Annals of Oncology Advance Access originally published online on September 19, 2006
Annals of Oncology 2006 17(11):1677-1686; doi:10.1093/annonc/mdl289
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2006 European Society for Medical Oncology
urogenital tumors |
Assessing interactions between mdm-2, p53, and bcl-2 as prognostic variables in muscle-invasive bladder cancer treated with neo-adjuvant chemotherapy followed by locoregional surgical treatment
1 The Genitourinary Oncology Service, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center and the Department of Medicine, Joan and Sanford Weill Medical College of Cornell University, New York
2 Division of Urology, Hospital das Clínicas, University of São Paulo and Department of Medical Oncology, Hospital Sírio-Libanês, São Paulo–SP–Brazil
3 Department of Pathology, Joan and Sanford Weill Medical College of Cornell University, New York, USA
4 Department of Epidemiology and Biostatistics, Joan and Sanford Weill Medical College of Cornell University, New York, USA
5 Department of Urology, Memorial Sloan-Kettering Cancer Center New York and the Department of Surgery, Joan and Sanford Weill Medical College of Cornell University, New York, USA
* Correspondence to: Dr D. F. Bajorin, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. Tel: (646) 422-4333; Fax: (212) 988-1079; E-mail: bajorind{at}mskcc.org
 |
Abstract |
|---|
 Top Abstract introductionmaterial and methodsresultsdiscussionReferences |
|---|
Background: Tumor proliferation and apoptosis may be influenced by the mdm-2 gene product, which can block the antiproliferative effects of p53. bcl-2, one of a family of related genes that regulates the apoptotic pathway, exhibits a negative influence. Both individual and cooperative effects of these gene products may affect the biological behavior of primary bladder cancers and long-term outcome to standard therapy.
Methods: This study retrospectively evaluated the association with survival of mdm-2, p53, and bcl-2 expression in 59 patients with muscle-invasive, node-negative transitional cell carcinoma (TCC) treated with neo-adjuvant chemotherapy followed by locoregional surgery. Each marker was defined as an altered phenotype if 
20% malignant cells in the primary tumor exhibited staining; normal or minimal expression was defined as <20% cells exhibiting staining.
Results: Altered mdm-2, p53, and bcl-2 expression was observed in 37%, 54%, and 46% of patients, respectively. In single marker analysis, altered p53 expression correlated with long-term survival (P = 0.05) but mdm-2 (P = 0.42) or bcl-2 (P = 0.17) did not. In the multiple-marker analysis, a prognostic index simultaneously assessing mdm-2, p53, and bcl-2 correlated with survival (P = 0.01). The 5-year survival for patients in which all markers were normally expressed was 54% compared with 25% in those with all three markers aberrantly expressed. Patients with aberrant expression of either one or two markers had an intermediate 5-year survival (49%). There was no association of molecular markers either alone or in combination with pathologic downstaging after neo-adjuvant chemotherapy.
Conclusion: The cooperative effects of phenotypes determined by mdm-2, p53, and bcl-2 expression may predict survival in patients with muscle-invasive TCC of the bladder.
Key words: bcl-2, mdm-2, p53, prognosis, transitional cell carcinoma, urothelial tract cancer
|
introduction |
|---|
|
TopAbstractintroductionmaterial and methodsresultsdiscussionReferences |
|---|
Muscle-invasive transitional cell carcinoma (TCC) is associated with a significant potential for metastatic disease at the time of diagnosis. In even the best trials exploring optimal therapy consisting of neo-adjuvant chemotherapy, cystectomy, and pelvic lymph node dissection,
50% of patients will relapse and succumb to the disease [1]. Patients whose tumors exhibit a complete response to chemotherapy appear to have the best prognosis [1]. Prior to any chemotherapy, there are no reliable clinical and pathological factors besides clinical stage of disease [1], presence or absence of ureteral obstruction [2], and ultimately rare tumor histologic subtypes such as sarcomatoid variant and small-cell carcinomas [3, 4] that can predict relapse and survival. Clinical tools that determine prognosis and delineate those patients who may or may not need chemotherapy would be of immense value. Other than clinical stage and response to therapy, tumor-specific factors that have the ability to prognostically categorize patients with muscle-invasive TCC into low- and high-risk groups for recurrence and survival, and ultimately, delineate specific therapies, are needed.
Genes and their proteins involved in cell cycle regulation and apoptosis may represent potential prognostic tools. Among the identified cell cycle regulatory molecules, p53, Rb, and p21 (waf1/cip1) have been evaluated most extensively. p53, a tumor suppressor gene, is critical for normal cell growth, development, and programmed cell death. Altered p53 gene protein expression is observed in 50% of all human cancers [5]. p21 (waf1/cip1) exerts positive regulation of p53 leading ultimately to cell cycle arrest [6, 7]. The expression of the Rb, p53, and p21 (waf1/cip1) gene products has been correlated with survival in both superficial and muscle-invasive disease [8–14]. The risk of TCC progression has also been correlated with a prognostic index, i.e. altered expression of >1 protein, such as the cooperative effects of altered expression of p53 and Rb gene proteins [13, 14]. These observations indicate that survival outcomes may be related to the expression of one or more molecular markers.
A recent meta-analysis of 117 studies of superficial and muscle-invasive tumors comprising 10 026 patients evaluated the prognostic role of either p53 gene protein overexpression (96% of the studies) assessed by immunohistochemistry (IHC) or p53 gene mutation [15]. The overall risk of recurrence and progression for those patients with p53 gene protein overexpression were 1.61 [95% confidence interval (CI) 1.22–2.13] and 3.06 (95% CI 1.91–4.92), respectively. In the same meta-analysis, the overall risk of mortality was 1.43 (95% CI 1.21–1.69). This meta-analysis had some limitations including long periods of patient recruitment, patient heterogeneity, different procedures for tissue fixation, different primary antibodies (Abs), and variation in cut points for positive versus negative staining. The correlation of p53 protein status and clinical parameters might also be overestimated as a result of publication and reporting biases. Thus, the authors concluded that prospective multicenter studies with centralized immunohistochemical analysis in a more homogenous patient population are necessary to draw definite conclusions about the role of p53 protein expression to predict prognosis and direct specific bladder-cancer-management strategies [15]. However, it is not known which molecular markers, either alone or in combination, can most reliably predict tumor biology.
In instances where the p53 gene appears intact, regulation of its function by other cell cycle regulation genes may affect tumor biology. Mouse double minute-2 (mdm-2), a gene that is under the transcriptional control of p53, inhibits p53-related antiproliferative effect as part of an autoregulatory loop by binding the transcriptional activation domain of p53 or causing enhanced proteasome-dependent p53 protein degradation [16, 17]. The expression of mdm-2 gene protein can be detected by IHC in superficial and invasive TCC specimens [18–23]. Clinical studies in solid tumors, including breast, ovarian carcinoma, and squamous cell carcinoma of the head and neck [24–26] have demonstrated correlation with mdm-2-altered protein expression and shorter survival. Conversely, no correlation with survival has been evident with cervical and renal cell carcinoma [27, 28]. Several studies seeking associations between mdm-2 and p53 gene products expression and survival in TCC have shown divergent results possibly explained by the inclusion of clinically different patient populations, tumor characteristics, clinical management, or different methodologies to characterize gene expression [18–23].
bcl-2, initially discovered in human B-cell lymphoma, is a proto-oncogene intrinsically involved in the apoptosis cascade [29]. bcl-2 gene protein overexpression in myeloid malignancies is a consequence of the translocation from its normal location at 18q21 to the immunoglobulin-heavy chain locus at 14q32, resulting in increased production of bcl-2 mRNAs and their encoded proteins [30]. bcl-2 belongs to a family of related genes that regulates the apoptotic pathway, with bcl-2 promoting a negative influence. bcl-2 gene protein overexpression is associated with a poor prognosis and aggressive biological behavior in prostate cancer [31], non-small-cell lung cancer [32], and breast cancer [33]. The role of bcl-2 protein gene expression in TCC is controversial. Retrospective studies have demonstrated a correlation between bcl-2 gene protein overexpression (on the basis of a score for the strength of staining, i.e. strong versus weak or negative) and poorer overall survival (OS) in patients with muscle-invasive disease treated with chemoradiotherapy (P = 0.03) [34]. Conversely, one of the largest retrospective series involving 119 patients with superficial or locally advanced disease has shown an unexpected association between bcl-2 protein expression and favorable prognosis in muscle-invasive TCC of the bladder (P = 0.04) [35].
The data of combining these three critical gene proteins involved in both cell cycle regulation and apoptosis and seeking for prognostic associations is scanty and at the same time controversial. In a retrospective study including 119 patients with muscle-invasive TCC, there was an association between co-overexpression of p53/mdm-2 and poor outcome (hazard ratio 0.367; P = 0.04). Conversely, the authors found a favorable association between overexpression of the p53/bcl-2 phenotype and outcome (hazard ratio 3.487; P = 0.01) [35]. Therefore, the role of these three genes proteins either isolated or in combination in delineating prognosis in muscle-invasive TCC remains to be defined.
This study sought to define the incidence of altered patterns of either of the three genes proteins (i.e. mdm-2, p53, and bcl-2) that may be critical to define the biologic behavior and chemotherapy response/resistance in muscle-invasive TCC of the bladder treated with neo-adjuvant chemotherapy followed by locoregional treatment and to examine possible associations between altered expression of mdm-2, p53, and bcl-2, and any associations with clinical outcome. We also sought to evaluate whether proteins involved in cell cycle regulation as well as those involved in regulation of programmed cell death could complement each other in predicting survival.
|
material and methods |
|---|
|
TopAbstractintroductionmaterial and methodsresultsdiscussionReferences |
|---|
patients and treatment characteristics
Tumor specimens were retrospectively obtained from a previously reported study of neo-adjuvant chemotherapy with M-VAC (methotrexate, vinblastine, doxorubicin and cisplatin) followed by definitive locoregional treatment carried out at Memorial Sloan-Kettering Cancer Center (MSKCC) in 111 patients with muscle-invasive, node-negative TCC of the bladder [8]. Fifty-nine of 111 patients were included in this study on the basis of tumor availability. Their initial staging and evaluation included cystoscopy with bimanual exam under anesthesia and transurethral resection of the bladder tumor (TURBT), computed tomography (CT) scan of the abdomen and pelvis, or an abdominal and pelvic ultrasound, and chest X-ray (CXR). All patients had histologically confirmed muscle-invasive TCC of the bladder. Eligibility criteria included no clinical or radiographic evidence of lymph node and distant metastases (N0 M0). Normal hematologic, renal, hepatic, and cardiac functions were also required. All patients had been treated with the M-VAC regimen as previously described [8, 36] for a total of four cycles of therapy.
Staging was defined according to the TNM (tumor–node–metastasis) Staging Classification [37]. Response was assessed using both clinical and pathological staging. Downstaging after chemotherapy required a complete or partial response. In brief, complete response required resolution of disease. Partial response was defined as previously published, with a required reduction of at least two grades of the T stage (T4 
T0-2, or T3 T0-1, or T2 Ta) [31]. Definitive surgery followed neo-adjuvant chemotherapy; radical cystectomy with bilateral pelvic lymph node dissection was routinely recommended. However, since not all patients agreed to radical cystectomy, alternative primary tumor surgical management in patients requesting bladder preservation included partial cystectomy or a secondary TURBT to resect any residual disease. Downstaging analysis after chemotherapy was carried out primarily in patients who underwent a radical or a partial cystectomy and in those who had full-bladder wall evaluation on TURBT, as less intensive clinical evaluations frequently underestimate the true pathological stage after the administration of neo-adjuvant chemotherapy.
All patients were followed every 3 months for the first 2 years. Each visit involved a physical examination, laboratory tests, CT scan of the abdomen and pelvis, and CXR. These studies were extended to 6-month intervals for the following 2 years, and then yearly. For those patients treated with bladder-preservation surgery, follow-up cystoscopies were carried out every 3 months for the first 2 years and less frequently thereafter.
immunohistochemical studies
IHC analysis for p53 protein expression was carried out using standard avidin–biotin technique with mouse monoclonal antibody (mAb) Pab1801 (Ab2, IgG1; Oncogene Science Uniondale, NY). mdm-2 protein expression was assessed by anti-mdm-2 mAb 2A10 (provided by Dr Arnold Levine) using 3T3-BALB/c and 3T3-DM cells [19]. Clone 124, a mouse mAb against human bcl-2, was used to assess for bcl-2 expression (DAKO Corp., Carpenteria, CA). Briefly, 5-µm thick sections were deparaffinized and incubated in 10% normal horse serum for 15 min at room temperature, followed by 2-h incubation with appropriately diluted primary Abs, i.e. 2A10 was used at 1 : 1000 dilution; PAb1801 was used at 200 ng/ml at 4 °C; and Clone 124 was used at 2.5 µ/ml. Sections were immersed in boiling 0.01% citric acid (pH 6.0) for 15 min to enhance antigen retrieval, allowed to cool, and incubated with primary Abs overnight at 40°C. After the sections were washed extensively, they were subsequently incubated for 30 min with biotinylated horse anti-mouse IgG Abs (Vector Laboratories, Burlingame, CA) at the different dilutions according to each specific marker, i.e. 1 : 200 for mdm-2, 1 : 100 for p53, and 1 : 500 for bcl-2; then incubated for 30 min with avidin–biotin peroxidase complexes (1 : 25 dilution; Vector Laboratories). Diaminobenzidine 0.06% was used as the chromogen and 1% modified Harris hematoxylin as the counterstain. Immunoreactivities were classified in two categories: negative (no tumor cells displaying cytoplasmic, granular staining-bcl-2 or no tumor-cell-nuclei staining for p53/mdm-2) and positive (percent tumor cells displaying cytoplasmic, granular staining or tumor-cell-nuclei staining for p53/mdm-2). Approximately 100 cells per slide were evaluated for each of these markers, and the actual number of cells stained with the Ab was reported in deciles. Both histological confirmation of TCC and IHC analysis were carried out by the pathologists at MSKCC.
statistical analysis
All pathologic and IHC studies were completed, analyzed, and recorded blind to clinical information. It is important to emphasize that to circumvent any potential bias secondary to the degree of response to neo-adjuvant chemotherapy, patients' samples were obtained before exposure to chemotherapy during TURBT. It is also important to mention that the IHC studies involving the three molecular markers were carried out after the systemic and locoregional treatment was fully completed. The expression of mdm-2 and bcl-2 protein was analyzed as a continuous variable, and as no difference in survival was observed between the 10% and 20% cut points, the highest value was chosen for tumor cells defined with a high expression for either one of these two markers. A similar cut point, previously described [8, 38], was used to define p53 protein overexpression. Thus, the results were categorized according to the percentage of tumor cell staining for mdm-2, p53, and bcl-2 as follows: no or minimal expression (<20%) or altered expression (20%). The following prognostic variables were examined to determine whether they were significantly associated with OS time: clinical stage (T2 versus >T2), multifocality (yes versus no), papillary disease (yes versus no), ureteral obstruction (yes versus no), palpable mass (yes versus no), de novo presentation (yes versus no), downstaging after chemotherapy (yes versus no), bladder preservation (yes versus no), p53 (<20% versus 20%), mdm-2 (<20% versus 20%), and bcl-2 (<20% versus 20%). The Fisher's exact test was used to determine whether there was any association between the three molecular markers and clinical variables, including pathologic downstaging after neo-adjuvant chemotherapy. The survival interval was defined as the interval between the date of the first TURBT at MSKCC and the date of death or censoring. The causes of death during the first 6 years of follow-up were identified as either cancer- or noncancer related [36], but deaths occurring beyond 6 years of follow-up could not be similarly coded in a reliable fashion. Therefore, we considered the 6-year landmark to specify those patients who died from cancer-related causes rather than unrelated etiologies. The Kaplan–Meier method was used to estimate the survival distributions. Since the follow-up was >10 years and we were specifically interested in events in the first 6 years, we used the Wilcoxon test to detect any significant difference in median survival time according to the molecular and clinical prognostic variables in a univariate analysis.
|
results |
|---|
|
TopAbstractintroductionmaterial and methodsresultsdiscussionReferences |
|---|
A total of 59 patients were evaluated—49 males and 10 females (Table 1). The median follow-up time was 12.7 years. The median survival was 4.2 years. Approximately 90% of the patients presented with a Karnofsky performance status
80%. The median age was 59 years (range 32–78). As expected for patients with muscle-invasive disease, 53 out of 59 patients (90%) presented with high-grade tumors. A total of 16 patients (27%) presented with multifocal tumors, and in 43 cases, the bladder tumor was unifocal. A palpable mass or ureteral obstruction was disclosed in 31% and 22% of the cases, respectively. The bladder was preserved in 34 patients (58%).
|
Pathological response to chemotherapy was available in the 45 patients, 16 of whom had a complete or a partial response. Fourteen patients, most of whom underwent TURBT (eight patients), were not assessable for pathologic response analysis because of insufficient tumor samples or biopsies that did not entirely represent the full bladder wall thickness. Five other patients in whom pathologic response was not available in our database underwent a radical (two patients) or a partial (four patients) cystectomy. Lack of ureteral obstruction (P = 0.01) and pathologic downstaging after M-VAC chemotherapy (P = 0.003) appeared as the only clinical variables associated with prolonged survival in the univariate analysis (Table 2).
|
Altered expression of mdm-2 protein, defined as
20% cells staining positive, was seen in 22 patients (37%) (Table 3). Thirty-five cases (59%) had minimal or no expression, defined as <20% cells staining for mdm-2. Two patients (4%) were not assessable for mdm-2 due to an insufficient specimen available for immunohistochemical staining. No association was found between mdm-2 and tumor stage (P = 0.50), pathologic downstaging after M-VAC chemotherapy (P = 0.55), or p53 status (P = 0.21). The median survival time for patients presenting with tumors with altered mdm-2 protein expression was 2.7 years compared with 4.1 years for patients with tumors that had minimal or no mdm-2 protein expression (P = 0.42) (Table 4) (Figure 1A).
|
|
|
The rationale for the cut point used (
20%) for defining positive or negative p53 staining was previously described by our group and was reproducible across studies [8, 9]. Altered expression of p53 was observed in 54% of the patients. Twenty-six patients presented with no or minimal p53 protein expression (44%) (Table 3). The univariate analysis revealed that p53-altered protein expression was associated with poorer survival compared with those who had no or minimal protein expression (P = 0.006) (Table 4) (Figure 1B). However, no correlation with initial tumor stage (P = 0.34) or pathologic downstaging after M-VAC chemotherapy (P = 0.21) was observed.
Altered expression of bcl-2, defined as 20% cells staining positive, was seen in 27 patients (46%). Thirty-two tumors (54%) had a minimal or negative expression, defined as <20% cells staining for bcl-2 (Table 3). No correlation was found between bcl-2 and tumor stage, downstaging after chemotherapy, or p53 status. The median survival time for patients presenting with altered bcl-2 expression was 3 years compared with 5.2 years for those with minimal or no bcl-2 expression (P = 0.14). This difference was not statistically significant, although the survival curve showed that those with bcl-2 altered expression had a consistently shorter survival (Table 4) (Figure 1C).
We analyzed possible associations between molecular phenotypes and chemotherapy response/resistance. Three groups of patients were identified on the basis of distinct patterns of p53 and mdm-2 expression: both p53 and mdm-2 with altered protein expression; both p53 and mdm-2 with no or minimal altered protein expression; p53 or mdm-2 altered protein expression. There was no correlation between mdm-2/p53 phenotypes and chemotherapy-induced pathologic downstaging (P = 0.77). Similarly, three distinct molecular phenotypes existed for p53 and bcl-2 staining (double positive, double negative, and mixed) and no correlations were found between these phenotypes and pathologic downstaging after neo-adjuvant chemotherapy (P = 0.21). We also sought to analyze for possible associations between the cooperative effects of cell cycle and apoptotic regulatory gene proteins using phenotypes on the basis of mdm-2, p53, and bcl-2 expression and response to neo-adjuvant chemotherapy. Three groups of patients were identified on the basis of distinct patterns of p53, mdm-2, and bcl-2 expression: all three markers with altered protein expression; three markers with no or minimal altered protein expression; and either one or two aberrantly expressed markers. There was no correlation between mdm-2/p53/bcl-2 phenotypes with pathologic downstaging after neo-adjuvant chemotherapy (P = 0.79).
An attempt was made to evaluate the expression of these gene products involved in cell cycles as part of a ‘prognostic index’, i.e. the prognostic ability of a phenotype using p53 and mdm-2 expression (Table 3). Three groups of patients with different 5-year survival rates were identified. Patients in whom both p53 and mdm-2 were altered had a 5-year survival of only 36%. Conversely, prolonged 5-year survival of 53% was observed for patients who presented with no or minimal mdm-2 and p53 protein expression. Patients who had altered expression of either marker presented with an intermediate 5-year survival of 48%. The difference in survival of the three groups was statistically significant (Table 4) (P = 0.046) (Figure 2).
|
Similarly, we evaluated the prognostic ability as a result of the expression of gene products involved in apoptosis pathway using a phenotype containing both p53 and bcl-2 staining (Table 3). Patients in whom both p53 and bcl-2 were altered had a 5-year survival of 33%. Conversely, when neither marker had altered expression, the 5-year survival was 65%. Patients who had altered expression of either marker presented with a 5-year survival of 48%. The difference among groups 1, 2, and 3 was statistically significant (Table 4) (Figure 3).
|
Also assessed were the cooperative effects of cell cycle and apoptotic regulatory gene proteins using phenotypes on the basis of mdm-2, p53, and bcl-2 expression (Table 3). The survival in patients in whom all three markers were normally expressed was more favorable (5-year survival of 54% and a median survival of 9.9 years) in contrast to those patients in whom all three markers were aberrantly expressed (5-year survival of 25% and a median survival of 1.1 years). An intermediate outcome (5-year survival of 49% and a median survival of 4.7 years) was observed for patients with either one or two aberrantly expressed markers. The difference in survival distributions of the three groups was statistically significant (P = 0.01) (Table 4) (Figure 4).
|
|
discussion |
|---|
|
TopAbstractintroductionmaterial and methodsresultsdiscussionReferences |
|---|
This study demonstrates that mdm-2 protein altered expression, defined as
20% cells immunohistochemical staining of the primary tumor, is present in a minority of patients (37%) with muscle-invasive, node-negative TCC of the bladder. The frequency reported is similar to that of previously published studies, in which the frequency of altered mdm-2 gene product expression in invasive TCC ranges from 9% to 61% [19, 22, 39]. For example, Schmitz-Drager et al. [23], using a different mAb (IF-2) in a retrospective study of 92 patients, demonstrated mdm-2 protein overexpression in 18%, 49%, and 22% of carcinoma in situ, T1, and advanced tumors, respectively. Our results are also in accordance with those reported by Lianes et al. [19], who used a similar technique and cut point to define altered mdm-2 protein expression. Differences in expression might be a consequence of the heterogeneity in patient populations and management, different staining techniques, Abs, and cut points used to assess normal versus aberrant expression, thus making any comparison difficult.
bcl-2 protein expression was a frequent finding in high-grade, muscle-invasive TCC evidenced by IHC expression in 46% of primary tumors. This high degree of bcl-2 protein detection was not surprising, as 80% of our study population was high grade and/or had disease extending deeply into the bladder wall or outside the bladder. Other studies have reported an association between greater bcl-2 protein expression, with both higher tumor grade [40, 41] and/or higher-stage disease [41, 42]. Pollack et al. [43] examined a similar cohort of patients with muscle-invasive TCC treated with radiation therapy, and found an almost identical 47% staining with the same Ab used in this study.
In the present study, altered mdm-2 protein expression was not a predictive marker for extent of disease or pathologic downstaging after M-VAC chemotherapy. The lack of association between mdm-2 protein expression and clinical markers that delineate extent of disease is also supported by other studies [21–23]. Previous reports have shown increased frequency of mdm-2 altered protein expression in low-grade and early-stage tumors compared to high-grade and advanced lesions [18, 19, 22], indicating that mdm-2 altered protein expression may be commonly involved in early-TCC tumorigenesis. Since all patients in this study had high-grade, muscle-invasive disease, it is not surprising that no association was observed with stage. Other studies that include high-grade and invasive lesions also failed to show association of mdm-2 protein expression with tumor stage and histology [21, 23].
No association between altered mdm-2 and p53 gene product expression was observed in our patient population. Other studies that included patients with high-grade and invasive lesions also failed to demonstrate an association between both markers [21, 22]. Pfister et al. [44] evaluated both molecular markers in 269 patients with superficial TCC of the bladder, and a positive association between mdm-2 and p53 was found only in low-grade and very early stage lesions. Perhaps the complex interactions of multiple markers precludes association of just two gene products, or simply this study was not robust enough to detect a significant correlation.
Our findings indicate that mdm-2 protein expression may not be an independent marker for delineating DNA aberrations and tumor biology in advanced and high-grade TCC. However, the cooperative effects of mdm-2/p53 phenotypes appeared to be potentially useful in predicting survival time. The outcome was poorer when both markers were aberrantly expressed, in contrast to those with intact expression of either one or both genes (36% versus 48% versus 53%, respectively). The prognostic value of altered expression of both mdm-2 and p53 expression has been confirmed by other studies in both superficial and muscle-invasive TCC [20, 22, 35]. Lianes et al. have postulated a hypothesis to explain the impact of four distinct phenothypes on the basis of p53 and mdm-2 protein expression. A double-negative phenotype, i.e. p53 and mdm-2 with normal or minimal protein expression, may be associated with virus infections such as those caused by human papillomavirus, particularly viral subtypes 16 and 18, or it may also be found in cases with minimal chromosomal changes or genetic instability. Conversely, a double-positive phenotype may be associated with p53 gene mutations, particularly at exon 8, that are capable of maintaining certain functions such as transactivation of the mdm-2 gene leading to mdm-2 protein overexpression and potential inhibition of coexistent wild-type p53 gene. Phenotypes consisting of mdm-2 protein overexpression in conjunction with p53 normal protein expression may be associated with relevant chromosomal changes and microsatellite alterations that would lead to an increase in wild-type p53 protein producing mdm-2 protein overexpression. Wild-type p53 proteins have a very short half-life, thus these products may not be detectable using IHC-based assays; on the other hand, mdm-2 proteins appear to be chemically more stable and thus identifiable. The phenotype consisting of normal mdm-2 protein expression in conjunction with p53 protein overexpression is thought to be associated with specific mutations affecting the p53 gene, such as those occurring at exons 5 and 7, leading to nonactive p53 products not capable of producing transactivation of the mdm-2 gene [19].
bcl-2 protein expression, as an independent marker, was not significantly associated with outcome at the P = 0.05 level. Of substantial interest, however, was the observation that the bcl-2 and p53 status of the primary tumor is potentially complementary in predicting survival. Three cohorts with varying 5-year survival could be distinguished by their bcl-2 and p53 protein expression. Patients with wild-type p53 and minimal or no bcl-2 expression had the most favorable prognosis with a 5-year survival of 65%. In contrast, patients who had both altered p53 and high expression of bcl-2 had markedly inferior 5-year survival of 33%, and those with either altered p53 or high expression of bcl-2 had an intermediate prognosis (5-year survival of 48%). It is of interest that these findings are similar to those previously reported by Bilim et al. [42] in which a similar survival correlation with p53 and bcl-2 status was observed in TCC patients treated with surgery alone. Similar to our findings, different phenotypes on the basis of p53 and bcl-2 protein expression may have implications in tumor behavior. Miyashita et al. [45] demonstrated that p53 induces a decrease in the expression of the apoptosis-suppressing gene bcl-2 in the murine leukemia cell M1, while simultaneously stimulating increases in the expression of bax, a gene that encodes a dominant-inhibitor of the bcl-2 protein. Wang et al. [46] showed that bcl-2 overexpression is probably one of the most important mechanisms by which tumor cells escape p53-mediated apoptosis. A double-positive phenotype, i.e. altered mdm-2 and p53 protein expression, would be expected to have a poor prognosis (as observed in our findings) due to p53 mutations, resulting in an absence of an important inductor of apoptosis in conjunction with bcl-2 overexpression facilitating escape of p53-mediated apoptosis. This concept is supported by the association of p53 and bcl-2 protein overexpression with high-grade lesions [40, 41, 47] and more advanced disease [41, 42, 47, 48]. Conversely, the double-negative phenotype would translate into improved disease-free survival rates, as was also observed in our study.
Most importantly, the complementary effects of cell cycle and apoptotic regulatory gene proteins, including mdm-2, p53, and bcl-2, seemed to be a more powerful prognostic in delineating survival than the phenotypes on the basis of two-gene products or isolated gene proteins. Patients with all markers intact had a favorable outcome, with a 5-year survival of 54% and a median survival of 9.9 years. Conversely, the outcome of patients with all markers aberrantly expressed was poor, with a 5-year survival of 25% and a median survival of 1.1 years. Patients with either one or two markers aberrantly expressed had an intermediate survival outcome.
We could not demonstrate an association between p53 protein expression and chemotherapy sensitivity despite the fact that p53 altered protein expression was associated with poorer survival. p53 correlation with response to treatment is controversial, and studies report conflicting correlations despite a recent meta-analysis indicating that p53 protein status may play a critical role in TCC biology and outcome [15]. For patients receiving bacille Calmette-Guerin therapy, studies report both a higher rate of treatment failure and poorer prognosis for those patients with p53 protein overexpression [49–51]. For patients receiving chemotherapy, Garcial de Muro et al. [52] demonstrated that altered p53 protein expression as well as aberrant p53 and p21 protein expression were independent predictors of decreased survival after neo-adjuvant chemotherapy and bladder preservation. Sarkis et al. [8] reported similar results in patients treated with neo-adjuvant M-VAC followed by surgery. Conversely, in vitro studies have reported that alterations in p53 may lead to increased sensitivity to DNA-damaging agents. Supporting this concept in the clinic, Cote et al. [53] found no benefit of adjuvant chemotherapy in wild-type p53 expression tumors in contrast with those that had altered p53 protein expression. These findings are the basis for a national randomized trial in which patients with organ-confined tumors and altered p53 immunoreactivity are randomized to observation or to three cycles of chemotherapy in order to assess for differences in survival.
mdm-2 expression did not predict chemotherapy sensitivity in our study. Reported data indicate, similar to our findings, that mdm-2 is not a useful marker for predicting chemotherapy response either to neo-adjuvant chemotherapy in locally advanced disease or to palliative chemotherapy in patients with distant metastases or unresectable disease [54]. Conversely, the combination of mdm-2 protein expression with other markers involved in cell regulation and apoptosis such as p21 (waf1/cip1) may predict chemotherapy resistance in breast cancer [55].
The data regarding the role of bcl-2 protein expression as a marker of chemotherapy resistance are limited despite that a preclinical model in which Miyake et al. [56] showed that in vitro and in vivo KoTCC-1 cells transfected with mutant-type bcl-2 and/or p53 exhibited higher resistance to cisplatin treatment. Cells transfected with both mutant-type p53 and bcl-2 had significantly higher resistance than cells transfected with either mutant-type p53 or bcl-2 alone. Clinically, studies by Rodel et al. [57] and Matsumoto et al. [58] were unable to demonstrate an association between the status of bcl-2 protein expression with either complete response or local control after cisplatin radiation for bladder preservation. In the latter study, a bax/bcl-2 ratio had a significant association with the complete response rate (P = 0.0289), indicating that expression of functionally opposite proteins may be a potential surrogate marker for chemotherapy (and radiation) sensitivity [58]. Lastly, Weiss et al. [59] studied patients treated with TURBT followed by chemotherapy for bladder preservation and failed to show an association between the apoptotic index and complete response, local control, and cause-specific survival.
Our study indicates that one marker alone, or multiple phenotypes on the basis of p53/mdm-2, p53/bcl-2, and p53/mdm-2/bcl-2, may not be a sufficiently powerful marker for chemotherapy response/resistance in a small cohort of patients despite the fact that these phenotypes may have a profound effect on intrinsic tumor biology and outcome. Given the large number of candidate markers, a study assessing independent significance will require a greater number of patients than was possible in this study. Future directions may not be restricted to just a few markers nor just protein expression. Gene transcript ‘signatures’ assessed by hierarchical clustering have now been used to predict biological behavior [60]. A study from our center has reported an accuracy rate of 90% to delineate OS for patients with invasive bladder cancer using this methodology. Patients with positive lymph nodes and poor survival were aligned with a particular genetic profile consisting of 174 probes. These results were confirmed by IHC analysis on tissue arrays. Interestingly, the signaling network analyses pointed out the relevance of p53 gene pathway in bladder cancer progression, lymph node metastases, and poor survival as one of the top targets identified in patients with invasive tumors was the p53 activated protein 1.
There are several limitations to this study that should be pointed out. These include: (i) the small sample size; (ii) tissue sample availability of only 59 out of 111 patients for evaluation of the three molecular markers; (iii) the lack of uniform surgical management after systemic chemotherapy; and (iv) the arbitrary choice of the 20% cut point for segregating altered versus nonaltered mdm-2 and bcl-2 expression. The small study size (59 patients for whom tumor samples were available) precluded a multivariate analysis of the prognostic value related to these three molecular markers on survival time. Similarly, we felt it was inappropriate to carry out a multivariate analysis of conventional pathological and clinical parameters and their association with OS. Importantly, one could argue that there was a potential bias related to the conservative surgical management and the availability of only 59 out of 111 patients' tumor samples and therefore that our results might be different if all 111 patients were analyzed. However, this assumption is unlikely as our survival data as well as the correlation between p53 and OS were superimposable among our subset of patients and the prior publications including all 111 patients and those managed with partial rather than complete cystectomy after neo-adjuvant chemotherapy [8, 36, 61]. Cut points for immunohistochemical correlations are controversial in p53 clinical correlation studies. However, this definition of altered staining characteristics of tumors has been previously correlated with TP53 mutations by our group, and therefore provided the basis for our choice of the 20% cut point [38]. Not surprisingly, no consensus exists for the 20% cut point used to define mdm-2 and bcl-2 altered expression, although several studies that used a similar cut point to define altered expression have shown correlations between mdm-2 expression and prognosis [19].
In summary, our results should be interpreted—not as definitive—but as hypothesis generating. Future studies of these markers, including a larger sample size, will be needed to clarify the optimal cut points to discriminate survival. They should be designed prospectively to ensure optimal characterization such as the Cancer and Leukemia Group B study (CALGB 90104) that was designed to prospectively validate the three markers from this study. Only through hypothesis testing of putative markers can the oncology investigative community provide the necessary foundation for the use of such markers in clarifying prognosis and developing clinical strategies to specifically target the gene abnormalities responsible for tumor biology, such as antisense oligonucleotides against mdm-2 [62, 63] or anti-bcl-2 antisense [64–66].
Received for publication March 30, 2006. Revision received July 3, 2006. Accepted for publication July 4, 2006.
|
References |
|---|
|
TopAbstractintroductionmaterial and methodsresultsdiscussionReferences |
|---|
1. Grossman HB, Natale RB, Tangen CM, et al. (2003) Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. N Engl J Med 349:859–866.
2. Leibovitch I, Ben-Chaim J, Ramon J, et al. (1993) The significance of ureteral obstruction in invasive transitional cell carcinoma of the urinary bladder. J Surg Oncol 52:31–35.[Web of Science][Medline]
3. Dahm P and Gschwend JE. (2003) Malignant non-urothelial neoplasms of the urinary bladder: a review. Eur Urol 44:672–681.[CrossRef][Web of Science][Medline]
4. Cheng L, Pan CX, Yang XJ, et al. (2004) Small cell carcinoma of the urinary bladder: a clinicopathologic analysis of 64 patients. Cancer 101:957–962.[CrossRef][Web of Science][Medline]
5. Jr Kaelin WG. (1999) The emerging p53 gene family. J Natl Cancer Inst 91:594–598.
6. Ferreira CG, Tolis C, Giaccone G. (1999) p53 and chemosensitivity. Ann Oncol 10:1011–1021.
7. Cayrol C, Knibiehler M, Ducommun B. (1998) p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells. Oncogene 16:311–320.[CrossRef][Web of Science][Medline]
8. Sarkis A, Bajorin D, Reuter V, et al. (1995) Prognostic value of p53 nuclear overexpression in patients with invasive bladder cancer treated with neoadjuvant MVAC. J Clin Oncol 13:1384–1390.[Abstract]
9. Sarkis AS, Dalbagni G, Cordon-Cardo C, et al. (1993) Nuclear overexpression of p53 protein in transitional cell bladder carcinoma: a marker for disease progression. J Natl Cancer Inst 85:53–59.
10. Stein JP, Ginsberg DA, Grossfeld GD, et al. (1998) Effect of p21WAF1/CIP1 expression on tumor progression in bladder cancer. J Natl Cancer Inst 90:1072–1079.
11. Logothetis CJ, Xu H, Ro JY, et al. (1992) Altered expression of retinoblastoma protein and known prognostic variables in locally advanced bladder cancer. J Natl Cancer Inst 84:1256–1261.
12. Cordon-Cardo C, Wartinger D, Petrylak D, et al. (1992) Altered expression of the retinoblastoma gene product: prognostic indicator in bladder cancer. J Natl Cancer Inst 84:1251–1256.
13. Cordon-Cardo C, Zhang Z-F, Dalbagni G, et al. (1997) Cooperative effects of 53 and pRb alterations in primary superficial bladder tumors. Cancer Res 57:1217–1221.
14. Chatterjee SJ, Datar R, Youssefzadeh D, et al. (2004) Combined effects of p53, p21, and pRb expression in the progression of bladder transitional cell carcinoma. J Clin Oncol 22:1007–1013.
15. Malats N, Bustos A, Nascimento CM, et al. (2005) P53 as a prognostic marker for bladder cancer: a meta-analysis and review. Lancet Oncol 6:678–686.[Medline]
16. Haupt Y, Maya R, Kazaz A, Oren M. (1997) Mdm2 promotes the rapid degradation of p53. Nature 387:296–299.[CrossRef][Medline]
17. Kubbutat MH, Jones SN, Vousden KH. (1997) Regulation of p53 stability by Mdm2. Nature 387:299–303.[CrossRef][Medline]
18. Pfister C, Moore L, Allard P, et al. (1999) Predictive value of cell cycle markers p53, MDM2, p21, and Ki-67 in superficial bladder tumor recurrence. Clin Cancer Res 5:4079–4084.
19. Lianes P, Orlow I, Zhang ZF, et al. (1994) Altered patterns of MDM2 and TP53 expression in human bladder cancer. J Natl Cancer Inst 86:1325–1330.
20. Barbareschi M, Girlando S, Fellin G, et al. (1995) Expression of mdm-2 and p53 protein in transitional cell carcinoma. Urol Res 22:349–352.[CrossRef][Web of Science][Medline]
21. Shiina H, Igawa M, Shigeno K, et al. (1999) Clinical significance of mdm2 and p53 expression in bladder cancer. A comparison with cell proliferation and apoptosis. Oncology 56:239–247.[CrossRef][Web of Science][Medline]
22. Jahnson S and Karlsson MG. (2000) Tumor mapping of regional immunostaining for p21, p53, and mdm2 in locally advanced bladder carcinoma. Cancer 89:619–629.[CrossRef][Web of Science][Medline]
23. Schmitz-Drager BJ, Kushima M, Goebell P, et al. (1997) p53 and MDM2 in the development and progression of bladder cancer. Eur Urol 32:487–493.[Web of Science][Medline]
24. Jiang M, Shao ZM, Wu J, et al. (1997) p21/waf1/cip1 and mdm-2 expression in breast carcinoma patients as related to prognosis. Int J Cancer 74:529–534.[CrossRef][Web of Science][Medline]
25. Tanner B, Hengstler JG, Laubscher S, et al. (1997) mdm 2 mRNA expression is associated with survival in ovarian cancer. Int J Cancer 74:438–442.[CrossRef][Web of Science][Medline]
26. Osman I, Sherman E, Singh B, et al. (2002) Alteration of p53 pathway in squamous cell carcinoma of the head and neck: impact on treatment outcome in patients treated with larynx preservation intent. J Clin Oncol 20:2980–2987.
27. Dellas A, Schultheiss E, Almendral AC, et al. (1997) Altered expression of mdm-2 and its association with p53 protein status, tumor-cell-proliferation rate and prognosis in cervical neoplasia. Int J Cancer 74:421–425.[CrossRef][Web of Science][Medline]
28. Moch H, Sauter G, Gasser TC, et al. (1997) p53 protein expression but not mdm-2 protein expression is associated with rapid tumor cell proliferation and prognosis in renal cell carcinoma. Urol Res 25:(Suppl 1)S25–S30.[Medline]
29. Hockenbery D, Nunez G, Milliman C, et al. (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348:334–336.[CrossRef][Medline]
30. Nowell PC and Croce CM. (1990) Philip Levine award lecture. Chromosome translocations and oncogenes in human lymphoid tumors. Am J Clin Pathol 94:229–237.[Web of Science][Medline]
31. McDonnell TJ, Troncoso P, Brisbay SM, et al. (1992) Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res 52:6940–6944.
32. Pezzella F, Turley H, Kuzu I, et al. (1993) bcl-2 protein in non-small-cell lung carcinoma. N Engl J Med 329:690–694.
33. Neri A, Marrelli D, Roviello F, et al. Bcl-2 expression correlates with lymphovascular invasion and long-term prognosis in breast cancer. Breast Cancer Res Treat 200:6 In press.
34. Hussain SA, Ganesan R, Hiller L, et al. (2003) BCL2 expression predicts survival in patients receiving synchronous chemoradiotherapy in advanced transitional cell carcinoma of the bladder. Oncol Rep 10:571–576.[Web of Science][Medline]
35. Uchida T, Minei S, Gao JP, et al. (2002) Clinical significance of p53, MDM2 and bcl-2 expression in transitional cell carcinoma of the bladder. Oncol Rep 9:253–259.[Web of Science][Medline]
36. Schultz P, Herr HW, Zhang ZF, et al. (1994) Neoadjuvant chemotherapy for invasive bladder cancer: prognostic factors for survival of patients treated with M-VAC with 5-year follow-up. J Clin Oncol 12:1394–1401.[Abstract]
37. TNM classification of malignant tumors. (1987) International Union Against Cancer. 4th edition (SpringerIn Hermanek P and Sobin LH (Eds.). , New York)143.
38. Cordon-Cardo C, Dalbagni G, Saez GT, et al. (1994) p53 mutations in human bladder cancer: genotypic versus phenotypic patterns. Int J Cancer 56:347–353.[Web of Science][Medline]
39. Ow K, Delprado W, Fisher R, et al. (2000) Relationship between expression of the KAI1 metastasis suppressor and other markers of advanced bladder cancer. J Pathol 191:39–47.[CrossRef][Web of Science][Medline]
40. Ye D, Li H, Qian S, et al. (1998) bcl-2/bax expression and p53 gene status in human bladder cancer: relationship to early recurrence with intravesical chemotherapy after resection. J Urol 160:2025–2029.[CrossRef][Web of Science][Medline]
41. Lipponen PK, Aaltomaa S, Eskelinen M. (1996) Expression of the apoptosis suppressing bcl-2 protein in transitional cell bladder tumors. Histopathology 28:135–140.[CrossRef][Web of Science][Medline]
42. Bilim V, Tomita Y, Kawasaki T, et al. (1996) Prognostic value of Bcl-2 and p53 expression in urinary tract transitional cell cancer. J Natl Cancer Inst 88:686–688.[Web of Science][Medline]
43. Pollack A, Wu C, Czerniak B, et al. (1997) Abnormal bcl-2 and pRb expression are independent correlates of radiation response in muscle-invasive bladder cancer. Clin Cancer Res 3:1823–1829.[Abstract]
44. Pfister C, Larue H, Moore L, et al. (2000) Tumorigenic pathways in low-stage bladder cancer based on p53, MDM2 and p21 phenotypes. Int J Cancer 89:100–104.[CrossRef][Web of Science][Medline]
45. Miyashita T, Krajewski S, Krajewska M, et al. (1994) Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9:1799–1805.[Web of Science][Medline]
46. Wang Y, Szekely L, Okan I, et al. (1993) Wild-type p53-triggered apoptosis is inhibited by bcl-2 in a v-myc-induced T-cell lymphoma cell line. Oncogene 8:3427–3431.[Web of Science][Medline]
47. Esrig D, Spruck CH III, Nichols PW, et al. (1993) p53 nuclear protein accumulation correlates with mutations in the p53 gene, tumor grade, and stage in bladder cancer. Am J Pathol 143:1389–1397.[Abstract]
48. Moch H, Sauter G, Moore D, et al. (1993) p53 and erbB-2 protein overexpression are associated with early invasion and metastasis in bladder cancer. Virchows Arch 423:329–334.
49. Lacombe L, Dalbagni G, Zhang ZF, et al. (1996) Overexpression of p53 protein in a high-risk population of patients with superficial bladder cancer before and after bacillus Calmette-Guerin therapy: correlation to clinical outcome. J Clin Oncol 14:2646–2652.
50. Caliskan M, Turkeri LN, Mansuroglu B, et al. (1997) Nuclear accumulation of mutant p53 protein: a possible predictor of failure of intravesical therapy in bladder cancer. Br J Urol 79:373–377.[Web of Science][Medline]
51. Pfister C, Flaman JM, Dunet F, et al. (1999) P53 mutations in bladder tumors inactivate the transactivation of the p21 and Bax genes and have a predictive value for the clinical outcome after bacillus Calmette-Guerin therapy. J Urol 162:69–73.[CrossRef][Web of Science][Medline]
52. del Muro Garcia X, Condom E, Vigues F, et al. (2004) p53 and p21 expression levels predict organ preservation and survival in invasive bladder carcinoma treated with a combined-modality approach. Cancer 100:1859–1867.[CrossRef][Web of Science][Medline]
53. Cote RJ, Esrig D, Groshen S, et al. (1997) p53 and treatment of bladder cancer. Nature 385:123–125.[Medline]
54. Kakehi Y, Ozdemir E, Habuchi T, et al. (1998) Absence of p53 overexpression and favorable response to cisplatin-based neoadjuvant chemotherapy in urothelial carcinomas. Jpn J Cancer Res 89:214–220.[CrossRef][Web of Science]
55. Sjostrom J, Blomqvist C, Heikkila P, et al. (2000) Predictive value of p53, mdm-2, p21, and mib-1 for chemotherapy response in advanced breast cancer. Clin Cancer Res 6:3103–3110.
56. Miyake H, Hara I, Yamanaka K, et al. (1999) Synergistic enhancement of resistance to cisplatin in human bladder cancer cells by overexpression of mutant-type p53 and Bcl-2. J Urol 162:2176–2181.[CrossRef][Web of Science][Medline]
57. Rodel C, Grabenbauer GG, Rodel F, et al. (2000) Apoptosis, p53, bcl-2, and Ki-67 in invasive bladder carcinoma: possible predictors for response to radiochemotherapy and successful bladder preservation. Int J Radiat Oncol Biol Phys 46:1213–1221.[CrossRef][Web of Science][Medline]
58. Matsumoto H, Wada T, Fukunaga K, et al. (2004) Bax to Bcl-2 ratio and Ki-67 index are useful predictors of neoadjuvant chemoradiation therapy in bladder cancer. Jpn J Clin Oncol 34:124–130.
59. Weiss C, Rodel F, Wolf I, et al. (2005) Combined-modality treatment and organ preservation in bladder cancer. Do molecular markers predict outcome? Strahlenther Onkol 181:213–222.
60. Sanchez-Carbayo M, Socci ND, Lozano J, et al. (2006) Defining molecular profiles of poor outcome in patients with invasive bladder cancer using oligonucleotide microarrays. J Clin Oncol 24:778–789.
61. Herr HW, Bajorin DF, Scher HI. (1998) Neoadjuvant chemotherapy and bladder sparing surgery for invasive bladder cancer: ten-year outcome. J Clin Oncol 16:1298–1301.
62. Wang H, Zeng X, Oliver P, et al. (1999) MDM2 oncogene as a target for cancer therapy: an antisense approach. Int J Oncol 15:653–660.[Web of Science][Medline]
63. Sato N, Mizumoto K, Maehara N, et al. (2000) Enhancement of drug-induced apoptosis by antisense oligodeoxynucleotides targeted against Mdm2 and p21WAF1/CIP1. Anticancer Res 20:837–842.[Web of Science][Medline]
64. Miyake H, Monia BP, Gleave ME. (2000) Inhibition of progression to androgen-independence by combined adjuvant treatment with antisense BCL-XL and antisense Bcl-2 oligonucleotides plus taxol after castration in the Shionogi tumor model. Int J Cancer 86:855–862.[CrossRef][Web of Science][Medline]
65. Gleave M, Tolcher A, Miyake H, et al. (1999) Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumor model. Clin Cancer Res 5:2891–2898.
66. Schaaf A, Sagi S, Langbein S, et al. (2004) Cytotoxicity of cisplatin in bladder cancer is significantly enhanced by application of bcl-2 antisense oligonucleotides. Urol Oncol 22:188–192.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Veerakumarasivam, H. E. Scott, S.-F. Chin, A. Warren, M. J. Wallard, D. Grimmer, K. Ichimura, C. Caldas, V. P. Collins, D. E. Neal, et al. High-Resolution Array-Based Comparative Genomic Hybridization of Bladder Cancers Identifies Mouse Double Minute 4 (MDM4) as an Amplification Target Exclusive of MDM2 and TP53 Clin. Cancer Res., May 1, 2008; 14(9): 2527 - 2534. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||