Affiliations of authors: C. F. Eisenberger, S. Hortopan, N.-H. Chow (Department of Urology, James Buchanan Brady Urological Institute), M. Schoenberg, F. F. Marshall (Department of Urology, James Buchanan Brady Urological Institute, and The Johns Hopkins Oncology Center), C. Enger (The Johns Hopkins Oncology Center), S. Shah (Department of OtolaryngologyHead and Neck Surgery, Division of Head and Neck Cancer Research), D. Sidransky (Department of OtolaryngologyHead and Neck Surgery, Division of Head and Neck Cancer Research, and The Johns Hopkins Oncology Center), The Johns Hopkins Medical Institutions, The Johns Hopkins University School of Medicine, Baltimore, MD.
Correspondence to: David Sidransky, M.D., The Johns Hopkins University School of Medicine, OtolaryngologyHead and Neck Surgery, Head and Neck Cancer Research, 818 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2196 (e-mail: dsidrans{at}jhmi.edu).
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ABSTRACT |
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INTRODUCTION |
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The specific clinical signs and symptoms of malignant renal disease are not usually helpful in making an early diagnosis. The classic triad of pain, hematuria, and a palpable flank mass is encountered in only 10% of patients and is usually associated with the presence of advanced disease (3). The widespread use of noninvasive axial imaging (e.g., computed tomography or magnetic resonance imaging) and diagnostic ultrasound has resulted in the incidental discovery of an increasing number of small asymptomatic renal tumors. Estimates suggest that as many as two thirds of organ-confined tumors are identified by serendipity (4). Nevertheless, 50% of patients with renal cancers are not curable by surgical resection at the time of presentation. Consequently, the development of a reliable, noninvasive method for the early detection of kidney cancer could represent an important clinical advance in the management of this patient population.
Microsatellite analysis is a polymerase chain reaction (PCR)-based technique that permits the detection of cancer-specific DNA alterations (loss of heterozygosity [LOH] and microsatellite instability) in neoplastic tissue. Recent application of this approach to the evaluation of body fluids has shown that squamous cell carcinoma of the aerodigestive tract and bladder cancer can be detected through the analysis of saliva and urine, respectively (5-7). Since the renal parenchyma is highly vascular and lies in close physical proximity to the renal collecting system and since the tubular epithelium from which most malignant renal neoplasms arise contributes directly to urine formation, we hypothesized that DNA alterations characteristic of malignancy could be identified by microsatellite analysis of either serum or voided urine specimens obtained from patients with renal malignancies. To test this hypothesis, we studied, by use of microsatellite analysis, preoperative urine and serum specimens obtained from patients with a variety of renal neoplasms.
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MATERIALS AND METHODS |
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Microsatellite analysis. Based on high rates of informativity and known patterns of
LOH and microsatellite instability in renal cancer, 28 microsatellite markers (Research Genetics,
Huntsville, AL) were identified for use in this series of experiments. Microsatellite markers (and
their chromosomal locations) are as follows: D1S251 (1pq), HTPO (2p), D3S1317 (3p), D3S587
(3p), D3S1560 (3p), D3S1289 (3p), D3S1286 (3p), D3S1038 (3p), D4S243 (4pq), FGA(4) (4q),
CSF (5q), ACTBP2 (5p), D8S348 (8q), D8S307 (8p), D9S747 (9p), D9S242 (9p), IFN a (9p),
D9S162 (9p), D11S488 (11q), THO (11p), vWA (12p), D13S802 (13q), MJD (14q), D17S695
(17p), D17S654 (17p), D18S51 (18q), MBP (18q), and D21S1245 (21q). Primer sequences and
locations were obtained from the Genome Database (The Johns Hopkins University, Baltimore,
MD). One primer from each pair was end-labeled with [-32P]adenosine triphosphate (Amersham Life Science Inc., Arlington Heights, IL) with the use
of bacteriophage T4-polynucleotide kinase (Life Technologies, Inc. [GIBCO BRL],
Gaithersburg, MD). Genomic DNA (50 ng) was subjected to 35 PCR cycles at a denaturing
temperature of 95 °C for 1 minute, followed by varying annealing temperatures (52
°C-60 °C, depending on the primer sequence) for 1 minute, an extension step at
72 °C for 1 minute, and a final extension step at 72 °C for 5 minutes by use of a
thermocycler (Hybaid, Teddington, U.K.). PCR products were then separated in denaturing
7% polyacrylamide-urea-formamide gels, followed by autoradiography for 12-36 hours on
X-omat film (Eastman Kodak Co., Rochester, NY) (9). LOH was
determined by a comparison of the intensity of the allelic bands from nonmalignant (lymphocyte)
DNA with that of the allelic bands from the target sample (from tumor, urine, or serum). A
reduction in the intensity of one allele in the target sample of more than 50% (30%
in serum as a result of a greater dilution with normal DNA), as assessed by two independent
observers (C. F. Eisenberger and D. Sidransky), was considered to represent LOH and the
presence of new "shifted" alleles (appearance of new bands) as microsatellite
instability. Clinical data were obtained from the patient charts. Every marker alteration (LOH or
microsatellite instability) was confirmed by reamplification of the starting material and
independent verification by two separate observers.
Statistical analysis. The sensitivity and specificity of marker alterations in urine and serum were calculated as follows: sensitivity = number of positive tests/number of cancer cases, and specificity = number of negative tests/number of cases without cancer. The proportion of patients showing a microsatellite alteration was compared between tumor stages with the use of Fisher's exact test. P values reported are two-sided.
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RESULTS |
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There was no association between evidence of either microsatellite instability or LOH in
serum and the stage of tumor (Table 2). Fifteen (60%) of 25
patients treated for malignant renal neoplasms in this study had evidence of either LOH or
microsatellite instability in serum samples obtained preoperatively. Of these 15 patients, three had
tumors of stage T1, eight had tumors of stage T2, and four had tumors of stage T3, and there was
no difference in serum detection between any of these tumor stages (P = .39,
Fisher's exact test). Similarly, the urine samples were positive in 19 patients, including four
patients with stage T1 tumor, 11 with stage T2 tumor, and four with stage T3 tumor. Again, there
was no association between stage of tumor and evidence of microsatellite alterations in urine
specimens (P = .39, Fisher's exact test). Patients with lesions of low
malignant potential failed to show evidence of serum alterations in this analysis.
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DISCUSSION |
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Advances in basic research have shed light on some events that putatively contribute to the development of renal neoplasia. Detailed studies of pathology (10) have underscored the morphologic heterogeneity of renal cancers. Genetic studies employing a variety of technologies (11-13) have shown that renal cancers are characterized by specific chromosomal abnormalities, the most common of which include subchromosomal losses on 3p in sporadic clear cell carcinomas, aneuploidy of chromosomes 7 and 17 in papillary renal tumors, and small deletions on chromosomal arm 1p in oncocytomas and on distal 1q in collecting duct carcinomas. Elegant familial studies of von Hippel-Lindau (VHL) disease have led to the identification of the gene putatively responsible for that disease located at 3p25 (14). Additional work (15) has shown that LOH at the VHL locus may be characteristic of most sporadic renal cancers. However, these advances in basic research have not yet translated into the development of reliable diagnostic markers of renal cancer.
The central clinical problem facing surgeons and oncologists who care for patients with renal cancer is that this cancer is unresponsive to conventional systemic adjuvant therapies, unlike other genitourinary cancers for which successful adjuvant therapies have been developed (i.e., cis-platinum-based chemotherapeutic regimens for bladder cancer and nonseminomatous testicular neoplasms) (16,17). Radioresistance is also characteristic of renal tumors, leaving surgery as the sole, consistently successful form of therapy. Surgical removal of the kidney has a limited role in the treatment of patients with advanced disease and is often not curative in patients with tumors that extend beyond Gerota's fascia or that involve regional lymphatics. Although prospective trials have not yet confirmed the value of early renal cancer detection, stage-specific survival data suggest that, if renal neoplasms could be consistently detected at the organ-confined stage, the disease-specific survival rate could be expected to increase. It is interesting to note that a recent retrospective autopsy series identified renal cancer as one of the most common undiagnosed tumors that contributed to death in U.S. patients who underwent postmortem examination (18).
The ease with which microsatellite analysis can be performed on a variety of DNA sources continues to increase. The molecular diagnosis of renal cancer by urinalysis is remarkable in that specimens from all patients contained a virtual absence of cellular material or only scant cellular fragments. Further enrichment of neoplastic cell (or cell fragment) populations in the urine with antibodies to cancer-specific antigens such as MN/CA9 protein (19) may further increase the sensitivity of microsatellite analysis. Evolving knowledge of the genetic changes that drive kidney cancer progression and the ability to distinguish malignant and benign tumors of the kidney will also lead to further improvements (20). We have demonstrated the ability to perform multiple analyses using a new high-throughput microcapillary array that can markedly enhance the potential clinical utility of this molecular diagnostic approach (21). Translation of this type of technology to the clinical laboratory may hold the key to broader application and eventual use of microsatellite-based cancer diagnostics.
Urologic malignancies have well-studied patterns of anatomic spread, the best characterized of which are testis and prostate cancers (3). Both of these tumor types are known for their propensity to metastasize by lymphatic routes. In contrast, renal cancer probably spreads by a combination of lymphatic and hematogenous mechanisms, a fact underscored by the finding that circulating cancer cells have been readily extracted from the blood of patients with various stages of malignant renal disease (22). The finding of serum microsatellite alterations in greater than 50% of the patients studied for this report probably reflects the hematogenous mechanism by which certain renal tumors spread. A recent report (23) confirmed a similar frequency of microsatellite alterations in the serum of patients with clear cell renal tumors. While it is not yet possible to say to what extent patterns of microsatellite alterations are related to the malignant and metastatic potential of individual renal tumors, further study of these tumors with different panels of markers may reveal patterns of LOH and microsatellite instability with prognostic as well as diagnostic potential. Coupled with high-throughput technologies, genome-wide search strategies, and the opportunity to study a larger population of patients in a multi-institutional venue, microsatellite analysis may permit the identification of patients with early and potentially resectable disease. Microsatellite alterations in serum predicted a poor prognosis in patients with head and neck cancer (24) and may also identify kidney cancer patients at risk for disease progression for whom experimental adjuvant therapies may be beneficial.
We have demonstrated that microsatellite analysis of urine can frequently detect the presence of malignancy in patients with clinically organ-confined renal cancer. Patients with renal lesions of lower malignant potential, such as oncocytomas, demonstrated DNA alterations in matched urine samples but not in preoperative serum samples. Individuals with nephrolithiasis and control subjects without genitourinary complaints or symptoms did not have positive microsatellite analyses in this study. Larger multicenter trials utilizing this assay for the diagnosis and potential staging of renal cancer patients are warranted to determine the ultimate clinical utility of this molecular approach.
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NOTES |
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Supported by Public Health Service grant P01CA77664-02 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, and by a grant from the Deutsche Forschungsgemeinschaft (to C. F. Eisenberger); by grant 96-78 from the American Cancer Society (to M. Schoenberg); and by a grant from the National Science Council, Taiwan, ROC (to N.-H. Chow).
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Manuscript received July 16, 1999; revised September 28, 1999; accepted October 1, 1999.
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