Urologic Oncology: Seminars and Original Investigations ] (2015) ∎∎∎–∎∎∎

http://dx.doi.org/10.1016/j1078-1439/r 2015 Elsev

Presented in part as a p2014.This study was supporte

League.* Corresponding author.E-mail address: r_seile

Original article

Bcl-2 predicts response to neoadjuvant chemotherapy and isoverexpressed in lymph node metastases of urothelial cancer of

the bladder

Bernhard Kiss, M.D.a, Veronika Skuginna, M.D.a, Achim Fleischmann, M.D.b, Robert H. Bellc,Colin Collins, Ph.D.d, George N. Thalmann, M.D.a, Roland Seiler, M.D.a,d,*

a Department of Urology, University of Bern, Bern, Switzerlandb Institute of Pathology, University of Bern, Bern, Switzerland

c Department of Bioinformatics, University of British Columbia, Vancouver, Canadad Department of Urologic Sciences, University of British Columbia, Vancouver, Canada

Received 31 August 2014; received in revised form 9 December 2014; accepted 10 December 2014

Abstract

Purpose: To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuvant chemotherapy in patientswith urothelial cancer of the bladder (UCB).Methods: Bcl-2 expression was analyzed in 2 different tissue microarrays (TMAs). One TMA was constructed of primary tumors and

their corresponding lymph node (LN) metastases from 152 patients with chemotherapy-naive UCB treated by cystectomy and pelviclymphadenectomy (chemotherapy-naive TMA cohort). The other TMA was constructed of tumor samples obtained from 55 patients withUCB before neoadjuvant chemotherapy (transurethral resection of the bladder cancer) and after cystectomy with pelvic lymphadenectomy(residual primary tumor [ypTþ], n ¼ 38); residual LN metastases [ypNþ], n ¼ 24) (prechemotherapy/postchemotherapy TMA cohort).Bcl-2 overexpression was defined as 10% or more cancer cells showing cytoplasmic immunoreactivity.Results: In both TMA cohorts, Bcl-2 overexpression was significantly (P o 0.05) more frequent in LN metastases than in primary

tumors (chemotherapy-naive TMA group: 18/148 [12%] in primary tumors vs. 39/143 [27%] in metastases; postchemotherapy TMA:ypTþ 7/35 [20%] vs. ypNþ 11/19 [58%]). In the neoadjuvant setting, patients with Bcl-2 overexpression in transurethral resection of thebladder cancer specimens showed significantly (P ¼ 0.04) higher ypT stages and less regression in their cystectomy specimens than did thecontrol group, and only one-eighth (13%) had complete tumor regression (ypT0 ypN0). In survival analyses, only histopathologicalparameters added significant prognostic information.Conclusions: Bcl-2 overexpression in chemotherapy-naive primary bladder cancer is related to poor chemotherapy response and might

help to select likely nonresponders. r 2015 Elsevier Inc. All rights reserved.

Keywords: Bladder cancer; Neoadjuvant chemotherapy; Response prediction; Bcl-2

1. Introduction

Although neoadjuvant chemotherapy is a standard treat-ment for different types of cancer, its use in urothelial cancer

.urolonc.2014.12.005ier Inc. All rights reserved.

roffered paper at the EAU Meeting, Stockholm,

d by Grant 36 - 418 (R.S.) from the Bern Cancer

Tel.: þ41-31-632-2045; fax: þ41-31-632-2181.r@gmx.ch (R. Seiler).

of the bladder (UCB) has long been controversial. Impor-tantly, mature data from the recently published trials [1,2]show a significant survival benefit in patients receivingneoadjuvant chemotherapy, a finding that could shift clinicalpractice to this treatment strategy for muscle-invasive UCB[3]. However, because a number of patients do not respond toneoadjuvant chemotherapy and are thus overtreated, person-alization of this approach is mandatory.

Alterations in the apoptotic cascade are a hallmark ofhuman cancers and have been shown to be related to

B. Kiss et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–82

chemoresistance [4]. Apoptosis is precisely controlled along2 different pathways: the receptor (intrinsic) pathway bydeath receptor signaling and the mitochondrial (extrinsic)pathway via the mitochondria [5]. The Bcl-2 family plays apivotal role in the mitochondrial pathway and can bedivided into proapoptotic (e.g., BAX, BAK, and BclXS) andantiapoptotic members (e.g., Bcl-2, Bcl-xl, and Bcl-w) [5].Bcl-2 is the most investigated member of the Bcl-2 family; itplays a pivotal role in human cancer [6] and has been shownto interfere with chemotherapy [7,8].

The predictive value of a biomarker may differ betweentypes of cancer [9,10] and treatment regimens [11], and alsoBcl-2 overexpression has been shown to be a marker ofboth a favorable [9,12] and unfavorable [8,10,13,14]response to chemotherapy. Interestingly, a relation betweenBcl-2 overexpression and unfavorable response is partic-ularly seen in platinum-based treatment [8,10,13,14] whichconstitutes the backbone of neoadjuvant chemotherapy inUCB. However, the predictive value of Bcl-2 expression inthis setting is unknown.

2. Methods and materials

2.1. Patient selection and follow-up

For construction of the 2 tissue microarrays (TMA), 2cohorts of patients were enrolled. To construct thechemotherapy-naive TMA, between January 1985 and April2008, 152 consecutive patients with UCB preoperativelystaged cN0cM0 (physical examination, chest x-ray, abdomi-nal and pelvic computed tomography and bone scan) butwith lymph node (LN) metastases on pathological exami-nation were enrolled. They underwent a standardizedextended pelvic lymphadenectomy (PLND) with cystec-tomy in a curative intent, and none received neoadjuvantchemotherapy.

To construct the prechemotherapy/postchemotherapyTMA, from January 2000 to August 2011, 55 consecutivepatients with muscle-invasive UCB were staged after initialdiagnosis by chest x-ray, bone scan, and computed tomog-raphy scan of the abdomen and pelvis. Radiologically, LNmetastasis was presumed in the presence of pathologicallyenlarged LNs 41 cm in diameter. Whenever enlarged LNswere accessible to biopsy, histological/cytological proofwas obtained. All 55 patients of this cohort receivedneoadjuvant chemotherapy for locally advanced primarytumors (cT4, n = 15), radiological suspicion of LNmetastases (n = 38, 21 confirmed by biopsy), or otherreasons (n = 2). All patients underwent cystectomy withstandardized bilateral extended PLND as a second proce-dure with curative intent 6 weeks after termination ofchemotherapy at the Department of Urology, Universityof Berne.

Both cohorts were followed according to a standardprotocol at 3 and 6 months postoperatively, at 6-month

intervals until the fifth year postoperatively, and then yearlythereafter.

2.2. Neoadjuvant chemotherapy in the prechemotherapy/postchemotherapy TMA cohort

In all cases, recommendation for neoadjuvant chemo-therapy was made by a multidisciplinary tumor board.Standard treatment consisted of a median of 4 cycles ofcisplatin and gemcitabine (n = 47). In patients withcontraindications to cisplatin (creatinine clearance o60ml/min, poor performance status, clinically relevant hearingloss or tinnitus, or neuropathy), carboplatin was used(n = 8).

2.3. Surgical technique

Pelvic LNs were uniformly dissected as follows: fromthe crossing of the ureter with the common iliac artery alongthe external iliac vessel to the inguinal ligament, alllymphatic tissue was meticulously removed. The antero-lateral border of the dissection field was the genitofemoralnerve (external iliac region). All tissue except the obturatornerve was removed from the obturator fossa (obturatorregion). Lymphatic tissue on the internal iliac vessels andtheir branches were dissected similarly. Subsequent todividing the vascular (dorsolateral) pedicle of the bladder,lymphatic tissue located medially to the internal iliac arterywas also removed (internal iliac region). The dissectedtissue was fixed separately in neutral buffered formalin(4%) and submitted for pathological evaluation. The proto-col for the PLND procedure was described in detailearlier [15].

2.4. Pathology

All tissue slides from both cohorts were re-evaluatedindependently for this study by 2 investigators experiencedin uropathology (A.F. and R.S.) who were blinded to theoutcome data.

Transurethral resection of the bladder cancer (TURB)specimens were examined microscopically to determine thetumor grade and the histological subtypes. All cystectomyspecimens were processed and evaluated at the Institute ofPathology, University of Bern. All tissues were fixed inneutral buffered formalin (4%) for approximately 24 hours.Features characteristic of bladder lesions (residual tumors,scars) were documented (location, size, relation to bladderwall and surrounding tissues, and multifocality). Tissuesamples were taken for histological examination from theselesions and were routinely taken from the bladder neck withtrigone, the dome, and the anterior and posterior walls.Whenever no residual tumor was detected, the area suspi-cious for the tumor bed was totally embedded. PerivesicalLNs and resection margins of ureters and urethra weretaken. Microscopically tumor grade and tumor stage were

B. Kiss et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8 3

noted. LN specimens from each anatomical location wereseparately examined visually and by palpation, and all LNsthus detected were completely embedded. Whenever noLNs were found, the entire tissue was embedded forhistological examination. Features of interest were thenumber, largest total diameter, and extranodal extension(ENE) of LN metastases. All tumors and LN metastaseswere staged according to the Seventh International UnionAgainst Cancer classification of 2009 [16].

In the cystectomy and PLND specimens of the preche-motherapy/postchemotherapy TMA cohort, tumor regres-sion grades (TRG) after neoadjuvant chemotherapy weredetermined as previously described [17]:

TRG 1 (complete response): absence of histologicallyidentifiable residual cancer cells and extensive fibrosis ofthe tumor bed,

TRG 2 (strong response): predominant fibrosis of thetumor bed and residual tumor cells occupying less than 50%of this area, and

TRG 3 (weak or no response): abundant residual cancercells outgrowing tumor bed fibrosis or absence ofregression.

This grading system was applied separately to primarytumors and LN metastases, and the “dominant” TRG (thehigher TRG of the 2 components) was determined for eachpatient.

2.5. Construction of the TMAs

The chemotherapy-naive TMA was constructed from152 patients with chemotherapy-naive UCB treated bycystectomy and PLND. In total, 4 samples were taken per

A

200 µm

Fig. 1. Immunostains of tumors with negative expression (A) and overexpressionor more of cancer cells. (Color version of figure is available online.)

patient (diameter ¼ 0.6 mm), 2 from primary tumors and 2from corresponding LN metastases.

The prechemotherapy/postchemotherapy TMA was con-structed from patients with UCB before and after neo-adjuvant chemotherapy. A maximum of 6 samples wastaken per patient, 2 from the primary tumor before neo-adjuvant chemotherapy (TURB, n ¼ 55) and, if stillpresent, 2 after neoadjuvant chemotherapy (ypTþ, n ¼ 38)as well as 2 from corresponding LN metastasis (ypNþ,n ¼ 25).

2.6. Immunohistochemistry

Freshly cut TMA sections were analyzed within a day in1 experiment for Bcl-2. Immunohistochemistry was per-formed using a Bond RX (Leica Biosystems). Pretreatmentof TMA sections and epitope retrieval were performed withTris buffer at 951C for 30 minutes. Incubation with primaryantibody (DAKO, clone BCL-2 124, dilution 1:100) wasfollowed by incubation with rabbit anti–mouse antibody for8 minutes and anti–rabbit polymer for 8 minutes. Boundprimary antibodies were visualized using a polymer-basedvisualization system (Envisionþ; Dako, Glostrup, Den-mark), with 3,30-diaminobenzidine as chromogen. Theprimary antibody replaced by an irrelevant antibody servedas negative control, and lymphocytes within the spots of theTMA served as positive control (Fig. 1). Additionally, acontrol staining with pancytokeratin was used to determinethe Bcl-2 expression in cancer cells. Interpretation of allimmunostainings was blinded for clinicopathologicalparameters and outcome data. Bcl-2 overexpression wasdefined as cytoplasmic immunoreactivity in 10% or more of

200 µm

B

(B) of Bcl-2. Overexpression was defined as staining of cytoplasm in 10%

Table 2Clinicopathological data of the 55 patients receiving neoadjuvantchemotherapy

Patient data (n ¼ 55)Age (median, range) at surgery, y 64 (35–78)Female/male, n 15/41Follow-up (median), y 5.5

Histopathological dataTumor stage, nypT0 17 (31%)ypT1/2 16 (29%)ypT3/4 22 (40%)

Lymphadenectomy dataEvaluated nodes per patient (median, range), n 31 (8–85)Positive nodes per patient (median, range), n 0 (0–37)Lymph node stage, nypN0 31 (56%)ypN1 7 (13%)ypN2–3 17 (31%)

B. Kiss et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–84

cancer cells, as described by others [18]. Staining intensitywas not considered.

2.7. Statistical analysis

McNemar test and Cohen κ were used to measure thedifferences and concordances of Bcl-2 expression betweenthe evaluated tumor components (chemotherapy naive:primary tumor vs. LN metastasis; prechemotherapy/post-chemotherapy: TURB vs. ypTþ vs. ypNþ). Bcl-2 expres-sion in relation to histopathological tumor features (primarytumor and LN stage; number, total diameter and ENE of LNmetastases) were analyzed using a 2-sided Wilcoxon rank-sum test (noncategorical data) and chi-square test (catego-rical data). Kaplan-Meier plots were used to estimate overallsurvival from surgery to the date of death. Patients whowere still living were censored. Differences in survivalbetween the subgroups were assessed using log-rank test.To identify independent prognostic factors, Cox propor-tional hazards models were applied. A significance level of0.05 was used for all tests. All statistical analyzes wereperformed using R software package, version 3.0.3.

3. Results

3.1. Patient data

Clinicopathological data of the 2 TMA cohorts are givenin Tables 1 and 2.

3.2. Technical issue

The number of eligible tumor samples on the TMAslides was as follows: 148 primary tumors and 143 LN

Table 1Clinicopathological data of 152 lymph node–positive patients withurothelial cancer of the bladder

Patient data (n ¼ 152)Age (median, range) at surgery, y 67 (35–89)Female/male, n 29/123Follow-up (median), y 7.2

Histopathological dataTumor stage, npT1 4 (3%)pT2 17 (11%)pT3 93 (61%)pT4 38 (25%)

Lymphadenectomy dataEvaluated nodes per patient (median, range), n 27 (10–56)Positive nodes per patient (median, range), n 3 (1–46)Lymph node stage, npN1 44 (29%)pN2/3 108 (71%)

Extranodal extension (ENE), nNo 71 (47%)Yes 81 (53%)

metastases in chemotherapy-naive TMA and 49 cases ofTURB, 35 residual primary tumors, and 19 residual LNmetastases in prechemotherapy/postchemotherapy TMA.

3.3. Chemotherapy-naive TMA: Bcl-2 expression inprimary tumors and corresponding LN metastases

Overexpression of Bcl-2 was significantly (P ¼ 0.001)more frequent in LN metastases (39/143, 27%) than inprimary tumors (18/148, 12%) (Fig. 2A). The concordancebetween Bcl-2 overexpression in primary tumors andcorresponding LN metastases was low (κ ¼ 0.19). Neitherin primary tumors nor in LN metastases was Bcl-2 over-expression significantly related to histopathological tumorcharacteristics (primary tumor and LN stages and number,total diameter, and ENE of LN metastases).

In univariate analysis, Bcl-2 expression in both tumorcomponents was not a prognosticator (Table 3). In multi-variate analysis, only advanced primary tumor stage (pT3–4)and presence of ENE were independent adverse risk factors(Table 3).

3.4. Prechemotherapy/postchemotherapy TMA: Bcl-2expression in TURB, residual primary tumor (ypTþ), andLN metastasis (ypNþ)

Bcl-2 overexpression was significantly (P o 0.02) morefrequent in ypNþ (11/19, 58%) than in primary tumorsbefore (TURB: 8/49, 16%) and after (ypTþ: 7/35, 20%)neoadjuvant chemotherapy (Fig. 2B). The concordancebetween Bcl-2 overexpression in primary tumors before(TURB) and after (ypTþ) neoadjuvant chemotherapy waslow (κ ¼ 0.18).

Bcl-2 overexpression in TURB was not related topretherapeutic tumor characteristics (clinical tumor stage,tumor grade, and histological subtype). LN metastases withBcl-2 overexpression showed more advanced LN stages,

1839

130104

0%

20%

40%

60%

80%

100%

Primarytumor

Metastasis

Bcl-2 negative

Bcl-2 overexpression

p=0.001

A

8 7

11

41 28

8

0%

20%

40%

60%

80%

100%

TURB ypT+ ypN+

Bcl-2 negative

Bcl-2 overexpression

p<0.02

B

Fig. 2. Column chart indicating Bcl-2 overexpression vs. negative Bcl-2 expression in different tumor components. Chemotherapy-naive TMA (A): Bcl-2overexpression was significantly more frequent in corresponding LN metastasis than in primary tumors. Prechemotherapy/postchemotherapy TMA (B): in LNmetastases, Bcl-2 overexpression was significantly more frequent than in primary tumors before (TURB) and after (ypTþ) neoadjuvant chemotherapy.

B. Kiss et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8 5

more positive LNs, and larger total diameter of LN metastasesthan did Bcl-2–negative LN metastases (Table 4). However,this trend was not significant (P 4 0.5).

Table 3Results of the univariate and multivariate analyses of Bcl-2 expression in primaryand in TURB, residual primary tumor (ypTþ) and LN metastases (ypNþ) in the

RL

Chemotherapy-naive TMAUnivariate analysisBcl-2 overexpression in primary tumors aBcl-2 overexpression in LN metastases a

Multivariate analysispT3/4 pT1/2Extracapsular extension of metastases withoutpN2/3 pN1Bcl-2 overexpression in primary tumors a

pT3/4 pT1/2Extracapsular extension of metastases withoutpN2/3 pN1Bcl-2 overexpression in metastases a

Prechemotherapy/postchemotherapy TMAUnivariate analysisBcl-2 overexpression in TURB aBcl-2 overexpression in ypTþ aBcl-2 overexpression in ypNþ a

Multivariate analysisypT3/4 ypT0 vs. ypT1/2ypN 2/3 ypN0 vs. ypN1Bcl-2 overexpression in TURB a

ypT3/4 ypT0 vs. ypT1/2ypN 2/3 ypN0 vs. ypN1Bcl-2 overexpression in ypTþ a

ypT3/4 ypT0 vs. ypT1/2ypN 2/3 ypN0 vs. ypN1Bcl-2 overexpression in ypNþ a

HR ¼ hazard ratio; RL ¼ reference level.aReference level: less than 10% of cancer cells showing cytoplasmic immuno

In univariate and multivariate survival analyses, onlyprimary tumor and LN stages (ypT and ypN) addedprognostic information (Table 3).

tumors and corresponding LN metastases in the chemotherapy-naive TMAprechemotherapy/postchemotherapy TMA, respectively

Overall survival

P HR CI

0.080.4

0.03 1.9 1.1–3.60.001 2.0 1.4–3.10.6 1.1 0.7–1.70.2 1.5 0.9–2.6

0.009 2.4 1.2–4.80.001 2.0 1.3–3.00.9 1.0 0.6–1.60.08 0.7 0.5–1.1

0.20.090.2

0.005 2.6 1.3–5.10.006 2.2 1.3–3.90.3 0.5 0.1–2.1

0.3 2.0 0.6–7.20.2 1.5 0.8–2.50.6 0.6 0.1–3.7

0.04 8.5 1.1–64.60.2 0.3 0.1–1.90.2 0.4 0.1–1.5

reactivity.

Table 4Relations of Bcl-2 expression in LN metastases after neoadjuvant chemotherapy (ypN) and pathological LN characteristics

Bcl-2 expression in ypN

Bcl-2 overexpression Bcl-2 negative P

Lymph node stage, n 0.3ypN1 2 (18%) 3 (38%)ypN2/3 9 (82%) 5 (62%)

Positive nodes per patient (median, range), n 6 (1–37) 3 (1–17) 0.2Total diameter of metastases per patient (median, range), cm 3 (0.05–21.4) 2.3 (0.2–5.1) 0.4

B. Kiss et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–86

3.5. Bcl-2 expression in chemotherapy-naive TURB andresponse to neoadjuvant chemotherapy

Patients with Bcl-2 overexpression in TURB (8/49) hadsignificantly (P ¼ 0.04) more advanced ypT stages and lesstumor response to neoadjuvant chemotherapy as indicatedby higher TRG (TRG 2–3 in 87% of cases, P ¼ 0.04)(Table 5) in cystectomy specimens. Only one-eighth (13%)of patients with Bcl-2 overexpression in chemotherapy-naiveTURB completely responded (ypT0 ypN0) to neoadjuvantchemotherapy (Fig. 3). Although patients with Bcl-2 over-expression in TURB showed less tumor response to neo-adjuvant chemotherapy as indicated by higher “dominant”TRG, this trend was not significant (P ¼ 0.4; Table 5).

4. Discussion

Recent data on the benefit from neoadjuvant chemo-therapy in UCB [1,2] might shift the paradigm in its favor[3]. To avoid overtreatment of nonresponders, however,better prediction of chemotherapeutic response is needed.Bcl-2 is the most powerful inhibitor of the apoptoticcascade and can protect cancer cells from cytotoxic stimulithat promote apoptosis [10]. Not surprisingly, Bcl-2 over-expression has been identified as a marker of unfavorableresponse to apoptosis-inducing platinum-based chemother-apy in various types of cancer [8,10,13,14]. Our data are inline to these findings: UCB patients with Bcl-2 over-expression in TURB showed significantly fewer signs of

Table 5Cross table of Bcl-2 overexpression and tumor regression owing to neoadjuvant chby the “dominant” TRG

P ¼ 0.04

Primary tumors after neoadjuvant chemotherapy (P = 0.04) TRG 1TRG 2TRG 3

“Dominant” TRG (P = 0.4) TRG 1TRG 2TRG 3

regression to neoadjuvant chemotherapy (rate of completeresponse = 13%) when compared with patients without Bcl-2 expression in TURB (rate of complete response = 34%).Therefore, in UCB as well Bcl-2 appears to be a marker ofunfavorable response to platinum-based chemotherapy.Indeed, in UCB cell lines, Bcl-2 overexpression has beenshown to generate cisplatin resistance, whereas Bcl-2 down-regulation restored chemosensitivity [19]. Bcl-2 overexpres-sion is known to promote chemoresistance [5]; however,knowledge about the mechanisms of the relation betweenBcl-2 and inhibition of cisplatin-mediated apoptosis isscarce. Bcl-2 is only one component in the interactingapoptotic/antiapoptotic cascades. Another key element inthis control circuit is p53. Both mediators interact in asynergistic or also antisynergistic mode [18,20]. Bcl-2overexpression is one of the most important mechanismsby which tumor cells escape p53-mediated apoptosis [21]and can neutralize targets, up-regulated by p53. Contrarily,p53 can induce a decrease of Bcl-2 expression and mask theantiapoptotic effect of Bcl-2 [20]. Importantly, p53 over-expression may also reflect a partially or totally nonfunc-tional protein. The effect of p53 on cisplatin sensitivity isnot uniform, differs in various cancers, and p53 can alsoaugment cisplatin-induced apoptosis [5,22]. Biomarkers thatpromote chemoresistance are of utmost clinical interest, andcontributors of the aptoptotic cascade could play a dominantrole in this issue [23,24].

We evaluated Bcl-2 expression in primary tumors andcorresponding LN metastases of chemotherapy-naive UCBto test hypothesis that Bcl-2 might play a role in metastasis

emotherapy in the primary tumor and in both tumor components, indicated

TURB

Bcl-2 positive (n ¼ 8) Bcl-2 negative (n ¼ 41)

1 (13%) 15 (37%)5 }7 (87%) 8 }26 (63%)2 18

1 (13%) 14 (34%)3 }7 (87%) 9 }27 (66%)4 18

1

14

7

27

0%

20%

40%

60%

80%

100%

Bcl-2overexpression

Bcl-2 negative

non-/partial responderscomplete responders

p=0.4

Fig. 3. Distribution of response to neoadjuvant chemotherapy according toBcl-2 overexpression in TURB. Only one patient with Bcl-2 overexpres-sion in TURB showed complete response (P ¼ 0.4).

B. Kiss et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8 7

and identified more frequent overexpression in the latter(chemotherapy-naive cohort: 27% LN metastases vs. 12%primary tumor). In addition, the concordance of Bcl-2expression between the 2 tumor components was low(κ = 0.19). Differences in biomarker expression between the2 tumor components have been found for other biomarkers[11] and cancers [25,26], but not for Bcl-2. In-vitro, Bcl-2overexpression seems to support metastatic progressionwithout influencing the behavior of the primary tumor[27]. The authors concluded that Bcl-2 has a differentialeffect on the 2 tumor components [27]. Because patients aremore likely to die of metastases than of primary tumors, it isof high clinical relevance to understand the mechanismsunderlying metastatic progression. This discrepancybetween Bcl-2 expression in primary tumors vs. that inmetastases underlines the need for a comparative assess-ment of its expression in the 2 tumor components.

The prognostic effect of Bcl-2 remains controversial.Although Bcl-2 overexpression in UCB was associated withincreased tumor stages and a marker of adverse prognosis insome series [18,28,29], it was not in others [20,30]. Wecould also not identify a prognostic significance of Bcl-2overexpression. This controversy persists, when Bcl-2 over-expression is combined with other apoptosis-related bio-markers. It remained a prognosticator and reduced the effectof other biomarkers [18] or lost its prognostic information[20]. Therefore, the effect of Bcl-2 on survival may belimited and only detectable in larger series.

Drawbacks of our study are mostly related to itsretrospective character. In addition, although the 2 cohortswere homogeneous and underwent uniform surgical treat-ment and follow-up, they were both of limited size. Thepatient numbers in the Cox regression models of Bcl-2expression in ypTþ (n ¼ 35) and ypNþ (n ¼ 19) are low,these results have to be interpreted with caution. Moreover,the reported rate of Bcl-2 positivity in UCB in former andthe present series varies considerably (from 12%–63%)[18,20,28–30]. Thus, comparison of immunohistochemicaldata must be made with caution owing to the heterogeneityof the protocols and immunoassay kits applied as well as to

variances in the evaluated cohorts, some of which alsoincluded noninvasive bladder cancers.

5. Conclusions

Bcl-2 overexpression in chemotherapy-naive primarytumors is related to poor response to neoadjuvant chemo-therapy, which might help discriminate likely nonrespond-ers and thus avoid unnecessary therapy. Higher expressionof Bcl-2 in LN metastases than in primary tumors suggestsBcl-2 may play different roles and underlines the impor-tance of analyzing both components.

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