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Cancer Therapy: Clinical

Perioperative COX-2 and b-Adrenergic BlockadeImproves Metastatic Biomarkers in Breast CancerPatients in a Phase-II Randomized TrialLee Shaashua1, Maytal Shabat-Simon1, Rita Haldar1, Pini Matzner1, Oded Zmora2,Moshe Shabtai2, Eran Sharon3, Tanir Allweis4, Iris Barshack5, Lucile Hayman6,Jesusa Arevalo7, Jeffrey Ma7, Maya Horowitz1, Steven Cole7, andShamgar Ben-Eliyahu1

Abstract

Purpose: Translational studies suggest that excess perioperativerelease of catecholamines and prostaglandins may facilitatemetastasis and reduce disease-free survival. This trial tested thecombined perioperative blockade of these pathways in breastcancer patients.

Experimental Design: In a randomized placebo-controlledbiomarker trial, 38 early-stage breast cancer patients received11 days of perioperative treatment with a b-adrenergic antagonist(propranolol) and a COX-2 inhibitor (etodolac), beginning 5days before surgery. Excised tumors and sequential blood sampleswere assessed for prometastatic biomarkers.

Results: Drugs were well tolerated with adverse event ratescomparable with placebo. Transcriptome profiling of the primarytumor tested a priori hypotheses and indicated that drug treatmentsignificantly (i) decreased epithelial-to-mesenchymal transition,

(ii) reduced activity of prometastatic/proinflammatory transcrip-tion factors (GATA-1, GATA-2, early-growth-response-3/EGR3,signal transducer and activator of transcription-3/STAT-3), and(iii) decreased tumor-infiltrating monocytes while increasingtumor-infiltrating B cells. Drug treatment also significantly abro-gated presurgical increases in serum IL6 and C-reactive proteinlevels, abrogated perioperative declines in stimulated IL12 andIFNg production, abrogated postoperative mobilization ofCD16� "classical" monocytes, and enhanced expression ofCD11a on circulating natural killer cells.

Conclusions: Perioperative inhibition of COX-2 and b-adren-ergic signaling provides a safe and effective strategy for inhibitingmultiple cellular and molecular pathways related to metastasisand disease recurrence in early-stage breast cancer. Clin Cancer Res;23(16); 4651–61. �2017 AACR.

IntroductionThe removal of a primary tumor, and the abolition of its

potential immunosuppressive and metastasis-promoting effects(1), presents a window of opportunity to eliminate or control anyremaining minimal residual disease. Unfortunately, the periop-erative period and the excision of a primary tumor also trigger avariety of physiologic processes thatmaypotentially accelerate the

progression of pre-existing micrometastases and promote theinitiation of new metastases (2, 3). As such, the perioperativeperiod plays a critical role in determining long-term cancer out-comes, disproportionally to its short duration (4). Importantly,preclinical animal models of cancer suggest that pharmacologicmodification of perioperative physiology could be exploited toreduce the burden of residual disease (4).

Specifically, animal studies using syngeneic or human xeno-graft models of cancer have implicated peri-surgical high levelsof catecholamines and prostaglandins in mediating many ofthe prometastatic effects of surgery and perioperative stress(2, 4, 5). Catecholamines and prostaglandins are released bytumor cells, stromal cells within the tumor microenvironment,and by host physiologic systems as a result of physiologic andpsychologic stress responses to coping with cancer, tissuedamage, pain, and a variety of surgical impacts (6). Thesesignaling pathways can act directly on tumor cells to enhancetheir proliferation, motility, invasive capacity, resistance toanoikis, secretion of angiogenic factors (5, 7–9), and epitheli-al-to-mesenchymal transition (EMT; refs. 7, 10). Catechola-mines and prostaglandins can also indirectly promote metas-tasis by suppressing cell-mediated immunity (2), increasingprometastatic cytokines (e.g., IL8; ref. 11), and inducing inflam-mation, which is a hallmark of cancer progression (12).

During the last decade, we and others have found that phar-macologic inhibition of b-adrenoceptors and/or prostaglandin

1Sagol School of Neuroscience and School of Psychological Sciences, Tel AvivUniversity, Tel Aviv, Israel. 2Department of Surgery and Transplantation, ShebaMedical Center, Ramat Gan, Israel. 3Department of Surgery, Rabin MedicalCenter, Beilinson Hospital, Petach-Tikva, Israel. 4Department of Surgery, KaplanMedical Center, Rehovot, Israel. 5Department of Pathology, Sheba MedicalCenter, Ramat Gan, Israel. 6Department of Pathology, Rabin Medical Center,Petah Tikva, Israel. 7Departments of Medicine and Psychiatry and BiobehavioralSciences, David Geffen School of Medicine at UCLA, Los Angeles, California.

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

L. Shaashua and M. Shabat-Simon contributed equally to this article.

Corresponding Author: Shamgar Ben-Eliyahu, Tel Aviv University, Tel Aviv69978, Israel. Phone: 972-3640-7266; Fax: 972-3640-9520; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-0152

�2017 American Association for Cancer Research.

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synthesis can reduce the prometastatic and immune-suppressiveeffects of stress and surgery (9, 13–18). In these preclinical studies,the simultaneous administration of a b-blocker (propranolol)and a COX-2 inhibitor (etodolac) in combination (rather thaneach drug alone) has generally proven most effective, and some-times constitutes the only effective approach (19–21). The syn-ergistic effect of combined treatment protocolsmay stem from thefact that catecholamines and prostaglandins are both elevatedperioperatively and each can increase metastatic propensitythrough converging pathways (i.e., activation of the cAMP-Pro-tein Kinase A signaling system). Simultaneous inhibition of COX-2 and b-adrenergic signaling has reduced postoperativemetastasis(and in some cases improved overall survival) in multiple pre-clinical tumor models including breast, colon, lung, melanoma,and leukemia (19, 20, 22–24). Consistent with these preclinicalstudies, several pharmaco-epidemiologic studies have also docu-mented reductions in breast cancer progression or recurrence inpatients who happened to be taking b-blockers at or before initialdiagnosis (25, 26). Long-term use of COX-inhibiting NSAIDs wasalso associated with reduced risk of colorectal cancer (27). Toassess the potential biological impact of combined perioperativeCOX-2 and b-adrenergic inhibition in human breast cancer, weherein conducted a randomized placebo-controlled biomarkertrial employing etodolac and propranolol. Primary outcomeanalyses tested whether this combined drug treatment wouldreduce proinflammatory and prometastatic transcriptome pro-files in the malignant tissue.

Three specific transcriptome signatures were targeted a prioribased on previous research implicating them in COX-2 and/orb-adrenergic influences on breast cancer progression and metas-tasis. (i) The primary tumor's EMT profile was assessed as mes-enchymal polarization has been shown to promote intravasationand extravasation of epithelial tumor cells (28), and because bothCOX-2 activation and b-adrenergic signaling can promote EMT[COX-2 by inducing matrix metalloproteinase-1 (MMP-1) andMMP-2 (10) and inhibiting Smad signaling (29), and b-adren-ergic signaling by upregulating SNAIL and TWIST transcriptionfactors (7)]. (ii) Transcriptome signatures of tumor-infiltrating

leukocyte subpopulations were assessed based on data-linkingmonocyte/macrophage infiltration to breast cancer metastasisand B lymphocyte infiltration to reduced progression. (iii)Proinflammatory and prometastatic transcription control path-ways previously implicated in breast cancer progression werealso assessed [nuclear factor-kappaB (NF-kB)/cRel, activator-protein-1 (AP-1), GATA family, STAT family, NRF-2, EGRfamily transcription factors, and the glucocorticoid receptor(GR)]. Secondary outcome analyses addressed peripheralimmune parameters, including serum and ex-vivo–stimulatedTh1 and inflammatory cytokines (IL12, IFNg), NK-cell activa-tion markers, and circulating leukocyte populations (with aparticular focus on monocytes due to their involvement inmetastasis). This is the first clinical trial to test the efficacy ofa combined perioperative treatment with a b-blocker and aCOX-2 inhibitor in breast cancer patients.

Materials and MethodsPatients and inclusion/exclusion criteria

Thirty-eight women (age 33–70) diagnosed with stage I–IIIbreast cancer were enrolled from three medical centers in Israel.Exclusion criteria included (i) any contraindication for the drugs,such as diabetes, asthma, cardiovascular disease, or low bloodpressure, (ii) chronic use of any b-blocker or COX inhibitor, and(iii) chronic autoimmune disease. The study protocol (Clinical-Trials.gov Identifier: NCT00502684) was approved by Institu-tional Review Boards at each study site, and written informedconsent was obtained from patients before performing any study-related procedures.

Study design and drug treatmentThismulticenter double-blind placebo-controlled randomized

biomarker trial employed two equal-sized arms of drug- andplacebo treatment (Figs. 1A and 1B). Patient randomization wasstratified by age within each medical center (below or above 50).

Drug/placebo was administered for 11 consecutive days, start-ing 5 days before resection of the primary tumor (Fig. 1B). OralBID etodolac (400 mg) was administered throughout the treat-ment period. Propranolol was administered orally using extendedrelease formulations: 20 mg BID during the 5 days precedingsurgery; 80mg on the morning of surgery and on the evening andmorning following surgery; and 20 mg BID thereafter during 5postoperative days. Identical schedules were used for placebo andmedication.

Endpoints and assessmentsExcised tumor tissuewasfixed in 4% formaldehyde and stored as

a formalin-fixed paraffin-embedded (FFPE) block. Five 5-mm sec-tions were used for gene expression profiling as described below.Four blood samples were obtained between 7 and 11 AM. The firstwas taken before medication initiation (T1); the second and thirdwere taken on the mornings before and after surgery (T2 and T3,respectively), at least 1hourafter themorningmedicationdose; andthe fourth was taken at least 2 days after treatment cessation (T4;median of 16 days postmedication; Fig. 1B).

Gene expression profiling and bioinformatic analysisDetailed methods and references for gene expression profiling

and bioinformatic analysis are presented in the SupplementaryMethods. Briefly, RNAwas extracted from five 5-mmFFPE sections

Translational Relevance

The clinical trial reported here supports the safety andefficacy of pharmacologically inhibiting b-adrenergic andCOX-2pathways during the perioperative period in early-stagebreast cancer. Preclinical studies have shown that simulta-neous blockade of these two pathways improves long-termsurvival rates in several models of primary tumor excision.Moreover, this treatment is inexpensive and clinically feasiblefor patients without contraindications for the medicationsused (�50% of patients). Given the positive biomarker indi-cations reported in the present proof-of-concept trial, futurestudies assessing clinical impact on disease progress/recur-rence and overall survival are justified. This treatment mayalso be beneficial in a variety of cancer types and does notcontraindicate other cancer therapies. As such, brief perioper-ative inhibition of b-adrenergic and COX-2 signaling mayprovide a novel strategy for improving long-term canceroutcomes.

Shaashua et al.

Clin Cancer Res; 23(16) August 15, 2017 Clinical Cancer Research4652




Figure 1.

A, CONSORT diagram of clinical trial enrollment and treatment. B, Schematic presentation of the design and time schedule of the study. A double-blindplacebo-controlled biomarker trial was conducted in early-stage breast cancer patients, treating patients with placebo or with propranolol and etodolac for 11consecutive days, starting 5 days before surgery. Propranolol doses were increased on the day of surgery. Of the 38 patients recruited, 1 from each groupself-withdraw before surgery. Blood samples were collected before drug initiation (T1), on the morning before surgery (T2), on the morning after surgery (T3), andseveral days after cessation of drug treatment (T4). Tumor tissue was collected during surgery.

Perioperative COX-2 and b-Adrenergic Blockade in Breast Cancer

www.aacrjournals.org Clin Cancer Res; 23(16) August 15, 2017 4653




of breast tumors, tested for sufficient mass, and subjected togenome-wide transcriptional profiling using Illumina HumanHT-12 v4 Expression BeadChips (Illumina Inc.) with quantilenormalization (30). Linear model analyses of log2-transformedexpression values quantified the difference in average expres-sion between groups (drug treatment vs. placebo) after con-trolling for tumor stage. A priori hypotheses regarding EMTpolarization and tumor-associated leukocyte transcriptomeswere tested using Transcript Origin Analyses to relate all genesshowing � 1.25-fold differential expression in this study topreviously published reference transcriptome profiles derivedfrom mesenchymal- versus epithelial-polarized breast cancercells (GSE13915) or isolated leukocyte subsets (GSE1133). Apriori hypotheses regarding activity of breast cancer-relevanttranscription control pathways were tested using TELiS bioin-formatic analysis of transcription factor binding motifs in thepromoters of all genes showing � 1.25-fold differential expres-sion, using TRANSFAC position-specific weight matrices forinflammation-related pathways (NF-kB/cRel, AP-1), GATAfamily factors GATA1-GATA3, cytokine response factors STAT1and STAT3, the oxidative stress response factor NRF-2, theneuroendocrine response factor GR, and EGR family transcrip-tion factors EGR1-EGR4/NGFIC, as previously described. Sta-tistical testing of bioinformatics results was based on standarderrors derived from bootstrap resampling of linear modelresidual vectors over all genes assayed (which accounts for anypotential correlation across genes).

Blood collection, ELISA, flow cytometry, and induced cytokineproduction

The Supplementary Methods detail the standard proceduresused to assess serum IL6, C-reactive protein (CRP), IL10, andcortisol; ex-vivo lipopolysaccharide- (LPS) and phytohaemagglu-tinin- (PHA) stimulated production of IFNg and IL12; and flowcytometric analyses of NK-cell activation markers and leukocytesubset prevalence.

Statistical analysisAll analyses were two-sided and conducted based on a priori

hypotheses. Our primary hypothesis was that drug treatmentwould reduce three progression-related transcriptome profilesin malignant tissue (EMT, tumor-associated monocyte/macro-phage transcriptomes, and proinflammatory/prometastatictranscription factors). Our secondary hypothesis was that drugtreatment would shift circulating immune parameters towardlower inflammatory and higher antimetastatic immunity asindicated by serum and ex-vivo–stimulated cytokine levels, NK-cellactivation markers, and circulating "classical" (CD14þþCD16�)monocytes.

For tumor transcriptome analyses, the statistical significance ofbioinformatic result ratios (Drugs/Placebo) was tested by theStudent t test. For blood-measures analyses, a planned contrastwas used to compare the impact of drug treatment (average ofT2 and T3) with untreated levels (average of T1 and T4) (i.e.,Drugs [(T2þT3)� (T1þT4)]�Placebo [(T2þT3)� (T1þT4)]),and post-hoc comparisons were performed to assess group differ-ences at specific time points. For serum cytokine levels and geneexpression assessments, data were log transformed to stabilizevariance. Blood sample data during treatment were expressed as apercentage of the average value at no-treatment time points (i.e.,average of T1 and T4).

ResultsDemographics, adverse events, and drug compliance

The two groups did not differ on any demographic or cancer-related characteristic assessed (Tables 1 and 2). Two patientsreported physical discomfort within the first 2 days of treatment(before hospitalization): one placebo-treated patient reportedanxiety and showed increased heart rate and blood pressure; asecond drug-treated patient reported nausea. Both self-withdrewwithout further medical examination, and no additional sampleswere collected from these patients. The other 36 women reportedno adverse events and consumed at least 95%of theirmedication/placebo doses.

Tumor gene expressionGenome-wide transcriptional profiling of tumor tissues iden-

tified 163 genes showing >1.25-fold upregulation in tumorsfrom drug-treated patients versus placebo-treated controls, and141 genes were equivalently down regulated. A priori–specifiedbioinformatic analyses, using previously published mesenchy-mal and epithelial breast cancer cell transcriptomes as referencepoints, showed that drug treatment reduced the extent ofmesenchymal polarization (diagnosticity z-score: mean ¼–0.43 � SE 0.09, P < 0.0001) but had no significant effect onepithelial-characteristic gene expression (þ0.13 � 0.11, P ¼0.106; Fig. 2A). Similar a priori–specified tumor transcriptomeanalyses using isolated leukocyte subpopulations as referencepoints (Fig. 2B) indicated that drug treatment reduced expres-sion of CD14þ monocyte-related transcripts (–0.42 � 0.15, P ¼0.0036) and increased expression of genes characteristic ofCD19þ B cells (þ0.45 � 0.17, P ¼ 0.0033). We also tested apriori hypotheses regarding specific transcription control path-ways that are linked to prometastatic processes of inflamma-tion, tissue invasion, and EMT. Promoter-based bioinformaticanalyses (Fig. 2C) indicated downregulated activity of GATA-1(log2 fold difference in promoter binding site prevalence:mean ¼ –0.48 � 0.10, P < 0.0001), GATA-2 (–0.40 � 10,P ¼ 0.0001), STAT3 (–1.61 � 0.66, P ¼ 0.0154), EGR-3 (–0.70� 0.35, P ¼ 0.048), and GRE (–0.85 � 0.42, P ¼ 0.043) intumors from drug-treated patients.

Serum levels of soluble factorsAs expected, given that psychologic and physiologic stress

responses intensify in the lead-up to surgery (31), serum levelsof IL6 increased by 24% � 12.1% from T1 to T2 in the placebo-treated group and CRP levels similarly increased by 41.5% �20.5% (Fig. 3A and B). However, this pattern was significantlyreversed in the drug-treated group (11.3%� 5.5% decline for IL6,P ¼ 0.0009; 10% � 10.7% decline for CRP, P ¼ 0.034). On themorning after surgery (T3), both placebo- and drug-treatedgroups showed increases in IL6 and CRP above presurgical levels(IL6: þ573% � 97% and þ442% � 70% for placebo- and drug-treated groups, respectively; CRP:þ828%� 285% andþ635%�281%; all P < 0.001). A planned contrast of drug- versus placebo-treated groups during treatment (average of T2 and T3) versus offtreatment (averageof T1 andT4) showed a significant reduction inIL6 for the drug-treated group (P ¼ 0.011), and a marginallysignificant reduction in CRP. Drug treatment did not significantlyaffect serum cortisol or IL10 concentrations at any time point(Fig. 3C and D).

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Immune indices in blood samplesIn the placebo-treated group, ex-vivo LPS- and PHA-stimu-

lated production of IL12 and IFNg decreased progressively fromT1 to T3 (IL12: –38% � 10%; IFNg : –30.2% � 9.1%; both P <0.0001), as previously reported (31). Drug treatment blockedthis decrease, resulting in higher levels of these cytokines at T2and T3 (i.e., for the planned contrast described above, IL12:þ50.8% � 15.2%, P ¼ 0.028; IFNg : þ31% � 8.6%, P ¼0.024; Fig. 4A and B).

Drug treatment also blocked an influx of CD14þþCD16�

classical monocytes into circulation on the morning after surgery(T3; difference ¼ 85% � 15%, P¼ 0.032; Fig. 4C), and increasedexpression of the activation marker CD11a on NK cells(CD3�CD56þCD16þ) during treatment (average of T2 and T3)versus off treatment (average of T1 and T4; difference ¼þ16% �6.3%, P ¼ 0.024; Fig. 4D).

DiscussionThese data show that perioperative administration of the

b-adrenergic antagonist propranolol and the COX-2 inhibitoretodolac inducesmultiple favorable impacts on (i) primary tumorgene expression profiles (bioinformatic indications of reducedEMT; reduced activity ofGATA-1, GATA-2, EGR3,GRE, and STAT3transcription factors; and reduced tumor-associated monocytesand increased tumor-associated B cells) and on (ii) circulatingimmune parameters (serum and ex-vivo–induced cytokine levels,reduced classical monocyte influx, and increased NK-cell activa-tion markers). Each of these outcomes has previously beenlinked to reduced tumor progression in preclinical animalmodelsand/or human clinical studies. This study was designed solely as arandomized controlled test of perioperative propranolol andetodolac effects on biomarker outcomes, and involved no

Table 1. Baseline patient demographic and clinical characteristics (36 patients providing blood samples)

Control group (n ¼ 18) Treatment group (n ¼ 18) P

Age 55.2 (33–70) 55.3 (41–70) 0.97Mean (min–max)

BMI 25.7 (20.3–32.0) 26.3 (19.4–36.5) 0.73Mean (min–max)

Weight mean (min, max) 68.1 (52–86) 69.5 (50–103) 0.75

Smoking—present No 15 No 9 0.06Yes (<5 cigarettes per day) 1 Yes (<5 cigarettes per day) 2Yes (>5 cigarettes per day) 1 Yes (>5 cigarettes per day) 6NA 1 NA 1

T staging Tis 2 Tis 0 0.33T1 9 T1 13T2 5 T2 3T3 0 T3 0NA 2 NA 2

Histologic grade HG1 5 HG1 3 0.74(HG) HG2 6 HG2 10

HG2/3 1 HG2/3 1HG3 2 HG3 1DCIS/LCIS 4 DCIS/LCIS 3

Surgical resection Lumpectomy 13 Lumpectomy 15 0.56Mastectomy 2 Mastectomy 2Othera,b,c 3 Otherd 1

Metastatic spread No 18 No 16 0.34NA 0 NA 1

Axillary metastasis 1

ER status Negative 2 Negative 1 0.83Positive 16 Positive 17

PR status Negative 6 Negative 5 0.93Positive 12 Positive 13

HER2/neu status Negative 9 Negative 8 0.76NA 4 NA 3Positive 5 Positive 7

Tumor max. diameter 1.6 cm 1 cm 0.11

Carcinoma Invasive 13 Invasive 15 0.56Noninvasive 3 Noninvasive 1NA 2 NA 2

Abbreviations: DCIS, ductal carcinoma in situ; LCIS, lobular carcinoma in situ; NA, not available.aMastectomy þimmediate reconstruction with silicone.bLumpectomy (double- Lt&Rt).cLumpectomy (þIntraoperative radiation).dMastectomy with axillary sentinel lymph node excision.






long-term assessment of clinical outcomes. However, the favor-able safety profile and favorable impacts on tumor transcriptomeprofiles and immune parameters provide a rationale for futureclinical trials employing more robust sample sizes and long-termfollow-up to assess impacts of perioperative COX-2 and b-adren-ergic inhibition on clinical outcomes in early-stage breast cancer.

Tumor molecular characteristicsDrug treatment reduced tumor molecular biomarkers of EMT.

This finding corresponds well with animal studies indicating thatCOX-2 inhibitors and b-adrenergic antagonists can inhibit EMT inhuman tumor xenografts (5, 32). In metastatic breast cancer, amesenchymal phenotype is more prevalent in circulating tumorcells than in the primary tumor (33), suggesting the clinicalsignificance of EMT for metastasis. Moreover, the EMT profile ofthe primary tumor predicts long-term cancer outcomes, includingoverall survival (34), in several cancer types.

Drug treatment also decreased intra-tumoral molecular indi-cators of several pro-metastatic transcription control pathways,including GATA-1, GATA-2, and EGR3. GATA-1 exerts antiapop-

totic activity (35) and promotes EMT by downregulating E-cad-herin (5), and GATA-2 can inhibit the tumor-suppressor gene,phosphatase and tensin homolog (PTEN; ref. 36). Both factorspromote breast cancer development and progression (37). EGR3is induced by estrogen signaling (38) and has been linked tolymph node status, metastatic spread, and poor prognosis (39).Drug treatment also reduced indicated activity of the GR, whichcan enhance tumor cell survival by inducing Bcl-xL (40), inducingantiapoptotic signaling (41, 42), suppressing p53 activity (43),and inducing chemoresistance (8).

Inflammatory indicators in the tumor and circulationPrevious studies have reported that psychologic and surgery-

related sympathetic nervous system stress responses can elevatelevels of proinflammatory ligands, including IL6 and CRP (44).In the current study, both IL6 and CRP levels increased beforesurgery in the placebo group (from T1 to T2), but drug treat-ment reversed this effect and reduced levels of both inflamma-tory indicators at T2. IL6 and CRP are associated with tumorprogression and poor prognosis in multiple solid tumor types,

Table 2. Baseline patient demographic and clinical characteristics for transcriptome profilinga

Control group (n ¼ 15) Treatment group (n ¼ 10) P

Age 57 (43–70) 57.3 (46–70) 0.93Mean (min–max)

BMI mean (min–max) 25.1 (20.3–32.0) 26.4 (19.4–34.5) 0.48

Weight 66.1 (52–86) 67.7 (50–94) 0.76

Mean (min–max)

Smoking—present No 13 No 5 0.08Yes (<5 cigarettes per day) 1 Yes (<5 cigarettes per day) 2Yes (>5 cigarettes per day) 0 Yes (>5 cigarettes per day) 2NA 1 NA 1

T Staging Tis 2 Tis 0 0.40T1 8 T1 9T2 4 T2 1T3 1 T3 0

Histologic grade HG1 5 HG1 2 0.79(HG) HG2 6 HG2 6

HG2/3 1 HG2/3 0HG3 1 HG3 1DCIS/LCIS 2 DCIS/LCIS 1

Surgical resection Lumpectomy 11 Lumpectomy 9 0.31Mastectomy 1 Mastectomy 1Otherb,c,d 3 Other 0

Metastatic spread No 15 No 10 1

ER status Negative 0 Negative 1 0.45Positive 15 Positive 9

PR status Negative 3 Negative 3 0.84Positive 12 Positive 7

HER2/neu status Negative 8 Negative 6 0.30NA 3 NA 0Positive 4 Positive 4

Tumor max. diameter 1.8 cm 1.2 cm 0.32

Carcinoma Invasive 12 Invasive 8 0.93Noninvasive 2 Noninvasive 1NA 1 NA 1

Abbreviations: DCIS, ductal carcinoma in situ; LCIS, lobular carcinoma in situ; NA, not available.aTwenty-five tumors yielded RNA quality assured for sufficient mas.bMastectomy þ immediate reconstruction with silicone.cLumpectomy (double- Lt&Rt).dLumpectomy (þIntraoperative radiation).

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including breast, lung, and prostate, and hematopoietic malig-nancies (37, 45). IL6 activates the janus-kinase-STAT signalingpathway, which is well known to promote tumor cell prolifer-ation, survival, and invasion, as well as immunosuppressionand inflammation. STAT3 and STAT5 are strongly associatedwith cancer progression (37). Here, both plasma IL6 levels andindicators of tumor STAT3 activity were reduced by the drugtreatment. Drug treatment also effectively blocked a markedpostoperative (T3) mobilization of "classical" proinflammatoryCD14þþCD16� monocytes. Thus, combined perioperativeb-blockade and COX-2 inhibition may inhibit stress-inducedinflammatory and metastatic processes through multiple cellu-lar and molecular pathways.

Immune status in the tumor and circulationTranscriptome profiling of the primary tumor also indicated

potential effects of the combined perioperative drug treatment inincreasing tumor-infiltrating B cells and decreasing tumor-asso-ciated monocyte/macrophages. Tumor-infiltrating B cells com-prise up to60%of the tumor-associated lymphocytes (46, 47) andpredict increased survival rates in breast cancer (47, 48). Mono-cyte recruitment by tumors was shown in several animal modelsto be enhanced by b-adrenergic signaling, and to promote cancerprogression (5). In human cancers, tumor-infiltratingmonocytes,which often transform into M2-macrophages, are correlated withdecreased survival inmany solid tumors including breast, thyroid,and bladder cancers (49, 50). Thus, the profile of immune cell

Figure 2.

Effect of drug treatment on primary tumor transcriptomeindicators of EMT, tumor-associated leukocytes, andprometastatic transcription factors. Twenty-five tumorsyielded RNA of sufficient quality for transcriptomeprofiling (10 drug-treated and 15 placebo). A, Effects ofdrug treatment on primary tumor EMT gene expressionwere quantified by Transcript Origin Analysis (58) of 163genes showing >1.25-fold upregulation and 141 genesshowing equivalent downregulation in tumors fromdrug-treated patients versus controls, using referencetranscriptome profiles derived from mesenchymal-versus epithelial-polarized breast cancer cells (59). B,Transcript origin analysis also assessed the effects ofdrug treatment on expression of genes derived frommonocytes, dendritic cells, CD4þ and CD8þ T cells, Bcells, and NK cells, using reference data derived fromisolated samples of each cell type (58). C, Effect of drugtreatment on transcription control pathways as indicatedby bioinformatics analysis of transcription factor–bindingmotifs in promoters of differentially expressed genes.Data are presented as mean � SEM. Group differencesare indicated by � , P < 0.05; �� , P < 0.01; or ��, P < 0.001.






alterations reflected in whole-tumor transcriptome profiling indi-cates favorable effects of this drug regimen on local immune cellmediators of disease progression.

Consistent with previous reports in animal models, (31) stressand surgery also decreased LPS- and PHA-induced production ofIFNg and IL12 by circulating leukocytes. However, this suppres-sion was abrogated by drug treatment. These Th1 cytokines areprominent activators of antitumor CTL andNK cells (2). The drugtreatment also increased expression of CD11a (lymphocyte func-tion-associated antigen-1; LFA-1) on circulating NK cells. Thismembrane glycoprotein is a marker of NK-cell activation andinteracts with intercellular adhesion molecule-1 (ICAM-1) andother tumor ligands to promote tumor lysis by NK cells (51). In apreclinical model, where a spontaneously metastasizing ortho-topic primary tumor was excised, surgery reduced CD11a expres-sion on NK cells and our perioperative drug treatment (propran-olol and etodolac) blocked that effect and improved long-termsurvival rates (20).

Perioperative significance of the treatment and safetyconcerns

Combined administration of propranolol and etodolac hadfavorable impacts on multiple biomarkers assessed before,during, and following surgery. Given that both catecholamines

and prostaglandins are abundant throughout the perioperativeperiod, and that micrometastases and residual disease mayexist before and following surgery, these data suggest thattreatment throughout the entire perioperative period isoptimal.

The safety of this drug regimen is discussed in greater detailin the Supplementary Methods. Briefly, for patients withoutcontraindications, the safety profiles of propranolol and eto-dolac are well established, especially for the short durationemployed here (52, 53). Concerns and variable findings havebeen reported regarding the perioperative use of b1-selectiveantagonists (54), but no evidence indicates a risk associatedwith use of nonselective b-adrenergic antagonists such as pro-pranolol. Tissue healing was shown in animal studies not to beadversely affected by either drug or by their combined use (55).In the current study and in a previous study in cancer patients(56), we observed no serious or moderate adverse eventsassociated with this treatment regimen. This favorable safetyprofile is important in evaluating the overall cost–benefit ratiofor the present drug regimen, especially as both chronic andperioperative uses of COX inhibitors or b-adrenergic blockershave been associated with improved cancer outcomes (4, 7,57). Thus, any hypothetical long-term risk associated with thecombined use of propranolol and etodolac appears empirically

Figure 3.

Effect of drug treatment on circulating levels of IL6, CRP, IL10, and cortisol levels (n¼ 18 per group). Serum levels of IL6 (A), C-reactive protein (CRP) (B), IL10 (C),and cortisol (D) were assessed by commercial enzyme-linked immunosorbent assay (high-sensitivity ELISA kits for IL6 and IL10). Data represent mean � SEM.Group differences at a specific time point are indicated by � , P < 0.05; �� , P < 0.001. A significant contrast between drug and placebo conditions duringtreatment (T2 þ T3) vs. off treatment (T1 þ T4) is indicated by #.

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unlikely and should be weighed against the positive outcomesreported by translational, epidemiologic, and clinical studies,as well as by the favorable profile of molecular and cellularbiomarkers observed in this trial.

LimitationsThis study was designed solely as a randomized controlled

proof-of-concept test of perioperative propranolol and etodolaceffects on metastasis-related biomarkers in early-stage breastcancer. This study was powered only to detect those biomarkeroutcomes (based on effect sizes previously observed in preclinicalstudies), and it provides no information about long-term clinicaloutcomes (e.g., effects on relapse-free or overall survival). Thegenerality of these results also needs to be examined in futurestudies beyond the present single-nation context, and possiblyexamining alternative treatment durations and regimens (includ-ing the use of each agent alone in addition to combined use) andselective targeting of high-risk disease settings (e.g., ER�/PR�/her2� breast cancer) and other cancer types. It is also important tonote that the biomarkers examined in this trial were selected apriori as outcomes (based on previous clinical and preclinical

research) and are not intended to provide a prognostic biomarkerfor clinical disease progression.

SummaryData from this first clinical trial of perioperative treatment with

a COX-2 inhibitor and a b-adrenergic antagonist in early-stagebreast cancer find a favorable safety profile and favorable impacton multiple tumor and circulating biomarkers associated withcancer progression and metastasis. These findings provide abiological rationale for future clinical trials to assess the impactof this easily implemented, safe, and inexpensive treatmentregimen on long-term clinical outcomes (e.g., overall and recur-rence-free survival).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Shaashua, R. Haldar, O. Zmora, M. Horowitz,S. Cole, S. Ben-Eliyahu

Figure 4.

Effect of drug treatment on ex vivo–stimulated production of IL12 and IFNg , on numbers of circulating CD16� monocytes, and on CD11a (LFA-1) expression levelson NK cells (n ¼ 18 per group). Venipuncture blood samples were assayed for induced cytokine levels following 21-hour LPS and PHA-stimulation, assessed inculture supernatant by ELISA (A and B); circulating frequency of CD14þþCD16� "classical" monocytes (C); and expression levels of the activation markerCD11a on NK cells (CD3�CD56þCD16þ lymphocytes), assessed by flow cytometry (D). Data represent mean � SEM. Group differences at a specific time point areindicated by � , P < 0.05. A significant contrast between drug and placebo treatment (T2 þ T3) vs. off treatment (T1 þ T4) is indicated by #. A significantdecrease from T1 to T3 within the placebo group is indicated by ¥.






Development of methodology: L. Shaashua, R. Haldar, P. Matzner, O. Zmora,L. Hayman, M. Horowitz, S. Cole, S. Ben-EliyahuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Shaashua, M. Shabat-Simon, R. Haldar, P. Matzner,O. Zmora,M. Shabtai, E. Sharon, T. Allweis, I. Barshack, J. Arevalo, J.Ma, S. Cole,S. Ben-EliyahuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Shaashua,M. Shabat-Simon, R.Haldar, P.Matzner,O. Zmora, I. Barshack, J. Arevalo, S. Cole, S. Ben-EliyahuWriting, review, and/or revision of the manuscript: L. Shaashua, R. Haldar,O. Zmora, I. Barshack, M. Horowitz, S. Cole, S. Ben-EliyahuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Shaashua, M. Shabat-Simon, R. Haldar,O. Zmora, E. Sharon, I. Barshack, J. Ma, S. Cole, S. Ben-EliyahuStudy supervision: M. Shabat-Simon, R. Haldar, O. Zmora, E. Sharon,S. Ben-EliyahuOther (Clinical PI): O. Zmora

AcknowledgmentsWe thank Ella Rosenne, Hagar Lavon, and Eli Elbaz for their devoted

technical work supporting this project, and for Dr. Patricia Ganz for her criticalevaluation of this article.

Grant SupportThis work was supported by the National Cancer Institute Network on

Biobehavioral Pathways in Cancer (grant number 13XS084; to S. Ben-Eliyahu);the Israel Science Foundation (grant1406/10; to S.Ben-Eliyahu); and theNationalInstitutesofHealth/National InstituteonAging (grant P30AG017265; toS.Cole).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 17, 2017; revised March 14, 2017; accepted May 3, 2017;published OnlineFirst May 10, 2017.

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2017;23:4651-4661. Published OnlineFirst May 10, 2017.Clin Cancer Res Lee Shaashua, Maytal Shabat-Simon, Rita Haldar, et al. Randomized TrialMetastatic Biomarkers in Breast Cancer Patients in a Phase-II

-Adrenergic Blockade ImprovesβPerioperative COX-2 and

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