brm promoter polymorphisms and survival of advanced non … · introduction: brm, a key catalytic...

Personalized Medicine and Imaging

BRM Promoter Polymorphisms and Survival ofAdvanced Non–Small Cell Lung Cancer Patientsin the Princess Margaret Cohort and CCTGBR.24 TrialGeoffrey Liu1,2, Sinead Cuffe1, Shermi Liang3, Abul Kalam Azad1,2, Lu Cheng1,Yonathan Brhane1, Xin Qiu1, David W. Cescon1,2, Jeffrey Bruce1,2, Zhuo Chen1,2,Dangxiao Cheng1,2, Devalben Patel1,2, Brandon C.Tse1,2, Scott A. Laurie4,GlenwoodGoss4,Natasha B. Leighl1, Rayjean Hung5, Penelope A. Bradbury1, Lesley Seymour6,Frances A. Shepherd1,2, Ming Sound Tsao1,2, Bingshu E. Chen6,Wei Xu1,2, andDavid N. Reisman3

Abstract

Introduction: BRM, a key catalytic subunit of the SWI/SNFchromatin remodeling complex, is a putative tumor susceptibilitygene that is silenced in 15% of non–small cell lung cancer(NSCLC). Two novel BRM promoter polymorphisms (BRM-741 and BRM-1321) are associated with reversible epigeneticsilencing of BRM protein expression.

Experimental Design: Advanced NSCLC patients from thePrincess Margaret (PM) cohort study and from the CCTGBR.24 clinical trial were genotyped for BRM promoter poly-morphisms. Associations of BRM variants with survival wereassessed using log-rank tests, the method of Kaplan andMeier, and Cox proportional hazards models. Promoterswap, luciferase assays, and chromatin immunoprecipitation(ChIP) experiments evaluated polymorphism function.In silico analysis of publicly available gene expression datasetswith outcome were performed.

Results: Carrying the homozygous variants of both poly-morphisms ("double homozygotes", DH) when compared withthose carrying the double wild-type was associated with worseoverall survival, with an adjusted hazard ratios (aHR) of 2.74(95% CI, 1.9–4.0). This was confirmed in the BR.24 trial (aHR,8.97; 95% CI, 3.3–18.5). Lower BRM gene expression (by RNA-Seq ormicroarray) was associatedwith worse outcome (P < 0.04).ChIP and promoter swap experiments confirmed binding ofMEF2D and HDAC9 only to homozygotes of each polymor-phism, associated with reduced promoter activity in the DH.

Conclusions: Epigenetic regulatory molecules bind to twoBRM promoter sequence variants but not to their wild-typesequences. These variants are associated with adverse overall andprogression-free survival. Decreased BRM gene expression, seenwith these variants, is also associated with worse overall survival.Clin Cancer Res; 23(10); 2460–70. �2016 AACR.

IntroductionLung cancer is the leading cause of cancer deaths in the

industrialized world, even in this new era of screening (1, 2). The

majority of non–small cell lung cancer (NSCLC) patients presentat an advanced stage for which treatment is met with limitedsuccess. However, with considerable interindividual variability inlung cancer development, outcomes, and treatment response,heritable polymorphisms may play roles in lung cancer suscep-tibility/risk (3, 4) and outcome (5).

The SWI/SNF (SWItch/sucrose non-fermentable) complex is amultimeric chromatin remodeling complex that plays a key rolein regulating multiple cellular processes, including gene expres-sion, differentiation,DNA repair, and cell-cycle control (6–9). Thecomplex, consisting of a catalytic subunit with helicase ATPaseactivity [either Brahma (BRM) or BRM-related gene 1 (BRG1)] isrequired for the function of a variety of signal transductionpathways and anticancer protein activities [retinoblastoma (Rb),p53 and BRCA1, refs. 7, 9–11]. BRM regulates the expression of4% to 7% of mammalian genes, many of which have anticancerroles, among them, vimentin, E-cadherin, N-cadherin, estrogenreceptor, progesterone receptor, and CD44 (9, 12–16).

BRMprotein expression is lost in 15% to 40%ofmany primarysolid tumors, including in 17% to 30%ofNSCLC (17, 18). Unlikemost tumor suppressor genes, however, BRM is reversibly

1Princess Margaret Cancer Centre and University Health Network, University ofToronto, Toronto, Ontario, Canada. 2Ontario Cancer Institute, Toronto, Ontario,Canada. 3University of Florida, Gainesville, Florida. 4Ottawa Hospital CancerCentre, Ottawa, Ontario, Canada. 5Lunenfeld Research Institute andMount SinaiHospital, Toronto, Ontario, Canada. 6Canadian Cancer Trials Group, QueensUniversity, Kingston, Ontario, Canada.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

G. Liu and S. Cuffe share first authorship of this article.

W. Xu and D.N. Reisman share senior authorship of this article.

Corresponding Author: Geoffrey Liu, University of Toronto, 610 UniversityAvenue, 7th Floor Room 7-125, Toronto, Ontario, Canada, M5G 2M9. Phone:416-946-3428, 4501 ext; Fax: 416-946-6546; E-mail: [email protected];

doi: 10.1158/1078-0432.CCR-16-1640

�2016 American Association for Cancer Research.

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epigenetically silenced (18–20). Initially, pan-histone deacetylase(HDAC) inhibitors were identified as compounds that couldrestore BRM, but these agents were also found to inactivate BRMby inducing its acetylation (21). As these compounds showed thatBRM is regulated by HDACs, further analysis showed that BRMsilencing is regulated specifically by class 1 HDAC (HDAC3) andclass 2 HDAC (HDAC9) enzymes. Further, HDAC9 is overex-pressed in both BRM-deficient (>500-fold) cancer cell lines and(>20-fold) primary tumors (22, 23).

To determine how BRM was silenced, we previouslysequenced the BRM promoter in cancer cell lines and identifiedthe presence of two insertional/deletional polymorphisms(BRM-741:TTAAA) and (BRM-1321:TATTTTT) that correlatedstrongly with loss of BRM protein expression in cancer celllines and confirmed in primary lung cancers. The variantinsertion alleles of both polymorphisms produce sequencevariants that are highly homologous to myocyte enhancerfactor-2 (MEF2) transcription factor binding sites, where MEF2is known to recruit HDACs and silence genes (24). shRNAknockdown of HDAC9, MEF2D in BRM-deficient cell linesresults in the induction of BRM (24, 25). BRM demonstratesattributes of a tumor susceptibility gene, as the BRM-nullmouse does not develop spontaneous tumors, but showsdistinct abnormalities in cell-cycle control; when combinedwith a carcinogen, tumor development is potentiated (18). Wereasoned, therefore, that the promoter insertion variants maybe associated with cancer risk and have confirmed that indi-viduals carrying both BRM homozygous promoter insertionvariants have a 2-fold increase in the risk of lung cancer (24),particularly early-stage lung cancer (25), and in other cancers(26–28).

Prognostically, loss of BRM protein expression has beenlinked to adverse outcome across a varied mix of NSCLC stages(17, 29). However, the relationships between BRM promoterpolymorphisms, BRM gene expression, and outcome have notbeen documented previously. Potential prognostic implicationsare important, as involvement of the BRM pathway in tumorsmay identify a subset of individuals with worse outcome thatpotentially could benefit from focused development of a class ofdrugs targeting BRM reexpression. That the BRM polymorph-isms are associated with epigenetic factors is unique, but even

more so when such epigenetic silencing has been reversed by avariety of compounds and drugs such as certain nonsteroidalanti-inflammatory drugs (indoprofen) and flavonoids (genis-tein; refs. 30, 31).

As cancer drug development focuses on advanced disease, weundertook this study to evaluate the role of these polymorphismson survival outcomes in two independent patient cohorts. How-ever, we first evaluated the functional significance of the poly-morphisms through promoter swap and chromatin immunopre-cipitation (ChIP) experiments; these analyses can provide impor-tant adjunctive evidence supporting a potential clinical role of thisgene and specifically these two polymorphisms. Finally, weassessed the significance of BRM gene expression on clinicaloutcome, using publicly available databases, to provide the firstevidence of a link between our putative biological mechanismand the associations found in this study.

Materials and MethodsStudy cohorts

The studywas approvedby the institutional researchethicsboardand by the Lung Cancer Correlative Science and Tissue BankingSubcommittee of the Canadian Cancer Trials Group. Figure 1shows the flowof patient specimens in the polymorphism-survivalanalyses.

Princess Margaret (PM) cohort studyCases were incident (diagnosis � 6 months prior to enroll-

ment) stage III–IV NSCLC patients who participated in a pro-spective study evaluating the molecular epidemiology of lungcancer at thePMCancer Centre, Toronto, between2006 and2010.Eligibility included histological confirmation of NSCLC andprovision of written consent; exclusions were inability to com-municate in English and cognitive deficits interfering with abilityto understand consent. For cohort analysis of survival, cases werederived from the same underlying pool of advanced, incurablestage III–IV NSCLCs as in the case–control analysis, with thefollowing exceptions: smoking status was no longer part of theeligibility criteria, and all adult patients (� 18 years old) wereeligible. Available treatment, covariate, and survival data for atleast 6 months after diagnosis were an additional inclusioncriterion.

BR.24 clinical trialThe Canadian Cancer Trials Group (CTG) led the BR.24 clinical

trial, a randomized double blinded, international phase II trialthat evaluated carboplatin (area under the curve, AUC 6) andpaclitaxel (200 mg/m2) every 3 weeks for 6 to 8 cycles in com-binationwith either daily oral cediranib or placebo in thefirst-linetreatment of advanced stage IIIB–IV NSCLC patients, conductedbetween 2005 and 2008. Cediranib/placebo monotherapy after-ward in the absence of intolerable toxicity or disease progressionwas allowed. A preplanned interim analysis determined an imbal-ance in the number of investigator-designated cause of deaths,and the trial was terminated without entering the phase IIIportion.

DNA extraction and genotypingGermline DNA was extracted from the lymphocytes of whole

blood of all patient cohorts using a commercially available DNAisolation kit (5Primer, Cat#2300740). Genotyping for BRM-741

Translational Relevance

The role of germline biomarkers of chromatic remodelinghas not been well defined. The identification of two promoterpolymorphisms in the BRM gene has focused previously onlung cancer risk associations. In this study, we first demon-strate using expression data and functional assays, the poten-tial importance of this pathway and these specific polymorph-isms to the clinical outcomes of lung cancer patients. Wedemonstrate a novel and consistent prognostic association ofthese polymorphisms with overall and progression-free sur-vival across clinical trial and observational datasets. Theseresults help identify high-risk poor prognosis patients thatpotentially could benefit from future targeting of this pathwayby reversing the epigenetic phenomenon, a process we havedemonstrated previously using in vitro models.

BRM Polymorphisms and Lung Cancer Prognosis

www.aacrjournals.org Clin Cancer Res; 23(10) May 15, 2017 2461




and BRM-1321 was performed using two custom-designed Taq-man assays (24, 25). For quality control, positive and negativecontrols and blinded duplicate samples were included.

ChIP, promoter luciferase, and swap experimentsTo demonstrate the proteins that bind the BRM polymorphic

sequences and altered promoter activity levels by these polymor-phic variant, ChIP, promoter luciferase, and swap experimentswere performed. The insertion variants of the BRM polymorph-isms areMEF2binding sites (with>92%homology; ref. 24).Uponbinding, MEF2D recruits class II HDACs, specifically HDAC9,which results in targeted gene silencing (32, 33). To demonstratethis,ChIPexperimentswereperformed todetermine the specificityof MEF2D/HDAC9 binding to BRM insertion alleles, and not towild-type deletion alleles. To further demonstrate that the spec-ificity is polymorphism dependent and not cell-line dependent,isogenic BRM promoter constructs (forming double-homozygousand double-wild-type genotypes) were placed stably in the SW13cell line via homologous recombination. In these constructs(Fig. 3), a luciferase reporter IRES-neomycin gene was insertednear exon 2 of the BRM gene, which effectively disrupted BRMexpression, and allowed the inserted luciferase reporter gene to beunder the control of the BRM promoter. These isogenic promoterswapped constructs were stably integrated into the SW13 cell line,and then assessed using ChIP assays. These same constructs werethen used to determine the relative level of promoter activitythrough measurement of luciferase.

Statistical analysesFor patient cohorts, baseline demographic and clinicopath-

ologic data were described and cross-tabulated. Additionally,for the BR.24 trial, baseline demographic, clinical information,and survival outcome were compared between individuals withdata available for genetic analysis and those without. Departurefrom Hardy–Weinberg equilibrium (HWE) was tested using thePearson c2 test. All models assumed codominant genetic inher-itance for both polymorphisms, and combined polymorphismanalyses were performed using previously described categor-izations (24, 25).

Survival was defined as the time from date of pathologicaldiagnosis of stage III/IV (PM cohort) or randomization (BR.24cohort) to either thedate of death fromany cause (overall survival,OS), or date of first disease progression/recurrence or death fromany cause (progression-free survival, PFS). Patients were censoredwhen they were last known to be alive (for OS) and last assessedfor progression/recurrence (for PFS).

Survival rates and median survival times were estimated usingthe Kaplan–Meiermethod. Cox proportional hazardsmodels andthe log-rank test were used to test associations between thesequence variants and survival. Multivariate models were con-structed with genetic markers after adjusting for individual cov-ariates found to be associated with survival (at the P < 0.10 level).P values, adjusted hazard ratios (aHR) and their 95% confidenceintervals (95% CI) for survival were reported. All statistical testswere two-sided and were conducted using the SAS software

Figure 1.

CONSORT diagram of the PM cohort(left) and the Canadian Cancer TrialsGroup, BR.24 trial (right).

Liu et al.

Clin Cancer Res; 23(10) May 15, 2017 Clinical Cancer Research2462




(version9.2, SAS Institute Inc.) and theR software (version2.11.0,R Development Core Team). The Wald test was used for all thegenetic models of inheritance. A two-sided P value of less than<0.05 was considered statistically significant.

For ChIP experiments, protein–DNA sequence binding affinityresults were reported as specific anti-HDAC9 and anti-MEF2Dantibody binding, normalized against background, non-specificIgG, which resulted in a measurement of fold change (against thebackground). Log-transformed triplicate fold change data wereanalyzed using Student t tests. Similar fold-change data wereanalyzed for the promoter luciferase activity, comparing differentpromoter constructs.

Gene expression analysis of publicly available databases: we used apublicly available tool (www.kmplot.com/lung) to evaluate theprognostic effect of low BRM (gene symbol SMARCA2) geneexpression on OS in patients with NSCLC, using a lung cancerdataset encompassing 1,715 samples (1,405 NSCLC) with geneexpression and survival data from 10 independent studies (Jan-uary, 2015); analyses compared the bottom quartile of BRMexpression with the top three quartiles. RNA-Seq lung cancer datawere available from TCGA (n ¼ 431 adenocarcinomas; n ¼ 323squamous cell carcinomas), and survival analysis by quartiles wasperformed. In all cases, the method of Kaplan–Meier was used,and log-rank tests were performed.

ResultsPatient cohort demographics

Patient demographics for each study cohort are shown in Table1. Patients from BR.24 included only those suitable for first-linechemotherapy, rather than surgery or combined chemotherapyand radiation. Patients from the PM cohort included primarilyunresectable patients. Compared with BR.24 participants, PMcases weremore likely to have adenocarcinoma and have a higherfraction of the stage III patients. When comparing BR.24 patients

analyzed with those not analyzed due to lack of sample, excludedpatientsweremore likely of later stage, have adenocarcinoma, andhave unknown weight loss prior to randomization, leading todecisions to adjust for these variables in the final BR.24 model.Despite these differences, the analyzed population had similarsurvival outcomes and derived similar benefit from treatmentcompared with the total BR.24 cohort (test of homogeneitybetween treatment arm and availability of biospecimens P ¼0.93). In comparison, the analyzed and non-analyzed PMcohorts, however, were very similar (Supplementary Table 1).

BRM polymorphisms and survival analysesBoth polymorphisms were in the Hardy–Weinberg equilibri-

um (P > 0.05), and in mild linkage disequilibrium (D'¼ 0.48 forPMcohort, and0.39 for BR.24 trial). Genotypingwas successful in>99.8% of cases. Although �20% carried at least one homozy-gous variant of these twopolymorphisms, 11% to 15%of patientscarried homozygous variants of both polymorphisms (termed the"double homozygote" or DH). Because of a higher proportion ofstage III patients and the inclusion of unresectable stage IIIApatients, the median OS for the PM cohort is substantially higherthan that of BR.24 (Table 1).

Table 2 (OS), Table 3 (PFS), and Fig 2 (Kaplan–Meier curves)report strongly significant survival relationships with the individ-ual polymorphisms, and further specifically with the DH variantsof these two BRM polymorphisms (when compared with thedouble-wild-type polymorphisms). The strongest survival rela-tionship was found in the placebo (conventional therapy) arm ofBR.24, where there was uniform treatment of all patients withcarboplatin–paclitaxel, and adjusted hazard ratios exceeded 8(PFS) and 16 (OS). Subgroup prognostic analyses (Supplemen-tary Table S2) showed similar results in never-smokers andsmokers, in patients with adenocarcinomas versus squamous cellcarcinomas, by age and gender, for both the PM cohort and the

Table 1. Clinicodemographic and genotyping information

Survival analysis Comparison

Characteristic Category PM cohortBR.24 cohortanalyzed

BR.24 notanalyzed

Analyzed vs. notanalyzed P value

Total number 548 219 77Age (years) Median (range) 63 (31–90) 60 (38–82) 57 (36–72) 0.10Sex Male/female 52%/48% 59%/41% 56%/44% 0.74Ethnicity Caucasian/Asian/other 67%/15%/18% Not reportedc Not reportedSmoking status Current/former/never-smoker 30%/44%/26% Not reported Not reportedPack-yearsa Median (smokers) 38 (1–216) Not reported Not reportedECOG performance status 0/1–2/3þ 15%/84%/1% 23%/77%/0 27%/73%/0 0.59Weight loss � 5%/< 5%/unknown Not reported 28%/58%/13% 18%/39%/43% <0.001Histological Adenocarcinoma 67% 46% 64% 0.01Subtype Squamous cell 16% 26% 12%

Other 17% 28% 25%Stage III/IV 46%/54% 17%/83% 6%/94% 0.02Platinum agent Cisplatin/carboplatin based therapy 82% 100% 100%

No-platinum chemotherapy 10% 0% 0%Neither/unknown 8% 0% 0%

Follow-up time Median (months) 42 18 17 0.91Overall survival Median (months) 19 9 10 0.64Time to randomizationb <6 months/ �6 months 89%/11% 91%/9% 0.89Dose cohort 30 mg/ 45 mg of cediranib/placebo 84%/16% 86%/14% 0.94Treatment arm Conventional (placebo) arm/

experimental (cediranib) arm50%/50% 51%/49% 0.99

aCalculated in ever-smokers only.bFrom date of diagnosis.cMajority are expected to be of Caucasian descent.






BR.24 trial participants. For the PM cohort, subset analysis of the82% of patients treated with platinum-based chemotherapyfound virtually identical survival associations with these twoBRMpolymorphisms as the entire PM cohort; further subset analysesfound consistent associations regardless of whether the therapywas cisplatin- or carboplatin-based doublet therapy.

ChIP and promoter swap experimentsFigure 3 shows ChIP experiments of representative cell lines

(including several lung cancer cell lines) carrying different BRMpolymorphism combinations. In brief, of the cell lines evalu-ated, all BRM wild-type genotypes resulted in little to nobinding of MEF2/HDAC9, while there was significant binding

observed in all homozygous variants (P < 0.0001, all compar-isons), including homozygous variants created through thepromoter construct swap experiment of the SW13 cell line. Inaddition, the same promoter constructs demonstrated a 7.8-fold increased luciferase expression when these insertion allelesare absent as compared with when they are present (Fig. 3),thereby demonstrating that these BRM polymorphic variantsbind specific proteins and are functionally involved in regu-lating BRM expression.

BRM gene expression and survivalGene expression data frompublicly available sources of expres-

sion microarrays (Fig. 2, middle) and RNA-Seq (Fig. 2, right) of

Table 2. BRM polymorphisms and OS in advanced-stage NSCLC

PM cohort(N ¼ 548)

BR.24 conventionalarm (N ¼ 109)

BR.24 experimentalarm (N ¼ 110)

BR.24 all patients(N ¼ 219)

Polymorphism Category % aHR (95% CI)a % aHR (95% CI)b % aHR (95% CI)b % aHR (95% CI)b

BRM-741 Wild-type 29% Reference 30% Reference 27% Reference 28% ReferenceHeterozygote 48% 1.60 (1.2–2.2) 43% 2.81 (1.5–5.4) 49% 2.21 (1.1–4.5) 45% 2.75 (1.7–4.4)Homozygote 23% 2.49 (1.9–3.3) 29% 5.52 (2.7–11.2) 25% 3.91 (1.8–8.4) 27% 4.98 (3.0–8.3)P valuec <0.001 <0.001 0.002 <0.001Minor allele frequency 46% 50% 49% 50%

BRM-1321 Wild-type 34% Reference 28% Reference 30% Reference 29% ReferenceHeterozygote 44% 1.18 (0.9–1.5) 55% 3.14 (1.6–6.0) 46% 2.01 (1.1–3.8) 50% 2.53 (1.6–3.9)Homozygote 22% 1.99 (1.5–2.7) 18% 7.64 (3.5–16.7) 24% 3.54 (1.8–7.1) 21% 4.98 (3.0–8.3)P valuec <0.001 <0.001 0.002 <0.001

Minor allele frequency 44% 44% 46% 45%Combined polymorphisms Double wild-type 21% Reference 19% Reference 18% Reference 19% Reference

No homozygote 47% 1.40 (1.0–1.8) 46% 2.90 (1.3–6.7) 48% 3.46 (1.3–9.3) 47% 3.40 (1.8–6.3)One homozygote 21% 2.17 (1.6–3.1) 24% 5.05 (2.09–12.3) 18% 9.26 (3.2–27.0) 21% 6.62 (3.4–13.1)Two homozygotes 12% 2.74 (1.9–4.0) 11% 16.8 (6.0–46.7) 15% 6.36 (2.2–19.1) 13% 8.97 (3.3–18.5)P valuec <0.001 <0.001 0.001 <0.001

aAdjusted for sex, stage, ECOG performance status at diagnosis, number of lines of systemic therapy received, and prior surgical resection of lung primary or site ofoligometastases.bAdjusted for sex, stage, ECOG performance status, weight loss, and time from diagnosis to randomization; in the "all patients" analysis, there was also stratificationby treatment arm.CP values are from Cox proportional hazard models comparing BRM polymorphisms and survival.

Table 3. BRM polymorphisms and PFS in advanced-stage NSCLC

PM cohort (N ¼ 548)BR.24 conventional

arm (N ¼ 109)BR.24 experimental

arm (N ¼ 110)BR.24 all patients

(N ¼ 219)Polymorphism Category % aHR (95% CI)a % aHR (95% CI)b % aHR (95% CI)b % aHR (95% CI)b

BRM-741 Wild-type 29% Reference 30% Reference 27% Reference 28% ReferenceHeterozygote 48% 1.57 (1.2–1.9) 43% 1.63 (0.9–3.0) 49% 1.45 (0.8–2.7) 45% 1.69 (1.1–2.6)Homozygote 23% 2.05 (1.6–2.7) 29% 3.75 (1.9–7.4) 25% 2.25 (1.1–4.5) 27% 3.02 (1.9–4.8)P valuec <0.001 <0.001 0.07 <0.001Minor allele frequency 46% 50% 49% 50%

BRM-1321 Wild-type 34% Reference 28% Reference 30% Reference 29% ReferenceHeterozygote 44% 1.22 (1.0–1.5) 55% 2.04 (1.1–3.7) 46% 1.10 (0.6–2.0) 50% 1.48 (1.0–2.2)Homozygote 22% 1.86 (1.5–2.4) 18% 3.37 (1.6–7.2) 24% 1.96 (1.0–3.9) 21% 2.57 (1.6–4.2)P valuec <0.001 0.006 0.07 <0.001Minor allele frequency 44% 44% 46% 45%

Combined polymorphisms Double wild-type 21% Reference 19% Reference 18% Reference 19% ReferenceNo homozygote 47% 1.52 (1.2–1.9) 46% 1.71 (0.8–3.7) 48% 1.27 (0.6–2.7) 47% 1.62 (1.0–2.7)One homozygote 21% 1.86 (1.4–2.5) 24% 2.87 (1.3–6.6) 18% 2.96 (1.2–7.0) 21% 3.11 (1.7–5.6)Two homozygotes 12% 2.71 (1.9–3.8) 11% 8.30 (3.2–21.9) 15% 2.08 (0.8–5.3) 13% 3.75 (1.9–7.3)P valuec <0.001 0.001 0.02 <0.001

aAdjusted for sex, stage, ECOG performance status at diagnosis, number of lines of systemic therapy received, and prior surgical resection of lung primary or site ofoligometastases.bAdjusted for sex, stage, ECOG performance status, weight loss, and time from diagnosis to randomization; in the "all patients" analysis, there was also stratificationby treatment arm.cP values are from Cox proportional hazard models comparing BRM polymorphisms and survival.

Liu et al.





mainly early and locally advanced stage NSCLC are presented.Significant association was observed between lower BRM expres-sion levels andpoorer survival in expressionmicroarray data, witha HR of 0.42 (95% CI, 0.32–0.84; P ¼ 0.02). Lower BRMexpression was associated inversely with prognosis consistentlyin adenocarcinomas regardless of the method of assessing BRM

gene expression: from RNA-Seq data, P ¼ 0.04 and expressionmicroarray data, P ¼ 0.01. In contrast, two different sets of dataobtained through two different assessments of BRM gene expres-sion differed on the relationships with survival in squamouscell carcinoma, where there were significant associations in theRNA-Seq data (P ¼ 0.04) but not in expression microarray data

Figure 2.

Kaplan–Meier survival curves by BRMpolymorphisms and BRM geneexpression. P values are derived fromlog-rank tests. Where reported, HRswith 95% CIs are from univariable Coxproportional hazard models. N ¼number of patients assessed in eachanalysis. Top, OS and PFS of the PMcohort [463/548 (84%) had died at amedian follow-up of 42 months] andBR.24 trial patients [153/219, 70%)had died at a median follow-up of18 months] by BRM polymorphismcombinations. Middle, Overall survivalcurves of NSCLC patients by BRMgene expression levels in tumor tissuefrom 10 publicly available microarraygene expression datasets (www.kmplot.com/lung). Bottom, TCGAlung adenocarcinoma and squamouscell carcinoma datasets using RNA-Seq data (February 2014). To avoidduplication of results, 67 squamouscell carcinoma cases were removedfrom the TCGA analysis, as thesesamples were already included in themicroarray gene expression analysis.






Figure 3.

Functional assays. Top, ChIP experiments demonstrate that binding of MEF2D and HDAC9 occurs only in the presence of the homozygous variants (P < 0.001; Student ttest) and not the wild-types (P > 0.05). Vertical axes show the binding affinity relative to background binding by nonspecific IgG in the C33A (cervical carcinoma),H552 (lung adenocarcinoma), and A547 (lung squamous carcinoma) cell lines (log-transformed ratio þ standard error). (Continued on the following page.)

Liu et al.





(P ¼ 0.68). Separate analyses of expression microarray data bysmoking status suggested that BRM expression were stronglyassociated with NSCLC in never-smokers but not in smokers;however, further multivariable analyses determined that thisdifference was largely driven by histology (P < 0.02) rather thanby smoking status (P ¼ 0.62).

DiscussionTwo novel BRM promoter insertion deletion polymorph-

isms, BRM-741 and BRM-1321, are linked to epigenetic silenc-ing of BRM expression and have been linked to lung cancerrisk (24, 25). The presence of both homozygous variantsstrongly correlates with loss of BRM expression in lung cancer,a loss that, in turn, has been linked to adverse clinical out-comes (17, 29, 34). While previously we had documentedthese polymorphisms as being associated with risk of early-stage NSCLC (27), in the present analysis, we further describethat individuals carrying the homozygous variants of one orboth polymorphisms had substantially worse survival out-comes compared with their wild-type counterparts in twoindependent datasets, one an observational cohort, and theother, a clinical trial. We performed molecular experiments toexplain the functional significance of these polymorphisms,demonstrating that the presence of these polymorphic variantsis physically connected to binding of regulatory proteinsimportant in BRM expression. Finally, we reported that BRMgene expression was also prognostic for NSCLC survival,through analysis of publicly available data. Each of thesefindings incrementally builds on our understanding of theseBRM polymorphisms and its clinical relevance. These findingsinclude the mechanism of polymorphism function, and con-sistent findings across DNA and RNA levels of BRM withprognosis, compatible with proposed mechanisms of BRMfunction. Such cumulative evidence supports the importanceof BRM in NSCLC outcome.

BRM germline polymorphisms may be useful not only forrisk stratification but also for prognostication. This is of impor-tance because our prior research suggests that BRM is a poten-tially druggable target (21). In one clinical setting, CT screeningof lung cancer may benefit from further refinement by incor-poration of molecular risk markers (35). Yet in a contrasting setof circumstances, other markers such as somatic ALK transloca-tions already help identify patients with aggressive (poor prog-nosis) tumors that respond well to drugs that target the rear-ranged ALK protein (36). If a somatic genomic alteration suchas ALK can lead to drug targets, perhaps the concept of agermline epigenetic target may not be so far-fetched. In thepast, a barrier to adoption of polymorphic variants in risk/prognostic models has been a lack of clear biological or func-

tional significance of such polymorphisms, a flaw that isaddressed in this study. In fact, putting all the available evi-dence from the literature and the results of this study together,truly functional consequences of these polymorphisms mayaffect tumor biology and drug sensitivity, while our priorresearch suggested BRM is a druggable target (21, 24).

To better understand why these polymorphisms mightunderlie clinically relevant associations, we examined potentialroles of BRM in cancer. Loss of BRM expression has beenimplicated in the progression of lung adenocarcinoma intosolid predominant tumors with features of epithelial–mesen-chymal transition (EMT), which is consistent the fact the SWI/SNF regulates key genes involved in EMT changes, such asE-cadherin, N-cadherin, and vimentin, and loss of bronchialepithelial phenotype (34). Similarly, a link between BRM lossand cancer progression has been suggested among patients withgastric (37) and skin cancer (38). Loss of BRM protein expres-sion has been linked to poorer outcome among patients withNSCLC (17, 29), and other cancers (39).

BRM can impact cancer evolution in a number of ways. Itsfunctional linkage to such proteins as Rb and p53 may in partexplain how BRM silencing can result in the loss of cellulargrowth control, a central mechanism by which BRM is thoughtto potentiate the risk of cancer (12, 40, 41). However, BRM andSWI/SNF are also involved in DNA repair, and the aberration ofDNA repair proteins, such as GADD45, BRCA1, and p53,functionally linked to BRM and SWI/SNF, are known to poten-tiate cancer development (10, 42). Loss of BRG1 and BRM hasbeen linked to the EMT, thought to be a major step towardmore aggressive disease; BRM and SWI/SNF have been linked toE-cadherin, N-cadherin, vimentin, CD44, CEACAM1, andintegrin expression, which are also linked to EMT (12, 34,43, 44). In addition, SWI/SNF in general has been shown toplay roles in cellular differentiation and organ development(45–49). Interestingly, the SWI/SNF complex may also increasesensitivity to cisplatin (50) but increase resistance to EGFRinhibitors. Indeed, there is no shortage of feasible mechanismsby which BRM loss could impact cancer development andclinical outcomes.

Studies to evaluate lung cancer risk or prognosis as a functionof a given germline polymorphism have been pursued vigor-ously. Prototypical polymorphisms that lie within the codingregion of a protein are surmised to alter the protein's functionby changing its primary sequence. This change in proteinsequence alters the function of the protein, the cell, and theorganism, thus yielding a significant clinical association. Whenthese polymorphisms are not specifically related to a proteinsequence, the value of a given polymorphism is strengthenedby evidence showing that it affects gene function. To this end,the ChIP and luciferase data presented herein show that two key

(Continued.) Second, a BRM reporter construct was placed in the SW13 (adrenal cortical carcinoma) cell line via homologous recombination, where a luciferase(luc) gene linked to an internal ribosime entry site (IRES)-neomycin gene was placed at the beginning of the transcriptional start site; the endogenous BRMgene is disrupted such that it is no longer expressed. Instead, the luciferase gene, now under the control of the BRM promoter, is expressed as a measureof the BRM promoter activity. Third panel, We obtained a number of these SW13 daughter cell lines (with BRM reporter constructs) derived from single cells, bydilutional cloning both with and without these polymorphisms. By comparing luciferase activity from six clonal SW13 daughter cell lines (three each) whicheither did or did not harbor these BRM insertion variants, a 5-fold higher luciferase activity was observed in the daughter cell lines harboring the BRM promoterwild-type construct when compared with the cell lines harboring the homozygous variants (P < 0.001). Bottom, Using the same isogenic SW13 celllines, ChIP evaluation of the swapped promoters demonstrated that MEF2D and HDAC9 bind only to the variant alleles (log-transformed data; P < 0.001), andnot to the wild-type alleles (P > 0.05). Axes are the same as those in the top panel.






BRM-regulating proteins, MEF2D and HDAC9, bind in or nearthe polymorphisms, in the presence of the variant insertionallele. Luciferase assays suggest that the BRM variants are alsoassociated with lower BRM expression.

In our study design, the consistency of our BRM findingsacross different study populations of risk (24, 25) and prog-nosis (herein) is a strength of our findings. Limitations arepresent, though. First, because we lacked sufficient quantities ofresearch tumor specimens in our advanced stage cohorts, wewere unable to perform matched polymorphism-gene expres-sion–protein expression assessments of BRM. Previously pub-lished protein expression-outcome analyses (17, 29) and thecurrent gene expression-outcome analyses confirm a consistentassociation with clinical outcome, but utilized samples fromearlier stage NSCLC patients. Gene expression analysis ofpublic databases may be confounded by differences in stageand other prognostic variables, possibly explaining the signif-icant relationship in squamous cell carcinoma by RNA-Seq butnot by expression microarray. Second, we are pursuing aparallel analysis of these polymorphisms in a randomizedclinical trial of adjuvant cisplatin chemotherapy in early-stageNSCLC, where we will be able to answer questions of potentialBRM–platinum drug interactions (50; all BR.24 patients weretreated with carboplatin) and to evaluate a uniformly cisplatin-treated patient cohort. Nonetheless, the PM cohort was treatedwith a platinum doublet in 82% of patients, of which 61%werecisplatin-based doublets (primarily cisplatin–etoposide forstage III and cisplatin–vinorelbine for stage IV), where subsetanalyses found similar results regardless of use of platinumagents or the specific platinum agent itself. Third, the interplaybetween EGFR, ALK, and other somatic changes with BRM onprognosis is unknown; we had too few patients with thesemolecular alterations to perform molecular-specific analyses ofBRM relationships. We also acknowledge that the dual roles ofBRM polymorphisms on risk and prognosis may not be con-sistent: in other tumors and settings, a risk association may nottranslate into a survival association, and vice versa; evaluationin each specific cancer disease site is necessary (51). We did nothave the power or tumor samples to explore the effect of thesepolymorphisms in difference milieu (e.g., different BRG expres-sion patterns, modifying effect of other chromatin remodelingmembers). In the TCGA analysis, survival data could have beenconfounded by other known prognostic variables (e.g., stage);however, TCGA clinical data quality is heterogeneous, and thusthese data are offered only as weak supporting evidence. None-theless, the totality of our findings has opened additionalroutes of exploration.

In summary, the same two BRM promoter polymorphismsthat were previously associated with increased risk of develop-ing NSCLC are also strongly associated with adverse survival intwo different advanced NSCLC patient samples. This is aclinically important discovery given the high magnitude ofprognostic association found, with values that could conceiv-ably lead to clinically meaningful germline molecular prog-nostication. Further, pharmacologic reversal of the epigeneticsilencing of BRM has been shown to be a potentially viabletherapeutic strategy (18, 21). With one in five patients homo-zygous for either BRM-741 or BRM-1321, and one in tenhomozygous for both, a sizeable population of NSCLC maybenefit either from future targeted therapy or prognostic strat-ification to allow decision-making on utilizing more aggressive

or different therapeutic strategies. Additional studies are need-ed to further validate our study findings in other lung cancerpopulations, specifically the relationships between these poly-morphisms and somatic lung cancer molecular alterations, andbetween these polymorphisms and other members of thesechromatin remodeling complexes.

Disclosure of Potential Conflicts of InterestG. Liu is a consultant/advisory boardmember for AstraZeneca, Novartis, and

Pfizer. G. Goss reports receiving speakers bureau honoraria from AstraZeneca,Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, and Pfizer. D.N. Reismanholds ownership interest (including patents) in Zenagene. No potential con-flicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Liu, X. Qiu, G. Goss, M.S. Tsao, B.E. Chen, W. Xu,D.N. ReismanDevelopment ofmethodology:G. Liu, S. Cuffe, S. Liang, D.W. Cescon, J. Bruce,D. Cheng, B.E. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Liu, S. Cuffe, S. Liang, A.K. Azad, X. Qiu,S.A. Laurie, N.B. Leighl, R. Hung, P.A. Bradbury, L. Seymour, F.A. Shepherd,M.S. TsaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Liu, S. Cuffe, A.K. Azad, L. Cheng, Y. Brhane,X. Qiu, D.W. Cescon, J. Bruce, R. Hung, L. Seymour, B.E. Chen, W. Xu, D.N.ReismanWriting, review, and/or revision of themanuscript:G. Liu, S. Cuffe, Y. Brhane,X. Qiu, J. Bruce, B.C. Tse, S.A. Laurie, N.B. Leighl, R. Hung, P.A. Bradbury,L. Seymour, M.S. Tsao, B.E. Chen, W. Xu, D.N. ReismanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Liu, Z. Chen, D. PatelStudy supervision: G. Liu, G. Goss, W. Xu

AcknowledgmentsWe acknowledge the support of Alan B. Brown Chair in Molecular

Genomics (to G. Liu), the Scott Taylor Chair in Lung Cancer Research (toF.A. Shepherd), the Ontario Institute for Cancer Research High ImpactClinical Trials Network (to G. Liu), the OSI Pharmaceuticals FoundationChair in Cancer New Drug Development (to N.B. Leighl), the Qasim ChoksiChair of Lung Cancer Translational Research (to M.S. Tsao), and RachelDresbeck for her contribution in editing. This work was supported by theCanadian Cancer Trials Group Tumour Tissue Data Repository (TTDR), andin particular, Shakeel Virk and Lois E. Shepherd. The CCTG TTDR is amember of the Canadian Tumour Repository Network. We also acknowledgeuse of data from The Cancer Genome Atlas (TCGA) lung adenocarcinomaand lung squamous cell carcinoma projects.

Grant SupportCanadian Cancer Trials Group conduct of this trial was supported by

funding received from the Canadian Cancer Society Research Institute (grant#021039 and #015469) and through a contract with AstraZeneca. Pharma-cogenetic analyses were also supported by the Ontario Institute for CancerResearch High Impact Clinical Trials Program and the Cancer Care OntarioChair in Experimental Therapeutics and Population Studies (to G. Liu).Support was also provided by the Princess Margaret Cancer Centre LusiWong Early Detection of Lung Cancer Program, Alan Brown Chair inMolecular Genomics, and the Posluns Family Fund. The functional evalua-tions were supported by 1R01CA136683-01A1 (PI: D.N. Reisman) and1R01CA127636-01 (PI: D.N. Reisman).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 7, 2016; revised October 4, 2016; accepted October 23, 2016;published OnlineFirst November 8, 2016.


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2017;23:2460-2470. Published OnlineFirst November 8, 2016.Clin Cancer Res Geoffrey Liu, Sinead Cuffe, Shermi Liang, et al. and CCTG BR.24 TrialSmall Cell Lung Cancer Patients in the Princess Margaret Cohort

−BRM Promoter Polymorphisms and Survival of Advanced Non

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