Association of Angiogenesis Related Markers With Bladder
Cancer Outcomes and Other Molecular Markers
Shahrokh F. Shariat,* Ramy F. Youssef, Amit Gupta, Daher C. Chade,Pierre I. Karakiewicz, Hendrik Isbarn, Claudio Jeldres, Arthur I. Sagalowsky,Raheela Ashfaq and Yair LotanFrom the Departments of Urology (SFS, RFY, AG, DCC, AIS, YL) and Pathology (RA), University of Texas Southwestern Medical Center,Dallas, Texas, and Cancer Prognostics and Health Outcomes Unit, University of Montreal, Montreal, Quebec, Canada (PIK, HI, CJ)
Abbreviations
and Acronyms
bFGF � basic fibroblast growthfactor
LN � lymph node
MVD � microvessel density
RC � radical cystectomy
TSP-1 � thrombospondin 1
UCB � urothelial carcinoma ofthe bladder
VEGF � vascular endothelialgrowth factor
Submitted for publication September 21,2009.
Study received institutional review board ap-proval.
Supplementary material for this article can beobtained at http://www.utsouthwestern.edu/utsw/cda/dept10807/files/572811.html.
* Correspondence and current address: Divi-sion of Urology, Sidney Kimmel Center for Pros-tate and Urologic Cancer, Memorial Sloan-Ket-tering Cancer Center, 1275 York Ave., Box 27,New York, New York 10065 (e-mail: [email protected]).
See Editorial on page 1667.
Purpose: We tested whether the altered immunohistochemical expression ofangiogenesis related markers is associated with outcomes of patients withurothelial carcinoma of the bladder, and assessed the correlation of angiogenesisrelated markers with molecular markers commonly altered in urothelial bladdercarcinoma.Materials and Methods: Vascular endothelial growth factor, basic fibroblastgrowth factor and thrombospondin 1 expression data were collected, as weremicrovessel density data. Immunohistochemical staining was performed on spec-imens from 204 patients treated with radical cystectomy for urothelial carcinomaof the bladder. We also stained serial sections of the specimens for cyclin E1,cyclin D1, p53, p21, p27, pRB, Ki-67, Bcl-2, caspase-3, survivin and cyclooxygen-ase-2. We measured time to disease recurrence and cancer specific mortality, aswell as the association with clinical and pathological features and other molec-ular markers.Results: The altered expression of vascular endothelial growth factor (over ex-pression), basic fibroblast growth factor (over expression) and thrombospondin 1(decreased expression) was 86%, 79% and 63%, respectively. Median microvesseldensity was 20. All 4 markers were associated with established clinicopatholog-ical features of aggressive urothelial carcinoma of the bladder (such as stage,lymphovascular invasion and lymph node metastasis) and other molecular mark-ers. On multivariable analyses that adjusted for standard pathological featuresbasic fibroblast growth factor and thrombospondin 1 were independent predictorsof disease recurrence (HR 3.6, p � 0.002 and HR 2.2, p � 0.001, respectively) andcancer specific mortality (HR 2.8, p � 0.02 and HR 2.3, p � 0.003, respectively).When all 4 markers were included in 1 model basic fibroblast growth factor andthrombospondin 1 retained their independent association with disease recur-rence (HR 2.9, p � 0.014 and HR 1.8, p � 0.022, respectively) and only throm-bospondin 1 was independently associated with cancer specific mortality (HR 1.9,p � 0.031).Conclusions: Angiogenesis related molecular markers are commonly altered inurothelial carcinoma of the bladder, making them a target for therapy. Down-regulation of thrombospondin 1 and up-regulation of basic fibroblast growthfactor are independent predictors of clinical outcomes of patients with urothelialcarcinoma of the bladder.
ANGIOGENESIS MARKER EXPRESSION IN PATIENTS WITH BLADDER CANCER 1745
UROTHELIAL carcinoma of the bladder is the fourthmost common malignancy in men and the ninthmost common in women in the United States.1 Rad-ical cystectomy is the mainstay of treatment formuscle invasive and refractory noninvasive UCB.However, up to 50% of patients experience diseaserelapse and eventual death despite adequately per-formed surgery.2,3 Current neoadjuvant and adju-vant chemotherapy regimens may improve out-comes in select patients by a limited margin.4,5
These disappointing outcomes are partially due tosuboptimal patient selection for the administrationof routine therapeutics. Indeed standard clinical andpathological characteristics such as tumor gradeand stage provide only limited insight into the biol-ogy of the tumor. Identification of molecular alter-ations involved in UCB tumorigenesis and progres-sion will lead to more reliable prognostication andtreatment selection. Thus, there is an imminentneed for molecular markers associated with UCBprognostication and prediction of response to noveltargeted therapies.
Improved understanding of molecular biology andpathogenesis has opened avenues for the use of mo-lecularly targeted therapies. Currently several noveldrugs seem particularly promising such as inhibi-tors of tumor angiogenesis. Angiogenesis is impor-tant for tumor growth and metastasis. Angiogenesiscontributes to the tumor promoting extracellular mi-lieu by providing oxygen, nutrients and growth fac-tors to the neoplastic cells.6 Development of reliablemolecular predictive markers is expected to improvetreatment decisions, therapy development and out-comes in UCB. Smaller studies have shown thatVEGF, bFGF and TSP-1 levels as well as MVD-CD31 have prognostic value in UCB.7–10
To validate and further test the association ofVEGF, bFGF and TSP-1 expression, and MVD withUCB characteristics and outcomes, we stained spec-imens from 204 patients treated with RC. Moreoverwe evaluated the association of these angiogenesisrelated markers with cell cycle, proliferation andapoptosis related molecular markers.
MATERIALS AND METHODS
Patient PopulationThe study was performed after approval by a local HumanInvestigations Committee, and in accordance with an as-surance filed with and approved by the Department ofHealth and Human Services. Informed consent was ob-tained from each subject. The study cohort was composedof a consecutive sample of 204 patients treated with RCwho had specimens available and whose surgery was per-formed between July 1995 and July 2006 at a teachinghospital at the University of Texas Southwestern MedicalCenter, Dallas, Texas. None of the patients received neo-
adjuvant therapy.
Histology, tumor grade, stage and presence of carci-noma in situ were confirmed by blinded reevaluation ofthe original pathology slides according to strict criteria.For each patient comprehensive clinical and pathologicaldata elements were collected, and entered into an institu-tional review board approved database. Staff pathologistswith expertise in genitourinary pathology examined allspecimens according to previously published protocols.2
The 2002 TNM classification was used for pathologicalstaging and the 2002 WHO classification was used forpathological grading. The followup protocol was describedelsewhere.2 Patients were seen postoperatively at leastevery 3 to 4 months for the first year, semiannually for thesecond year and annually thereafter. Detection of cancersin the ureter and/or the urethra was coded as secondprimaries and not as recurrence.
Tissue Microarray,
Immunohistochemistry and ScoringThe tissue microarrays comprised 3 replicate 0.6 mm corediameter samples from the primary, index cancer. Thiswas done with a precision instrument (Beecher Instru-ments, Silver Spring, Maryland). Multiple sausage inter-nal controls were also placed with the standard controls.Sections (5�m) were obtained from the microarray andstained with hematoxylin and eosin to confirm the pres-ence of tumor as well as to assess tumor histology. Tumorsamples were randomly arranged on the blocks.
Immunostaining for the testing cohort was performedin a single laboratory on the Dako Autostainer (Carpinte-ria, California). Staining and scoring protocols for antibod-ies can be accessed online. We used bright field microscopyimaging coupled with advanced color detection software(Automated Cellular Imaging System, Clarient, Inc., AlisoViejo, California). We obtained the mean, maximum,range and standard deviation of the staining intensity aswell as percent positive nuclei/area measurements by us-ing random hot spots within each specimen. The meanwas calculated for data analysis.
The expressions of VEGF, bFGF and TSP-1 were scoredby assigning a proportion score and an intensity score.The proportion score was assigned and represented theestimated proportion of positive staining cells (0—0% to24% cells, 1—25% to 49%, 2—50% to 74% and 3—75% to100%). The intensity score was assigned and representedthe average intensity of positive cells (0—none, 1—weak,2—intermediate and 3—strong). The proportion and in-tensity scores were combined to obtain a total score whichranged from 0 to 6. We categorized the score into 0 to 2 vs3 to 6. VEGF and bFGF expression were considered al-tered when the combined score was 3 to 6. For TSP-1 thereis an inverse relationship and scores of 0 to 2 were con-sidered altered. For MVD we counted the vessels in 10random hot spots. Vessel area was calculated as the cu-mulative area occupied by the vessels in the scoring re-gion. MVD was calculated by dividing the mean vesselnumber per mm2 tissue area.
Statistical AnalysesUnivariable recurrence and survival probabilities wereestimated using the Kaplan-Meier method. Univariable
and multivariable Cox regression models addressed time
ANGIOGENESIS MARKER EXPRESSION IN PATIENTS WITH BLADDER CANCER1746
to recurrence and mortality. In all models proportionalhazards assumptions were verified using the Grambsch-Therneau residual based test. The change in predictiveaccuracy resulting from the addition of markers to stan-dard predictors was quantified with Harrell’s concordanceindex.11 The area under the curve was quantified using200 bootstrap resamples. All reported p values are 2-sidedand statistical significance was set at 0.05. All statisticaltests were performed with S-Plus® Professional.
RESULTS
Association of VEGF, bFGF, TSP-1 and MVD
With Clinical and Pathological Characteristics
Figure 1 shows the representative expression statusfor VEGF, bFGF, TSP-1 and CD31 (for MVD calcu-lations). The expression of VEGF was altered in 86%of patients, bFGF in 79% and TSP-1 in 63%. MedianMVD was 20. Altered VEGF expression was associ-ated with advanced pathological stage, lymphovas-cular invasion and LN metastasis (p �0.001). Al-tered bFGF expression was associated with hightumor grade (p � 0.007), advanced pathologicalstage, lymphovascular invasion and LN metastasis(p �0.001). Altered TSP-1 expression was associatedwith advanced pathological stage (p � 0.0043), lym-phovascular invasion (p � 0.014) and LN metastasis(p � 0.013). Patients with LN metastasis had higherMVD in the primary tumor compared to those withnegative lymph nodes (p � 0.020).
Figure 1. Representative expression status for VEGF, bFGF,
Association of VEGF, bFGF,
TSP-1 and MVD With Clinical Outcomes
Disease recurred in 102 patients (50%), 83 died ofmetastatic UCB (40.7%) and 19 (9.3%) died of nonUCBrelated causes. All patients who died of UCB hadwidely metastatic disease. Overall 2, 5 and 10-yearrecurrence-free survival estimates were 60% (stan-dard error 3), 56% (3) and 52% (3), respectively.UCB specific survival probability estimates at 2, 5and 8 years were 65% (3), 60% (3) and 54% (4),respectively. Median followup for censored caseswas 48 months (IQR 40 to 56).
On univariable analysis VEGF, bFGF, TSP-1 andMVD were associated with disease recurrence andcancer specific mortality (p �0.05, figs. 2 and 3,respectively). On multivariable analyses only bFGFand TSP-1 were independent predictors of diseaserecurrence (HR 3.6, p � 0.002 and HR 2.2, p � 0.001,respectively). When all 4 markers were included inthe same model bFGF and TSP-1 retained theirindependent association with disease recurrence(HR 2.9, p � 0.014 and HR 1.8, p � 0.022, respec-tively). The accuracy of a base model that includedstandard pathological features (ie grade, lympho-vascular invasion, pathological stage and LN status)for the prediction of disease recurrence was 72.7%.Addition of FGF and TSP-1 improved the accuracy ofthe base model by 1.6% and 0.9%, respectively. Ad-dition of VEGF or MVD did not improve the accu-racy of the base model.
TSP-1 levels and CD31 for MVD. Reduced from �20.
ANGIOGENESIS MARKER EXPRESSION IN PATIENTS WITH BLADDER CANCER 1747
On multivariable analysis only bFGF and TSP-1were independent predictors of cancer specific mor-tality (HR 2.8, p � 0.02 and HR 2.3, p � 0.003,respectively). When all 4 markers were included inthe same model only TSP-1 retained its independentassociation with cancer specific mortality (HR 1.9,p � 0.031). The accuracy of a base model that in-cluded standard pathological features (ie tumorgrade, lymphovascular invasion, pathological stageand LN status) for the prediction of cancer specificmortality was 72.8%. Addition of TSP-1 improvedthe accuracy of the base model by 0.6%. Addition ofVEGF, FGF or MVD did not improve the accuracy ofthe base model.
Association of VEGF, bFGF, TSP-1
and MVD With Other Molecular Markers
Altered VEGF expression was associated with al-tered p21 status (p � 0.003), altered p27 status(p �0.001), altered pRB status (p �0.001), alteredcyclin E1 expression (p �0.001) and altered Ki-67expression (p � 0.015). Altered bFGF expressionwas associated with altered p27 status (p � 0.003),altered pRB status (p � 0.005) and altered Ki-67expression (p � 0.025). Altered TSP-1 expression
Figure 2. Probability of bladder cancer recurrence-free survival6), bFGF (green line, altered when combined score was 3 to 6), TMVD-CD31 (blue line, altered when MVD count above median).
was associated with altered cyclooxygenase-2 status
(p �0.001), altered p21 status (p � 0.002) and al-tered p27 status (p � 0.002). MVD was higher inpatients with altered p53 status (p � 0.028) andaltered cyclin E1 status (p � 0.03).
DISCUSSION
In this study we investigated the usefulness of 4angiogenesis related molecular markers in the pre-diction of the clinical outcome of UCB. VEGF wasaltered in more than 85% of patients treated withradical cystectomy. However, only TSP-1 and bFGFwere independent predictors of disease recurrenceand cancer specific mortality on multivariable anal-yses after adjusting for standard clinicopathologicalfeatures. When all 4 markers were included in 1model, TSP-1 and bFGF retained their independentassociation with disease recurrence but only TSP-1was independently associated with cancer specificmortality.
Down-regulation of TSP-1 expression was inde-pendently associated with pathological features ofadvanced disease as well as with cancer recurrenceand mortality. TSP-1, an important component ofthe extracellular matrix, has been implicated in the
ing to VEGF (green line, altered when combined score was 3 togreen line, altered when combined score was 0 to 2) levels and
accordSP-1 (
regulation of cell growth and proliferation, cell mo-
ANGIOGENESIS MARKER EXPRESSION IN PATIENTS WITH BLADDER CANCER1748
tility, cytoskeletal organization, inflammation andwound healing, and the development and differenti-ation of cell types. Recently it has been shown to bea potent inhibitor of angiogenesis in vitro and invivo.12,13 Grossfeld et al previously reported thataltered TSP-1 expression was independently associ-ated with an increased risk of disease recurrenceand all cause mortality in 163 patients treated withRC.10 However, the prognostic value of TSP-1 wasnot independent of p53 expression status since these2 molecular markers were strongly correlated. Inour study we could not demonstrate a correlationbetween TSP-1 and p53 expression. However, loss ofTSP-1 expression was associated with alterations inother cell cycle regulators such as p21, which is ap53 dependent and independent downstream regu-lator of p53 activity.14,15 In addition, altered TSP-1expression was associated with altered p27 expres-sion, a Cip/Kip family of cyclin dependent kinaseinhibitor protein that controls cell cycle progressionat G1.16,17
bFGF was associated with established features ofbiologically aggressive UCB such as pathologicalstage, lymphovascular invasion, LN metastasis, mo-lecular markers commonly altered in UCB (ie p27,
Figure 3. Probability of bladder cancer specific survival accordin(green line, altered when combined score was 3 to 6), TSP-1 (gree(blue line, altered when MVD count above median).
pRb and Ki-67) and disease recurrence. It has been
hypothesized that during wound healing and tumordevelopment the action of heparan sulfate degrad-ing enzymes activates bFGF, thus mediating theformation of new blood vessels. bFGF may behave asa transforming/oncogenic factor inducing cell prolif-eration and motility. The interaction with specificreceptors leads to unchecked proliferation via theRas-MAPK pathway. The most common oncogenesand some of the tumor suppressor genes relevant toUCB are components of this pathway.18 Clinicalstudies have shown that there is a strong correlationamong bFGF, VEGF, and vascular density and met-astatic potential, and poor survival with human tu-mors.19 Moreover altered bFGF expression has beenassociated with resistance to cisplatin in humanUCB cell lines.20 Since bFGF also contributes toUCB progression via its extracellular matrix in-volvement, bFGF could be a candidate for therapeu-tic targeting.21
Of the angiogenic factors VEGF has been identi-fied as a crucial regulator of normal and pathologicalangiogenesis. VEGF produces a number of impor-tant biological effects such as endothelial mitogene-sis and migration, extracellular matrix remodelingvia induction of proteinases, increased vascular per-
GF (green line, altered when combined score was 3 to 6), bFGF, altered when combined score was 0 to 2) levels and MVD-CD31
g to VEn line
meability and maintenance of newly formed vascu-
ANGIOGENESIS MARKER EXPRESSION IN PATIENTS WITH BLADDER CANCER 1749
lature.6,22 VEGF was over expressed in 86% of pa-tients with UCB, supporting its role in bladdertumorigenesis and identifying it as a potential tar-get for therapy in patients with UCB. In addition,VEGF over expression was associated with patho-logical features, and altered expression of p21, p27,pRB, cyclin E1 and Ki-67, suggesting complex inter-actions between different UCB pathways. Whilethese associations are important, in our study VEGFexpression had no prognostic value in patientstreated with RC. Increased VEGF levels can resultin increased vascular permeability and interstitialfluid pressure, impairing chemotherapy delivery.Adding anti-VEGF to the chemotherapy of UCBmight lead to improved responses.22,23
We showed that MVD was higher in patients withLN metastasis. Jaeger et al stained 41 muscle invasivetumors following RC, demonstrating higher MVD inthe primary tumor if LN metastases were present.24
MVD was the only molecular marker associated withp53 alterations out of the 4 markers. However, inagreement with some and in contrast with other pre-vious studies we could not find an association betweenMVD and outcomes after RC.8,25,26 Explanations forthese discrepancies include differences in the choice ofantibody, as well as immunohistochemical stainingand scoring protocols. For example, the endothelium oftumor and normal tissues is heterogeneous. Thus,pan-endothelial antibodies such as CD31 may notstain tumor vessels to the same degree and may gen-erally react better with larger vessels.27
The importance of angiogenic factors in the biol-ogy of UCB (preclinical and retrospective clinicaldata), together with the limited efficacy and safety ofcurrent chemotherapy and the activity of antiangio-genic agents in other cancers, support the need forphase I/II clinical trials of antiangiogenic agents in
UCB. Meanwhile understanding the mechanisms of
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The retrospective design, short followup and useof immunohistochemistry are among the limitationsof our study. Immunohistochemistry may be affectedby choice and concentration of antibody, specimenhandling and technical procedures, variability in in-terpretation and stratification criteria, and scoringsystems. To adjust for some of these limitations weused tissue microarrays and an automated au-tostainer to eliminate differences in staining, and toreduce the variables in analysis. We also used areproducible and accurate standardized, automatedscoring system to assess molecular marker expres-sion in tissue sections based on bright field micros-copy imaging coupled with advanced color detectionsoftware. The improvements in predictive accuracyfor outcome with angiogenesis related molecularmarkers were limited. However, the association ofthese markers with prognosis and the high propor-tion of tumors that express these markers makethem attractive targets for targeted therapies.
CONCLUSIONS
Angiogenesis is a hallmark of cancer. In this initialstudy VEGF was commonly altered in UCB speci-mens, thereby serving as a possible target for ther-apy. Down-regulation of TSP-1 and up-regulation ofbFGF are independent predictors of UCB recur-rence, and down-regulation of TSP-1 is also associ-ated with UCB specific mortality. This integratedunderstanding of the biology and its translationalrelevance may lead to the development of novel
treatment approaches for UCB.
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EDITORIAL COMMENT
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Several small studies have demonstrated the overexpression of angiogenesis related markers such asVEGF and bFGF in bladder cancer as well as theassociation with aggressive tumor phenotypes.1,2
However, this study of more than 200 patients offersthe most convincing evidence of this phenomenon.Although bladder cancers may be sensitive to che-motherapy, durable responses are the exception andlong-term survival remains poor. The results of thisstudy suggest that therapy specifically targeted toangiogenesis pathways may serve as an alternative
Multiple phase 2 and 3 trials are under way to studythe efficacy of bevacizumab and multikinase inhibi-tors such as sunitinib and sorafenib, although earlyresults of the former have been somewhat disap-pointing (reference 22 in article). These findings un-derscore the importance of further investigation intothe biology of bladder cancer if we hope to improvethe survival of patients with advanced disease.
Tin C. Ngo and Mark L. Gonzalgo
Department of UrologyStanford University School of Medicine
or adjunct to current chemotherapeutic regimens. Stanford, California
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2. Allen LE and Maher PA: Expression of basic fibroblast growth factor and its receptor in an invasive bladder carcinoma cell line. J Cell Physiol 1993; 155: 368.
