Urologic Oncology: Seminars and Original Investigations 33 (2015) 111.e1–111.e7
Original article
Role of fibroblast growth factor in squamous cell carcinoma of the bladder: Prognostic biomarker and potential therapeutic target Ramy F. Youssefa,b,*, Payal Kapurc, Ahmed Mosbahb, Hassan Abol-Eneinb, Mohamed Ghoneimb, Yair Lotand a
b
Department of Urology, University of California, Irvine, CA Department of Urology, Urology and Nephrology Center, Mansoura University, Mansoura, Egypt c Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX d Department of Urology, University of Texas Southwestern medical center, Dallas, TX
Received 6 June 2014; received in revised form 29 September 2014; accepted 30 September 2014
Abstract Background: We evaluated the association of fibroblast growth factor (FGF2) expression with pathologic features and clinical outcomes of squamous cell carcinoma (SCC) of the urinary bladder. Methods: Immunohistochemistry of FGF2 was performed on radical cystectomy specimens with pure SCC from 1997 to 2003. The relationship between FGF2 and pathologic parameters and oncological outcome was assessed. Results: The study included 151 patients with SCC (98 men) with a median age of 52 years (range: 36–74 y). Schistosomal infection was found in 81% of patients. Pathologic category was T2 and T3 in 88% of patients and the grade was low in 450%. Lymph node invasion and lymphovascular invasion were found in 30.5% and 16%. Altered FGF2 was associated with tumor grade (P ¼ 0.014), lymph node invasion, and lymphovascular invasion (P ¼ 0.042). Altered FGF2 was associated with both disease recurrence and cancer-specific mortality (P r 0.001) in Kaplan-Meier analyses and was an independent predictor of cancer recurrence (hazard ratio ¼ 2.561, P ¼ 0. 009) and cancer-specific mortality (hazard ratio ¼ 2.679, P ¼ 0. 033) in multivariate Cox regression analyses. Adding FGF2 to a model including standard clinicopathologic prognostics (pathologic T category, lymph node status, and grade) showed a significant improvement (6%) in accuracy of prediction poor oncological outcome. Conclusions: FGF2 overexpression is associated with aggressive pathologic features and worse outcomes after radical cystectomy for SCC, suggesting a good prognostic and possible therapeutic role. r 2015 Elsevier Inc. All rights reserved.
Keywords: Bladder cancer; Biomarkers; Squamous cell carcinoma; FGF2
1. Introduction Egypt has one of the highest incidences of bladder cancer (BC) worldwide [1]. BC is the most prevalent cancer in Egyptian men who have cumulative risk of 2.6% [2]. Chronic bilharzial cystitis is the most important risk factor for BC in Egypt and the cause of higher incidence of squamous cell carcinoma (SCC). Patients with schistosomal-associated SCC Disclosure: Supported by a grant from the Egyptian Ministry of Higher Education, Egypt via the Egyptian Cultural and Educational Bureau (ECEB) in Washington, DC. * Corresponding author. Tel.: þ1-214-4-970-571. E-mail address: [email protected]. http://dx.doi.org/10.1016/j.urolonc.2014.09.020 1078-1439/r 2015 Elsevier Inc. All rights reserved.
are usually younger and can present with locally advanced stage despite the more frequent low-grade tumors encountered [3–5]. Prolonged inflammation due to schistosomiasis can lead to squamous metaplasia, dysplasia, and eventually carcinoma [3–5]. Inflammation creates an environment abundant in growth factors–like fibroblast growth factor (FGF2) that favor cell proliferation, migration, angiogenesis, and the suppression of apoptosis [4–6]. Basic FGF or FGF2 is one of the 23 members of the FGF family (FGFs). It has been implicated in angiogenesis, cellular proliferation, invasiveness, and metastatic potential of BC [7–12]. It was found to be associated with established features of aggressive urothelial carcinoma of the bladder (UCB) such as pathologic category, lymphovascular
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invasion (LVI), lymph node invasion (LNI), disease recurrence, and poor survival [7,8]. There have been reports about involvement of FGF2 in tumorigenesis, and its role as a prognostic marker in multiple cancers including SCC of different organs [7,13–19]. However, there have been no studies evaluating the prognostic role of FGF2 in SCC of the urinary bladder, mostly owing to rarity of the disease. More importantly, the relative rarity of SCC of the bladder and sparse information about its biology has limited the ability to design clinical trials addressing management. Radical surgery alone is inadequate to control the disease [20]. External irradiation and systemic chemotherapy are less efficient in SCC compared with UCB [20]. Efficient multimodal treatment approaches using systemic therapies and risk stratification tools to help patients' selection for these treatments are lacking. Incorporating prognostic biomarkers with classic pathologic features is needed to improve management and outcomes of SCC of the bladder. The goal of this study was to evaluate the association of FGF2 overexpression with pathologic characteristics of SCC and clinical outcome after radical cystectomy (RC). The molecular signature of SCC with FGF2 alteration and poor oncological outcome was determined. 2. Material and methods 2.1. Patient population The study included 151 patients treated by RC and pelvic lymphadenectomy in Mansoura, Egypt, from 1997 to 2003 for pure SCC. Patients with mixed histology, inadequate follow-up, or insufficient paraffin-embedded archival pathologic specimens were excluded. A comprehensive clinicopathologic database was constructed after an institutional review board approval. 2.2. Pathologic evaluation All surgical specimens were processed according to standard pathologic procedures. Histology, tumor grade, stage, and histological evidence of schistosomal infection were confirmed by blinded review by experienced genitourinary pathologists. Tumor grade was assigned according to the 1973 World Health Organization grading system (graded from 1 ¼ well differentiated to 3 ¼ poorly differentiated). Pathologic category was reassigned according to the 2010 American Joint Committee on Cancer TNM staging system. In addition, LVI was evaluated and defined as the unequivocal presence of tumor cells within an endothelium-lined space without underlying muscular walls. 2.3. Patients follow-up All patients were followed up with every 2 months in the first 6 months and at 6-month intervals thereafter. Follow-up
evaluation routinely comprised a medical history assessment and physical examination, laboratory tests (including measurement of creatinine and alkaline phosphatase levels, liver function parameters, and blood count), urine analysis, urinary exfoliative cytology, abdominal ultrasonography, and chest x-ray. Computerized tomography, magnetic resonance imaging, and bone scans were done when findings suggested disease progression, defined as any emerging local or distant tumor. 2.4. Construction of tissue microarray blocks Detailed description of tissue microarray (TMA) construction has been described before [4]. Overall, 3 representative areas for each tumor were identified. For each case, 3 representative samples of 1.0-mm core diameter were obtained and randomly arranged on the TMA blocks. Serial sections of 3 to 4 mm were obtained from the microarray. Each case had at least 2 core punches with sufficient tumor for evaluation. 2.5. Immunohistochemistry and scoring We performed immunohistochemical (IHC) staining of FGF2 and other markers belonging to different cancer pathways using serial sections from the same paraffinembedded TMA blocks. These markers included cell cycle regulatory markers (p53, p21, p27, and cyclin E), angiogenesis markers (vascular endothelial growth factor [VEGF] and thrombospondin [TSP]-1), apoptotic markers (Caspase3, Bcl-2, and Bax), inflammatory markers (COX-2), markers of cell proliferation (Ki-67), and markers of signal transduction pathways (epidermal growth factor receptor [EGFR] and extracellular signal-regulated kinases). Immunostaining was performed in a single laboratory on the Dako Autostainer (Carpinteria, California). Bright-field microscopy imaging coupled with advanced color detection software (Automated Cellular Imaging System, Clarient, Inc, Aliso Viejo, CA) was used. Scoring of IHC staining for FGF2 was scored as 0 ¼ no staining, 1 ¼ focal or diffuse, weak staining, 2 ¼ focal or diffuse moderate staining or focal strong staining, and 3 ¼ diffuse strong staining. FGF2 was considered positive when the score was Z2. Scoring systems for other markers have been described in detail before [4,5,7,21,22]. 2.6. Statistical analysis The Pearson chi-square test was performed to examine the relationships of FGF2 alterations with pathologic parameters. Outcomes were measured as time to disease recurrence or to BC–specific mortality. Disease recurrence was defined as local failure in the cystectomy bed, regional lymph nodes (LNs), or distant metastasis after RC. The period of disease-free survival (DFS) was defined as the time between the date of RC and the development of local
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recurrence or distant metastasis. Censored survival values represent patients who were alive without clinical evidence of disease at the last follow-up. Cause of death was determined by chart review or treating physicians. The period of cancer-specific survival (CSS) was defined as the time between the date of RC and death due to cancer. Univariate recurrence and survival probabilities after RC were estimated using the Kaplan–Meier analysis, and differences were assessed by the log-rank test. Multivariate Cox Regression analysis addressed time to recurrence and the cancer-specific mortality after RC. Accuracy in prediction of poor oncological outcome (disease recurrence or cancer-specific mortality was evaluated by receiver operating characteristic curve). All reported P values are 2-sided, and statistical significance was set at 0.05. All statistical tests were performed with SPSS version 21.0. 3. Results 3.1. Patient demographics and clinicopathologic findings Of the 151 patients in our study, 98 (65%) were men and 53 (35%) were women. The median age at diagnosis was 52 years (range: 36–74 y). The pathologic category was T2 in 50%, T3 in 38%, and T1 and T4 in 6% of the patients. The grade was low in 450% of the patients. Bilharzial infection was found in 81% of patients (n = 122). LNI was found in 30.5% (n = 46), and LVI was found in 16% of patients (n = 24). Median LNs removed during RC was 22 (range: 4–70). Positive surgical margin was found in 3% of cases (n = 5). Table 1 describes the patients' characteristics including clinicopathologic parameters and relation to FGF2 alterations. 3.2. Association of FGF2 with pathologic characteristics and other markers Fig. 1 shows representative IHC staining results for FGF2. Overall, FGF2 was altered in 28 (18.5%) patients. There was no difference in age, sex, pathologic T category, bilharzial infection, and number of LNs removed between patients with normal vs. those with altered expression of FGF2 (P 4 0.05). FGF2 was associated with tumor grade (P ¼ 0.014), LVI, and LNI (P ¼ 0.042) as detailed in Table 1. 3.3. Association of FGF2 with oncological outcome Median follow-up after RC was 63.2 months (range: 0–100 mo). The 5-year DFS rates and cancer-specific survival rates for the 151 patients included in the study were 67 ⫾ 4% and 78 ⫾ 4%, respectively. The Kaplan–Meier analyses showed that FGF2 is associated with both disease recurrence (Fig. 2) and BC–specific mortality (Fig. 3) (P o 0.05). The 5-year DFS and CSS rates were 76 ⫾ 4% and 84 ⫾ 4%,
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Table 1 Patient characteristics including FGF2 association with clincopathologic parameters No. of patients (%)
FGF2 Altered (%)
P value Nonaltered (%)
Total
151
28 (18.5) 123 (81.5)
Age, y Mean (median) Range
51.83 36–74
52 (52) 39–65
Gender Female Male
53 (35.1) 98 (64.9)
10 (35.7) 43 (35) 18 (64.3) 80 (65)
Tumor grade Low High
80 (53.0) 71 (47.0)
9 (32.1) 71 (57.7) 19 (67.9) 52 (42.3)
Pathologic category pT1 pT2 pT3 pT4
10 (6.6) 75 (49.7) 57 (37.7) 9 (6.0)
2 (7.1) 15 (53.6) 9 (32.1) 2 (7.1)
0.902
0.940
0.014
0.922
Extravesical extension rT2 85 (56.3) 4T2 66 (43.7) Organ confinement Confined (T1/2 N0) 68 (45.0) Nonconfined (T3/4 83 (55.0) or Nþ) LN metastasis Absent Present
51.8 (51) 36–74
8 (6.5) 60 (48.8) 48 (39) 7 (5.7) 0.601
17 (60.7) 68 (55.3) 11 (39.3) 55 (44.7) 0.798 12 (42.9) 56 (45.5) 16 (57.1) 67 (54.5) 0.042
105 (69.5) 46 (30.5)
15 (53.6) 90 (73.2) 13 (46.4) 33 (26.8)
Lymphovascular invasion Absent Present
0.042 127 (84.1) 24 (15.9)
20 (71.4) 107 (87) 8 (28.6) 16 (13)
Bilharziasis Absent Present
29 (19.2) 122 (80.8)
7 (24.1) 21 (75)
DNA ploidy Diploid Nondiploid
82 (54.3) 69 (45.7)
15 (53.6) 67 (54.5) 13 (46.4) 56 (45.5)
0.388 22 (17.9) 101 (82.1) 0.931
respectively, in FGF2-negative vs. 39 ⫾ 1% and 49 ⫾ 11%, respectively, in FGF2-altered tumors. FGF2 was an independent predictor of disease recurrence (hazard ratio ¼ 2.561; 95% CI: 1.261–5.201; P ¼ 0.009) and BC–specific mortality (hazard ratio ¼ 2.679; 95% CI: 1.082–6.633; P ¼ 0.033) in multivariate Cox proportional hazards regression analyses; adjusted for the effects of pathologic tumor category, grade, LNI, and LVI (Table 2). Adding FGF2 to a model including standard clinicopathologic prognostics (pathological T category, LN status, and grade) showed an improvement (6%) in accuracy of prediction of poor oncological outcome.
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Fig. 1. Altered vs. unaltered expression of FGF2 in SCC of the urinary bladder. (Color version of figure is available online.)
3.4. Molecular signature of SCC with FGF2 alteration and poor oncological outcome
Inflammation and angiogenesis are linked to carcinogenesis [23]. Overexpression of FGF2 and its receptors was
found to cause aberrant cell proliferation in various cancers and many human tumor cell lines [11,13,14,24]. Moreover, the prognostic value of FGF2 was demonstrated in multiple cancers (e.g., lung, brain, breast, thyroid, bladder, kidney, liver, gastric, and esophageal cancers) including SCC of other organs (e.g., lung, head and neck, and esophageal SCCs) [7,13–18]. Expression of FGF2 has been implicated in the regulation of angiogenesis, cell survival, invasiveness, and subsequent metastases of UCB [7–12]. We have previously reported association of FGF2 with established features of aggressive UCB such as pathological category, LVI, LNI, molecular markers commonly altered in UCB (i.e., p27, pRb, and Ki-67), and disease recurrence [7]. The current study is the first to report the important prognostic role of FGF2 in SCC of the urinary bladder. Altered FGF2 was associated with aggressive pathologic features of SCC of
Fig. 2. Disease-free survival probabilities in 151 cases treated by radical cystectomy for SCC of the urinary bladder based on FGF2 alterations. (Color version of figure is available online.)
Fig. 3. Cancer-specific survival probabilities in 151 cases treated by radical cystectomy for SCC of the urinary bladder based on FGF2 alterations. (Color version of figure is available online.)
Of the 28 patients who had FGF2 alterations, 15 (54%) had recurrence or died due to cancer. Evaluation of other markers alterations in these 15 patients revealed the following: 15 (100%) patients had p27, Ki-67, Bcl-2, and VEGF; 14 (93%) had TSP-1 and caspase-3; 10 (67%) had EGFR and cyclin-E; and 9 (60%) had COX-2 alterations. Other markers were less frequently altered, including p21 (40%), p53 (33%), extracellular signal-regulated kinases (27%), and Bax (13%).
4. Discussion
R.F. Youssef et al. / Urologic Oncology: Seminars and Original Investigations 33 (2015) 111.e1–111.e7 Table 2 Multivariable Cox proportional hazards regression analyses to evaluate disease recurrence and cancer-specific mortality in 151 cases treated by radical cystectomy for SCC of the urinary bladder All patients
Disease recurrence Hazard 95% CI ratio
P value Hazard 95% CI ratio
P value
T category Grade LNþ LVI FGF2
2.244 1.411 1.708 1.537 2.561
0.025 0.387 0.178 0.304 0.009
0.113 0.881 0.295 0.104 0.033
1.108–4.544 0.646–3.081 0.784–3.718 0.677–3.492 1.261–5.201
Cancer-specific mortality
2.061 1.079 1.714 2.295 2.679
0.843–5.040 0.400–2.907 0.625–4.704 0.842–6.256 1.082–6.633
the urinary bladder such as high grade, LVI, and LNI. Moreover, FGF2 was associated with both disease recurrence and cancer-specific mortality in Kaplan–Meier analyses and was an independent predictor of both disease recurrence and cancer-specific mortality. The 5-year DFS and CSS rates almost doubled in patients with no FGF2 alterations in comparison with those with FGF2 alteration. The risk of cancer recurrence or death due to cancer was 42.6 times higher in FGF2-altered tumors in comparison with that of those without alteration. Adding FGF2 to a model including standard clinicopathologic prognostics showed a significant improvement in predictive accuracy. Moreover, tumors with altered FGF2 and poor oncological outcome were also more likely to have alterations in markers associated with angiogenesis, cell cycle regulation, apoptosis, and signal transduction cancer pathways. Approximately 20% to 40% of patients with organconfined disease have recurrence after RC [4,25]. Previous studies have shown that stage, grade, LNI, and LVI are insufficient in predicting which patients are most likely to have recurrence owing to biological heterogeneity among tumors with similar stage [4,25]. Because systemic chemotherapy is ineffective as an adjuvant therapy for SCC, there is need for improved understanding of tumor biology that may identify actionable targets for therapy [20]. Exploring molecular alterations can improve prognostication and help design of clinical trial, development of novel therapies, and tailoring aggressive and effective multimodal treatment approached for high-risk patients. FGFs are involved in a variety of cellular processes, such as stemness, proliferation, antiapoptosis, drug resistance, and angiogenesis [23,26–30]. Previous studies have shown that FGF2 can be produced by the tumor cells or by the surrounding stroma and promotes oncogenesis through both autocrine and paracrine modes [23,24,31,32]. Its expression is under the control of a variety of transcription factors that are known to contribute to oncogenesis. The interaction with specific FGF receptors leads to unchecked proliferation via multiple cancer pathways, including the phosphatidylinositol 3-kinase-Akt 1 pathway, Ras-MAP kinase pathway, and protein kinase C–dependent signal transduction pathway [7,16,23,32]. In this study, we found multiple
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alterations in other biomarkers belonging to different cancer pathways in patients who had poor oncological outcomes and FGF2 alterations. Most of these patients had alterations in angiogenesis markers (100% VEGF and 93% TSP-1), apoptotic markers (100% Bcl-2 and 93% caspase-3), cell cycle regulators (100% p27 and 67% cyclin E), markers of proliferation (100% Ki-67), inflammatory markers (60% COX-2), and markers of signal transduction (67% EGFR). It was shown from previous studies that FGF2 can induce a proinflammatory signature in endothelium through activation of multiple growth factors, cytokines, chemokines, membrane receptors, and adhesion molecules [23,32]. FGF2 regulates the expression of cadherins, integrins, metalloproteases, and various extracellular matrix components [23]. An intimate cross talk exists between inflammatory and angiogenic responses during FGF2-driven neovascularization [23,32]. Clinical studies have shown that there is a strong correlation among FGF2, VEGF, vascular density, metastatic potential, and poor survival with human tumors [23,33]. VEGF produces a number of important biological effects such as endothelial mitogenesis and migration, extracellular matrix remodeling via induction of proteinases, increased vascular permeability, and maintenance of newly formed vasculature [7,23]. Interestingly, the role of FGF2 is not limited to prognosis for multiple cancers, including SCC of the bladder, as shown in our study. Perhaps, more importantly, FGF2 might be implicated in response to different cancer treatment modalities and therapeutics. Expression of FGF2 had been correlated with resistance to paclitaxel in tumors in human patients and to cisplatin in a human BC cell line [16,34]. FGF2 may alter DNA function and apoptotic threshold in cells. Initial efforts to block FGF2-mediated drug resistance in animal models showed favorable results [35]. Thus the manipulation of FGF2 activities to increase the effectiveness of chemotherapeutics may represent a promising approach. Older studies had shown that interferon-α can inhibit FGF2, angiogenesis, and BC in mice [36]. Pentose polysulfate sodium reduced urinary FGF2 in enterocystoplasty patients [37]. Recently, small-molecule FGF receptor inhibitors were found to block FGFRdependent urothelial carcinoma in vitro and in vivo [38]. Another novel approach is combining FGF2-targeted adenovirus-mediated mutant-Rad 50 gene transfer to enhance SCC chemosensitization [19]. Another group has suggested the possibility of using FGF antisense mRNAs in esophageal SCC overexpressing FGF2 [17]. Radiotherapy is an integral part of different SCC treatment protocols. However, frequently, tumor response is insufficient. Irradiation might induce additional undesired effects such as the release of angiogenic growth factors, potentially impairing the expected irradiation effect. Novel strategies of cancer therapy combining irradiation and antiangiogenic therapy are under evaluation. The idea behind this concept is to normalize the dysfunctional tumor vasculature or to inhibit vessel regrowth or repair of the
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irradiation-damaged tumor vasculature. Increased FGF2 release and proliferation were noticed after combined irradiation/anti VEGF treatment in xerotransplanted SCC [30]. FGF2 upregulation is probably a compensatory reaction of the tumor owing to VEGF depletion. Similarly, therapies targeting VEGF or VEFGR were found to be hindered by drug resistance, as a result of up-regulation of other angiogenic factors such as FGF2 [26,27]. As an intimate cross talk exists between FGF2 and VEGF, targeting both FGF2 and VEGF might be superior to targeting VEGF alone [32]. Using targeted therapies with an additional anti-FGF2 effect might represent a potential strategy to compensate resistance to anti-VEGF only therapy [32]. Targeting EGFR only may have similar drug resistance problem [26,28,29]. Recent studies have shown involvement of FGF2 cancer pathway as a novel mechanism of acquired resistance to EGFR-targeted therapies [28,29]. It was shown that the FGF2 pathway might work independently or in combination with EGFR pathway and another intimate cross talk could exist between FGF2 and EGFR [24]. Dovitinib (TKI258), ponatinib (AP24534), and AZD4547 are multikinase inhibitors with multiple targets that have been investigated in clinical trials for various types of human cancer treated previously with other targeted therapies [26,27,39]. Future FGF-targeted therapies can have a potential role as a maintenance treatment following other modalities like RC, radiation, and other systemic therapies [39]. The present study has some limitations, particularly in retrospective data collection and the variability of IHC techniques. To limit this variability, we used a combination of automated methods and an experienced pathologist to confirm all IHC readings. Our findings need to be validated prospectively, and perhaps this can be achieved through a multicenter study. Internal validation was not tried as we are looking for external validation in other cohorts. To our knowledge, this is the first study that clearly shows an association of FGF2 with aggressive pathologic features and poor oncological outcome in patients with bladder SCC. Future prognostic tools combining biomarkers with classic pathologic prognostics can improve oncological outcome prediction, patient counseling, follow-up scheduling, use of adjuvant therapies, and design of clinical trials.
5. Conclusions FGF2 overexpression is associated with aggressive pathologic features and worse outcomes after RC for SCC, suggesting a potential prognostic role for FGF2 in bladder SCC. Alteration in FGF2 may happen among interactions between different cancer pathways involving inflammation, angiogenesis, inhibition of cell cycle regulation and apoptosis, and abnormal proliferation. Our findings support the need for evaluation of therapeutic strategies targeting FGF2 signaling in SCC of the urinary bladder.
References [1] Parkin DM. The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl 2008;218:12–20. [2] Parkin DM, Bray F, Ferlay J, Jemal A. Cancer in Africa 2012. Cancer Epidemiol Biomarkers Prev 2012;118(18):4372–84. [3] Shokeir AA. Squamous cell carcinoma of the bladder: pathology, diagnosis and treatment. BJU Int 2004;93:216–20. [4] Youssef RF, Shariat SF, Kapur P, Kabbani W, Mosbah A, Abol-Enein H, et al. Prognostic value of cyclooxygenase-2 expression in squamous cell carcinoma of the bladder. J Urol 2011;185:1112–7. [5] Youssef R, Kapur P, Kabbani W, Shariat SF, Mosbah A, Abol-Enein H, et al. Bilharzial vs non-bilharzial related bladder cancer: pathological characteristics and value of cyclooxygenase-2 expression. BJU Int 2011;108:31–7. [6] Leibovici D, Grossman HB, Dinney CP, Millikan RE, Lerner S, Wang Y, et al. Polymorphisms in inflammation genes and bladder cancer: from initiation to recurrence, progression, and survival. J Clin Oncol 2005;23:5746–56. [7] Shariat SF, Youssef RF, Gupta A, Chade DC, Karakiewicz PI, Isbarn H, et al. Association of angiogenesis related markers with bladder cancer outcomes and other molecular markers. J Urol 2010;183:1744–50. [8] Chikazawa M, Inoue K, Fukata S, Karashima T, Shuin T. Expression of angiogenesis-related genes regulates different steps in the process of tumor growth and metastasis in human urothelial cell carcinoma of the urinary bladder. Pathobiology 2008;75:335–45. [9] Okada-Ban M, Moens G, Thiery JP, Jouanneau J. Nuclear 24 kD fibroblast growth factor (FGF)-2 confers metastatic properties on rat bladder carcinoma cells. Oncogene 1999;18:6719–24. [10] Miyake H, Yoshimura K, Hara I, Eto H, Arakawa S, Kamidono S. Basic fibroblast growth factor regulates matrix metalloproteinases production and in vitro invasiveness in human bladder cancer cell lines. J Urol 1997;157:2351–5. [11] Zaravinos A, Volanis D, Lambrou GI, Delakas D, Spandidos DA. Role of the angiogenic components, VEGFA, FGF2, OPN and RHOC, in urothelial cell carcinoma of the urinary bladder. Oncol Rep 2012;28:1159–66. [12] Thomas-Mudge RJ, Okada-Ban M, Vandenbroucke F, VincentSalomon A, Girault JM, Thiery JP, et al. Nuclear FGF-2 facilitates cell survival in vitro and during establishment of metastases. Oncogene 2004;23:4771–9. [13] Rades D, Setter C, Dahl O, Schild SE, Noack F. Fibroblast growth factor 2–a predictor of outcome for patients irradiated for stage II-III nonsmall-cell lung cancer. Int J Radiat Oncol Biol Phys 2012;82:442–7. [14] Lefevre G, Babchia N, Calipel A, Mouriaux F, Faussat AM, Mrzyk S, et al. Activation of the FGF2/FGFR1 autocrine loop for cell proliferation and survival in uveal melanoma cells. Invest Ophthalmol Vis Sci 2009;50:1047–57. [15] Donnem T, Al-Shibli K, Al-Saad S, Busund LT, Bremnes RM. Prognostic impact of fibroblast growth factor 2 in non-small cell lung cancer: coexpression with VEGFR-3 and PDGF-B predicts poor survival. J Thorac Oncol 2009;4:578–85. [16] Gan Y, Wientjes MG, Au JL. Expression of basic fibroblast growth factor correlates with resistance to paclitaxel in human patient tumors. Pharm Res 2006;23:1324–31. [17] Barclay C, Li AW, Geldenhuys L, Baguma-Nibasheka M, Porter GA, Veugelers PJ, et al. Basic fibroblast growth factor (FGF-2) overexpression is a risk factor for esophageal cancer recurrence and reduced survival, which is ameliorated by coexpression of the FGF-2 antisense gene. Clin Cancer Res 2005;11:7683–91. [18] Chodak GW, Hospelhorn V, Judge SM, Mayforth R, Koeppen H, Sasse J. Increased levels of fibroblast growth factor-like activity in urine from patients with bladder or kidney cancer. Cancer Res 1988;48:2083–8. [19] Figures MR, Wobb J, Araki K, Liu T, Xu L, Zhu H, et al. Head and neck squamous cell carcinoma targeted chemosensitization. Otolaryngol Head Neck Surg 2009;141:177–83.
R.F. Youssef et al. / Urologic Oncology: Seminars and Original Investigations 33 (2015) 111.e1–111.e7 [20] Abol-Enein H, Kava BR, Carmack AJ. Nonurothelial cancer of the bladder. Urology 2007;69(Suppl. 1):93–104. [21] Youssef R, Kapur P, Shariat SF, Arendt T, Kabbani W, Mosbah A, et al. Prognostic value of apoptotic markers in squamous cell carcinoma of the urinary bladder. BJU Int 2012;110:961–6. [22] Youssef RF, Shariat SF, Kapur P, Kabbani W, Ghoneim T, King E, et al. Expression of cell cycle-related molecular markers in patients treated with radical cystectomy for squamous cell carcinoma of the bladder. Hum Pathol 2011;42:347–55. [23] Presta M, Andres G, Leali D, Dell'Era P, Ronca R. Inflammatory cells and chemokines sustain FGF2-induced angiogenesis. Eur Cytokine Netw 2009;20:39–50. [24] Marshall ME, Hinz TK, Kono SA, Singleton KR, Bichon B, Ware KE, et al. Fibroblast growth factor receptors are components of autocrine signaling networks in head and neck squamous cell carcinoma cells. Clin Cancer Res 2011;17:5016–25. [25] Ghoneim MA, Abdel-Latif M, el-Mekresh M, Abol-Enein H, Mosbah A, Ashamallah A, et al. Radical cystectomy for carcinoma of the bladder: 2,720 consecutive cases 5 years later. J Urol 2008;180:121–7. [26] Katoh M, Nakagama H. FGF receptors: cancer biology and therapeutics. Med Res Rev 2014;34:280–300. [27] Katoh M. Therapeutics targeting angiogenesis: genetics and epigenetics, extracellular miRNAs and signaling networks (Review). Int J Mol Med 2013;32:763–7. [28] Ware KE, Hinz TK, Kleczko E, Singleton KR, Marek LA, Helfrich BA, et al. A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop. Oncogenesis 2013;2:e39. [29] Terai H, Soejima K, Yasuda H, Nakayama S, Hamamoto J, Arai D, et al. Activation of the FGF2-FGFR1 autocrine pathway: a novel mechanism of acquired resistance to gefitinib in NSCLC. Mol Cancer Res 2013;11:759–67. [30] Fruth K, Weber S, Okcu Y, Noppens R, Klein KU, Joest E, et al. Increased basic fibroblast growth factor release and proliferation in xenotransplanted squamous cell carcinoma after combined irradiation/
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
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anti-vascular endothelial growth factor treatment. Oncol Rep 2012;27: 1573–9. Cao Y, Cao R, Hedlund EM. R Regulation of tumor angiogenesis and metastasis by FGF and PDGF signaling pathways. J Mol Med (Berl) 2008;86:785–9. Alessi P, Leali D, Camozzi M, Cantelmo A, Albini A, Presta M. AntiFGF2 approaches as a strategy to compensate resistance to anti-VEGF therapy: long-pentraxin 3 as a novel antiangiogenic FGF2-antagonist. Eur Cytokine Netw 2009;20:225–34. Billottet C, Janji B, Thiery JP, Jouanneau J. Rapid tumor development and potent vascularization are independent events in carcinoma producing FGF-1 or FGF-2. Oncogene 2002;21:8128–39. Miyake H, Hara I, Gohji K, Yoshimura K, Arakawa S, Kamidono S. Expression of basic fibroblast growth factor is associated with resistance to cisplatin in a human bladder cancer cell line. Cancer Lett 1998;123:121–6. Coleman AB. Positive and negative regulation of cellular sensitivity to anti-cancer drugs by FGF-2. Drug Resist Updat 2003;6: 85–94. Dinney CP, Bielenberg DR, Perrotte P, Reich R, Eve BY, Bucana CD, et al. Inhibition of basic fibroblast growth factor expression, angiogenesis, and growth of human bladder carcinoma in mice by systemic interferon-alpha administration. Cancer Res 1998;58: 808–14. Barrington JW, Fulford S, Fraylin L, Fish R, Shelley M, Stephenson TP. Reduction of urinary basic fibroblast growth factor using pentosan polysulphate sodium. Br J Urol 1996;78:54–6. Lamont FR, Tomlinson DC, Cooper PA, Shnyder SD, Chester JD, Knowles MA. Small molecule FGF receptor inhibitors block FGFRdependent urothelial carcinoma growth in vitro and in vivo. Br J Cancer 2011;104:75–82. Gozgit JM, Wong MJ, Moran L, Wardwell S, Mohemmad QK, Narasimhan NI, et al. Ponatinib (AP24534), a multitargeted panFGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models. Mol Cancer Ther 2012;11:690–9.
Original article
Role of fibroblast growth factor in squamous cell carcinoma of the bladder: Prognostic biomarker and potential therapeutic target Ramy F. Youssefa,b,*, Payal Kapurc, Ahmed Mosbahb, Hassan Abol-Eneinb, Mohamed Ghoneimb, Yair Lotand a
b
Department of Urology, University of California, Irvine, CA Department of Urology, Urology and Nephrology Center, Mansoura University, Mansoura, Egypt c Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX d Department of Urology, University of Texas Southwestern medical center, Dallas, TX
Received 6 June 2014; received in revised form 29 September 2014; accepted 30 September 2014
Abstract Background: We evaluated the association of fibroblast growth factor (FGF2) expression with pathologic features and clinical outcomes of squamous cell carcinoma (SCC) of the urinary bladder. Methods: Immunohistochemistry of FGF2 was performed on radical cystectomy specimens with pure SCC from 1997 to 2003. The relationship between FGF2 and pathologic parameters and oncological outcome was assessed. Results: The study included 151 patients with SCC (98 men) with a median age of 52 years (range: 36–74 y). Schistosomal infection was found in 81% of patients. Pathologic category was T2 and T3 in 88% of patients and the grade was low in 450%. Lymph node invasion and lymphovascular invasion were found in 30.5% and 16%. Altered FGF2 was associated with tumor grade (P ¼ 0.014), lymph node invasion, and lymphovascular invasion (P ¼ 0.042). Altered FGF2 was associated with both disease recurrence and cancer-specific mortality (P r 0.001) in Kaplan-Meier analyses and was an independent predictor of cancer recurrence (hazard ratio ¼ 2.561, P ¼ 0. 009) and cancer-specific mortality (hazard ratio ¼ 2.679, P ¼ 0. 033) in multivariate Cox regression analyses. Adding FGF2 to a model including standard clinicopathologic prognostics (pathologic T category, lymph node status, and grade) showed a significant improvement (6%) in accuracy of prediction poor oncological outcome. Conclusions: FGF2 overexpression is associated with aggressive pathologic features and worse outcomes after radical cystectomy for SCC, suggesting a good prognostic and possible therapeutic role. r 2015 Elsevier Inc. All rights reserved.
Keywords: Bladder cancer; Biomarkers; Squamous cell carcinoma; FGF2
1. Introduction Egypt has one of the highest incidences of bladder cancer (BC) worldwide [1]. BC is the most prevalent cancer in Egyptian men who have cumulative risk of 2.6% [2]. Chronic bilharzial cystitis is the most important risk factor for BC in Egypt and the cause of higher incidence of squamous cell carcinoma (SCC). Patients with schistosomal-associated SCC Disclosure: Supported by a grant from the Egyptian Ministry of Higher Education, Egypt via the Egyptian Cultural and Educational Bureau (ECEB) in Washington, DC. * Corresponding author. Tel.: þ1-214-4-970-571. E-mail address: [email protected]. http://dx.doi.org/10.1016/j.urolonc.2014.09.020 1078-1439/r 2015 Elsevier Inc. All rights reserved.
are usually younger and can present with locally advanced stage despite the more frequent low-grade tumors encountered [3–5]. Prolonged inflammation due to schistosomiasis can lead to squamous metaplasia, dysplasia, and eventually carcinoma [3–5]. Inflammation creates an environment abundant in growth factors–like fibroblast growth factor (FGF2) that favor cell proliferation, migration, angiogenesis, and the suppression of apoptosis [4–6]. Basic FGF or FGF2 is one of the 23 members of the FGF family (FGFs). It has been implicated in angiogenesis, cellular proliferation, invasiveness, and metastatic potential of BC [7–12]. It was found to be associated with established features of aggressive urothelial carcinoma of the bladder (UCB) such as pathologic category, lymphovascular
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invasion (LVI), lymph node invasion (LNI), disease recurrence, and poor survival [7,8]. There have been reports about involvement of FGF2 in tumorigenesis, and its role as a prognostic marker in multiple cancers including SCC of different organs [7,13–19]. However, there have been no studies evaluating the prognostic role of FGF2 in SCC of the urinary bladder, mostly owing to rarity of the disease. More importantly, the relative rarity of SCC of the bladder and sparse information about its biology has limited the ability to design clinical trials addressing management. Radical surgery alone is inadequate to control the disease [20]. External irradiation and systemic chemotherapy are less efficient in SCC compared with UCB [20]. Efficient multimodal treatment approaches using systemic therapies and risk stratification tools to help patients' selection for these treatments are lacking. Incorporating prognostic biomarkers with classic pathologic features is needed to improve management and outcomes of SCC of the bladder. The goal of this study was to evaluate the association of FGF2 overexpression with pathologic characteristics of SCC and clinical outcome after radical cystectomy (RC). The molecular signature of SCC with FGF2 alteration and poor oncological outcome was determined. 2. Material and methods 2.1. Patient population The study included 151 patients treated by RC and pelvic lymphadenectomy in Mansoura, Egypt, from 1997 to 2003 for pure SCC. Patients with mixed histology, inadequate follow-up, or insufficient paraffin-embedded archival pathologic specimens were excluded. A comprehensive clinicopathologic database was constructed after an institutional review board approval. 2.2. Pathologic evaluation All surgical specimens were processed according to standard pathologic procedures. Histology, tumor grade, stage, and histological evidence of schistosomal infection were confirmed by blinded review by experienced genitourinary pathologists. Tumor grade was assigned according to the 1973 World Health Organization grading system (graded from 1 ¼ well differentiated to 3 ¼ poorly differentiated). Pathologic category was reassigned according to the 2010 American Joint Committee on Cancer TNM staging system. In addition, LVI was evaluated and defined as the unequivocal presence of tumor cells within an endothelium-lined space without underlying muscular walls. 2.3. Patients follow-up All patients were followed up with every 2 months in the first 6 months and at 6-month intervals thereafter. Follow-up
evaluation routinely comprised a medical history assessment and physical examination, laboratory tests (including measurement of creatinine and alkaline phosphatase levels, liver function parameters, and blood count), urine analysis, urinary exfoliative cytology, abdominal ultrasonography, and chest x-ray. Computerized tomography, magnetic resonance imaging, and bone scans were done when findings suggested disease progression, defined as any emerging local or distant tumor. 2.4. Construction of tissue microarray blocks Detailed description of tissue microarray (TMA) construction has been described before [4]. Overall, 3 representative areas for each tumor were identified. For each case, 3 representative samples of 1.0-mm core diameter were obtained and randomly arranged on the TMA blocks. Serial sections of 3 to 4 mm were obtained from the microarray. Each case had at least 2 core punches with sufficient tumor for evaluation. 2.5. Immunohistochemistry and scoring We performed immunohistochemical (IHC) staining of FGF2 and other markers belonging to different cancer pathways using serial sections from the same paraffinembedded TMA blocks. These markers included cell cycle regulatory markers (p53, p21, p27, and cyclin E), angiogenesis markers (vascular endothelial growth factor [VEGF] and thrombospondin [TSP]-1), apoptotic markers (Caspase3, Bcl-2, and Bax), inflammatory markers (COX-2), markers of cell proliferation (Ki-67), and markers of signal transduction pathways (epidermal growth factor receptor [EGFR] and extracellular signal-regulated kinases). Immunostaining was performed in a single laboratory on the Dako Autostainer (Carpinteria, California). Bright-field microscopy imaging coupled with advanced color detection software (Automated Cellular Imaging System, Clarient, Inc, Aliso Viejo, CA) was used. Scoring of IHC staining for FGF2 was scored as 0 ¼ no staining, 1 ¼ focal or diffuse, weak staining, 2 ¼ focal or diffuse moderate staining or focal strong staining, and 3 ¼ diffuse strong staining. FGF2 was considered positive when the score was Z2. Scoring systems for other markers have been described in detail before [4,5,7,21,22]. 2.6. Statistical analysis The Pearson chi-square test was performed to examine the relationships of FGF2 alterations with pathologic parameters. Outcomes were measured as time to disease recurrence or to BC–specific mortality. Disease recurrence was defined as local failure in the cystectomy bed, regional lymph nodes (LNs), or distant metastasis after RC. The period of disease-free survival (DFS) was defined as the time between the date of RC and the development of local
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recurrence or distant metastasis. Censored survival values represent patients who were alive without clinical evidence of disease at the last follow-up. Cause of death was determined by chart review or treating physicians. The period of cancer-specific survival (CSS) was defined as the time between the date of RC and death due to cancer. Univariate recurrence and survival probabilities after RC were estimated using the Kaplan–Meier analysis, and differences were assessed by the log-rank test. Multivariate Cox Regression analysis addressed time to recurrence and the cancer-specific mortality after RC. Accuracy in prediction of poor oncological outcome (disease recurrence or cancer-specific mortality was evaluated by receiver operating characteristic curve). All reported P values are 2-sided, and statistical significance was set at 0.05. All statistical tests were performed with SPSS version 21.0. 3. Results 3.1. Patient demographics and clinicopathologic findings Of the 151 patients in our study, 98 (65%) were men and 53 (35%) were women. The median age at diagnosis was 52 years (range: 36–74 y). The pathologic category was T2 in 50%, T3 in 38%, and T1 and T4 in 6% of the patients. The grade was low in 450% of the patients. Bilharzial infection was found in 81% of patients (n = 122). LNI was found in 30.5% (n = 46), and LVI was found in 16% of patients (n = 24). Median LNs removed during RC was 22 (range: 4–70). Positive surgical margin was found in 3% of cases (n = 5). Table 1 describes the patients' characteristics including clinicopathologic parameters and relation to FGF2 alterations. 3.2. Association of FGF2 with pathologic characteristics and other markers Fig. 1 shows representative IHC staining results for FGF2. Overall, FGF2 was altered in 28 (18.5%) patients. There was no difference in age, sex, pathologic T category, bilharzial infection, and number of LNs removed between patients with normal vs. those with altered expression of FGF2 (P 4 0.05). FGF2 was associated with tumor grade (P ¼ 0.014), LVI, and LNI (P ¼ 0.042) as detailed in Table 1. 3.3. Association of FGF2 with oncological outcome Median follow-up after RC was 63.2 months (range: 0–100 mo). The 5-year DFS rates and cancer-specific survival rates for the 151 patients included in the study were 67 ⫾ 4% and 78 ⫾ 4%, respectively. The Kaplan–Meier analyses showed that FGF2 is associated with both disease recurrence (Fig. 2) and BC–specific mortality (Fig. 3) (P o 0.05). The 5-year DFS and CSS rates were 76 ⫾ 4% and 84 ⫾ 4%,
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Table 1 Patient characteristics including FGF2 association with clincopathologic parameters No. of patients (%)
FGF2 Altered (%)
P value Nonaltered (%)
Total
151
28 (18.5) 123 (81.5)
Age, y Mean (median) Range
51.83 36–74
52 (52) 39–65
Gender Female Male
53 (35.1) 98 (64.9)
10 (35.7) 43 (35) 18 (64.3) 80 (65)
Tumor grade Low High
80 (53.0) 71 (47.0)
9 (32.1) 71 (57.7) 19 (67.9) 52 (42.3)
Pathologic category pT1 pT2 pT3 pT4
10 (6.6) 75 (49.7) 57 (37.7) 9 (6.0)
2 (7.1) 15 (53.6) 9 (32.1) 2 (7.1)
0.902
0.940
0.014
0.922
Extravesical extension rT2 85 (56.3) 4T2 66 (43.7) Organ confinement Confined (T1/2 N0) 68 (45.0) Nonconfined (T3/4 83 (55.0) or Nþ) LN metastasis Absent Present
51.8 (51) 36–74
8 (6.5) 60 (48.8) 48 (39) 7 (5.7) 0.601
17 (60.7) 68 (55.3) 11 (39.3) 55 (44.7) 0.798 12 (42.9) 56 (45.5) 16 (57.1) 67 (54.5) 0.042
105 (69.5) 46 (30.5)
15 (53.6) 90 (73.2) 13 (46.4) 33 (26.8)
Lymphovascular invasion Absent Present
0.042 127 (84.1) 24 (15.9)
20 (71.4) 107 (87) 8 (28.6) 16 (13)
Bilharziasis Absent Present
29 (19.2) 122 (80.8)
7 (24.1) 21 (75)
DNA ploidy Diploid Nondiploid
82 (54.3) 69 (45.7)
15 (53.6) 67 (54.5) 13 (46.4) 56 (45.5)
0.388 22 (17.9) 101 (82.1) 0.931
respectively, in FGF2-negative vs. 39 ⫾ 1% and 49 ⫾ 11%, respectively, in FGF2-altered tumors. FGF2 was an independent predictor of disease recurrence (hazard ratio ¼ 2.561; 95% CI: 1.261–5.201; P ¼ 0.009) and BC–specific mortality (hazard ratio ¼ 2.679; 95% CI: 1.082–6.633; P ¼ 0.033) in multivariate Cox proportional hazards regression analyses; adjusted for the effects of pathologic tumor category, grade, LNI, and LVI (Table 2). Adding FGF2 to a model including standard clinicopathologic prognostics (pathological T category, LN status, and grade) showed an improvement (6%) in accuracy of prediction of poor oncological outcome.
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Fig. 1. Altered vs. unaltered expression of FGF2 in SCC of the urinary bladder. (Color version of figure is available online.)
3.4. Molecular signature of SCC with FGF2 alteration and poor oncological outcome
Inflammation and angiogenesis are linked to carcinogenesis [23]. Overexpression of FGF2 and its receptors was
found to cause aberrant cell proliferation in various cancers and many human tumor cell lines [11,13,14,24]. Moreover, the prognostic value of FGF2 was demonstrated in multiple cancers (e.g., lung, brain, breast, thyroid, bladder, kidney, liver, gastric, and esophageal cancers) including SCC of other organs (e.g., lung, head and neck, and esophageal SCCs) [7,13–18]. Expression of FGF2 has been implicated in the regulation of angiogenesis, cell survival, invasiveness, and subsequent metastases of UCB [7–12]. We have previously reported association of FGF2 with established features of aggressive UCB such as pathological category, LVI, LNI, molecular markers commonly altered in UCB (i.e., p27, pRb, and Ki-67), and disease recurrence [7]. The current study is the first to report the important prognostic role of FGF2 in SCC of the urinary bladder. Altered FGF2 was associated with aggressive pathologic features of SCC of
Fig. 2. Disease-free survival probabilities in 151 cases treated by radical cystectomy for SCC of the urinary bladder based on FGF2 alterations. (Color version of figure is available online.)
Fig. 3. Cancer-specific survival probabilities in 151 cases treated by radical cystectomy for SCC of the urinary bladder based on FGF2 alterations. (Color version of figure is available online.)
Of the 28 patients who had FGF2 alterations, 15 (54%) had recurrence or died due to cancer. Evaluation of other markers alterations in these 15 patients revealed the following: 15 (100%) patients had p27, Ki-67, Bcl-2, and VEGF; 14 (93%) had TSP-1 and caspase-3; 10 (67%) had EGFR and cyclin-E; and 9 (60%) had COX-2 alterations. Other markers were less frequently altered, including p21 (40%), p53 (33%), extracellular signal-regulated kinases (27%), and Bax (13%).
4. Discussion
R.F. Youssef et al. / Urologic Oncology: Seminars and Original Investigations 33 (2015) 111.e1–111.e7 Table 2 Multivariable Cox proportional hazards regression analyses to evaluate disease recurrence and cancer-specific mortality in 151 cases treated by radical cystectomy for SCC of the urinary bladder All patients
Disease recurrence Hazard 95% CI ratio
P value Hazard 95% CI ratio
P value
T category Grade LNþ LVI FGF2
2.244 1.411 1.708 1.537 2.561
0.025 0.387 0.178 0.304 0.009
0.113 0.881 0.295 0.104 0.033
1.108–4.544 0.646–3.081 0.784–3.718 0.677–3.492 1.261–5.201
Cancer-specific mortality
2.061 1.079 1.714 2.295 2.679
0.843–5.040 0.400–2.907 0.625–4.704 0.842–6.256 1.082–6.633
the urinary bladder such as high grade, LVI, and LNI. Moreover, FGF2 was associated with both disease recurrence and cancer-specific mortality in Kaplan–Meier analyses and was an independent predictor of both disease recurrence and cancer-specific mortality. The 5-year DFS and CSS rates almost doubled in patients with no FGF2 alterations in comparison with those with FGF2 alteration. The risk of cancer recurrence or death due to cancer was 42.6 times higher in FGF2-altered tumors in comparison with that of those without alteration. Adding FGF2 to a model including standard clinicopathologic prognostics showed a significant improvement in predictive accuracy. Moreover, tumors with altered FGF2 and poor oncological outcome were also more likely to have alterations in markers associated with angiogenesis, cell cycle regulation, apoptosis, and signal transduction cancer pathways. Approximately 20% to 40% of patients with organconfined disease have recurrence after RC [4,25]. Previous studies have shown that stage, grade, LNI, and LVI are insufficient in predicting which patients are most likely to have recurrence owing to biological heterogeneity among tumors with similar stage [4,25]. Because systemic chemotherapy is ineffective as an adjuvant therapy for SCC, there is need for improved understanding of tumor biology that may identify actionable targets for therapy [20]. Exploring molecular alterations can improve prognostication and help design of clinical trial, development of novel therapies, and tailoring aggressive and effective multimodal treatment approached for high-risk patients. FGFs are involved in a variety of cellular processes, such as stemness, proliferation, antiapoptosis, drug resistance, and angiogenesis [23,26–30]. Previous studies have shown that FGF2 can be produced by the tumor cells or by the surrounding stroma and promotes oncogenesis through both autocrine and paracrine modes [23,24,31,32]. Its expression is under the control of a variety of transcription factors that are known to contribute to oncogenesis. The interaction with specific FGF receptors leads to unchecked proliferation via multiple cancer pathways, including the phosphatidylinositol 3-kinase-Akt 1 pathway, Ras-MAP kinase pathway, and protein kinase C–dependent signal transduction pathway [7,16,23,32]. In this study, we found multiple
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alterations in other biomarkers belonging to different cancer pathways in patients who had poor oncological outcomes and FGF2 alterations. Most of these patients had alterations in angiogenesis markers (100% VEGF and 93% TSP-1), apoptotic markers (100% Bcl-2 and 93% caspase-3), cell cycle regulators (100% p27 and 67% cyclin E), markers of proliferation (100% Ki-67), inflammatory markers (60% COX-2), and markers of signal transduction (67% EGFR). It was shown from previous studies that FGF2 can induce a proinflammatory signature in endothelium through activation of multiple growth factors, cytokines, chemokines, membrane receptors, and adhesion molecules [23,32]. FGF2 regulates the expression of cadherins, integrins, metalloproteases, and various extracellular matrix components [23]. An intimate cross talk exists between inflammatory and angiogenic responses during FGF2-driven neovascularization [23,32]. Clinical studies have shown that there is a strong correlation among FGF2, VEGF, vascular density, metastatic potential, and poor survival with human tumors [23,33]. VEGF produces a number of important biological effects such as endothelial mitogenesis and migration, extracellular matrix remodeling via induction of proteinases, increased vascular permeability, and maintenance of newly formed vasculature [7,23]. Interestingly, the role of FGF2 is not limited to prognosis for multiple cancers, including SCC of the bladder, as shown in our study. Perhaps, more importantly, FGF2 might be implicated in response to different cancer treatment modalities and therapeutics. Expression of FGF2 had been correlated with resistance to paclitaxel in tumors in human patients and to cisplatin in a human BC cell line [16,34]. FGF2 may alter DNA function and apoptotic threshold in cells. Initial efforts to block FGF2-mediated drug resistance in animal models showed favorable results [35]. Thus the manipulation of FGF2 activities to increase the effectiveness of chemotherapeutics may represent a promising approach. Older studies had shown that interferon-α can inhibit FGF2, angiogenesis, and BC in mice [36]. Pentose polysulfate sodium reduced urinary FGF2 in enterocystoplasty patients [37]. Recently, small-molecule FGF receptor inhibitors were found to block FGFRdependent urothelial carcinoma in vitro and in vivo [38]. Another novel approach is combining FGF2-targeted adenovirus-mediated mutant-Rad 50 gene transfer to enhance SCC chemosensitization [19]. Another group has suggested the possibility of using FGF antisense mRNAs in esophageal SCC overexpressing FGF2 [17]. Radiotherapy is an integral part of different SCC treatment protocols. However, frequently, tumor response is insufficient. Irradiation might induce additional undesired effects such as the release of angiogenic growth factors, potentially impairing the expected irradiation effect. Novel strategies of cancer therapy combining irradiation and antiangiogenic therapy are under evaluation. The idea behind this concept is to normalize the dysfunctional tumor vasculature or to inhibit vessel regrowth or repair of the
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irradiation-damaged tumor vasculature. Increased FGF2 release and proliferation were noticed after combined irradiation/anti VEGF treatment in xerotransplanted SCC [30]. FGF2 upregulation is probably a compensatory reaction of the tumor owing to VEGF depletion. Similarly, therapies targeting VEGF or VEFGR were found to be hindered by drug resistance, as a result of up-regulation of other angiogenic factors such as FGF2 [26,27]. As an intimate cross talk exists between FGF2 and VEGF, targeting both FGF2 and VEGF might be superior to targeting VEGF alone [32]. Using targeted therapies with an additional anti-FGF2 effect might represent a potential strategy to compensate resistance to anti-VEGF only therapy [32]. Targeting EGFR only may have similar drug resistance problem [26,28,29]. Recent studies have shown involvement of FGF2 cancer pathway as a novel mechanism of acquired resistance to EGFR-targeted therapies [28,29]. It was shown that the FGF2 pathway might work independently or in combination with EGFR pathway and another intimate cross talk could exist between FGF2 and EGFR [24]. Dovitinib (TKI258), ponatinib (AP24534), and AZD4547 are multikinase inhibitors with multiple targets that have been investigated in clinical trials for various types of human cancer treated previously with other targeted therapies [26,27,39]. Future FGF-targeted therapies can have a potential role as a maintenance treatment following other modalities like RC, radiation, and other systemic therapies [39]. The present study has some limitations, particularly in retrospective data collection and the variability of IHC techniques. To limit this variability, we used a combination of automated methods and an experienced pathologist to confirm all IHC readings. Our findings need to be validated prospectively, and perhaps this can be achieved through a multicenter study. Internal validation was not tried as we are looking for external validation in other cohorts. To our knowledge, this is the first study that clearly shows an association of FGF2 with aggressive pathologic features and poor oncological outcome in patients with bladder SCC. Future prognostic tools combining biomarkers with classic pathologic prognostics can improve oncological outcome prediction, patient counseling, follow-up scheduling, use of adjuvant therapies, and design of clinical trials.
5. Conclusions FGF2 overexpression is associated with aggressive pathologic features and worse outcomes after RC for SCC, suggesting a potential prognostic role for FGF2 in bladder SCC. Alteration in FGF2 may happen among interactions between different cancer pathways involving inflammation, angiogenesis, inhibition of cell cycle regulation and apoptosis, and abnormal proliferation. Our findings support the need for evaluation of therapeutic strategies targeting FGF2 signaling in SCC of the urinary bladder.
References [1] Parkin DM. The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl 2008;218:12–20. [2] Parkin DM, Bray F, Ferlay J, Jemal A. Cancer in Africa 2012. Cancer Epidemiol Biomarkers Prev 2012;118(18):4372–84. [3] Shokeir AA. Squamous cell carcinoma of the bladder: pathology, diagnosis and treatment. BJU Int 2004;93:216–20. [4] Youssef RF, Shariat SF, Kapur P, Kabbani W, Mosbah A, Abol-Enein H, et al. Prognostic value of cyclooxygenase-2 expression in squamous cell carcinoma of the bladder. J Urol 2011;185:1112–7. [5] Youssef R, Kapur P, Kabbani W, Shariat SF, Mosbah A, Abol-Enein H, et al. Bilharzial vs non-bilharzial related bladder cancer: pathological characteristics and value of cyclooxygenase-2 expression. BJU Int 2011;108:31–7. [6] Leibovici D, Grossman HB, Dinney CP, Millikan RE, Lerner S, Wang Y, et al. Polymorphisms in inflammation genes and bladder cancer: from initiation to recurrence, progression, and survival. J Clin Oncol 2005;23:5746–56. [7] Shariat SF, Youssef RF, Gupta A, Chade DC, Karakiewicz PI, Isbarn H, et al. Association of angiogenesis related markers with bladder cancer outcomes and other molecular markers. J Urol 2010;183:1744–50. [8] Chikazawa M, Inoue K, Fukata S, Karashima T, Shuin T. Expression of angiogenesis-related genes regulates different steps in the process of tumor growth and metastasis in human urothelial cell carcinoma of the urinary bladder. Pathobiology 2008;75:335–45. [9] Okada-Ban M, Moens G, Thiery JP, Jouanneau J. Nuclear 24 kD fibroblast growth factor (FGF)-2 confers metastatic properties on rat bladder carcinoma cells. Oncogene 1999;18:6719–24. [10] Miyake H, Yoshimura K, Hara I, Eto H, Arakawa S, Kamidono S. Basic fibroblast growth factor regulates matrix metalloproteinases production and in vitro invasiveness in human bladder cancer cell lines. J Urol 1997;157:2351–5. [11] Zaravinos A, Volanis D, Lambrou GI, Delakas D, Spandidos DA. Role of the angiogenic components, VEGFA, FGF2, OPN and RHOC, in urothelial cell carcinoma of the urinary bladder. Oncol Rep 2012;28:1159–66. [12] Thomas-Mudge RJ, Okada-Ban M, Vandenbroucke F, VincentSalomon A, Girault JM, Thiery JP, et al. Nuclear FGF-2 facilitates cell survival in vitro and during establishment of metastases. Oncogene 2004;23:4771–9. [13] Rades D, Setter C, Dahl O, Schild SE, Noack F. Fibroblast growth factor 2–a predictor of outcome for patients irradiated for stage II-III nonsmall-cell lung cancer. Int J Radiat Oncol Biol Phys 2012;82:442–7. [14] Lefevre G, Babchia N, Calipel A, Mouriaux F, Faussat AM, Mrzyk S, et al. Activation of the FGF2/FGFR1 autocrine loop for cell proliferation and survival in uveal melanoma cells. Invest Ophthalmol Vis Sci 2009;50:1047–57. [15] Donnem T, Al-Shibli K, Al-Saad S, Busund LT, Bremnes RM. Prognostic impact of fibroblast growth factor 2 in non-small cell lung cancer: coexpression with VEGFR-3 and PDGF-B predicts poor survival. J Thorac Oncol 2009;4:578–85. [16] Gan Y, Wientjes MG, Au JL. Expression of basic fibroblast growth factor correlates with resistance to paclitaxel in human patient tumors. Pharm Res 2006;23:1324–31. [17] Barclay C, Li AW, Geldenhuys L, Baguma-Nibasheka M, Porter GA, Veugelers PJ, et al. Basic fibroblast growth factor (FGF-2) overexpression is a risk factor for esophageal cancer recurrence and reduced survival, which is ameliorated by coexpression of the FGF-2 antisense gene. Clin Cancer Res 2005;11:7683–91. [18] Chodak GW, Hospelhorn V, Judge SM, Mayforth R, Koeppen H, Sasse J. Increased levels of fibroblast growth factor-like activity in urine from patients with bladder or kidney cancer. Cancer Res 1988;48:2083–8. [19] Figures MR, Wobb J, Araki K, Liu T, Xu L, Zhu H, et al. Head and neck squamous cell carcinoma targeted chemosensitization. Otolaryngol Head Neck Surg 2009;141:177–83.
R.F. Youssef et al. / Urologic Oncology: Seminars and Original Investigations 33 (2015) 111.e1–111.e7 [20] Abol-Enein H, Kava BR, Carmack AJ. Nonurothelial cancer of the bladder. Urology 2007;69(Suppl. 1):93–104. [21] Youssef R, Kapur P, Shariat SF, Arendt T, Kabbani W, Mosbah A, et al. Prognostic value of apoptotic markers in squamous cell carcinoma of the urinary bladder. BJU Int 2012;110:961–6. [22] Youssef RF, Shariat SF, Kapur P, Kabbani W, Ghoneim T, King E, et al. Expression of cell cycle-related molecular markers in patients treated with radical cystectomy for squamous cell carcinoma of the bladder. Hum Pathol 2011;42:347–55. [23] Presta M, Andres G, Leali D, Dell'Era P, Ronca R. Inflammatory cells and chemokines sustain FGF2-induced angiogenesis. Eur Cytokine Netw 2009;20:39–50. [24] Marshall ME, Hinz TK, Kono SA, Singleton KR, Bichon B, Ware KE, et al. Fibroblast growth factor receptors are components of autocrine signaling networks in head and neck squamous cell carcinoma cells. Clin Cancer Res 2011;17:5016–25. [25] Ghoneim MA, Abdel-Latif M, el-Mekresh M, Abol-Enein H, Mosbah A, Ashamallah A, et al. Radical cystectomy for carcinoma of the bladder: 2,720 consecutive cases 5 years later. J Urol 2008;180:121–7. [26] Katoh M, Nakagama H. FGF receptors: cancer biology and therapeutics. Med Res Rev 2014;34:280–300. [27] Katoh M. Therapeutics targeting angiogenesis: genetics and epigenetics, extracellular miRNAs and signaling networks (Review). Int J Mol Med 2013;32:763–7. [28] Ware KE, Hinz TK, Kleczko E, Singleton KR, Marek LA, Helfrich BA, et al. A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop. Oncogenesis 2013;2:e39. [29] Terai H, Soejima K, Yasuda H, Nakayama S, Hamamoto J, Arai D, et al. Activation of the FGF2-FGFR1 autocrine pathway: a novel mechanism of acquired resistance to gefitinib in NSCLC. Mol Cancer Res 2013;11:759–67. [30] Fruth K, Weber S, Okcu Y, Noppens R, Klein KU, Joest E, et al. Increased basic fibroblast growth factor release and proliferation in xenotransplanted squamous cell carcinoma after combined irradiation/
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
111.e7
anti-vascular endothelial growth factor treatment. Oncol Rep 2012;27: 1573–9. Cao Y, Cao R, Hedlund EM. R Regulation of tumor angiogenesis and metastasis by FGF and PDGF signaling pathways. J Mol Med (Berl) 2008;86:785–9. Alessi P, Leali D, Camozzi M, Cantelmo A, Albini A, Presta M. AntiFGF2 approaches as a strategy to compensate resistance to anti-VEGF therapy: long-pentraxin 3 as a novel antiangiogenic FGF2-antagonist. Eur Cytokine Netw 2009;20:225–34. Billottet C, Janji B, Thiery JP, Jouanneau J. Rapid tumor development and potent vascularization are independent events in carcinoma producing FGF-1 or FGF-2. Oncogene 2002;21:8128–39. Miyake H, Hara I, Gohji K, Yoshimura K, Arakawa S, Kamidono S. Expression of basic fibroblast growth factor is associated with resistance to cisplatin in a human bladder cancer cell line. Cancer Lett 1998;123:121–6. Coleman AB. Positive and negative regulation of cellular sensitivity to anti-cancer drugs by FGF-2. Drug Resist Updat 2003;6: 85–94. Dinney CP, Bielenberg DR, Perrotte P, Reich R, Eve BY, Bucana CD, et al. Inhibition of basic fibroblast growth factor expression, angiogenesis, and growth of human bladder carcinoma in mice by systemic interferon-alpha administration. Cancer Res 1998;58: 808–14. Barrington JW, Fulford S, Fraylin L, Fish R, Shelley M, Stephenson TP. Reduction of urinary basic fibroblast growth factor using pentosan polysulphate sodium. Br J Urol 1996;78:54–6. Lamont FR, Tomlinson DC, Cooper PA, Shnyder SD, Chester JD, Knowles MA. Small molecule FGF receptor inhibitors block FGFRdependent urothelial carcinoma growth in vitro and in vivo. Br J Cancer 2011;104:75–82. Gozgit JM, Wong MJ, Moran L, Wardwell S, Mohemmad QK, Narasimhan NI, et al. Ponatinib (AP24534), a multitargeted panFGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models. Mol Cancer Ther 2012;11:690–9.
