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Prognostic significance of clinicopathologic and deoxyribonucleic acid flow cytometric variables in non-metastatic renal cell carcinoma in the modern era
Urologic Oncology: Seminars and Original Investigations 23 (2005) 328 –332
Original article
Prognostic significance of clinicopathologic and deoxyribonucleic acid flow cytometric variables in non-metastatic renal cell carcinoma in the modern era Peter E. Clark, M.D.a,*, Joseph A. Veys, M.D.a, Matthew R. Eskridge, M.D.a, Ralph D. Woodruff, M.D.a,b, M. Craig Hall, M.D.a a b
Department of Urology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA Department of Pathology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA
Received 10 November 2004; received in revised form 18 January 2005; accepted 19 January 2005
Abstract Objective: The prognostic value of deoxyribonucleic acid (DNA) ploidy in renal cell carcinoma (RCC) is not well-defined among modern surgical nephrectomy series. We sought to determine which variables correlated with overall survival and recurrence-free survival in the modern era. Methods: We reviewed all patients from 1992 to 2000, who prospectively had DNA ploidy analysis of their primary tumor determined at the time of nephrectomy for nonmetastatic RCC. Variables examined included age, gender, ethnicity, presentation (incidental vs. symptomatic), preoperative laboratory studies, American Society for Anesthesiology class, tumor size, tumor-nodes-metastasis stage, histology, Fuhrman grade, and diploid versus nondiploid tumor. Statistical analyses of overall survival and recurrence-free survival were performed using the Kaplan-Meier method, log-rank test, and Cox regression model using commercially available software. Results: Sixty men and 41 women, median age 61 years (range, 23– 85), were included. Pathologic stage included T1 (54 patients), T2 (14), and T3 (33). Eighty-four patients had conventional RCC. A total of 58 patients had well-differentiated (Fuhrman Grade 1 [12] or Grade 2 [46]), 28 had moderately differentiated (Grade 3), 12 had poorly differentiated tumors (Grade 4), and 3 were not specified. There were 52 patients who had diploid tumors, and 49 had aneuploid tumors. Median follow-up was 39 months (range, 0 –109). Actuarial 5-year overall survival was 70%, and 5-year recurrence-free survival was 76%. Diploid tumors were significantly associated with better recurrence-free survival (P ⫽ 0.02) but not overall survival (P ⫽ 0.17). On multivariate analysis, the American Society for Anesthesiology class (P ⫽ 0.01), abnormal preoperative platelet count (P ⫽ 0.03), and tumor differentiation (P ⫽ 0.01) were independent predictors of overall survival, whereas only tumor differentiation (P ⫽ 0.05) was an independent predictor of recurrence-free survival. Conclusions: In the modern era, DNA ploidy is not an independent predictor of either overall survival or recurrence-free survival in patients with nonmetastatic RCC. The most important predictor of recurrence-free survival is tumor differentiation. © 2005 Elsevier Inc. All rights reserved. Keywords: Renal cell carcinoma; Prognosis; Deoxyribonucleic acid ploidy
1. Introduction Approximately 35,000 newly diagnosed cases of renal cell carcinoma (RCC) are expected in 2004, and more than 12,000 people will die of the disease [1]. RCC is known for its heterogeneity, and numerous studies have been published at* Corresponding author. Tel. ⫹1-336-716-5702; fax: ⫹1-336-7165711. E-mail address: [email protected] (P. Clark). 1078-1439/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2005.01.017
tempting to define prognostic variables for this unpredictable disease [2– 4]. In the 1980s, several studies suggested that tumor nuclear deoxyribonucleic acid (DNA) content or DNA ploidy is an important predictor of outcome in RCC [5– 8]. As a result, starting in 1992, we prospectively began assessing tumor DNA ploidy for all patients who underwent partial or radical nephrectomy for RCC. We report on the use of prospectively determined DNA ploidy for predicting outcome among patients who underwent extirpative surgery for clinically localized RCC in the modern era.
P.E. Clark et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 328 –332
2. Materials and methods Under an institutional review board approved protocol, all patients who underwent a radical or partial nephrectomy for RCC between 1992 and 2000 were retrospectively reviewed. Only patients who did not have evidence for distant metastatic disease at the time of nephrectomy were included in the analysis. All histologic cell types of RCC were included. All patients underwent partial or radical nephrectomy using standard open techniques through either a flank, extended anterior subcostal, or thoracoabdominal approach. Data collected included patient age, gender, race, American Society of Anesthesiology (ASA) class, mode of presentation (incidental or symptomatic), preoperative laboratory values (hemoglobin, platelet count, serum creatinine, serum calcium, and serum albumin), laterality (solitary or bilateral synchronous tumors), histologic type, nuclear grade, pathologic stage using the 1997 tumor-nodes-metastasis (TNM) guidelines, and DNA ploidy status. DNA ploidy status of the tumors was determined prospectively as a routine part of specimen processing and in the pathology department at our institution. Ploidy was determined by image analysis of tumor imprint slides. The slides were stained with a standard Feulgen technique using a Feulgen Stain Kit (Bacus Labs, Lombard, IL). The Feulgen stained sections were analyzed using a CAS200, a video based interactive image cytometer (Bacus Labs). The resulting histograms were interpreted for ploidy determination [9]. Nuclear grade was assigned by one pathologist (R.D.W.) according to the criteria outlined by Fuhrman et al. [10] The ASA class was assigned by the staff anesthesiologist at the time of radical nephrectomy. The ASA class consists of: class 1: healthy patient; class 2: mild systemic disease; class 3: severe systemic disease that is not incapacitating but does limit activity; class 4: severe illness that is incapacitating and a constant threat to survival; class 5: moribund patient who is not expected to survive more than 24 hours, with or without an operation [11]. Patients were followed at regular intervals with history and physical examination, laboratory studies, chest x-ray, and periodic abdominal imaging with computerized tomography. Patient status at last follow-up was recorded as was the date of any disease recurrence. Survival was determined using the Kaplan Meier method. Overall survival was defined as the time from nephrectomy to the time of death. Patients who were alive were censored at the time of last follow-up. Recurrence-free survival was defined as the time from nephrectomy to the time of first documented recurrence. Patients without evidence of recurrence were censored at the time of last followup. There were no recurrences among the 12 patients with Fuhrman Grade 1 tumors and only 3 among the 46 patients with Grade 2 tumors. Therefore, for the purposes of analysis, Grades 1 and 2 tumors were combined as “well” differentiated tumors. Only one patient in the group had evidence of lymph node involvement on final pathology, which
329
was not suspected at nephrectomy. For the purposes of analysis, this patient was included in the highest T stage group (T3). There were no deaths among patients with chromophobe or collecting duct histologic tumors, and no recurrences in those with histologic subtypes other than clear cell or conventional RCC. Therefore, in the absence of “events,” these could not be included in either univariate or multivariate analyses. Univariate analysis of survival was performed using the log-rank test. Multivariate analysis was performed using the Cox regression model for overall and recurrence-free survival. All statistical analyses were performed using commercially available software (StatView, SAS Institute Inc., Cary, NC).
3. Results 3.1. Clinical/pathologic variables One hundred and one patients were included in the study (60 men and 41 women). Median patient age at the time of nephrectomy was 61 years old (range, 23– 85). All but 11 of the patients were Caucasian. Clinical variables are outlined in Table 1. Erythrocytosis and hypercalcemia were uncommon in our patient population. The majority of patients in this series underwent a radical nephrectomy, and most presented with unilateral tumors. Surprisingly, half of our patients presented with symptoms such as pain, hematuria, weight loss, or night sweats. Pathologic variables are outlined in Table 2. The majority of tumors were clear cell or conventional RCC. The ploidy status of the tumors was almost evenly split between diploid (52) and aneuploid (49) tumors. One patient with stage T2 disease was found at final pathology to also have N1 disease and, in subsequent analyses, was combined with patients who had stage T3 disease. 3.2. Overall survival Median follow-up for the entire cohort was 39 months (range, 0 –109). Five-year actuarial overall survival for the entire cohort was 69.6% (Fig. 1). On univariate analysis, the factors significantly associated with overall survival were ASA class (P ⫽ 0.0007), anemia (P ⫽ 0.02), platelet status (normal vs. thrombocytopenia vs. thrombocytosis, P ⫽ 0.0007), renal function (normal vs. chronic renal insufficiency vs. end-stage renal disease, P ⫽ 0.01), tumor size (⬎7 vs. ⱕ7 cm., P ⫽ 0.02), tumor stage (P ⫽ 0.004), and tumor differentiation (well [Grade 1 or 2] vs. moderate [Grade 3] vs. poor [Grade 4], P ⬍ 0.0001). Tumor DNA ploidy status was not associated with overall survival (P ⫽ 0.17). On multivariate analysis, the independent predictors of decreased overall survival were high ASA class (P ⫽ 0.01), abnormal platelet count (P ⫽ 0.03), and a less differentiated tumor (P ⫽ 0.01). Patients with poorly differentiated tumors had a 20.2-fold increased risk of dying
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P.E. Clark et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 328 –332 Table 2 Pathologic and tumor characteristics for all 101 patients*
Table 1 Clinical features for all 101 patients Feature ASA class 1 2 3 4 Presentation Incidental Symptomatic Unknown Solitary/bilateral Solitary Bilateral Type of surgery Partial nephrectomy Radical nephrectomy Both Preoperative hemoglobin Anemic (male ⬍14 g/dl, female ⬍12 g/dl) Normal Erythrocytosis (male ⬎18 g/dl, female ⬎16 g/dl) Unknown Preoperative platelet Thrombocytopenia (⬍160,000/l) Normal Thrombocytosis (⬎360,000/l) Unknown Preoperative creatinine Normal (⬍1.5 mg/dl) Renal insufficiency (⬎1.5 mg/dl) End stage renal disease Unknown Hypercalcemia No (⬍10.5 mg/dl) Yes (⬎10.5 mg/dl) Unknown
Number of patients 7 45 42 7 46 45 10 97 4 11 86 4 37 53 1 10 7 78 12 4 85 11 3 2 78 3 20
(95% confidence intervals 2.7–148.8) compared to patients with well-differentiated tumors.
Number of patients T Stage 1 2 3 Fuhrman Grade 1 2 3 4 Not specified Size (cm) ⬍7 ⬎7 DNA ploidy Diploid Aneuploid Histology Clear cell Papillary Chromophobe Collecting duct
54 14 33 12 46 28 12 3 71 30 52 49 84 12 3 1
* Of the total cohort, 1 patient had lymph node disease noted on final pathologic analysis.
determined tumor DNA ploidy status was associated with recurrence-free survival by univariate analysis but was not an independent predictor of either overall or recurrence-free survival. Differentiation (Fuhrman nuclear grade) was an independent predictor of both overall and recurrence-free survival. ASA class and an abnormal platelet count were independent predictors of overall survival but not recurrence-free survival. Despite the early enthusiasm for DNA ploidy as a predictor for outcome in RCC, more recent studies have yielded mixed results. A number of studies have shown that tumor aneuploidy is associated with worse outcome [12–
3.3. Recurrence-free survival 1
.8
Percent Surviving
Five-year actuarial recurrence-free survival for the entire cohort was 75.6% (Fig. 2). On univariate analysis, the factors associated with recurrence-free survival were anemia (P ⫽ 0.003), platelet status (P ⬍ 0.0001), tumor size (P ⬍ 0.0001), tumor stage (P ⬍ 0.0001), tumor differentiation (Fig. 3; P ⬍ 0.0001), and tumor DNA ploidy status (Fig. 4; P ⫽ 0.02). On multivariate analysis, only tumor differentiation was an independent predictor of recurrence-free survival (P ⫽ 0.05). Tumor DNA ploidy status was not an independent predictor of outcome (P ⫽ 0.71).
.6
.4
.2
0 0
4. Discussion In our cohort of patients who underwent radical or partial nephrectomy for clinically localized RCC, prospectively
20
40
60
80
100
120
Time (months) Fig 1. Overall survival in 105 patients after nephrectomy for renal cell carcinoma.
P.E. Clark et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 328 –332
331
1
Percent Surviving
.8
.6
.4 .2
0 0
20
40
60
80
100
120
Fig 4. Recurrence-free survival by tumor ploidy (diploid vs. aneuploid).
Time (months) Fig 2. Recurrence-free survival in all 105 patients with renal cell carcinoma.
18]. However, other studies have shown that DNA ploidy is not an independent predictor of outcome [19 –23], which is in agreement with our findings. One study has even reported that aneuploid status was associated with better outcome among patients with RCC [24]. The likely reasons for this discrepancy across reports are varied, and include problems of small sample size, failure to perform an adequate multivariate analysis, differences in patient selection, and the inherent biases present in any retrospective analysis. Another critical potential problem inherent in DNA ploidy analysis is the showing in several reports that tumor DNA ploidy is spatially heterogeneous within any given tumor. Thus, sampling error or inadequate sampling is a major impediment to the routine use of DNA ploidy status as a predictive marker for RCC [25–27]. Based on these problems and the results of our own experience reported here, we agree with the conclusion of the Union Internationale Contre le Cancer and the American Joint Committee on Cancer 1997 report that at present, there is insufficient evidence to support the routine use of tumor DNA ploidy status as a prognostic factor in RCC [3].
Nuclear Fuhrman grade has been a prognostic marker for RCC in a large number of studies [10,28 –32]. In our study, this was the only independent predictor of recurrence-free survival in our cohort of patients with clinically localized disease at nephrectomy (only one patient had positive lymph nodes on final pathology). Perhaps it is not surprising that a higher ASA class, an indirect measure of patient comorbidity, is an independent predictor of overall survival but not recurrence-free survival. Our results support the prognostic significance of a normal versus abnormal platelet count at nephrectomy for predicting clinical outcome, as has been previously reported [33,34]. This study has several limitations that need discussion. While the determination of DNA ploidy status was prospective, the clinical data were determined retrospectively and are, therefore, prone to the inherent potential biases of any such analysis. For example, performance status was not prospectively assessed in these patients. Although performance status has now been an important prognostic factor in RCC, it is not possible to assign reliably this retrospectively and is, therefore, not included in this analysis [28]. Although this study had 101 patients, this is still a relatively small number for certain analyses. For example, the low number of events (either death or tumor recurrence) in nonclear cell histologic tumors precluded an analysis of the effect of histologic subtype on outcome. This result may also explain the lack of association between T stage and clinical outcome, although all patients with clinical evidence of metastatic disease, including radiographic evidence of lymphadenopathy, were intentionally excluded from the analysis.
5. Conclusions
Fig 3. Recurrence-free survival by differentiation of the primary tumor. Well differentiated are Fuhrman Grade 1–2, moderately differentiated are Grade 3, and poorly differentiated are Grade 4 tumors.
In the modern era, tumor DNA ploidy status is not an independent predictor of either overall or recurrence-free survival in patients with nonmetastatic RCC. The most important predictors of overall survival are ASA class, preoperative platelet status, and tumor grade. The best independent predictor of recurrence-free survival is tumor grade.
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P.E. Clark et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 328 –332
References [1] Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CA Cancer J Clin 2004;54:8 –29. [2] Thrasher JB, Paulson DF. Prognostic factors in renal cancer. Urol Clin North Am 1993;20:247– 62. [3] Gelb AB. Renal cell carcinoma: Current prognostic factors. Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997;80:981– 6. [4] Van Brussel JP, Mickisch GH. Prognostic factors in renal cell and bladder cancer. BJU Int 1999;83:902– 8. [5] Grignon DJ, Ayala AG, el-Naggar A, et al. Renal cell carcinoma. A clinicopathologic and DNA flow cytometric analysis of 103 cases. Cancer 1989;64:2133– 40. [6] Grignon DJ, el-Naggar A, Green LK, et al. DNA flow cytometry as a predictor of outcome of stage I renal cell carcinoma. Cancer 1989; 63:1161–5. [7] Rainwater LM, Hosaka Y, Farrow GM, et al. Well differentiated clear cell renal carcinoma: Significance of nuclear deoxyribonucleic acid patterns studied by flow cytometry. J Urol 1987;137:15–20. [8] Ljungberg B, Stenling R, Roos G. Tumor spread and DNA content in human renal cell carcinoma. Cancer Res 1988;48:3165–7. [9] Bacus JW, Grace LJ. Optical microscope system for standardized cell measurements and analyses. Appl Opt 1987;26:3280. [10] Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982; 6:655– 63. [11] American Society of Anesthesiologists. New classification of physical status. Anesthesiology 1963;24:111. [12] Flint A, Grossman HB, Liebert M, et al. DNA and PCNA content of renal cell carcinoma and prognosis. Am J Clin Pathol 1995;103:14 –9. [13] Nativ O, Sabo E, Raviv G, et al. The impact of tumor size on clinical outcome in patients with localized renal cell carcinoma treated by radical nephrectomy. J Urol 1997;158:729 –32. [14] Ruiz-Cerda JL, Hernandez M, Gomis F, et al. Value of deoxyribonucleic acid ploidy and nuclear morphometry for prediction of disease progression in renal cell carcinoma. J Urol 1996;155:459 – 65. [15] Di Silverio F, Casale P, Colella D, et al. Independent value of tumor size and DNA ploidy for the prediction of disease progression in patients with organ-confined renal cell carcinoma. Cancer 2000;88: 835– 43. [16] Abou-Rebyeh H, Borgmann V, Nagel R, et al. DNA ploidy is a valuable predictor for prognosis of patients with resected renal cell carcinoma. Cancer 2001;92:2280 –5. [17] Di Silverio F, Sciarra A, Flammia GP, et al. Surgical enucleation for renal cell carcinoma (RCC). Prognostic significance of tumour stage, grade and DNA ploidy. Scand J Urol Nephrol 1997;31:123– 8.
[18] Bartoletti R, Selli C, Nicolo G, et al. Cytofluorometric and cytogenetic evaluation of renal cell carcinoma: Correlations with the clinical course. Urol Int 1997;58:1–7. [19] Gelb AB, Sudilovsky D, Wu CD, et al. Appraisal of intratumoral microvessel density, MIB-1 score, DNA content, and p53 protein expression as prognostic indicators in patients with locally confined renal cell carcinoma. Cancer 1997;80:1768 –75. [20] Katusin D, Uzarevic B, Petrovecki M, et al. Clinical, histopathological and flow-cytometric properties of incidental renal cell carcinomas. Urol Res 2000;28:52– 6. [21] Shalev M, Gdor Y, Leventis A, et al. The prognostic value of DNA ploidy in small renal cell carcinoma. Pathol Res Pract 2001;197:7–12. [22] Elfving P, Mandahl N, Lundgren R, et al. Prognostic implications of cytogenetic findings in kidney cancer. Br J Urol 1997;80:698 –706. [23] Papadopoulos I, Rudolph P, Weichert-Jacobsen K. Value of p53 expression, cellular proliferation, and DNA content as prognostic indicators in renal cell carcinoma. Eur Urol 1997;32:110 –7. [24] Eskelinen M, Lipponen P, Nordling S. Prognostic evaluation of DNA flow cytometry and histo-morphological criteria in renal cell carcinoma. Anticancer Res 1995;15:2279 – 83. [25] Ruiz-Cerda JL, Hernandez M, Sempere A, et al. Intratumoral heterogeneity of DNA content in renal cell carcinoma and its prognostic significance. Cancer 1999;86:664 –71. [26] Nenning H, Rassler J, Minh DH. Heterogeneity of DNA distribution pattern in renal tumours. Anal Cell Pathol 1997;14:9 –17. [27] Ljungberg B, Mehle C, Stenling R, et al. Heterogeneity in renal cell carcinoma and its impact no prognosis–A flow cytometric study. Br J Cancer 1996;74:123–7. [28] Zisman A, Pantuck AJ, Dorey F, et al. Improved prognostication of renal cell carcinoma using an integrated staging system. J Clin Oncol 2001;19:1649 –57. [29] Zisman A, Pantuck AJ, Figlin RA, et al. Validation of the ucla integrated staging system for patients with renal cell carcinoma. J Clin Oncol 2001;19:3792–3. [30] Green LK, Ayala AG, Ro JY, et al. Role of nuclear grading in stage I renal cell carcinoma. Urology 1989;34:310 –5. [31] Medeiros LJ, Gelb AB, Weiss LM. Renal cell carcinoma. Prognostic significance of morphologic parameters in 121 cases. Cancer 1988; 61:1639 –51. [32] Selli C, Hinshaw WM, Woodard BH, et al. Stratification of risk factors in renal cell carcinoma. Cancer 1983;52:899 –903. [33] Symbas NP, Townsend MF, El-Galley R, et al. Poor prognosis associated with thrombocytosis in patients with renal cell carcinoma. BJU Int 2000;86:203–7. [34] O‘Keefe SC, Marshall FF, Issa MM, et al. Thrombocytosis is associated with a significant increase in the cancer specific death rate after radical nephrectomy. J Urol 2002;168:1378 – 80.
Original article
Prognostic significance of clinicopathologic and deoxyribonucleic acid flow cytometric variables in non-metastatic renal cell carcinoma in the modern era Peter E. Clark, M.D.a,*, Joseph A. Veys, M.D.a, Matthew R. Eskridge, M.D.a, Ralph D. Woodruff, M.D.a,b, M. Craig Hall, M.D.a a b
Department of Urology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA Department of Pathology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA
Received 10 November 2004; received in revised form 18 January 2005; accepted 19 January 2005
Abstract Objective: The prognostic value of deoxyribonucleic acid (DNA) ploidy in renal cell carcinoma (RCC) is not well-defined among modern surgical nephrectomy series. We sought to determine which variables correlated with overall survival and recurrence-free survival in the modern era. Methods: We reviewed all patients from 1992 to 2000, who prospectively had DNA ploidy analysis of their primary tumor determined at the time of nephrectomy for nonmetastatic RCC. Variables examined included age, gender, ethnicity, presentation (incidental vs. symptomatic), preoperative laboratory studies, American Society for Anesthesiology class, tumor size, tumor-nodes-metastasis stage, histology, Fuhrman grade, and diploid versus nondiploid tumor. Statistical analyses of overall survival and recurrence-free survival were performed using the Kaplan-Meier method, log-rank test, and Cox regression model using commercially available software. Results: Sixty men and 41 women, median age 61 years (range, 23– 85), were included. Pathologic stage included T1 (54 patients), T2 (14), and T3 (33). Eighty-four patients had conventional RCC. A total of 58 patients had well-differentiated (Fuhrman Grade 1 [12] or Grade 2 [46]), 28 had moderately differentiated (Grade 3), 12 had poorly differentiated tumors (Grade 4), and 3 were not specified. There were 52 patients who had diploid tumors, and 49 had aneuploid tumors. Median follow-up was 39 months (range, 0 –109). Actuarial 5-year overall survival was 70%, and 5-year recurrence-free survival was 76%. Diploid tumors were significantly associated with better recurrence-free survival (P ⫽ 0.02) but not overall survival (P ⫽ 0.17). On multivariate analysis, the American Society for Anesthesiology class (P ⫽ 0.01), abnormal preoperative platelet count (P ⫽ 0.03), and tumor differentiation (P ⫽ 0.01) were independent predictors of overall survival, whereas only tumor differentiation (P ⫽ 0.05) was an independent predictor of recurrence-free survival. Conclusions: In the modern era, DNA ploidy is not an independent predictor of either overall survival or recurrence-free survival in patients with nonmetastatic RCC. The most important predictor of recurrence-free survival is tumor differentiation. © 2005 Elsevier Inc. All rights reserved. Keywords: Renal cell carcinoma; Prognosis; Deoxyribonucleic acid ploidy
1. Introduction Approximately 35,000 newly diagnosed cases of renal cell carcinoma (RCC) are expected in 2004, and more than 12,000 people will die of the disease [1]. RCC is known for its heterogeneity, and numerous studies have been published at* Corresponding author. Tel. ⫹1-336-716-5702; fax: ⫹1-336-7165711. E-mail address: [email protected] (P. Clark). 1078-1439/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2005.01.017
tempting to define prognostic variables for this unpredictable disease [2– 4]. In the 1980s, several studies suggested that tumor nuclear deoxyribonucleic acid (DNA) content or DNA ploidy is an important predictor of outcome in RCC [5– 8]. As a result, starting in 1992, we prospectively began assessing tumor DNA ploidy for all patients who underwent partial or radical nephrectomy for RCC. We report on the use of prospectively determined DNA ploidy for predicting outcome among patients who underwent extirpative surgery for clinically localized RCC in the modern era.
P.E. Clark et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 328 –332
2. Materials and methods Under an institutional review board approved protocol, all patients who underwent a radical or partial nephrectomy for RCC between 1992 and 2000 were retrospectively reviewed. Only patients who did not have evidence for distant metastatic disease at the time of nephrectomy were included in the analysis. All histologic cell types of RCC were included. All patients underwent partial or radical nephrectomy using standard open techniques through either a flank, extended anterior subcostal, or thoracoabdominal approach. Data collected included patient age, gender, race, American Society of Anesthesiology (ASA) class, mode of presentation (incidental or symptomatic), preoperative laboratory values (hemoglobin, platelet count, serum creatinine, serum calcium, and serum albumin), laterality (solitary or bilateral synchronous tumors), histologic type, nuclear grade, pathologic stage using the 1997 tumor-nodes-metastasis (TNM) guidelines, and DNA ploidy status. DNA ploidy status of the tumors was determined prospectively as a routine part of specimen processing and in the pathology department at our institution. Ploidy was determined by image analysis of tumor imprint slides. The slides were stained with a standard Feulgen technique using a Feulgen Stain Kit (Bacus Labs, Lombard, IL). The Feulgen stained sections were analyzed using a CAS200, a video based interactive image cytometer (Bacus Labs). The resulting histograms were interpreted for ploidy determination [9]. Nuclear grade was assigned by one pathologist (R.D.W.) according to the criteria outlined by Fuhrman et al. [10] The ASA class was assigned by the staff anesthesiologist at the time of radical nephrectomy. The ASA class consists of: class 1: healthy patient; class 2: mild systemic disease; class 3: severe systemic disease that is not incapacitating but does limit activity; class 4: severe illness that is incapacitating and a constant threat to survival; class 5: moribund patient who is not expected to survive more than 24 hours, with or without an operation [11]. Patients were followed at regular intervals with history and physical examination, laboratory studies, chest x-ray, and periodic abdominal imaging with computerized tomography. Patient status at last follow-up was recorded as was the date of any disease recurrence. Survival was determined using the Kaplan Meier method. Overall survival was defined as the time from nephrectomy to the time of death. Patients who were alive were censored at the time of last follow-up. Recurrence-free survival was defined as the time from nephrectomy to the time of first documented recurrence. Patients without evidence of recurrence were censored at the time of last followup. There were no recurrences among the 12 patients with Fuhrman Grade 1 tumors and only 3 among the 46 patients with Grade 2 tumors. Therefore, for the purposes of analysis, Grades 1 and 2 tumors were combined as “well” differentiated tumors. Only one patient in the group had evidence of lymph node involvement on final pathology, which
329
was not suspected at nephrectomy. For the purposes of analysis, this patient was included in the highest T stage group (T3). There were no deaths among patients with chromophobe or collecting duct histologic tumors, and no recurrences in those with histologic subtypes other than clear cell or conventional RCC. Therefore, in the absence of “events,” these could not be included in either univariate or multivariate analyses. Univariate analysis of survival was performed using the log-rank test. Multivariate analysis was performed using the Cox regression model for overall and recurrence-free survival. All statistical analyses were performed using commercially available software (StatView, SAS Institute Inc., Cary, NC).
3. Results 3.1. Clinical/pathologic variables One hundred and one patients were included in the study (60 men and 41 women). Median patient age at the time of nephrectomy was 61 years old (range, 23– 85). All but 11 of the patients were Caucasian. Clinical variables are outlined in Table 1. Erythrocytosis and hypercalcemia were uncommon in our patient population. The majority of patients in this series underwent a radical nephrectomy, and most presented with unilateral tumors. Surprisingly, half of our patients presented with symptoms such as pain, hematuria, weight loss, or night sweats. Pathologic variables are outlined in Table 2. The majority of tumors were clear cell or conventional RCC. The ploidy status of the tumors was almost evenly split between diploid (52) and aneuploid (49) tumors. One patient with stage T2 disease was found at final pathology to also have N1 disease and, in subsequent analyses, was combined with patients who had stage T3 disease. 3.2. Overall survival Median follow-up for the entire cohort was 39 months (range, 0 –109). Five-year actuarial overall survival for the entire cohort was 69.6% (Fig. 1). On univariate analysis, the factors significantly associated with overall survival were ASA class (P ⫽ 0.0007), anemia (P ⫽ 0.02), platelet status (normal vs. thrombocytopenia vs. thrombocytosis, P ⫽ 0.0007), renal function (normal vs. chronic renal insufficiency vs. end-stage renal disease, P ⫽ 0.01), tumor size (⬎7 vs. ⱕ7 cm., P ⫽ 0.02), tumor stage (P ⫽ 0.004), and tumor differentiation (well [Grade 1 or 2] vs. moderate [Grade 3] vs. poor [Grade 4], P ⬍ 0.0001). Tumor DNA ploidy status was not associated with overall survival (P ⫽ 0.17). On multivariate analysis, the independent predictors of decreased overall survival were high ASA class (P ⫽ 0.01), abnormal platelet count (P ⫽ 0.03), and a less differentiated tumor (P ⫽ 0.01). Patients with poorly differentiated tumors had a 20.2-fold increased risk of dying
330
P.E. Clark et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 328 –332 Table 2 Pathologic and tumor characteristics for all 101 patients*
Table 1 Clinical features for all 101 patients Feature ASA class 1 2 3 4 Presentation Incidental Symptomatic Unknown Solitary/bilateral Solitary Bilateral Type of surgery Partial nephrectomy Radical nephrectomy Both Preoperative hemoglobin Anemic (male ⬍14 g/dl, female ⬍12 g/dl) Normal Erythrocytosis (male ⬎18 g/dl, female ⬎16 g/dl) Unknown Preoperative platelet Thrombocytopenia (⬍160,000/l) Normal Thrombocytosis (⬎360,000/l) Unknown Preoperative creatinine Normal (⬍1.5 mg/dl) Renal insufficiency (⬎1.5 mg/dl) End stage renal disease Unknown Hypercalcemia No (⬍10.5 mg/dl) Yes (⬎10.5 mg/dl) Unknown
Number of patients 7 45 42 7 46 45 10 97 4 11 86 4 37 53 1 10 7 78 12 4 85 11 3 2 78 3 20
(95% confidence intervals 2.7–148.8) compared to patients with well-differentiated tumors.
Number of patients T Stage 1 2 3 Fuhrman Grade 1 2 3 4 Not specified Size (cm) ⬍7 ⬎7 DNA ploidy Diploid Aneuploid Histology Clear cell Papillary Chromophobe Collecting duct
54 14 33 12 46 28 12 3 71 30 52 49 84 12 3 1
* Of the total cohort, 1 patient had lymph node disease noted on final pathologic analysis.
determined tumor DNA ploidy status was associated with recurrence-free survival by univariate analysis but was not an independent predictor of either overall or recurrence-free survival. Differentiation (Fuhrman nuclear grade) was an independent predictor of both overall and recurrence-free survival. ASA class and an abnormal platelet count were independent predictors of overall survival but not recurrence-free survival. Despite the early enthusiasm for DNA ploidy as a predictor for outcome in RCC, more recent studies have yielded mixed results. A number of studies have shown that tumor aneuploidy is associated with worse outcome [12–
3.3. Recurrence-free survival 1
.8
Percent Surviving
Five-year actuarial recurrence-free survival for the entire cohort was 75.6% (Fig. 2). On univariate analysis, the factors associated with recurrence-free survival were anemia (P ⫽ 0.003), platelet status (P ⬍ 0.0001), tumor size (P ⬍ 0.0001), tumor stage (P ⬍ 0.0001), tumor differentiation (Fig. 3; P ⬍ 0.0001), and tumor DNA ploidy status (Fig. 4; P ⫽ 0.02). On multivariate analysis, only tumor differentiation was an independent predictor of recurrence-free survival (P ⫽ 0.05). Tumor DNA ploidy status was not an independent predictor of outcome (P ⫽ 0.71).
.6
.4
.2
0 0
4. Discussion In our cohort of patients who underwent radical or partial nephrectomy for clinically localized RCC, prospectively
20
40
60
80
100
120
Time (months) Fig 1. Overall survival in 105 patients after nephrectomy for renal cell carcinoma.
P.E. Clark et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 328 –332
331
1
Percent Surviving
.8
.6
.4 .2
0 0
20
40
60
80
100
120
Fig 4. Recurrence-free survival by tumor ploidy (diploid vs. aneuploid).
Time (months) Fig 2. Recurrence-free survival in all 105 patients with renal cell carcinoma.
18]. However, other studies have shown that DNA ploidy is not an independent predictor of outcome [19 –23], which is in agreement with our findings. One study has even reported that aneuploid status was associated with better outcome among patients with RCC [24]. The likely reasons for this discrepancy across reports are varied, and include problems of small sample size, failure to perform an adequate multivariate analysis, differences in patient selection, and the inherent biases present in any retrospective analysis. Another critical potential problem inherent in DNA ploidy analysis is the showing in several reports that tumor DNA ploidy is spatially heterogeneous within any given tumor. Thus, sampling error or inadequate sampling is a major impediment to the routine use of DNA ploidy status as a predictive marker for RCC [25–27]. Based on these problems and the results of our own experience reported here, we agree with the conclusion of the Union Internationale Contre le Cancer and the American Joint Committee on Cancer 1997 report that at present, there is insufficient evidence to support the routine use of tumor DNA ploidy status as a prognostic factor in RCC [3].
Nuclear Fuhrman grade has been a prognostic marker for RCC in a large number of studies [10,28 –32]. In our study, this was the only independent predictor of recurrence-free survival in our cohort of patients with clinically localized disease at nephrectomy (only one patient had positive lymph nodes on final pathology). Perhaps it is not surprising that a higher ASA class, an indirect measure of patient comorbidity, is an independent predictor of overall survival but not recurrence-free survival. Our results support the prognostic significance of a normal versus abnormal platelet count at nephrectomy for predicting clinical outcome, as has been previously reported [33,34]. This study has several limitations that need discussion. While the determination of DNA ploidy status was prospective, the clinical data were determined retrospectively and are, therefore, prone to the inherent potential biases of any such analysis. For example, performance status was not prospectively assessed in these patients. Although performance status has now been an important prognostic factor in RCC, it is not possible to assign reliably this retrospectively and is, therefore, not included in this analysis [28]. Although this study had 101 patients, this is still a relatively small number for certain analyses. For example, the low number of events (either death or tumor recurrence) in nonclear cell histologic tumors precluded an analysis of the effect of histologic subtype on outcome. This result may also explain the lack of association between T stage and clinical outcome, although all patients with clinical evidence of metastatic disease, including radiographic evidence of lymphadenopathy, were intentionally excluded from the analysis.
5. Conclusions
Fig 3. Recurrence-free survival by differentiation of the primary tumor. Well differentiated are Fuhrman Grade 1–2, moderately differentiated are Grade 3, and poorly differentiated are Grade 4 tumors.
In the modern era, tumor DNA ploidy status is not an independent predictor of either overall or recurrence-free survival in patients with nonmetastatic RCC. The most important predictors of overall survival are ASA class, preoperative platelet status, and tumor grade. The best independent predictor of recurrence-free survival is tumor grade.
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References [1] Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CA Cancer J Clin 2004;54:8 –29. [2] Thrasher JB, Paulson DF. Prognostic factors in renal cancer. Urol Clin North Am 1993;20:247– 62. [3] Gelb AB. Renal cell carcinoma: Current prognostic factors. Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997;80:981– 6. [4] Van Brussel JP, Mickisch GH. Prognostic factors in renal cell and bladder cancer. BJU Int 1999;83:902– 8. [5] Grignon DJ, Ayala AG, el-Naggar A, et al. Renal cell carcinoma. A clinicopathologic and DNA flow cytometric analysis of 103 cases. Cancer 1989;64:2133– 40. [6] Grignon DJ, el-Naggar A, Green LK, et al. DNA flow cytometry as a predictor of outcome of stage I renal cell carcinoma. Cancer 1989; 63:1161–5. [7] Rainwater LM, Hosaka Y, Farrow GM, et al. Well differentiated clear cell renal carcinoma: Significance of nuclear deoxyribonucleic acid patterns studied by flow cytometry. J Urol 1987;137:15–20. [8] Ljungberg B, Stenling R, Roos G. Tumor spread and DNA content in human renal cell carcinoma. Cancer Res 1988;48:3165–7. [9] Bacus JW, Grace LJ. Optical microscope system for standardized cell measurements and analyses. Appl Opt 1987;26:3280. [10] Fuhrman SA, Lasky LC, Limas C. Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 1982; 6:655– 63. [11] American Society of Anesthesiologists. New classification of physical status. Anesthesiology 1963;24:111. [12] Flint A, Grossman HB, Liebert M, et al. DNA and PCNA content of renal cell carcinoma and prognosis. Am J Clin Pathol 1995;103:14 –9. [13] Nativ O, Sabo E, Raviv G, et al. The impact of tumor size on clinical outcome in patients with localized renal cell carcinoma treated by radical nephrectomy. J Urol 1997;158:729 –32. [14] Ruiz-Cerda JL, Hernandez M, Gomis F, et al. Value of deoxyribonucleic acid ploidy and nuclear morphometry for prediction of disease progression in renal cell carcinoma. J Urol 1996;155:459 – 65. [15] Di Silverio F, Casale P, Colella D, et al. Independent value of tumor size and DNA ploidy for the prediction of disease progression in patients with organ-confined renal cell carcinoma. Cancer 2000;88: 835– 43. [16] Abou-Rebyeh H, Borgmann V, Nagel R, et al. DNA ploidy is a valuable predictor for prognosis of patients with resected renal cell carcinoma. Cancer 2001;92:2280 –5. [17] Di Silverio F, Sciarra A, Flammia GP, et al. Surgical enucleation for renal cell carcinoma (RCC). Prognostic significance of tumour stage, grade and DNA ploidy. Scand J Urol Nephrol 1997;31:123– 8.
[18] Bartoletti R, Selli C, Nicolo G, et al. Cytofluorometric and cytogenetic evaluation of renal cell carcinoma: Correlations with the clinical course. Urol Int 1997;58:1–7. [19] Gelb AB, Sudilovsky D, Wu CD, et al. Appraisal of intratumoral microvessel density, MIB-1 score, DNA content, and p53 protein expression as prognostic indicators in patients with locally confined renal cell carcinoma. Cancer 1997;80:1768 –75. [20] Katusin D, Uzarevic B, Petrovecki M, et al. Clinical, histopathological and flow-cytometric properties of incidental renal cell carcinomas. Urol Res 2000;28:52– 6. [21] Shalev M, Gdor Y, Leventis A, et al. The prognostic value of DNA ploidy in small renal cell carcinoma. Pathol Res Pract 2001;197:7–12. [22] Elfving P, Mandahl N, Lundgren R, et al. Prognostic implications of cytogenetic findings in kidney cancer. Br J Urol 1997;80:698 –706. [23] Papadopoulos I, Rudolph P, Weichert-Jacobsen K. Value of p53 expression, cellular proliferation, and DNA content as prognostic indicators in renal cell carcinoma. Eur Urol 1997;32:110 –7. [24] Eskelinen M, Lipponen P, Nordling S. Prognostic evaluation of DNA flow cytometry and histo-morphological criteria in renal cell carcinoma. Anticancer Res 1995;15:2279 – 83. [25] Ruiz-Cerda JL, Hernandez M, Sempere A, et al. Intratumoral heterogeneity of DNA content in renal cell carcinoma and its prognostic significance. Cancer 1999;86:664 –71. [26] Nenning H, Rassler J, Minh DH. Heterogeneity of DNA distribution pattern in renal tumours. Anal Cell Pathol 1997;14:9 –17. [27] Ljungberg B, Mehle C, Stenling R, et al. Heterogeneity in renal cell carcinoma and its impact no prognosis–A flow cytometric study. Br J Cancer 1996;74:123–7. [28] Zisman A, Pantuck AJ, Dorey F, et al. Improved prognostication of renal cell carcinoma using an integrated staging system. J Clin Oncol 2001;19:1649 –57. [29] Zisman A, Pantuck AJ, Figlin RA, et al. Validation of the ucla integrated staging system for patients with renal cell carcinoma. J Clin Oncol 2001;19:3792–3. [30] Green LK, Ayala AG, Ro JY, et al. Role of nuclear grading in stage I renal cell carcinoma. Urology 1989;34:310 –5. [31] Medeiros LJ, Gelb AB, Weiss LM. Renal cell carcinoma. Prognostic significance of morphologic parameters in 121 cases. Cancer 1988; 61:1639 –51. [32] Selli C, Hinshaw WM, Woodard BH, et al. Stratification of risk factors in renal cell carcinoma. Cancer 1983;52:899 –903. [33] Symbas NP, Townsend MF, El-Galley R, et al. Poor prognosis associated with thrombocytosis in patients with renal cell carcinoma. BJU Int 2000;86:203–7. [34] O‘Keefe SC, Marshall FF, Issa MM, et al. Thrombocytosis is associated with a significant increase in the cancer specific death rate after radical nephrectomy. J Urol 2002;168:1378 – 80.