, attention has been focused on the role of bladder stromal cells in tumorigenesis as these cells may be essential to the growth and progression of urothelial carcinoma.16, 17

To better understand the role of stromal cells in bladder carcinogenesis, we examined the prevalence of allelic loss at three microsatellite polymorphic markers on chromosomes 3p25-26 (D3S3050), 9q32-33 (D9S177), and 17p13 (TP53) in both the stroma surrounding the bladder carcinoma and corresponding tumors. In addition, the X-chromosome inactivation status of the stromal cells was examined by analyzing the DNA methylation pattern at a polymorphic site on the androgen receptor gene.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients

Eighteen women with urothelial carcinoma of the bladder underwent either transurethral resection (n = 2) or radical cystectomy (n = 16) at the Indiana University School of Medicine (Indianapolis, IN) and Case Western Reserve University (Cleveland, OH) from 1990 to 1998. All samples were procured after obtaining signed informed consent in accordance with the Institutional Committee for the Protection of Human Subjects. Patients had a mean age of 67 years (range, 32–83 years). Histologic grading was performed according to the 1973 World Health Organization (WHO) classification.18 All patients had 1973 WHO Grade 3 (high-grade) urothelial carcinoma. Pathologic staging was performed according to the TNM system.19 The pathologic stages are shown in Table 1. Four patients had T1 disease, five patients had T2 disease, eight patients had T3 disease, and one patient had T4 disease.

Table 1. Results of Loss of Heterozygosity and X-Chromosome Inactivation Analysis in 18 Female Patients with Bladder Carcinoma
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Tumor Samples and Microdissection

Histologic sections were prepared from formalin-fixed, paraffin-embedded blocks and stained with hematoxylin and eosin for histopathologic review. Tumor cells and stroma immediately adjacent to the tumor were microdissected (Fig. 1) using a laser microdissection protocol.12, 20, 21 Approximately 400–600 cells were microdissected from 5-μm histologic sections. Lymphoid or normal muscle tissues microdissected from the same specimen were used as control samples for each patient.

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Figure 1. Precise laser-assisted microdissection of bladder stroma from an area adjacent to the urothelial tumor. (A) Before microdissection. (B) After microdissection. (C) Laser-captured stroma cells.

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Detection of LOH

Polymerase chain reaction (PCR) and gel electrophoresis were performed as previously described22 on sequences containing highly polymorphic microsatellite repeat motifs at loci of interest on chromosomes 17p13 (TP53 locus), 3p25-26 (D3S3050 locus), and 9q32-33 (D9S177 locus). The criterion for allelic loss was complete or nearly complete absence of one allele in the stromal or tumor DNA, detected by direct visualization.23, 24 Results were reported as noninformative (NI) when visual inspection could not distinguish two distinct band forms in control DNA after PCR amplification. PCR reactions for each polymorphic microsatellite marker were repeated at least twice, and the same results were obtained.

Clonal Analysis Using X-Chromosome Inactivation

The microdissected tissue sample was placed in 15 μL of buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid, 1% Tween 20, and 0.2 mg/mL proteinase K [pH 8.3]) and incubated overnight at 37 °C for DNA extraction.25, 26 Eight-microliter aliquots of the DNA extract were digested overnight with 1 U of HhaI restriction endonuclease (New England Biolabs, Beverly, MA) in a total volume of 10 μL. Control reactions for each sample were incubated in the digestion buffer without HhaI endonuclease. Primers used in this reaction were: AR-sense: 5′TCC AGA ATC TGT TCC AGA GCG TGC3′ and AR-antisense: 5′GCT GTG AAG GTT GCT GTT CCT CAT3′.25, 26 PCR and gel electrophoresis were performed as described.25, 26

The clonality of the samples was evaluated on the basis of a polymorphism of the X-linked human androgen receptor gene (HUMARA) locus.25–28 This technique is dependent on digestion of DNA with the methylation-sensitive restriction enzyme, HhaI, PCR amplification of the HUMARA locus, and the detection of methylation at this locus. With this method, only the methylated HUMARA allele is amplified by PCR. The random inactive status of an X chromosome is established in all female somatic cells early in embryogenesis.29 Normal female tissue should be a cellular mosaic, with an equal distribution of cells containing maternal or paternal-derived inactivated X chromosomes. Nonrandom X-chromosome inactivation indicates a clonal process.27, 30

The cases were considered informative if the control sample displayed two alleles after PCR amplification without HhaI digestion. Nonrandom inactivation of the X chromosomes was defined as a complete or nearly complete absence of one or the other allele after HhaI digestion, indicating the predominance of one androgen receptor allele.25, 26

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

We analyzed the allelic loss and X-chromosome inactivation status in the stroma adjacent to urothelial carcinoma from 18 female patients with bladder carcinoma. Table 1 summarizes the findings. All patients had high-grade, invasive urothelial carcinoma of the bladder (Table 1). The stromal cells showed a high frequency of LOH on chromosomes 3p25-26 (61%), 9q32-33 (47%), and 17p13 (29%). Fourteen of 18 specimens (78%) showed LOH in the stroma in at least one of the three markers examined (Fig. 2). A high rate of allelic loss was also observed in corresponding tumors on chromosomes 3p25-26 (44%), 9q32-33 (67%), and 17p13 (47%). Comparison of the tumor cells to adjacent stromal cells reveals no clear concordance of patterns of allelic loss for all three loci. Tumor and stroma showed different patterns of allelic loss (Table 1).

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Figure 2. Representative results of allelic imbalance in the stroma adjacent to urothelial carcinoma of the bladder. N: normal control tissue specimen from the same patient; S: bladder stroma adjacent to urothelial carcinoma. The arrowheads indicate loss of either the upper or lower allele. Note the bottom three images, which highlight the nonrandom pattern of X-chromosome inactivation in the stromal cells. U: without HhaI digestion; D: with HhaI digestion.

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In 6 of 18 patients, the pattern of X-chromosome inactivation was non-informative in the stroma. Of the 12 informative patients, nonrandom X-chromosome inactivation was detected in the stroma in 50% of the patients.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

We studied the LOH in the stromal cells adjacent to high-grade, invasive urothelial carcinoma and corresponding tumors in 18 patients using laser-assisted tissue microdissection. We selected three polymorphic markers on chromosomes 3p25-26, 9q32-33, and 17p13 that are frequently deleted in bladder carcinoma16, 17, 31–34 and performed laser-assisted microdissection to avoid contamination from nearby carcinoma cells. We found high rates of LOH and nonrandom X-chromosome inactivation, indicating that the stroma of urothelial carcinoma may, in fact, be neoplastic and clonal rather than a reactive response to the carcinoma. In addition, comparison of the patterns of allelic loss encountered in the stromal and tumor cells revealed no clear concordance, suggesting that the LOH encountered in the adjacent stroma may be occurring independently of the genetic alterations in the tumor itself.

The presence of allelic imbalance in the adjacent normal-appearing tissue sections (including stromal cells) of epithelial tumors has been previously reported.16, 17, 35 Forsti et al.17 reported the results of LOH analysis of the adjacent, microscopically normal tissue samples from six patients with bladder carcinoma and concluded that the histologically normal urothelium adjacent to urothelial carcinoma already harbors genetic changes typical of bladder carcinoma. Similarly, Muto et al.16 examined the LOH at 10 microsatellite markers, in addition to methylation of the promotor region of the p16 tumor suppressor gene, in both multiple bladder tumors and morphologically normal urothelium from six patients with bladder carcinoma. They reported the high occurrence (77%) of either LOH or methylation in “normal” urothelium, supporting the “field effect” theory of bladder carcinogenesis.

Although the results of LOH analysis from three loci in our study did not demonstrate clear concordance of the pattern of allelic loss noted in the tumor and adjacent stroma, other researchers have reported the common occurrence of genetic changes in the surrounding normal epithelium similar to those found in the nearby carcinoma. Califano et al.36 demonstrated that areas of apparently benign mucosa in head and neck squamous cell carcinoma adjacent to premalignant lesions show LOH typical of squamous cell carcinoma, suggesting that these tissue sections are all derived from a common single progenitor cell. Similar findings (allelic loss on chromosomes 3p and 9p) have been reported in the tissue sections adjacent to lung carcinoma.37, 38

The interplay of neoplastic epithelium and the adjacent stroma has been described extensively in breast carcinoma. Moinfar et al.10 investigated LOH at 12 polymorphic DNA markers (chromosomes 2p, 3p, 11q, 16q, and 17q) in the epithelial and stromal cells of patients with either mammary ductal carcinoma in situ (n = 11) or invasive ductal carcinoma (n = 5). The authors reported a high rate of LOH in the morphologically normal stroma close to the tumor. The incidence of LOH ranged from 10% to 67% for ductal carcinoma in situ and from 20% to 75% of informative cases for invasive ductal carcinoma. In addition, analysis of breast tissue specimens from 10 women who underwent bilateral reduction mammoplasty revealed no LOH in either the epithelium or stroma. Moinfar et al.10 postulated that the mammary stroma may play a key role in inducing neoplastic transformation of epithelial cells. Unfortunately, each component of the breast carcinoma was microdissected manually and subject to cross-contamination, weakening the argument made by the authors. Further evidence for the role of the breast stroma in tumorigenesis was reported by Shekhar et al.11 They demonstrated in a novel in vitro model that the fibroblasts isolated from breast stroma adjacent to carcinoma will induce the proliferation of breast epithelial tumor cells whereas fibroblasts isolated from patients undergoing reduction mammoplasty inhibited or retarded the growth of carcinoma cells. These studies suggested an important role of the stroma in the carcinogenesis of epithelial neoplasia.

The role of the stroma in other epithelial tumors also has been studied previously. Macintosh et al.35 demonstrated that in the stroma surrounding foci of prostatic adenocarcinoma, there was a low (three of nine informative samples), but significant, incidence of LOH. The authors postulated that prostate carcinoma may not be simply an isolated epithelial proliferation but rather the result of a complex interrelationship in which stromal cells promote epithelial growth. Similarly, Wernert et al.9 demonstrated by laser-assisted microdissection that genetic alterations occur frequently in the adjacent fibroblastic tumor stroma of patients with nonhereditary invasive colon and breast carcinomas.9

In the current study, we found that a significant percentage of the stromal cells adjacent to invasive urothelial carcinoma harbored genetic changes commonly encountered in invasive urothelial carcinoma, supporting the field effect theory of carcinogenesis. In addition, the high incidence of nonrandom X-chromosome inactivation (50% of informative cases) in the stromal cells suggests a clonal origin of these cells. However, we did not encounter a clear concordance between the LOH pattern encountered in the tumor and its adjacent stroma, suggesting that the genetic changes taking place in the tumor and its adjacent stroma may be occurring independently. This finding is not unique, as other investigators have reported that the LOH identified in the fibrotic tissue surrounding pancreatic carcinoma is distinct from the pattern identified in the adenocarcinoma itself.39, 40 However, caution in interpreting this result is warranted because the laser-assisted microdissection used to obtain stromal cells reduces but does not eliminate the chance that the above finding of genetic alterations in the stromal cells is a result of cross-contamination. In addition, the possibility exists that the abnormal stromal cells may, in fact, represent sarcomatoid transformation of tumor cells that closely resemble bladder stromal cells.

The most consistently informative marker of the clonal composition of neoplastic and preneoplastic disorders in females is the nonrandom pattern of X-chromosome inactivation.25–27, 30 Benign proliferative lesions can be distinguished from neoplasms. Benign lesions are comprised of cells with randomly inactivated X chromosomes, whereas neoplasms originate from a single cell and are, therefore, composed of cells with activation of the same X chromosome.26, 27, 41, 42 The rate of nonrandom X-chromosome inactivation we encountered from the stromal cells in the current study is significant and in keeping with previous findings with other types of tumors,10, 25, 26, 43–45 suggesting a clonal origin of these cells. However, this result must be interpreted with the knowledge that previous investigators have demonstrated that in urothelial samples taken from bladder specimens harboring both benign and neoplastic disease, nonrandom X-chromosome inactivation was found to occur in patches of approximately 120 mm2 in size.46 Thus, samples from the adjacent stroma with the finding of nonrandom X-chromosome inactivation may represent a normally occurring phenomenon and origin from a common progenitor cell in these stromal cells. Finally, although cross-contamination of the specimens could be responsible for this finding, the purity of tissue collection with the laser-assisted microdissection makes this possibility unlikely.

The current study findings reveal that genetic changes that commonly occur with invasive urothelial carcinoma frequently are found in the adjacent stroma and suggest that the stroma of urothelial carcinoma may play an important role in bladder carcinogenesis. The stroma in bladder carcinoma may be neoplastic rather than a reactive response to the carcinoma.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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