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© 2003 American Society for Clinical Oncology Molecular Grading of Urothelial Cell Carcinoma With Fibroblast Growth Factor Receptor 3 and MIB-1 is Superior to Pathologic Grade for the Prediction of Clinical Outcome
From the Department of Pathology, Josephine Nefkens Institute, Erasmus University; the Department of Urology, Erasmus University and University Hospital; the Departments of Urology and Pathology, Sint Franciscus Gasthuis, Rotterdam; Department of Pathology, Westeinde Hospital, The Hague, The Netherlands; Laboratoire de Morphogenèse Cellulaire et Progression Tumorale, Institut Curie, Paris; and Service d’Urologie, Centre Hospitalier Universitaire Henri Mondor, Créteil, France. Address reprint requests to Ellen C. Zwarthoff, PhD, Department of Pathology, Josephine Nefkens Institute, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands; email: e.zwarthoff{at}erasmusmc.nl.
Purpose: Fibroblast growth factor receptor 3 (FGFR3) mutations were recently found at a high frequency in well-differentiated urothelial cell carcinoma (UCC). We investigated the relationship between FGFR3 status and three molecular markers (MIB-1, P53, and P27kip1) associated with worse prognosis and determined the reproducibility of pathologic grade and molecular variables. Patients and Methods: In this multicenter study, we included 286 patients with primary (first diagnosis) UCC. The histologic slides were reviewed. FGFR3 status was examined by polymerase chain reaction–single strand conformation polymorphism and sequencing. Expression levels of MIB-1, P53, and P27kip1 were determined by immunohistochemistry. Mean follow-up was 5.5 years (range, 0.4 to 18.4 years). Results: FGFR3 mutations were detected in 172 (60%) of 286 UCCs. Grade 1 tumors had an FGFR3 mutation in 88% of patient samples and grade 3 tumors in 16% of patient samples. Conversely, aberrant expression patterns of MIB-1, P53, and P27kip1 were seen in 5%, 2%, and 3% of grade 1 tumors and in 85%, 60%, and 56% of grade 3 tumors, respectively. In multivariate analysis with recurrence rate, progression, and disease-specific survival as end points, the combination of FGFR3 and MIB-1 proved independently significant in all three cases. By using these two molecular markers, three molecular grades (mGs) could be identified: mG1 (mutation; normal expression), favorable prognosis; mG2 (two remaining combinations), intermediate prognosis; and mG3 (no mutation; high expression), poor prognosis. The molecular variables were more reproducible than pathologic grade (85% to 100% v 47% to 61%). Conclusion: The FGFR3 mutation represents the favorable molecular pathway of UCC. Molecular grading provides a new, simple, and highly reproducible tool for clinical decision making in UCC patients.
UROTHELIAL CELL carcinoma (UCC) of the urinary bladder is a serious health problem. More than 260,000 cases are diagnosed worldwide each year, accounting for 3.2% of all new cancer cases.1 For 2002, 54,000 new cases and 12,000 deaths predicted for the United States make UCC the fifth most common cancer and the ninth leading cause of cancer death.2 The patients can be divided into two groups for clinical management, depending on the pathologic stage. In most patients, UCC is superficial (ie, pTa to pT1) at first presentation. After transurethral resection, these patients are intensively monitored by cystoscopy because up to 70% experience one or more recurrences and 10% to 15% will progress to invasive, potentially lethal UCC.3–5 Patients with intermediate or high-risk superficial UCC usually receive adjuvant intravesical instillations with, in most cases, bacille Calmette-Guérin.6,7 Cystectomy, radiotherapy, and systemic chemotherapy are the preferred treatment options in case of invasive (ie,  pT2) disease, depending on the presence of metastasis and the patient’s comorbidity and life expectancy. Clinical and pathologic variables for prediction of patient prognosis have been studied extensively.3,5,8 Although variability in pathologic assessment is a recognized problem,9,10 it is still one of the best predictors of prognosis. Research efforts of the last decades resulted in a long list of molecular investigations for a possible better assessment of UCC prognosis. Oncogenes, tumor suppressor genes and associated cell-cycle proteins, proliferation antigens, growth factors, cell adhesion molecules, and chromosomal alterations have been identified in UCC.11–21 Of these, the p53 gene is the most investigated molecular marker.12 Some molecular markers hold considerable promise to assess aggressive UCC behavior (eg, P53, RB, P27kip1, P21, Ki-67/MIB-1, and E-cadherin). Only a few studies have analyzed the performance of multiple molecular markers. Analogous to clinical and pathologic indices, combinations lead to a more tailored prediction of prognosis.3,8,11,16,18 Nevertheless, the value of molecular markers over clinicopathologic indices is still being questioned, and their clinical use is limited.12–15 Point mutations in the human fibroblast growth factor receptor 3 (FGFR3) gene are well documented in inherited skeletal anomalies, such as achondroplasia and thanatophoric dysplasia, that are associated in most cases with dwarfism.22–24 In addition, an oncogenic role has been proposed for mutant FGFR3.25,26 Recently, FGFR3 mutations were found in more than 40% of UCC patients,27–29 whereas a low frequency was observed in multiple myeloma30 and cervical carcinoma patients.31 Surprisingly, FGFR3 mutations in UCC were related to favorable disease: 84% of pTa grade 1 tumors had a mutation, as compared with 7% of pT2 grade 3 or higher tumors.29 Moreover, in a prospective study with a 1-year follow-up, we showed that FGFR3 mutations were associated with a low recurrence rate of superficial UCC.28 However, so far, nothing is known about the FGFR3 mutation in relation to long-term clinical follow-up or other molecular markers. On the basis of these initial observations, we designed this multicenter study to compare, in a large patient group, the relationships among three immunohistochemical markers associated with worse UCC prognosis: the proliferation marker MIB-1, P53 nuclear overexpression, reduced expression of the cell-cycle inhibitor P27kip1, and the FGFR3 mutation. In addition, we investigated the reproducibility of pathologic grading and molecular variables in the same series.
Patients This study consisted of 286 patients with papillary UCC. They were 23 to 90 years old (mean, 65.7 ± 12.0 years). None of them had a hereditary skeletal disorder documented. The patient and tumor characteristics at first diagnosis are listed in Table 1  .32–34 Of the 286 patients, 246 (86%) had superficial (ie, pTa or pT1) UCC. The patients were seen at the urology departments of the University Hospital Rotterdam (n = 139), at the Sint Franciscus Gasthuis (n = 121), or at one of three urology departments in the Rotterdam (the Netherlands) surroundings (n = 28). The medical-ethical committee of the Erasmus University and the University Hospital Rotterdam approved the study (MEC 168.922/1998/55). A paraffin-embedded, formalin-fixed tissue block was obtained. This was freshly cut into 4-µm-thick sections and mounted on amino alkyl silane–coated glass slides for immunohistochemistry. Standard hematoxylin and eosin slides were made from the last cut section. These slides were reviewed by a specialized genitourinary pathologist (T.H.v.d.K.) with the criteria of the 1998 World Health Organization/International Society of Urological Pathology classification system.34 Furthermore, two additional pathologists with a special interest in uropathology (A.C.J. and E.C.M.O.) graded 210 slides for reproducibility purposes. Fifty UCCs from the archive served as a teaching set to facilitate agreement on grading parameters.34 In case of multifocality, the papillary lesion with the highest grade or stage was taken. The largest tumor was taken if grade and stage were the same for multiple UCCs.
Follow-Up Clinical information and follow-up data were collected by chart review. Disease-specific survival was determined for all patients. For patients with primary superficial UCC, recurrence, recurrence rate, and progression were additional end points. Recurrence rate was defined as the number of histologically proven recurrences divided by years of follow-up. Hence, the recurrence rate takes into account the clinical course of patients during a longer interval and does not cover only the time to first recurrence. Progression was defined as development of invasive (ie, pT2) UCC. Mean follow-up was 5.5 years (range, 0.4 to 18.4 years) for all patients and was 5.9 years (range, 0.8 to 18.4 years) for patients with superficial UCC. After superficial UCC, 153 (62%) patients experienced one or more recurrences. The median time to first recurrence was 1.0 years (range, 0.2 to 9.7 years). An upper-tract (ureter or renal pelvis) recurrence was found in 15 (5.2%) patients. Sixty patients had a recurrence rate of more than 0.50 per year. Progression was found in 28 (11.4%) patients. Median time to progression was 3.1 years (range, 0.3 to 14.3 years). Twenty-six (9.1%) patients died as a result of UCC. Fifteen of these were initially diagnosed with superficial UCC. Median time to death was 3.8 years (range, 0.4 to 14.2 years). During follow-up, of the 160 patients who received intravesical treatment, 101 patients received bacille Calmette-Guérin. A cystectomy was performed in 22 patients. Eleven of these cystectomies were performed after progressive disease. Twenty-nine patients (of whom 22 were treated with curative intent) were treated by radiotherapy and 14 patients received systemic chemotherapy for metastatic UCC. The patients were censored at their last visit to the urology department or at the date of their death.
Immunohistochemistry
FGFR3 Mutation Analysis
Statistical Analysis
We studied the performance of four molecular markers in a series of 286 primary UCC FGFR3 mutations. We detected activating FGFR3 mutations in 172 (60%) of the 286 tumors. In the 246 superficial UCCs, 164 (67%) tumors had a mutation in the FGFR3 gene. Examples of these mutations determined by PCR-SSCP and sequencing are shown in Fig 1 . The mutations were found in six different codons (Table 2). In three patient samples, two concurrent mutations were found in one tumor. The mutation S249C accounted for 120 (69%) of the 175 mutations. We found no difference between S249C and other FGFR3 mutations in relation to clinical data (not shown). No activating FGFR3 mutations were found in 75 matched normal (blood) DNA samples or in normal tissue; this confirms the somatic nature of FGFR3 mutations in UCC.
Molecular Markers and Histopathology Examples of normal and aberrant expression of the three immunohistochemical markers are shown in Fig 2 . Table 3 shows the correlation of the molecular markers and histopathologic data. The highest percentage (88%) of FGFR3 mutations was seen in grade 1 tumors and the lowest percentage (16%) was found in grade 3 tumors. Conversely, aberrant expression patterns of MIB-1, P53, and P27kip1 were seen in 5%, 2%, and 3% of grade 1 tumors and in 85%, 60%, and 56% of grade 3 tumors, respectively. Therefore, the FGFR3 mutation is generally associated with a favorable histopathologic diagnosis, whereas aberrant expression of MIB-1, P53, and P27kip1 indicates disease with an unfavorable prognosis. When we compared the performance of the molecular markers in the 22 patients who underwent cystectomy, we retrospectively observed aberrant MIB-1 expression in all 22 patients, whereas an FGFR3 mutation was observed in only three of these patients. Combing two UCC-related molecular features—FGFR3 mutation and high MIB-1 expression—we could define 249 (87%) tumors. This combination defined the highest percentage of UCC.
A Molecular Grading Model for Prediction of Prognosis The first end point of interest was the prediction of disease progression for patients with superficial UCC. The corresponding Kaplan-Meier analyses for the performance of the four molecular markers are shown in Fig 3A through D. This figure illustrates, analogous to the histopathologic data in Table 3 , that UCC patients with an FGFR3 mutation have a significantly better prognosis than those without the FGFR3 mutation; the opposite is true for patients with aberrant expression of an immunohistochemical marker. These results prompted us to perform multivariate analysis for the prediction of progression with the single variables and combinations of variables listed in Table 4. The combination FGFR3/MIB-1 (P < .001) and the clinicopathologic index (P = .007) proved to be independent predictors of progression. Additional details on multivariate analyses are explained below. Figure 4A shows the Kaplan-Meier plot for the combination FGFR3/MIB-1. The difference in behavior was evident between the groups FGFR3 mutation/MIB-1 normal (n = 138) and FGFR3 wild-type/MIB-1 high (n = 47). It is interesting to note that the 61 remaining patients with tumors characterized by no FGFR3 mutation and normal MIB-1 expression or by an FGFR3 mutation and high MIB-1 expression behaved more or less the same, representing an intermediate prognosis with regard to disease progression. These results from Fig 4A led us to propose a molecular grading model for UCC that distinguishes three molecular grades: mG1 (favorable prognosis), mG2 (intermediate prognosis), and mG3 (poor prognosis), as shown in Fig 4B. This figure illustrates the different disease courses for the various molecular grades. The combinations of FGFR3 status with P53 and P27kip1 expression were significant predictors only in univariate analysis (not shown).
Multivariate Analyses Illustrate the Value of Molecular Grade We also performed multivariate analyses for the other study end points: recurrence, recurrence rate, and disease-specific survival. Table 5 shows the significant predictors in multivariate analysis for patients with superficial UCC. In addition to progression, the molecular grade was also an independent predictor for recurrence rate and disease-specific survival. The recurrence rate per year calculated for the independent variables (molecular and morphologic grade) is shown in Fig 5. Table 6 lists the significant predictors in multivariate analysis for all patients (N = 286) relating to disease-specific survival. Figure 6 shows the Kaplan-Meier plots for the independent predictors of disease-specific survival (pathologic stage and molecular grade). In addition, we investigated the possible influence of treatment variables (intravesical treatment for progression and cystectomy for disease-specific survival). When we added these covariates to the multivariate analyses, they were not significant. To clarify further the relation between the molecular grading and disease-specific survival as depicted in Table 6 and Fig 6B, we calculated the absolute numbers and time to death for patients in each molecular grade. Of the patients with mG1, two patients (1.4%) died as a result of UCC. Nine patients (13%) with mG2 and 15 patients (20%) with mG3 died as a result of UCC. The median time to death was 5.1, 6.2, and 2.3 years for mG1, mG2, and mG3, respectively. These data indicated two conclusions. First, the molecular variables were at least as predictive as clinicopathologic indices, and second, combinations of variables provided a more accurate prediction of prognosis than single variables.
FGFR3 Status and CIS The presence of CIS next to a papillary UCC generally indicates a worse prognosis. Therefore, we analyzed the incidence of FGFR3 mutations in patients with associated CIS. In the 19 patients with associated CIS, only five mutations were found (P = .003; two-sided Fisher’s exact test). In addition, only 14 mutations were detected in the 42 patients who had associated CIS with their primary UCC or who developed CIS during follow-up (P < .001). Taken together, FGFR3 mutations were, as expected, not frequent in UCC patients with associated CIS. These data are in accord with the absence of FGFR3 mutations in CIS itself.29
Reproducibility
Intense research efforts are being made to identify and characterize molecular markers for various malignancies. However, the value of these markers compared with traditional clinicopathologic indices for diagnosis and prognosis may still be questioned. This study shows that molecular markers can be used to assess patients’ prognosis for UCC more accurately and reproducibly than assessment with traditional clinicopathologic indices. In this respect, the identification of the FGFR3 mutation as a selective marker for favorable disease is of major importance. Multiple end points were determined for the prediction of prognosis in our series of patients. First, definitive cure reflected by a long recurrence-free follow-up is most likely to be achieved in patients with solitary tumors, as reported previously.5 The independent predictors for assessment of the biologic behavior of superficial UCC reflected by recurrence rate and disease progression were the molecular grade, the clinicopathologic index, and the morphologic grade, assessed by a single expert pathologist. For disease-specific survival, a longer follow-up may be needed, and radical treatment (cystectomy) may give an additional bias. Nevertheless, the molecular grade (FGFR3/MIB-1) again proved to be independently significant. The data obtained from multivariate analyses indicated that the molecular variables were at least as predictive as clinicopathologic indices and that combinations provided a more accurate prediction of prognosis than single variables. We were surprised to find that patients with aberrant expression of MIB-1, P53, or P27kip1 had a better prognosis when a coexisting FGFR3 mutation was present in their UCC; that is, the presence of an FGFR3 mutation was protective. Our finding that these patients had a similar prognosis as patients with no FGFR3 mutation and normal expression of these proteins made it feasible to combine these groups and to propose a molecular grading model for UCC on the basis of three molecular grades (mG1 to mG3). Because the combination of FGFR3 and MIB-1 proved, in contrast to other molecular combinations, to be of independent value for several of our study end points in multivariate analyses, we advocate this combination to be used for further validation of the molecular grade in clinical settings. The poor reproducibility of pathologic stage and grade is a recognized problem and a major concern for clinicians. Grade assessment may differ in 40% to 50% of cases.9,10 Our study confirmed this high interobserver variability. We also investigated the molecular markers and clearly demonstrated their superior reproducibility. To our knowledge, this study is the first to compare the reproducibility of pathologic grading and molecular variables in the same series of patients. McShane et al36 performed an elaborate study that addressed the reproducibility of P53 immunohistochemistry. They found a 91% agreement, which is comparable with our result of 88%. Furthermore, interobserver variability seems not to be an issue for the FGFR3 mutation because this analysis seemed highly reproducible, with 100% concordance. Moreover, the identification of FGFR3 mutations was a simple procedure, and more sophisticated and faster ways for detecting single nucleotide changes recently have been developed.37 With our method, the molecular grade may be provided in the same time frame that is needed for a routine pathologic examination. The intermediate grade 2 tumors are often bothersome for clinical decision making. Therefore, an additional advantage of molecular grade compared with morphologic grade was the relative low number of grade 2 tumors; that is, 24% mG2 versus 44% grade 2.33 In conclusion, molecular grading composed of three molecular grades is more reproducible than pathologic assessment.
FGFR3 mutations occur in 60% of primary UCC, and their presence was generally linked to a favorable disease course. As expected from previous studies, aberrant expression of MIB-1, P53, or P27kip1 indicated unfavorable disease characteristics.11,16–20 By combining the favorable FGFR3 mutation with the unfavorable high MIB-1 expression, 87% of UCC could be characterized. Only 12% of these UCC were positive for both markers. This supports a model in which the FGFR3 mutation and the MIB-1 marker define two pathways of UCC pathogenesis, covering almost 90% of the patients (Fig 7
From the molecular point of view, the FGFR3 mutation and its apparent positive identification of low-risk patients is also intriguing. It has been shown that mutant FGFR3 can transform NIH 3T3 cells when targeted to the cell membrane and that these cells are tumorigenic in nude mice.44,45 Chesi et al45 showed that, in multiple myeloma, FGFR3 and RAS mutations are mutually exclusive and that both mutations lead to activation of the same downstream pathway (mitogen-activated protein kinase). Thus, it would be interesting to analyze RAS mutations and FGFR3 mutations in the same UCC. So far, the frequency of RAS mutations in UCC has been controversial.21 Assuming that there is a link between favorable UCC and RAS mutations, some reported studies might have included too many patients with advanced disease.21 In addition to RAS mutations, it would be interesting to analyze other molecules of the FGFR3 signal-transduction pathway. The identification of the FGFR3 mutation as the representative of the favorable pathway in this series of 286 primary UCCs is of major importance for patients, clinicians, and scientists. Before this mutation and its apparent positive identification of low-risk patients were noted, only molecular indicators of worse prognosis were available in UCC. We proposed a simple, highly reproducible method to determine the molecular grade of UCC, and it proved superior to traditional clinicopathologic indices. These results further emphasize the need for molecular markers to predict accurately both worse and favorable disease courses and suggest that this approach will be of value for other types of malignancy as well. Our results indicate the need for future prospective trials, and these trials may confirm the idea that the molecular grade can be used in clinical decision making with regard to the frequency of cystoscopic follow-up, adjuvant intravesical instillations, or timing of radical treatments such as cystectomy. In addition, a major target for future research will be for scientists to unravel the mechanisms of action of the favorable FGFR3 mutation.
We thank the histotechnology and the immunohistochemical laboratories of the University Hospital Rotterdam for the preparation of slides and the immunostaining procedures, the urology departments of the Vlietland hospitals in Schiedam and Vlaardingen, and the urology department of the Ijsselland hospital in Capelle a/d Ijssel for cooperation.
Supported by the University Hospital Rotterdam as part of a top-down revolving fund project (FED 0930) and by grants from the Maurits and Anna de Kock foundation, the Comité de Paris Ligue Nationale Contre le Cancer (UMR 144, laboratoire associé), the Centre Nationale de Recherche Scientifique, and the Institut Curie.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
, high) or (
, low) MIB-1 staining. (B) Molecular grading was based on these parameters.
