Abstract

Context.—Molecular analysis of microsatellite alterations of biologically distinct tumor cell subpopulations from the same patient may aid in the determination of tumor origin and further our understanding of the genetic basis of cancer progression.

Design.—The authors examined the pattern of allelic loss with polymorphic microsatellite markers on chromosome 9p21 (D9S161, D9S171, IFNA), regions of putative tumor suppressor gene p16, and on chromosome 17p13 (TP53), the p53 locus, in matched primary and metastatic bladder cancers from 9 patients. All patients underwent cystectomy for bladder cancer and had regional lymph node metastases at the time of surgery. Genomic DNA was prepared from primary cancers and matched synchronous lymph node metastases using a microdissection method.

Results.—The overall frequency of allelic loss was 78% in primary cancer and 89% in paired metastatic cancer. The frequency of allelic loss in the primary cancer was 86% with D9S161, 67% with D9S171, 71% with IFNA, and 80% with TP53. The frequency of allelic loss in matched metastatic cancer was 100% with D9S161, 62% with D9S171, 71% with IFNA, and 80% with TP53. An identical pattern of allelic imbalance (allelic loss or retention) at multiple DNA loci was observed in matched primary and metastatic carcinoma in 8 (88%) cases. One case showed allelic loss in the metastasis, but not in the primary cancer.

Conclusions.—The pattern of allelic loss at chromosome 9p21 (p16) and 17p13 (p53) was generally maintained during cancer progression to metastasis, and identical allelic loss in primary cancer was conserved in paired metastatic carcinoma. These data suggest that these genetic changes may be useful in establishing a diagnosis and determining tumor origins in difficult cases.

It is estimated that 54 300 new cases of bladder carcinoma will be diagnosed and 12 400 people will die of bladder cancer in the United States in 2001.1 Progressive growth of distant metastases is responsible for the majority of cancer-related deaths. Very little is known about the molecular mechanisms underlying the metastatic progression of bladder cancer. Recent advances in tissue microdissection techniques permit the selective procurement of tumor cell populations from paraffin-embedded archival material for genetic analysis. Molecular analysis of microsatellite alterations of biologically distinct tumor cell subpopulations from the same patient may aid in the determination of tumor origin and further our understanding of the genetic basis of cancer progression.

Chromosome region 9p21 contains a tumor suppressor gene locus p16. p16 protein binds to cyclin-dependent kinase 4 (CDK4) and inhibits its interaction with cyclin D, subsequently leading to cell cycle arrest at the late G1 stage. A high frequency of allelic loss on chromosome 9p21 was observed in bladder cancer, implicating that p16 gene deletion may play an important role in the carcinogenesis and progression of bladder cancer.2,3 Similarly, p53 gene alterations are frequently encountered in different stages of bladder cancer development.4–7 In this study, we analyzed the pattern of allelic loss in these regions in 9 matched primary and metastatic bladder tumors.

MATERIALS AND METHODS

We studied 9 patients who had undergone radical cystectomy and bilateral lymphadenectomy between 1992 and 1995. All patients had regional lymph node metastases at the time of surgery. Patients ranged in age from 51 to 86 years (mean, 66 years). Grading of the primary tumor was performed according to the 1998 World Health Organization/International Society of Urologic Pathology classification.8 The 1997 TNM system was used for pathologic staging.9 Genomic DNA was prepared from primary cancers and matched synchronous lymph node metastases using a microdissection method.10,11 Normal control DNA was prepared from uninvolved lymph nodes in all cases. The following oligonucleotide primer pairs for the microsatellite DNA markers were used: D9S161, D9S171, IFNA, and TP53 (Research Genetics, Huntsville, Ala).12 Polymerase chain reaction amplification and gel electrophoresis were performed as previously described.10,11 The criterion for allelic loss was complete or nearly complete absence of 1 allele in tumor DNA, as defined by direct visualization.10,11 Polymerase chain reactions for each polymorphic microsatellite marker were repeated at least twice from the same DNA preparations, and the same results were obtained.

RESULTS

The frequency of allelic loss in the primary cancer was 86% with D9S161, 67% with D9S171, 71% with IFNA, and 80% with TP53. The frequency of allelic loss in matched metastatic cancer was 100% with D9S161, 62% with D9S171, 71% with IFNA, and 80% with TP53. The overall frequency of allelic loss was 78% in primary cancer and 89% in paired metastatic cancer. An identical pattern of allelic imbalance (allelic loss or retention) at multiple DNA loci was observed in matched primary and metastatic carcinoma in 8 (89%) of 9 cases (Table, Figure). For example, case 5 showed loss of the same allele in primary cancer and matched metastases at all 4 marker loci (D9S161, D9S171, IFNA, and TP53). One case (case 1) showed allelic loss in the metastasis, but not in the primary cancer.

Comparison of Allelic Loss Pattern in Matched Cases of Primary and Metastatic Bladder Cancer From 9 Patients

Comparison of Allelic Loss Pattern in Matched Cases of Primary and Metastatic Bladder Cancer From 9 Patients
Comparison of Allelic Loss Pattern in Matched Cases of Primary and Metastatic Bladder Cancer From 9 Patients

Representative cases (A, case 5; B, case 9) showing concordant loss of heterogeneity results with primers flanking the p16 gene and the p53 gene. Primary tumor (P) and metastasis (M) consistently show loss of the same allele, as compared to normal control tissue (N), with any of the informative markers used. Arrows point to the deleted alleles

Representative cases (A, case 5; B, case 9) showing concordant loss of heterogeneity results with primers flanking the p16 gene and the p53 gene. Primary tumor (P) and metastasis (M) consistently show loss of the same allele, as compared to normal control tissue (N), with any of the informative markers used. Arrows point to the deleted alleles

COMMENT

Detailed characterization and comparison of genetic alterations of biologically distinct tumor cell subpopulations may provide information about progression and clonal evolution of bladder cancer. In this study, we analyzed the pattern of allelic loss with polymorphic microsatellite markers (D9S104, D9S161, D9S171, and IFNA) on chromosome 9p21, which contains putative tumor suppressor gene p16 and on chromosome 17p13 (TP53), which contains p53 gene in matched primary and metastatic bladder tumors from 9 patients. We found an identical pattern of allelic loss in matched primary and metastatic carcinoma from the same patient, suggesting allelic loss of these chromosome regions occurred prior to metastatic progression. The relative constancy of these genetic changes in primary bladder cancer and paired metastatic lesions may aid in diagnosis and identification of tumor origins in difficult cases.

Previously, we compared the pattern of allelic loss in primary tumor and matched synchronous lymph node metastasis from prostate cancer.13 A heterogenous pattern of allelic loss was observed. Forty-two percent of cases showed discordant allelic loss of microsatellite DNA markers on chromosome 8p12-21, 8p22, and 17q21 in primary tumor and matched lymph node metastasis.13 Molecular analysis of whole mount sections from entirely embedded prostate glands have confirmed that separate tumors from the same patient have multiclonal (independent) origin.11,14 However, molecular studies of multiple separate foci of bladder carcinoma suggest a monoclonal origin of the tumor.15 

Miyao et al16 studied 14 patients with lymph node metastases from bladder cancer and found complete concordance between genetic defects in the primary and metastatic sites. Loss of heterogeneity at chromosome 9 was seen in 64% of cases, and loss of heterogeneity at 17p was observed in 78% of cases. However, the pattern of allelic loss (upper or lower allele loss) was not reported.16 Orlow et al2 reported a lower incidence of genetic alterations (18%) at the p16 gene locus in chromosome 9p21 in a large cohort of bladder tumors. The discrepant results among different studies may be attributed to differences in the selection of polymorphic microsatellite markers, tissue microdissection techniques, tumor stage, and patient populations.

Our data indicated that the pattern of allelic loss at chromosome 9p21 (p16) and 17p13 (p53) in primary bladder cancer was generally maintained during cancer progression to metastasis, and an identical pattern of allelic loss in primary cancer was conserved in paired metastatic carcinoma, suggesting that allelic loss of these chromosome regions most likely occurs prior to the metastatic sequence. It is possible that allelic loss at these loci may be used as bladder cancer markers when the primary tumor of metastasis is unknown.

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Author notes

Reprints: Liang Cheng, MD, Department of Pathology, UH 3465, Indiana University School of Medicine, 550 N University Blvd, Indianapolis, IN 46202 (lcheng@iupui.edu).