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Scandinavian Journal of Urology

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Is Bcl-2 a predictive marker of neoadjuvantchemotherapy response in patients with urothelialbladder cancer undergoing radical cystectomy?

Polat Turker, Ulrika Segersten, Per-Uno Malmström & Tammer Hemdan

To cite this article: Polat Turker, Ulrika Segersten, Per-Uno Malmström & Tammer Hemdan (2019)Is Bcl-2 a predictive marker of neoadjuvant chemotherapy response in patients with urothelialbladder cancer undergoing radical cystectomy?, Scandinavian Journal of Urology, 53:1, 45-50,DOI: 10.1080/21681805.2019.1575467

To link to this article: https://doi.org/10.1080/21681805.2019.1575467

© 2019 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup.

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ARTICLE

Is Bcl-2 a predictive marker of neoadjuvant chemotherapy response in patientswith urothelial bladder cancer undergoing radical cystectomy?

Polat Turker, Ulrika Segersten, Per-Uno Malmstr€om and Tammer Hemdan

Department of Surgical Sciences, Uppsala University, Uppsala, Sweden

ABSTRACTBackground: Response to neoadjuvant cisplatin treatment in bladder cancer has been linked toexpression of Bcl-2 protein by cancer cells. The objective of this study was to test Bcl-2 as a predictivemarker of neoadjuvant cisplatin chemotherapy response in a patient cohort from randomized cystec-tomy trials.Methods: Tumor samples were taken from 247 patients with T2–T4 bladder cancer enrolled in tworandomized trials comparing cystectomy with or without neoadjuvant chemotherapy. Tissue microar-rays from pre-intervention transurethral resection specimens were assessed for Bcl-2 protein status byimmunohistochemistry. Extension of staining above 10% was regarded as positive. Downstaging andsurvival ratios in relation to Bcl-2 immunoreactivity and neoadjuvant chemotherapy utilization werecalculated using the log rank test and multivariate Cox proportional hazards regression analyses.Results: Bcl-2 expression was positive in 38% and negative in 62% of the 236 evaluable patients. Bcl-2negative patients receiving neoadjuvant chemotherapy had a significant increase in survival(p¼ 0.009), while Bcl-2 positive patients showed no difference (p¼ 0.4). However, the interaction vari-able between neoadjuvant chemotherapy and biomarker status was not significant (p¼ 0.38). Whenthe prognostic value was assessed in the no-chemotherapy group, 5-year overall survival times weresignificantly better among Bcl-2 positive patients than among Bcl-2 negative patients (42 months vs33 months, p¼ 0.04), but again Bcl-2 status did not remain independent when other factors wereadjusted. Also, in a multivariate analysis with all patients, Bcl-2 was not significant.Conclusions: Bcl-2 status is not an independent predictor of neoadjuvant cisplatin chemotherapyresponse and is not prognostic in muscle-invasive bladder cancer.

Abbreviations: M.I.B.C.: muscle-invasive urothelial bladder cancer; O.S.: overall survival; N.C.S.: Nordiccollaborative study; T.U.R.: transurethral resection; F.F.P.E.: formalin-fixed paraffin-embedded; T.M.A.s:tissue microarrays; I.H.C.: immunohistochemistry; D.S.S.: disease-specific survival; M.-V.A.C.: methotrex-ate, vinblastine, doxorubicin and cisplatin

ARTICLE HISTORYReceived 27 November 2018Revised 16 January 2019Accepted 24 January 2019

KEYWORDSUrothelial bladder cancer;Bcl-2; neoadjuvantcisplatin; predictive

Introduction

Radical cystectomy is the standard treatment in muscle-inva-sive urothelial bladder cancer (M.I.B.C.). Unfortunately, thistreatment provides a 5-year survival rate of only �50% [1].Neoadjuvant cisplatin-containing combination chemotherapyhas been shown to improve overall survival (O.S.) by �5–8%at 5 years [2,3], and is recommended for patients with local-ized (T2–T4a) muscle-invasive bladder cancer and adequaterenal function [4]. However, the proportion of patients givenneoadjuvant treatment is low. The major reasons for this arethat the survival benefit is low when given to all patients,and patients not responding to neoadjuvant chemotherapywould be exposed to unnecessary treatment-related toxic-ities [3,5]. There is an urgent need for markers suitable forpredicting the outcome of this treatment.

Apoptosis is the programmed cell death process whichmaintains a healthy balance between cell survival and celldeath in the organism. It can be initiated by both intrinsicand extrinsic signals [6]. One of the basic biological mecha-nisms behind cancer chemotherapy is to drive tumor cellsinto apoptotic cascade. The mode of cisplatin action hasbeen linked to its ability to crosslink with the purine baseson D.N.A., thus interfering with D.N.A. repair mechanisms,causing D.N.A. damage, and subsequently inducing apoptosisin cancer cells [7]. The mechanism by which damage recog-nition results in apoptosis is unclear; however, the inductionof p53 following D.N.A. strand breaks leads to apoptosis viathe intrinsic pathway and contributes to the cytotoxicity ofcisplatin [8]. When proteins involved in the apoptosis processbecome non-functional, the result may be increased drugresistance and cancer progression.

CONTACT Polat Turker polat.turker@surgsci.uu.se Department of Surgical Sciences, Uppsala University, Uppsala, 75185, SwedenSupplemental data for this article is available online at on the publisher’s website.

� 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built uponin any way.

SCANDINAVIAN JOURNAL OF UROLOGY2019, VOL. 53, NO. 1, 45–50https://doi.org/10.1080/21681805.2019.1575467

Bcl-2 family proteins are central regulators of this intrinsicpathway [9]. Some of the members of this family (e.g. BAXand BAK) are pro-apoptotic, while others (e.g. Bcl-2 and Bcl-XL) are anti-apoptotic [10]. The development of apoptosisdepends on the balance between pro-apoptotic and anti-apoptotic proteins, and these members are involved in thedownstream action by cisplatin [8]. Bcl-2 protein is located atthe intracellular membranes such as the mitochondrial mem-brane, nuclear membrane, and endoplasmic reticulum, andso may act to control ion channels, modulating caspase acti-vation or inhibiting cytochrome c export [11]. It also repre-sents transcriptional targets for the p53 tumor suppressorprotein that induces termination of the cell cycle or apop-tosis as a reduction to D.N.A. damage.

Immunohistochemical Bcl-2 expression has been studiedfor its prognostic value and predictive role for patientsreceiving chemotherapy for various cancer types, with con-flicting results [12–14]. Similarly, conflicting results have beenreported for urothelial cancer. Bcl-2 has been suggested tocorrelate with response to neoadjuvant chemotherapybefore cystectomy.

In a previous study, our group showed that emmprincould be a potential marker predicting neoadjuvant cisplatinresponse in cystectomy patients [15]. In that study, thepatient material and dataset used were based on tworandomized trials comparing cystectomy with or withoutneoadjuvant cisplatin. Having a control arm without chemo-therapy made it possible to separate the predictive andprognostic values of the marker studied. In this study, usingthe same cohort of patients, we aimed to validate the pre-dictive value of Bcl-2 regarding neoadjuvant chemotherapyresponse that was recently reported in a study by Kiss et al.[16]. Our pre-specified hypothesis was that bladder cancerpatients with negative Bcl-2 expression in tumor tissuewould have an improved response to neoadjuvant chemo-therapy compared to patients with Bcl-2 positive tumors.

Methods

Patient datasets

This study used the patient datasets from two prospectiverandomized trials by the Nordic collaborative group [2] com-paring cystectomy with vs without neoadjuvant chemother-apy. The total dataset was composed of 642 patients withprimary T1G3 or T2–T4aNXM0 urothelial bladder cancer whowere operated on between 1985 and 1997. Briefly, in the firsttrial, Nordic collaborative study 1 (N.C.S. 1), the intendedchemotherapy was two cycles of cisplatin 70mg/m2 intraven-ously and doxorubicin 30mg/m2 intravenously. All patientsin both the neoadjuvant and the control arm were plannedto receive irradiation preoperatively, consisting of 4 Gy dailyfor 5 consecutive days. In the second trial (N.C.S. 2), threecycles of cisplatin 100mg/m2 intravenously and methotrexate250mg/m2 intravenously were planned. Cystectomy was per-formed with lymph node dissection of the obturator fossa.The selection of 250 patients for this study is explained indetail elsewhere [15] (see also Supplementary Figure 1).Today, neoadjuvant chemotherapy is not routinely applied

for T1G3 tumor disease going into radical cystectomy. Toprevent the possible influence on survival analyses, T1G3patients (n¼ 3) were excluded from these datasets, leaving247 patients with T2–T4aNXM0 disease for inclusion. All thematerials were collected with ethical committee approval(approval no. 2008: 136).

Bcl-2 staining

Tumor specimens obtained during pre-treatment transureth-ral resection (T.U.R.) were preserved as formalin-fixed paraf-fin-embedded (F.F.P.E.) tissues. Representative tissue areasfrom F.F.P.E. materials were identified by a pathologist (C.B.),and two tissue microarrays (T.M.A.s) consisting of 1mm coreswere obtained from these areas. The T.M.A.s were con-structed with an automated instrument (ATA-27, BeecherInstruments, Sun Prairie, W.I.). Immunohistochemistry (I.H.C.)was performed as previously reported using an Autostainer480 instrument (Lab Vision, Fremont, C.A.) [17]. Antibodytype and concentrations were: DAKO, clone BCL-2 124, dilu-tion 1:100. The I.H.C. method had previously been tested andevaluated by the core facility of SciLifeLab, UppsalaUniversity producing the Human Protein Atlas [18]. Negativecontrols were produced using Universal Negative ControlSerum (A. Menarini Diagnostics, Winnersh, U.K.) instead ofprimary antibody.

Assessment of staining

Staining quality and score criteria were determined by aboard-certified pathologist (M.A.) with experience of Bcl-2staining in routine daily practice. Two independent observersassessed all samples, and discordant data were evaluated bythe pathologist, who made the final decision. For eachpatient, two different Bcl-2 stained T.M.A.s were evaluatedfor assessment of the cytoplasmic staining extension in termsof percentage area of positive staining. Staining intensitywas not considered. Samples with a staining area of 10% ormore were considered Bcl-2 positive, as previously described[16,19] (Figure 1).

Statistical analyses

O.S. and disease-specific survival (D.S.S.) curves were con-structed according to the Kaplan-Meier method, and differen-ces between groups were tested using the log rank test.Univariate and multivariate Cox proportional hazards regres-sion analyses were performed adjusting for patient inclusionin one of the two trials, treatment allocation to cystectomywith or without neoadjuvant chemotherapy, gender, age,T-category, and marker status. The interaction between neo-adjuvant treatment and marker status was also tested forsignificance in Cox analyses. Chi-squared tests and unpairedt-tests were used to compare categorical and quantitativevariables. Variables in the statistical analyses were defined asfollows. Clinical staging was determined by T.U.R. and imag-ing studies, and pathological staging was determined bycystectomy pathology. Downstaging was defined as

46 P. TURKER ET AL

downstaging from clinical T2 or higher to pathological Ta,Tis, or T0 stages. Age was assessed as a continuous variable.Because there were some differences in the interventionsbetween the two trials, trial group was also assessed as acategorical variable. For assessment of neoadjuvant chemo-therapy response, both downstaging and survival ratios werecalculated, but survival was used as response criteria.Statistical analyses were performed using version 20 of theI.B.M. S.P.S.S. software package.

Results

I.H.C. analysis was performed on tissue samples from 247patients, 11 of whom were excluded after the staining pro-cedure because of the low number of tumor cells on theT.M.A. sample. The clinicopathological properties of theremaining 236 patients are shown in Table 1.

Of the eligible tumor samples, 38% (n¼ 90) were assessedas Bcl-2 positive and 62% (n¼ 146) as Bcl-2 negative. Thepredictive value of Bcl-2 status was assessed by comparing

patients treated with cystectomy combined with neoadju-vant chemotherapy (chemotherapy group) vs those treatedonly with cystectomy (no-chemotherapy group) both for Bcl-2 negative and positive patients. Among the Bcl-2 negativepatients, the chemotherapy group had statistically better 5-year O.S. compared to the no-chemotherapy group (meanO.S.¼43 months vs 33 months, respectively, p¼ 0.009), whileno statistically significant difference was observed for theBcl-2 positive patients (mean O.S.¼ 47 months vs 42 months,respectively, p¼ 0.4) (Figure 2).

In addition, the interaction between Bcl-2 status and neo-adjuvant treatment was not significant (p¼ 0.38).

Similar results were also obtained for 5-year D.S.S. for allof the above calculations (data not shown). Information fordownstaging was available in 106 (89%) patients thatreceived neoadjuvant cisplatin treatment. Downstaging wasobserved in 48% and 38% of Bcl-2 positive and negativepatients, respectively (p¼ 0.3).

The prognostic value of Bcl-2 protein expression by tumorcells was evaluated in the no-chemotherapy group. Bcl-2positive patients had significantly better 5-year O.S. com-pared to Bcl-2 negative patients (42 months vs 33 months,p¼ 0.04). This survival advantage was not independent whenage, gender, trial, and clinical T-category variables wereadjusted (H.R.¼ 1.69, 95% CI¼ 0.95–3.00, p¼ 0.07). When allthe patients were analyzed in a multivariate Cox modelincluding gender, age, trial, clinical T-category, and Bcl-2 sta-tus, Bcl-2 was not a significant independent factor (Table 2).

Discussion

Bladder cancer, with its high incidence and mortality rate, isregarded as a serious health threat around the world.Although a large number of studies have attempted to dis-cover biological markers for better patient management,none have entered the clinical routine. Proper selection ofpatients for neoadjuvant chemotherapy is one problem.Studies involving more sophisticated methods such as genesignature techniques [20,21] show promise, but they areexpensive and have no widespread utility yet. In contrast,I.H.C. has been used around the world in routine practice formany years, resulting in increased experience with applica-tion and assessment of these techniques.

In this study, we evaluated the predictive value of Bcl-2expression on tumor cells for response to neoadjuvant cis-platin treatment, using I.H.C. to identify Bcl-2 protein statuson tumor tissue. Our hypothesis was that Bcl-2 negativepatients would have an enhanced neoadjuvant chemother-apy response and improved survival following neoadjuvantchemotherapy. To our knowledge, this is the largest study inthe literature evaluating this specific topic of bladder cancer.Furthermore, patients were randomized into treatment andcontrol arms. Having a control arm with no neoadjuvanttreatment gave us the chance to determine if enhanced sur-vival was related to neoadjuvant cisplatin treatment, or ifthese patients would in fact have a better prognosis withoutneoadjuvant treatment. Our data did not demonstrate Bcl-2as a predictor of neoadjuvant cisplatin response. Although

Figure 1. Immunohistochemistry images (�20 magnification): (a) Negative Bcl-2 expression of tumor cells (lymphocytes are immunoreactive and can beobserved), (b) Positive Bcl-2 expression of tumor cells.

SCANDINAVIAN JOURNAL OF UROLOGY 47

Bcl-2 status seemed related to neoadjuvant treatmentresponse in the Kaplan Meier analysis, the interactionbetween Bcl-2 status and treatment was not significant.According to Ballman [22], the interaction between themarker status (Bcl-2 status) and the treatment must be sig-nificant to prove that the marker has an independent pre-dictive value.

Positive Bcl-2 status was correlated with improved survivalfor the cystectomy-only group in univariate analyses, but thisrelation did not remain independent in the multivari-ate analyses.

In bladder cancer, positive expression of Bcl-2 protein hasbeen correlated with poor neoadjuvant cisplatin treatmentresponse [16,23,24]. One of the earlier clinical studies eval-uated the predictive value of Bcl-2 among 51 patients withM.I.B.C. in a randomized trial of radiotherapy with or withoutcisplatin, and reported similar findings to ours. Bcl-2 negativepatients receiving neoadjuvant chemotherapy had longersurvival rates than Bcl-2 positive patients receiving the sametreatment (median survival of 72 months vs 17 months) [23].The conclusion was that Bcl-2 status could identify patientswho may benefit from neoadjuvant chemotherapy.

Unfortunately, there was no information about the inter-action of marker status and neoadjuvant treatment.

More recently, Kiss et al. [16] studied the possible predict-ive effect of Bcl-2 and concluded that over-expression isrelated to poor chemotherapy response in bladder cancerand might help to select likely non-responders. Patientsnegative for Bcl-2 had a tendency towards a better responseto neoadjuvant chemotherapy, but this did not reach statis-tical significance. In univariate and multivariate analyses, Bcl-2 had no significant prognostic information. The major limi-tation of that study was the low patient numbers for evalu-ation of the predictive value of Bcl-2.

Another study evaluated the prediction of neoadjuvantM.-V.A.C. (methotrexate, vinblastine, doxorubicin and

Figure 2. Kaplan-Meier overall survival curves for patients with or without neoadjuvant chemotherapy and different Bcl-2 status.

Table 1. Clinicopathological characteristics of all 236 eligible patients (no statistical differences were detectedbetween neoadjuvant and cystectomy groups).

Bcl-2 negative (n¼ 146) Bcl-2 positive (n¼ 90)

Neoadjuvant (n¼ 67) Cystectomy (n¼ 79) Neoadjuvant (n¼ 52) Cystectomy (n¼ 38)

Gender Male 53 (79%) 64 (81%) 43 (83%) 34 (89%)Female 14 (21%) 15 (19%) 9 (17%) 4 (11%)

Mean age 63 (±9) 63 (±9) 64 (±7) 63 (±8)T stage cT2 31 (46%) 32 (41%) 24 (46%) 12 (32%)

cT3–4 36 (54%) 47 (59%) 28 (54%) 26 (68%)

Table 2. Multivariate Cox regression analyses of all 236 patients evaluatingassociated co-factors for survival.

H.R. 95% C.I. p

Gender (F/M) 1.23 0.76–1.97 0.39Age 1.01 1.00–1.04 0.21T-category (T3–4/T2) 1.47 0.99–2.18 0.06Trial (N.C.S.2/N.C.S.1) 1.26 0.80–2.00 0.33Neoadjuvant chemotherapy (no/yes) 1.64 1.11–2.41 0.01Bcl-2 (neg/pos) 1.50 1.00–2.27 0.05

48 P. TURKER ET AL

cisplatin) response followed by definitive treatment formuscle-invasive and node-negative urothelial cancer in aretrospectively collected dataset of 59 patients [24].Univariate analyses showed no significant predictive value ofBcl-2 immunoreactivity. However, a decreased 5-year survivalwas reported when all three markers in their panel (mdm-2,p53, Bcl-2) had been over-expressed compared to normalexpression (25% vs 54%). No association between chemo-therapy response and marker status was found. The studywas limited by the absence of a control group receiving noneoadjuvant chemotherapy.

Studies evaluating the prognostic value of Bcl-2 havereported conflicting results [25]. Although one study foundthat a high level of Bcl-2 protein was associated with apoorer response to different treatment modalities in differentcancer types [26], the opposing view was also supported bystudies correlating Bcl-2 positivity with improved progno-sis [12,14].

Bcl-2 positivity was correlated with decreased D.S.S.(H.R.¼ 1.71, p¼ 0.013) for M.I.B.C. patients undergoing cyst-ectomy [27]. Conversely, positive expression of Bcl-2 has alsobeen correlated with a favorable prognosis (H.R.¼ 0.179,p¼ 0.0474) in stage T2–4 invasive urothelial bladder cancer[28]. Other studies failed to show any significant prognosticvalue of this protein [16,19,23].

The major limitation of our study stems from methodo-logical limitations attributed to I.H.C., such as subjective scor-ing and reliance on antibody quality [29]. Another limitationis related to the patient cohorts. Preoperative radiotherapywas given to all patients in the first Nordic trial, but was notused in the second trial. To examine the impact of these lim-itations, we adjusted trial factors in multivariate analyses,and no significant effect was noted. In addition, the chemo-therapy regimens used in these trials not only differedbetween the trials, but also are not in general use today.From these two trials we could only use Swedish patient tis-sue, but, since the trials were stratified by country, the risk ofconfounding was minimal. Lymph node status in the cystec-tomy specimen, which is considered one of the most import-ant prognostic variables in M.I.B.C. today, was also notavailable in our material. Finally, it is important to mentionthat we evaluated only one protein in the Bcl-2 family.Investigation of different proteins from this family couldbe crucial.

Conclusions

According to the results of this study, Bcl-2 is neither anindependent predictor of neoadjuvant chemotherapyresponse, nor a prognostic marker for M.I.B.C. patients.

Acknowledgments

We are grateful to pathologists Christer Busch (C.B.) and MaysaaAbdulsalam Abdulla (M.A.) for evaluating the tumor tissue and annotat-ing the immunohistochemical staining, and to statistician Lisa Wernrothfor skilful assistance with the statistical analysis. We are also grateful tothe members of the Nordic Urothelial Cancer group for their contribu-tion with follow-up and collection of cancer material.

Funding

The study was supported by the Swedish Cancer Society, the HilleviFries Research Fund, the Lions Fund for Cancer Research, theFoundation in Memory of Johanna Hagstrand and Sigfrid Linn�ers, andthe Erik, Karin, G€osta Selanders Foundation.

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