Bcl2 Blocks Cisplatin-Induced Apoptosis and Predicts Poor Outcome Following Chemoradiation Treatment...

Bcl-2 Blocks Cisplatin-Induced Apoptosis and Predicts PoorOutcome Following Chemoradiation Treatment in AdvancedOropharyngeal Squamous Cell CarcinomaWilliam A.Michaud,1,2 Anthony C. Nichols,6 Edmund A. Mroz,1,2 William C. Faquin,4

John R. Clark,3 Shahnaz Begum,7William H. Westra,7 Hiroshi Wada,1,2

Paul M. Busse,5 Leif W. Ellisen,1,3 andJames W. Rocco1,2,6

Abstract Purpose:This study aimed to test the hypothesis that elevated expression of antiapoptotic Bcl-2family proteins predicts a poor therapeutic response of oropharyngeal squamous cell carcinoma(OPSCC) to concurrent platinum-based chemoradiation therapy.Experimental Design: Levels of Bcl-2, Bcl-XL, and Bcl-wwere determined and correlatedwithresistance to cisplatin in a large panel of cell lines derived from squamous cell carcinoma of thehead and neck (HNSCC). Univariate and multivariate analyses were used to evaluate the relation-ship between Bcl-2 and Bcl-XL expression and disease-free survival following chemoradiationtherapy in a uniformly treated cohort of patients with OPSCC.Results: In HNSCC cell lines, high endogenous Bcl-2 expressionwas associated with increasedcisplatin resistance, and experimental overexpression of Bcl-2 promoted cisplatin resistance. Inpatients, tumors positive for Bcl-2 before treatment had greater risk of treatment failure (hazardratio, 5.99; 95% confidence interval, 1.73^20.8; P = 0.0014). In contrast, endogenous Bcl-XL

showed no correlation either with cisplatin sensitivity in the cell line panel in vitro, or with riskof recurrence in vivo (hazard ratio, 1.28; 95% confidence interval, 0.39^4.19; P = 0.68).Associations between Bcl-2 expression and other clinical characteristics did not account for thepredictive value of Bcl-2.Conclusions: Immunohistochemical assessment of Bcl-2 in pretreatment biopsy specimenscan predict response of advanced OPSCC to concurrent platinum-based chemoradiation. Astreatments targeting Bcl-2 and its family members become available, this immunohistochemicalassessment could help personalize therapy by identifying a subpopulation of patients with a poorprognosis who might benefit from such treatments.

Identifying pretreatment molecular markers that can predict

response to therapy is of great interest in head and neck

oncology and is required to develop personalized treatments

that maximize survival while minimizing morbidity. Numerous

approaches have been examined in head and neck squamous

cell carcinoma (HNSCC), including the use of individual and

combined markers and global genomic strategies, with some

limited success (1). In oropharyngeal squamous cell carcinoma

(OPSCC), for example, infection with human papilloma virus

(HPV) is associated with improved survival (2–6), whereas

high levels of the epidermal growth factor receptor are

associated with treatment resistance (6). Nevertheless, neither

of these markers has sufficient predictive power to individualize

treatment selection and direct therapy. Global genomic

approaches that identify prognostic gene signatures have not

cleared the important hurdle of reproducibility, because

different genomic marker sets share few genes (7–9). Conse-

quently, additional markers of response are needed to predict

outcome, guide the intensity and choice of therapy, and point

the way to novel therapeutic targets.Recent studies on the in vitro response to genotoxic agents in

HNSCC have described cellular pathways related to treatmentresistance and identified potential markers of therapeutic

response. One pathway thought to be important for cellular

survival in HNSCC involves two members of the p53 family,TAp73 and DNp63a (Fig. 1A; ref. 10). TAp73 isoforms, whichshare many of the proapoptotic functions of p53, are highly

Cancer Therapy: Preclinical

Authors’Affiliations: 1Massachusetts General Hospital (MGH) Cancer Center,Departments of 2Surgery, 3Medicine, 4Pathology, and 5Radiation Oncology,Massachusetts General Hospital, and 6Department of Otolaryngology,Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; and 7Department ofPathology, TheJohns Hopkins Medical Institutions, Baltimore, MarylandReceived10/7/08; revised11/11/08; accepted11/12/08.Grant support: Financial support was providedby the LaurenceMurphy Fund, theNorman Knight Fund, and the James Kelly Fund for Head and Neck Research, aClinical Investigator Award from the Flight Attendant Medical Research Institute(J.W. Rocco), and NIH grant DE015945 (L.W. Ellisen andJ.W. Rocco).The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: Supplementary data for this article are available at Clinical Cancer ResearchOnline (http://clincancerres.aacrjournals.org/).W.A. Michaud, A.C. Nichols, and E.A. Mroz contributed equally to themanuscript.Requests for reprints: JamesW. Rocco, GRJ-904, Mass. General Hospital, 55Fruit St., Boston, MA 02114. Phone: 617-726-5251; Fax: 617-726-8623; E-mail:[email protected].

F2009 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-08-2581

www.aacrjournals.org Clin Cancer Res 2009;15(5) March1, 20091645

expressed in cultured HNSCC cells and primary tumors, unlikein the normal epithelial cells of origin (11). Genotoxic treat-ments of these tumor cells activate a TAp73-dependent apop-totic transcriptional program that leads to cell death even in theabsence of functional p53 (12). Indeed, the association of somep53 mutants with treatment resistance in HNSCC is postulatedto arise from their inhibition of p73-dependent apoptosis (13).We have found (11, 14) that the proapoptotic effects of

TAp73 are often kept in check by DNp63a, the predominantform of p63 in normal epithelia and in HNSCC (15). DNp63a,normally restricted to basal cells of squamous epithelia, ishighly expressed in most HNSCC (15, 16). In HNSCC whereDNp63a inhibits TAp73 function, platinum-based chemo-therapeutic agents have been proposed to act by inducing

degradation of DNp63a (17) and phosphorylation of TAp73(18), allowing expression of the TAp73-induced proapoptoticpathway and cell death. Consistent with DNp63a as a target ofplatinum agents, one study showed that HNSCC not expressingDNp63a are resistant to such chemotherapy (19).This key role of DN63a in tumor cell survival (11, 14, 18)

led us to ask how HNSCC cells that do not express anti-apoptotic DNp63a manage to survive. Neither TAp73 nordownstream apoptotic triggers like p53-up-regulated modu-lator of apoptosis are typically lost in HNSCC (20). Ratherwe found that cell lines deficient in DNp63a overexpress theantiapoptotic protein Bcl-2, which acts downstream of TAp73to block cell death (Fig. 1A; ref. 14). Based on these findings,we hypothesized that if high Bcl-2 allows HNSCC cells to stayalive in the absence of endogenous DNp63a, high Bcl-2 mightalso enhance survival of cells subjected to chemotherapy thatleads to loss of DNp63a.Antiapoptotic members of the Bcl-2 family such as Bcl-2,

Bcl-XL, and Bcl-w control the integrity of the outer mito-chondrial membrane, thereby regulating the susceptibilityto apoptosis through the intrinsic pathway (21). In numerousstudies, elevated expression of antiapoptotic Bcl-2 familymembers has been associated with resistance to chemotherapy(reviewed in ref. 22). In HNSCC, both Bcl-2 and Bcl-XL expres-sion have been associated with resistance to genotoxic therapy,although there have been inconsistent reports about the relativecontribution of each protein, with several studies presentingconflicting results (23–25).In the present study we examined how these proapoptotic

and antiapoptotic proteins correlate with cisplatin resistancein vitro and, in a well-characterized cohort of patients havingOPSCC, with clinical outcome following chemoradiationtreatment. Consistent with our model, in vitro resistance tocisplatin correlated positively with Bcl-2 levels. In patients, highpretreatment Bcl-2 levels in tumors were strikingly correlatedwith poor clinical outcome. Bcl-XL, in contrast, showed little orno correlation with cisplatin sensitivity in vitro or with clinicaloutcome following chemoradiation treatment.

Translational Relevance

Previous studies have reported conflicting outcomesregarding the role of Bcl-2 family members in predictingtreatment outcome in squamous cell carcinomaof theheadand neck (HNSCC). Our prior studies had identified Bcl-2as critical for survival of cell lines derived from HNSCC. Inthis study we first showed that high Bcl-2 expression isspecifically correlated with resistance to cisplatin amongthese cell lines. In a uniformly treated cohort of patientswith advanced oropharyngeal squamous cell carcinoma,we showed that elevated pretreatment Bcl-2 levels predicttherapeutic resistance to platinum-based concurrentchemoradiation. This study defines a novel subgroup ofpatients who will require more aggressive or novel thera-pies to achieve survival rates comparable to patients havingBcl-2^ negative tumors. Furthermore, the elevated levels ofBcl-2 found in this subgroup of patients representan attractive therapeutic target for small-molecule Bcl-2inhibitors currently being developed.

Fig. 1. Roles of Bcl-2 family members in inhibiting celldeath after cisplatin treatment of cell lines derived fromHNSCC. A, diagram summarizing how genotoxictherapy such as chemoradiation affects the p53 familymembers p63 and p73 in HNSCC, and the downstreaminhibition of p73-induced apoptosis by antiapoptoticmembers of the Bcl-2 family. B throughD, scatter plotsof cisplatin IC50 versus endogenous mRNA levels ofBcl-2 family members for19 cell lines derived fromHNSCC. Logarithmic horizontal axis allows display ofcell lines differing by several orders of magnitude inexpression. Regression lines of IC50 against logarithmof mRNA level are plotted, along with correlationcoefficients and significance. B, Bcl-2; C, Bcl-XL;D, Bcl-w. Bcl-2 mRNA levels displayed in this figurewere highly correlated with protein expression seenonWestern blots; cf. Supplementary Fig. S3.


www.aacrjournals.orgClin Cancer Res 2009;15(5)March1, 2009 1646

Materials andMethods

Cell lines. Cell lines derived from HNSCC were generous gifts of

David Sidransky (Johns Hopkins University) or of Robert Ferris

(University of Pittsburgh Cancer Institute), or were obtained from the

American Type Culture Collection. Supplementary Table S1 provides

information on specific cell lines. Cells were maintained at 37jC with5% CO2 in either RPMI or DMEM supplemented with 10% fetal bovine

serum and with penicillin/streptomycin.Expression constructs. Reverse transcription PCR of human fibroblast

cDNA was used to subclone cDNA for Bcl-2, Bcl-XL, or Bcl-w into theBamHI/XhoI restriction sites of the pLPC retrovirus expression vector

(primer sequences available on request). Production of high-titer

amphotrophic retroviral stocks and retroviral infection was done asdescribed (14).

Protein and mRNA analysis. Methods of protein detection by Western

blot, RNA extraction, cDNA preparation, and quantitative real-time PCR

(qRT-PCR) were as in a previous study (14), except that HotStart-IT Taq

Master Mix (USB) supplemented with SybrGreen (Invitrogen) was used

for PCR. Expression plasmids for Bcl-2, Bcl-XL, Bcl-w, DNp63a, andTAp73h were used to prepare standard curves for qRT-PCR. PCR resultsare expressed as femtograms of coding-sequence DNA providing

qRT-PCR results equivalent to cDNA corresponding to 50 ng total RNA.

Primers for PCR of p63 and p73 (sequences available on request) were

specific for their DN and TA forms, respectively, but did not distinguish

COOH-terminal variants. The anti-p63 4A4 antibody (MS 1081; Neo-

Markers) recognizes the DNA-binding domain common to all forms

of p63.

Analysis of cisplatin sensitivity. Cells in 96-well plates were treated

with cisplatin for 48 h, followed by analysis of cell viability with

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;

ref. 18). Data for MTT signal versus cisplatin concentration were

analyzed with the drfit package of the R statistical software environ-

ment (http://www.r-project.org) to obtain the IC50, the concentration

at which the MTT signal was reduced by half.

Patient data. Human studies approval was obtained from theMassachusetts General Hospital Internal Review Board (Partners

Human Research Committee) to obtain archival tumor specimensand do retrospective chart review. Patients with squamous cell

carcinoma of the oropharynx were identified in the Massachusetts

General Hospital pathology database. Inclusion criteria were: (a)

biopsy-proven squamous cell carcinoma of the oropharynx treated

definitively with concurrent chemoradiation, with or without neck

dissection; (b) no prior history of head and neck squamous cell

carcinoma; (c) no prior history of head and neck irradiation; (d)

pretreatment biopsy paraffin block available; (e) adequate documented

clinical follow-up for at least 2 y or until a recurrence proven by biopsy;

and (f) use of a platinum-based agent for chemotherapy (cisplatin or

carboplatin with paclitaxel). Of 69 patients meeting the first three

criteria, tumor specimens were unavailable for 13, 8 did not meet the

minimum follow-up, and 10 received a different chemotherapy

regimen. Results are reported for the remaining 38 patients. Entry into

the study was set as the initial biopsy date (all from May 1996 to May

2005). During the course of radiation, chemotherapy was administered

either as cisplatin (100 mg/m2 i.v. over 1 h) every 3 wk for up to 3

cycles, or as weekly administrations of carboplatin (area under the

curve = 1.5 i.v. over 30 min) and paclitaxel (45 mg/m2 i.v. over 30 min)

for up to 7 treatments as tolerated. Radiation therapy was delivered

as intensity modulated radiation therapy five times weekly in daily

fractions of 1.8 to 2.12 Gy for a total of 33 to 40 fractions to the gross

tumor volume, or as a concomitant boost accelerated fractionation

schedule (72 Gy delivered to the gross tumor volume). Lymph nodes

received 45 to 60 Gy according to the level of risk. Patients were

evaluated closely each 4 to 6 wk during the first 2 y of follow-up. The

median follow-up since initial biopsy of the 26 patients both alive and

disease free at last follow-up was 46.5 mo (range, 31–126 mo). One

patient died without evidence of recurrent disease after 25 mo.

Statistical analysis was done in the R environment, including the

survival package for analysis of disease-free survival.Immunohistochemistry. BenchMark XT automated tissue staining

systems (Ventana Medical Systems, Inc.) were used, with protocolsprovided by the manufacturer. Endogenous peroxidase activity wasblocked by H2O2. A combination of EDTA and boric acid in Tris buffer(CC1 reagent; Ventana) was applied to the tissues for antigen retrievalfor 30 to 60 min prior to washing and primary antibody incubation.Primary antibodies and incubation times were: p63 (4A4; 1:200dilution; 8 min), Bcl-2 prediluted antibody (Ventana; 1 h 52 min),and Bcl-XL (1:40 dilution; NeoMarkers; 32 min). Secondary antibodieswere UltraView horseradish peroxidase–conjugated multimers (goatantimouse and rat Igs.; Ventana). Antigen detection was done usingUltraView diaminobenzidine chromogen (Ventana). Tissues werecounterstained with hematoxylin.A pathologist blinded to outcome (WCF) used the criteria of Jackel

et al. (26) to score Bcl-2 and Bcl-XL immunohistochemistry. An intensityscore (0, absent; 1, weak; 2, moderate; 3, strong) and a prevalencescore (0, <25% of tumor stained; 1, 25–75% stained; 2, >75%stained) were added; a total of z3 was called positive.HPV analysis. HPV-16 DNA was detected by in situ hybridization–

catalyzed signal amplification for biotinylated probes (GenPoint;Dako), as described previously (27). A HPV-16–positive tumorspecimen provided a positive control. A pathologist blinded to outcome(WHW) scored slides as positive for HPV-16 if there was a punctatesignal specific to tumor cell nuclei.

Results

Cisplatin resistance of cell lines derived from HNSCC wasrelated to Bcl-2 expression. Our and others’ previous worklinked the expression of both p63 and p73 to cisplatinsensitivity in HNSCC and other tumors (Fig. 1A; refs. 10, 18).Therefore, we first examined whether their endogenousexpression correlated with cisplatin sensitivity in a panel of19 HNSCC-derived cell lines. We used real-time quantitativeRT-PCR (qRT-PCR) to assay the major p63 and p73 isoformsexpressed in HNSCC, DNp63a, and TAp73. We comparedexpression levels with cisplatin resistance (IC50) determinedfrom a quantitative dose-response assay (MTT). BecauseDNp63a has been shown to promote survival of HNSCC cellsand to be targeted for degradation by cisplatin (14, 17), wepredicted that low DNp63a expression would be associatedwith cisplatin resistance. Indeed, low DNp63a expression washighly associated with cisplatin resistance (r = 0.70, P = 0.0008;Supplementary Fig. S1). Also as predicted, low endogenousexpression of TAp73, an effector of cell death in responseto cisplatin (28), was correlated with cisplatin resistance(Supplementary Fig. S2).We next examined correlation of Bcl-2 with cisplatin

resistance in these cell lines. We had previously shown thatBcl-2 expression correlates inversely with DNp63a levels andpotently blocks TAp73-dependent apoptosis in HNSCC cells,suggesting that Bcl-2 expression might be a strong predictor ofcisplatin resistance. As shown in Fig. 1B, we found in thepresent study that Bcl-2 expression was significantly correlatedto cisplatin resistance among these cell lines (r = 0.72, P =0.00055; Supplementary Table S1). We also examined expres-sion of two other antiapoptotic members of the Bcl-2 family,Bcl-XL, and Bcl-w, which are thought to have similar targetbinding specificity as Bcl-2 (22). Of these, Bcl-XL has beenassociated in some studies with cisplatin resistance (29) andwith poor outcome in HNSCC (6). In contrast to the results

Bcl-2 Levels Predict Chemoradiation Sensitivity


with Bcl-2, expression of these other antiapoptotic membersof the Bcl-2 family was not correlated to cisplatin resistance(Fig. 1C and D).To examine mechanisms that might account for this

correlation between Bcl-2 expression and cisplatin resistance,we investigated the effects of overexpressing Bcl-2 in a subset ofthese cell lines. Figure 2A shows results of such an experimenton the JHU029 cell line, a line highly sensitive to cisplatin.Cells were infected with control retrovirus or with virus codingfor Bcl-2, selected with puromycin for stably infected cells, andthen subjected to cisplatin. Control cells expressed high levelsof DNp63a and very low levels of Bcl-2 protein, as we reportedpreviously for this cell line (14). Cisplatin treatment led to lossof DNp63a, consistent with a previous report in other HNSCClines (17). The apoptotic program elicited by loss of DNp63a isevidenced by expression of the p53/p73 target gene NOXA andcleavage of poly ADP-ribose polymerase after exposure ofcontrol cells to cisplatin (Fig. 2A, leftmost two lanes).JHU029 cells overexpressing Bcl-2 showed loss of DNp63a

and induction of NOXA similar to that in control cells, soupstream steps in the response to cisplatin were intact despiteBcl-2 overexpression. Cells overexpressing Bcl-2, however, didnot show the classic downstream sign of apoptosis, cleavage ofpoly ADP-ribose polymerase (Fig. 2A; compare the second andfourth lanes from the left). This result was consistent with theposition of Bcl-2 along the apoptotic cascade; Bcl-2 inhibitsBH3-only proapoptotic proteins from initiating mitochondriallysis and the resulting activation of caspases, whose subsequentcleavage of poly ADP-ribose polymerase is a sign of apoptosis(22). Thus overexpression of Bcl-2 in this cell line allowed earlysteps in the response to cisplatin, but inhibited downstreamapoptotic mechanisms.The effect of Bcl-2 overexpression on cisplatin resistance in

this sensitive cell line is shown in Fig. 2B. In this experiment,control cells and cells overexpressing Bcl-2 were exposed to a

range of cisplatin concentrations covering those reached inclinical practice (30, 31), and cell viability was assessed withMTT. Bcl-2 overexpression shifted the cisplatin concentration-response curve to the right. After overexpression of Bcl-2, abouthalf of the cells escaped death after 48 hours’ exposure to3 Ag/mL of cisplatin, conditions that killed almost all controlcells. We did similar studies of Bcl-2 overexpression on eightadditional HNSCC cell lines, with both high and low DNp63aexpression. As shown in Fig. 2C, overexpression of Bcl-2 led to a50% increase in cisplatin IC50 over all nine lines tested. Thus,Bcl-2 overexpression could increase cisplatin resistance in thesecell lines regardless of DNp63a status.To determine whether reduction of Bcl-2 expression would

decrease resistance to cisplatin, we attempted to prepare celllines with stably reduced Bcl-2 levels by infecting cells withlentivirus coding for short hairpin RNAs targeting Bcl-2 forknockdown. In all our attempts, no cells expressing suchshRNAs survived puromycin selection for infected cells,whereas puromycin-resistant cells infected with controllentiviruses were readily obtained with high efficiency (notshown). These results suggest that basal expression ofendogenous Bcl-2 plays a significant role in survival of thesecell lines.Poor outcome following chemoradiation treatment of advanced

OPSCC was related to Bcl-2 expression in pretreatment tumors.Based on these cell-line studies, we decided to examine whetherpretreatment Bcl-2 expression might serve as a marker for pooroutcome after concurrent chemoradiation of HNSCC. Becauseprevious studies had suggested that Bcl-XL could serve as sucha marker (6, 29), we chose to include Bcl-XL expression inour analysis, although Bcl-XL expression did not correlate withcisplatin resistance among the cell lines (Fig. 1C). Bcl-w was notanalyzed in the tumors because it failed to correlate with cell-line cisplatin resistance and has not been reported as a markerof response in HNSCC. Given our findings in cell lines and

Fig. 2. Effects of Bcl-2 overexpression on cisplatin resistance of cell lines derived from HNSCC. A, cisplatin treatment led to loss of DNp63 and activation of upstreamproapoptotic targets regardless of Bcl-2 expression, whereas Bcl-2 inhibited the downstream apoptotic program.Western blots of protein extracts taken after 21h exposureof vector-control JHU029 cells (left) or cells overexpressing Bcl-2 (right) to 5 Ag/mL cisplatin.The p73 target gene NOXA is induced by cisplatin in both cases, whereasthe apoptotic program, illustrated by cleavage (two bands) of poly-ADP ribose polymerase, was inhibited by Bcl-2 overexpression. B, Bcl-2 overexpression increasedcisplatin resistance ofJHU029 cells as assessed by MTTviability assay. MTT absorbance, normalized to vehicle-only control absorbance, in vector-control cells or cellsoverexpressing Bcl-2, treated with the indicated concentrations of cisplatin for 48 h; meanF SE of four observations at each concentration. Dashed lines, shift in IC50,the concentration of cisplatin required to reduce viability to 50% of control. C, Bcl-2 overexpression increased cisplatin resistance of all cell lines examined. For each of ninecell lines, mean cisplatin IC50 in cells overexpressing Bcl-2 is plotted against mean IC50 in vector-control cells examined in parallel. Dashed line, line of identity, expected ifBcl-2 expression did not affect IC50; solid line, observed linear regression of IC50 for cells expressing Bcl-2 versus IC50 for control cells, indicating a 54% increase inresistance following Bcl-2 overexpression; P value is for difference of observed slope from line of identity.



a previous clinical report (19), we also examined expressionof DNp63a.Selection of patients; immunohistochemical evaluation. We

examined characteristics and outcomes of patients havingstage III or IV OPSCC treated with concurrent radiation andplatinum-based chemotherapy, the present standard of care forsuch patients (32). In addition to the well-defined anatomicalorigin of these tumors, essentially all patients presenting withthis disease receive the same treatment at our institution,minimizing pretreatment selection bias. Clinical characteristicsof the patients are presented in Table 1.To facilitate comparison with results from other institutions,

we used commercially available reagents and automated equip-ment for immunohistochemical staining of p63, Bcl-2, andBcl-XL in pretreatment biopsy specimens. The pathologistevaluating the staining was unaware of treatment outcomesand used a published technique, described in Materials andMethods, that combines both staining intensity and prevalenceto classify a tumor as positive for an antigen. With respect toBcl-2, particular care was taken to exclude lymphocytes fromimmunohistochemical scoring. Although infiltration by lym-phocytes, which are Bcl-2–positive, can be a positive prognos-tic sign (33), we wished to restrict analysis of Bcl-2 to the tumor

cells in which elevated Bcl-2 was predicted to be associated withpoor outcome following genotoxic therapy.Staining for p63 was not helpful in distinguishing among

tumors. All 38 tumors showed moderate to robust staining forDNp63a, although the staining patterns differed substantiallyamong tumors. Examples of DNp63a staining patterns areshown in Fig. 3A. Due to the large fraction of p63-positive tumorcells in all cases, we could not classify tumor DNp63a status in away that provided a useful marker for treatment outcome.Patients with tumors expressing Bcl-2 were at greater risk of

recurrence. Classification of tumors for Bcl-2 and Bcl-XLstaining, in contrast, was straightforward. Half of the tumorswere positive for Bcl-XL staining, and 11 of 38 (29%) werepositive for Bcl-2. Unlike some other studies that found inversecorrelations of these two antiapoptotic proteins amongindividual tumors (24, 34), we found examples of allcombinations of Bcl-2 and Bcl-XL staining, as illustrated inFig. 3B. In fact, the distributions of Bcl-2 and Bcl-XL amongthese tumors were independent of each other, with one half ofboth Bcl-2–positive and Bcl-2–negative tumors showing Bcl-XLexpression (Table 1; P = 1 by Fisher exact test).As we wished to test the hypothesis that Bcl-2 expression is

a marker of poor outcome of therapy, we examined whether

Table 1. Patient characteristics and associated recurrence hazards

Characteristic Total Bcl-XL Bcl-2 Recurrence at last follow-up Hazard*

Positive Negative Pc Positive Negative Pc No Yes Ratio P

Age (y) <50 12 8 4 0.29 4 8 1 7 5 1 0.2650-65 21 8 13 6 15 15 6 0.62>65 5 3 2 1 4 5 0 0

Gender Male 31 16 15 1 11 20 0.084 20 11 1 0.085Female 7 3 4 0 7 7 0 0

Site Tongue base 16 9 7 0.60 5 11 1 11 5 1 0.99Tonsil 18 9 9 5 13 13 5 0.95Other 4 1 3 1 3 3 1 0.83

T 1 8 6 2 0.16 3 5 0.95 5 3 1 0.322 18 8 10 5 13 15 3 0.433 8 2 6 2 6 5 3 1.074 4 3 1 1 3 2 2 2.04

N 0 5 2 3 0.73 0 5 0.13 4 1 1 0.131 8 4 4 1 7 8 0 02 23 11 12 10 13 13 10 2.113 2 2 0 0 2 2 0 0

Disease stage III 11 5 6 1 1 10 0.12 11 0 0 0.02IV 27 14 13 10 17 16 11 1

Smoking No 17 9 8 1 6 11 0.49 13 4 1 0.47Yes 21 10 11 5 16 14 7 1.57

Alcohol No 28 14 14 1 8 20 1 22 6 1 0.078Yes 10 5 5 3 7 5 5 2.79

HPV Positive 25 13 12 1 8 17 0.72 20 5 0.33 0.054Negative 13 6 7 3 10 7 6 1

Bcl-XL Positive 19 6 13 1 13 6 1.28 0.68Negative 19 5 14 14 5 1

Bcl-2 Positive 11 6 5 1 4 7 5.99 0.0014Negative 27 13 14 23 4 1

NOTE: Clinical characteristics and immunohistochemical scoring as noted in Materials and Methods. Smoking refers to any history of smoking.Alcohol refers to a history of alcohol abuse or dependence. Of the 38 patients, all of whom were followed for at least 31 mo or until recurrence, 11had recurrent disease.*Hazard ratios based on Cox proportional hazard analysis. Reference level of each characteristic is shown as hazard ratio of 1, with an increasedhazard ratio indicating increased risk of recurrence. P values for significant differences in hazard ratios among levels of each characteristic basedon log rank test.cP values by Fisher exact test for differences from independent joint distributions of each characteristic with respect to Bcl-XL or Bcl-2 staining.



Bcl-2 expression was correlated to other clinical characteristicsthat might affect outcome. As shown in Table 1, no otherclinical characteristics differed significantly (at P < 0.05) amongthe groups distinguished by Bcl-2 staining.We then did Cox proportional hazard analysis on disease-free

survival as a function of Bcl-2 status. Patients with pretreatmenttumors staining for Bcl-2 had much shorter disease-freesurvival, as predicted by our model. This univariate analysisfound a hazard ratio of 5.99 for patients with tumors expressingBcl-2 (95% confidence interval, 1.73–20.8; P = 0.0014 by log-rank test). Pretreatment Bcl-XL status, in contrast, bore norelation to disease-free survival (95% confidence interval forhazard ratio, 0.39–4.19; P = 0.68). Figure 4 shows Kaplan-Meier plots illustrating this difference between Bcl-2 (Fig. 4A)and Bcl-XL (Fig. 4B) status. (Analysis of overall survival wasuninformative, because only seven patients died, including onewith no sign of recurrent disease.) These relations of disease-free survival to pretreatment tumor Bcl-2 and Bcl-XL status wereconsistent with our cell-line results (Fig. 1B and C).Of the 11 patients whose tumors recurred, recurrence was at

a distant site in 4 of 7 Bcl-2 positive cases, versus 1 of 4 caseshaving pretreatment tumors negative for Bcl-2. This differencewas not statistically significant (P = 0.55, Fisher’s exact test).In addition to Bcl-2 status, univariate Cox proportional

hazard analyses for each clinical characteristic in Table 1 foundno significant relations to disease-free survival (at P < 0.05)except for disease stage, with no recurrences in 10 patients

presenting with stage III disease (of whom only 1 had a Bcl-2–positive tumor).Forward stepwise multivariate analysis identified low Bcl-2,

HPV positivity, disease stage III, female gender, site other thantonsil or tongue base, and age >65 y as significant positivepredictors for disease-free survival. Detailed analysis of theseadditional predictors showed that there were no recurrences inseven female patients (none Bcl-2 positive) or in five patients>65 y (with only 1 Bcl-2 positive). Out of four patients withtumors in oropharyngeal sites other than tonsil or tonguebase, the single patient with recurrence had a Bcl-2–positivetumor. Thus, multivariate analysis provided little additionalpredictive value over Bcl-2 expression alone, except for thealready known relationship between good outcome and HPVinfection.Finally, although we had found no clinical characteristics

significantly correlated to Bcl-2 status in this study, werecognized that the sample size of only 38 patients might limitthe power to detect true correlations of Bcl-2 with othercharacteristics. Thus, we also examined three clinical character-istics that both came close to significant correlations with Bcl-2expression and had significant or near-significant univariaterelations to disease-free survival (Table 1): gender, N score, anddisease stage. None of seven female patients had Bcl-2–positivetumors or recurrent disease. Ten of 11 Bcl-2–positive tumorswere associated with an N score of 2, and 10 of 11 patients withBcl-2–positive tumors presented with stage IV disease.

Fig. 3. Immunohistochemical staining oftumors for p63, Bcl-2, and Bcl-XL.Immunostaining for indicated antigens inbrown, with hematoxylin counterstain. A,heterogeneity of staining patterns for p63among tumors. Examples from four separatetumors. B, examples of p63, Bcl-2, andBcl-XL staining in pretreatment biopsies offour additional tumors. H+E is conventionalH&E staining. Each column, sections from asingle tumor, with time to recurrence noted.From left to right: positive for Bcl-2, notBcl-XL; positive for Bcl-2 and Bcl-XL;positive for Bcl-XL, not Bcl-2; negative forboth Bcl-2 and Bcl-XL. Recurrenceinformation only applies to panel B.



To examine the practical implications of interactions withthese characteristics for using Bcl-2 as a marker, we repeateddisease-free survival analysis while restricting analysis to males(31 patients), to N score 2 (23 patients), or to disease stage IV(28 patients). As shown in Fig. 5, Bcl-2 expression wasassociated with shorter disease-free survival in each of thesesubgroups. Thus pretreatment expression of Bcl-2 in tumorswas associated with substantially increased risk of pooroutcome following chemoradiation treatment of OPSCC, in away that was not substantially related to covariation of Bcl-2status with other clinical characteristics of the patients.

Discussion

Predictive value of p63. We expected that staining for p63would be at best of limited predictive usefulness, because thevast majority of primary HNSCC express high levels of p63 asassessed by routine immunohistochemistry. Our ability todetect a correlation between p63 expression and cisplatinresistance in HSNCC cell lines may be due in part to the largerdynamic range of mRNA analysis. Furthermore, our cell-linestudies showed that Bcl-2 overexpression could lead toincreased cisplatin resistance even in cells expressing DNp63a(Fig. 2C). Thus, some tumors resistant to therapy mightnevertheless be p63-positive.

Different approaches to classifying p63 expression or analysisof different HNSCC cohorts might yield more informativeresults. For example, use of Western blot analysis on freshprotein extracts from HNSCC primary tumors was able tocorrelate DNp63a expression with sensitivity to chemoradia-tion (19).Predictive value of Bcl-2. Our results support the hypothesis,

based on the underlying molecular mechanisms, that pretreat-ment Bcl-2 status is closely related to the success of chemo-radiation treatment of OPSCC. Elevated Bcl-2 expression in theprimary tumor is associated with significantly increased risk ofrecurrence. At the time of last follow-up, only 4 of 27 patients(15%) with tumors negative for Bcl-2 had recurrent disease,whereas nearly two thirds (7 of 11) of those with tumorspositive for Bcl-2 had recurrence. In contrast, pretreatmentBcl-XL staining was not related to outcome, with about onethird of patients in each Bcl-XL category having recurrence.Relation to previous reports. The present study points a way

toward understanding apparently incompatible previousreports of relations between Bcl-2 expression and patientoutcomes in HNSCC, which have ranged from good prognosisthrough no relation to poor prognosis (24, 25, 34–41). First,we chose a well-defined subset of HNSCC with a definedanatomical source and with essentially identical treatment in allcases. A defined tumor origin in this type of study is crucial,because tumors originating from different sources may differ

Fig. 4. Relations of recurrence to pretreatment Bcl-2 andBcl-XL expression (cf. Table1). Kaplan-Meier plots ofdisease-free survival, with times of last follow-upindicated by cross-marks. Bcl-2 expression was relatedto significantly increased risk of recurrence (A) whereasrisk of recurrence was not related to Bcl-XL (B). SeeTable 1and text for Cox proportional hazard analysis.

Fig. 5. Relations of Bcl-2 expression to other prognostic indicators did not account for the observed relation between Bcl-2 and recurrence. Restricting analysis to malepatients (A), patients with N score of 2 (B), or disease stage IV (C) resulted in similar relations between pretreatment Bcl-2 status and recurrence.



substantially in the selective pressures they have faced, theirmutational strategies before clinical presentation, and theirresponses to therapy (32, 42). The type of tumor we examinedposed few problems in terms of bias arising from selection oftreatment. There also was at most limited relation betweenpretreatment Bcl-2 status and other clinical characteristics ofthe patients. We thus could relate differences in Bcl-2 status todifferences in treatment outcome while minimizing confound-ing influences of other uncontrolled factors.Second, we used a standardized immunohistochemical

method and strict criteria for scoring tumors as positive forBcl-2, combining staining intensity and prevalence into a singlescore (26). In evaluating stained specimens, we only scoredstaining of tumor cells, explicitly ignoring lymphoid tissue orBcl-2–positive lymphocytes infiltrating the tumors.Third, we examined the relation of Bcl-2 expression to the

outcome of a treatment that should be influenced directly bythe antiapoptotic effects of Bcl-2, chemoradiation. In particular,we did not include patients subjected to primary surgery withcurative intent. Whereas simple correlations between a bio-marker and outcome can serve prognosis regardless of theunderlying mechanisms, the strongest relation between abiomarker and treatment outcome is expected for a markerdirectly related to the mechanism of the treatment. That wasthe case for the relation of Bcl-2 to protection from apoptosis inthe present study.Notably, prior studies of HNSCC that shared the strengths

of the present study – an adequate number of Bcl-2–positivetumors with well defined origin, no major relation betweenBcl-2 status and other clinical characteristics, Bcl-2 scoreslimited to tumor cells, and examining response to a standard-ized treatment that damaged DNA – also showed the relationwe predicted and found between Bcl-2 status and treatmentoutcome. Examples are studies on response to primaryradiation therapy of stage I and II tumors of the oral cavity,pharynx, and larynx (35) and in early-stage laryngeal tumors(25). Contrary reports of no prognostic value or a positiveprognostic value of pretreatment Bcl-2 expression in HNSCCoften involved studies with few Bcl-2–positive cases (43–45),not specifically examining the effects of treatments that damageDNA (24, 37, 39–41), having correlations of Bcl-2 withunrelated positive prognostic indicators (36, 38), or notexplicitly excluding tumor-associated lymphoid tissue in theBcl-2 scoring.Differences between the patient population in the present

study and those in other studies may also account for somediscrepancies. For example, almost all (88%) of the patients inthe study by Aebersold et al. (34) on responses of advancedoropharyngeal tumors to radiation therapy were smokers,versus only about one half (55%) in the present study.Furthermore, in the study by Aebersold et al., smoking wasassociated with low Bcl-2 expression, whereas we found noassociation of smoking history with Bcl-2. Another possibilityis that Bcl-2 might be more closely related to outcome afterplatinum-based chemotherapy or concurrent chemoradiationthan to outcome after primary radiation therapy. Althoughplatinum-based chemotherapy and radiation both damageDNA and lead to apoptosis, differences in the intensity andnature of the DNA damage and subsequent apoptotic pathwaysafter the two types of treatments might be associated withdifferences in the ability of Bcl-2 to prevent apoptosis, or

combination therapy might alter apoptotic thresholds so thatBcl-2 expression is more important to tumor cell survival.Although our results did not show a relation of Bcl-XL

expression and outcome following chemoradiation therapy ofOPSCC, they do not rule out roles of Bcl-XL in helping todetermine treatment responses in HNSCC. Studies examiningprimary radiation treatment (25, 35) have shown that elevatedBcl-XL is associated with worse outcome in some forms ofHNSCC. Furthermore, although we did not find a correlationof endogenous Bcl-XL expression with cisplatin resistance incell lines derived from HNSCC, we have found that exogenousoverexpression of Bcl-XL in these cell lines can increase theircisplatin resistance (not shown). It will be important tocharacterize the specific roles of Bcl-2 and Bcl-XL in differenttypes of HNSCC and in the responses to different types ofcytotoxic therapy. Differences in cellular mechanisms respond-ing to radiation versus platinum-based chemotherapy, orinteractions of p53 status with proapoptotic and antiapoptoticmechanisms, might explain some of the yet-unresolved differ-ences in prognostic significance between Bcl-2 and Bcl-XL.Dynamics of subcellular localization may also play a rolebecause Bcl-2 is constitutively membrane-bound to themitochondria, whereas Bcl-XL and Bcl-w are cytosolic andtranslocate to the mitochondria during apoptosis (46).Relation to HPV. Our identification of Bcl-2 as an important

prognostic marker in advanced OPSCC complements recentstudies identifying HPV as a predictor of therapeutic responseand overall survival (2–6). Patients with HPV-associatedHNSCC typically are younger, lack the traditional HNSCC riskfactors of alcohol and tobacco, and are more likely to have ahigher number or oral and vaginal sexual partners (3). Therewas no correlation between HPV infection and either Bcl-2 orBcl-XL status among the patients in the present study, indicatingthat HPV and Bcl-2 status might be independent predictorsof disease-free survival. A larger study would be required todetermine the nature of interactions between Bcl-2 and HPVstatus for predicting outcome of OPSCC.Implications for therapy. Patients having oropharyngeal

tumors with low Bcl-2 expression typically had favorableoutcomes following concurrent chemoradiation therapy, sothe major outstanding clinical problem is the poor outcometypical of patients with high Bcl-2 expression. In this subset ofpatients, upfront surgery followed by chemoradiation may be atherapeutic option when the oropharyngeal primary is amena-ble to resection with acceptable morbidity. In this regard,transoral robotic surgery for base of tongue neoplasms mightprovide a minimally invasive approach that leads to lessmorbidity than classic open or endoscopic transoral lasersurgical approaches (47). For patients with high Bcl-2expression in tumors where surgical resection is impossible orwould yield excess morbidity, clinically effective medicaltreatments must be identified. Options to explore for thissubgroup include non-platin chemotherapy (e.g. taxanes orfluorouracil) either as induction therapy prior to radiation (48)or concurrent with radiation (49). Most interesting for thissubset would be biologic therapies, such as anti–epidermalgrowth factor receptor monoclonal antibodies or small-molecule Bcl-2 inhibitors, with radiation. Retrospective ana-lyses of Bcl-2 expression in tumor specimens from publishedtrials of radiation therapy with or without combinations ofcetuximab and cisplatin (reviewed in ref. 32) could validate and



extend the results of this present study, and potentially clarifywhether cetuximab can limit the apparent resistance toradiation of tumors expressing high levels of Bcl-2.The correlation of Bcl-2 with therapeutic response to

chemoradiation in advanced OPSCC is consistent with itsknown role as a primary regulator of apoptosis. This inter-pretation is supported by our in vitro data relating bothendogenous Bcl-2 expression and overexpressed exogenousBcl-2 to cisplatin resistance in HNSCC cell lines. As previouslysuggested, Bcl-2 may thus be an attractive therapeutic target inOPSCC. The recent solution of the structure of antiapoptoticBcl-2 family members (reviewed in ref. 46) has resulted in thedesign of a number of novel small molecule inhibitorscurrently under active therapeutic evaluation (21, 50). Target-ing Bcl-2 with these agents in the clinical trial setting may be a

compelling approach in patients with OPSCC predicted to dopoorly due to elevated pretreatment Bcl-2 expression whenupfront resection offers unacceptable morbidity. Phase I-IIstudies of small molecule Bcl-2 inhibitors are early indevelopment. Studies of these agents in patients whose headand neck squamous cancers overexpress Bcl-2 are eagerlyawaited.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Robert Ferris and David Sidransky for gifts of cell lines.



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Bcl2 Blocks Cisplatin-Induced Apoptosis and Predicts Poor Outcome Following Chemoradiation Treatment...

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