targeting the subtypes of breast cancer: rethinking investigational drugs

1. Introduction

2. Luminal A and luminal B breast

cancer

3. New targeted therapies for the

treatment of HER2-positive

breast cancer

4. Ductal triple-negative breast

cancer

5. Conclusion

6. Expert opinion

Review

Targeting the subtypes of breastcancer: rethinking investigationaldrugsGiuseppe Curigliano†, Marzia Locatelli, Luca Fumagalli, Janaina Brollo,Elisabetta Munzone, Franco Nole, Carmen Criscitiello & Aron Goldhirsch†European Institute of Oncology, Division of Medical Oncology, Department of Medicine, Milan,

Italy

Introduction: The choice of adjuvant treatments for women with breast can-

cer is based on several features that take into account the heterogeneity of

the disease. Questions raised during the decision process include the follow-

ing: i) What leads to the use of endocrine therapy? ii) What leads to the use

of anti-HER2 therapy? iii) What justifies the use of chemotherapy?.

Areas covered: Choices of adjuvant treatment are based on parameters

defined by molecular characterization of breast cancer subtypes or by approx-

imations to this classification using traditional clinical--pathological features.

Clinicians should consider cases within the various distinct subpopulation

in order to properly select the most ‘personalized’ adjuvant therapeutic

approach. Sensitivity to chemotherapy and/or targeted agents in subtypes

of breast cancers are predictable based on gene pathway alterations and asso-

ciated gene products. This review covers several clinical data on several inves-

tigational agents for early-stage breast cancer molecular subtypes. We

selected from literature data prospective Phase I, II and III clinical trials of che-

motherapy (weekly or daily schedules), including indicators of activity and

toxicity and data on survival/mortality.

Expert opinion: The future of many investigational therapeutics in breast

cancer is linked to our ability to identify the most druggable target in

each subtype.

Keywords: breast cancer subtypes, early breast cancer, investigational drugs

Expert Opin. Investig. Drugs (2012) 21(2):191-204

1. Introduction

Molecular characterization tools allow the identification of multiple breast cancersubtypes [1-3]. Any subtype is characterized by specific molecular events and canbe also defined by a traditional immunohistochemical approach in breast cancersubtype approximations [4-7]. Molecular subtypes have different risk factors [8,9], nat-ural histories [10-12] and different sensitivity to systemic chemotherapy and targetedagents [13-16].

Clinical decisions in systemic adjuvant therapy for patients with early breast can-cer must address three distinct questions: i) what justifies the use of endocrine ther-apy, ii) what justifies the use of anti-HER2 therapy, and iii) what justifies the use ofchemotherapy? Integration of adjuvant investigational agents should address thesethree major questions.

The availability of next-generation human genomic sequencing tools and prog-ress in sequencing and bio-computational technologies will enable genome-wide investigation of somatic mutations in human breast cancers [17] at diagnosisand during their natural history. These studies have the potential to highlightunderlying mechanisms of metastasis and resistance to drugs. Genome-wide

10.1517/13543784.2012.651456 © 2012 Informa UK, Ltd. ISSN 1354-3784 191All rights reserved: reproduction in whole or in part not permitted

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sequencing studies will, therefore, identify two specifictypes of mutations: the ‘drivers,’ providing a survival- andproliferation-selective advantage, and the ‘passengers,’ neutralto the selection process [18-20]. It will be essential to identify allmolecular pathways that emphasize the heterogeneity andcomplexity of human breast cancer in order to explain mecha-nisms sustaining proliferation hallmarks of cancer and ‘drive’tumor progression and resistance to chemotherapy andtargeted agents.Disease segmentation in subtypes can offer insights to person-

alize treatment [21]. Subtypes defined by clinical--pathological cri-teria are similar to but not identical to intrinsic subtypes andrepresent a convenient approximation. The St. Gallen approachuses immunohistochemical definition of estrogen (ER) and pro-gesterone receptor (PgR), the detection of overexpression and/oramplification of the human epidermal growth factor receptor 2(HER2) oncogene, and Ki-67 labeling index, a marker of cellproliferation, as the means of identifying tumor subtypes [21].Surrogate definition of intrinsic subtype can identify i) luminalA breast cancer, ER and PgR positive with a low Ki-67;ii) luminal B/HER2 negative, ER and PgR positive with highKi-67; iii) luminal B/HER2 positive; iv) HER2 positive, withER and PgR absent and v) ductal triple negative, ER, PgR andHER2 negative. We will overview all new promising moleculesunder investigation in breast cancer subtypes (Figure 1).

2. Luminal A and luminal B breast cancer

Endocrine therapy is the standard of care for hormone receptor-positive breast cancer. Tamoxifen alone for postmenopausal, orovarian function suppression plus tamoxifen as reasonable in pre-menopausal, is the primary option for patient with luminal Abreast cancer [21]. A substantial proportion of patients, despitebeing ER and/or PR positive, are either primarily resistant to

hormone therapies or will develop hormone resistance duringthe course of their disease. Signaling through complex growthfactor receptor pathways, which activate the ER, is emerging asimportant causes of endocrine resistance.

Several new targeted agents in pipeline are actually in develop-ment for targeting signaling pathways in patients with luminal Bbreast cancer. Clinical clue mechanisms to understand resistanceto endocrine therapy can be related to loss of ER expression [22],decrease in ER level over time; gradual loss of E dependence [23]

and upregulation of several transcriptional pathways [24]. Inpatients with endocrine-responsive disease, a ‘real-time’ testingof tumor tissue for genotype sequencing could be ideal. Earlydrug response and development of acquired resistance shouldbe monitored by serial biopsy of the tumor or, noninvasively,by circulating tumor cell analysis [25]. Ongoing clinical trialswith signal transduction inhibitors in combination with hor-monalmanipulation as ameans to overcome endocrine resistancein patients with breast cancer will be reviewed.

2.1 EGFR pathwaySeveral early clinical trials have been conducted with theepidermal growth factor receptor (EGFR) tyrosine kinaseinhibitors (TKIs) gefitinib or erlotinib either alone or in com-bination with endocrine therapy. Results from the monother-apy Phase II studies with gefitinib in patients with advancedbreast cancer were all relatively disappointing [26-28]. Two otherPhase II studies explored the potential benefit for combiningeither gefitinib or erlotinib with an aromatase inhibitor in unse-lected patients with ER-positive advanced breast cancer withvery low clinical efficacy [29,30]. In the setting of neoadjuvanttherapy for ER-positive postmenopausal breast cancer, a ran-domized trial of anastrozole alone or in combination withgefitinib given for 3 months prior to surgery showed noimprovement in tumor response rate or antiproliferative effectas determined by Ki-67 [31]. On the other hand, a preoperativetrial of gefitinib versus gefitinib combined with anastrozole for4 -- 6 weeks prior to surgery in women with ER+ EGFR+ pri-mary breast cancer reported that combined treatment inducedthe greatest reduction in tumor cell proliferation [32]. A -double-blind placebo-controlled Phase II trial of tamoxifenwith or without gefitinib was conducted in 290 patients asfirst-line endocrine therapy in postmenopausal women withER-positive metastatic breast cancer (MBC) [33], with anincrease in progression-free survival (PFS) from 8.8 to10.9 months (hazard ratio 0.84, 95% confidence interval (CI)0.59 -- 1.18, p = 0.31) [33]. A second randomized trial ofgefitinib and anastrozole versus anastrozole alone in a similarfirst-line patient population of women with ER-positiveadvanced breast cancer reported a prolongation of PFSfrom a median of 8.2 months with anastrozole to 14.6 monthswith the combination (hazard ratio 0.55, 95% CI0.32 -- 0.94) [34]. A second randomized Phase II trial with thesame combination of gefitinib and anastrozole did not showany statistically significant benefit [35]. Table 1 summarizesmajor clinical trials with anti-EGFR-targeted agents.

Article highlights.

. Breast cancer is not a single disease. Genetic array toolscan define several subtypes.

. Specific biological processes and distinct gene pathwaysare associated with prognosis and sensitivity tochemotherapy and targeted agents in different subtypesof breast cancers.

. A primary challenge for future drug development inbreast cancer will be to distinguish genes and pathwaysthat ‘drive’ cancer proliferation (drivers) from genes andpathways that have no role in the development ofcancer (passengers).

. The identification of functional pathways that areenriched for mutated genes will select subpopulationof patients who will most likely be sensitive tobiology-driven targeted agents.

. The selection of driver pathways in resistant tumors willpermit to discover a biology-driven platform for newdrug development to overcome resistance.

This box summarizes key points contained in the article.

Targeting the subtypes of breast cancer: rethinking investigational drugs

192 Expert Opin. Investig. Drugs (2012) 21(2)

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A Phase II clinical trial of letrozole and the monoclonal anti-body trastuzumab in patients with ER+/HER2+ MBC demon-strated a clinical benefit rate (CBR; partial response and stabledisease) of 50% [36]. Subsequently, the randomized Phase IITAnDEM trial in patients with ER+/HER2+ MBC reporteda better PFS with the addition of trastuzumab over anastrozolealone (4.8 vs 2.4 months, p = 00.0016) [37]. Other trials haveconducted with lapatinib, a potent oral TKI of both EGFRand HER2. Lapatinib has been explored in combination withendocrine therapy within a Phase III trial of 1286 patientswith metastatic ER+ breast cancer who were randomized toreceive either letrozole alone or letrozole combined with lapati-nib [38]. In patients with known ER+/HER2+ breast cancer, theaddition of lapatinib to letrozole significantly reduced the riskof progression (hazard ratio 0.71, 95% CI 0.53 -- 0.96,p = 0.019), improving the median PFS form 3.0 months forletrozole to 8.2 months for the combination [38]. The doubletargeting of ER and HER2 may be effective in tumorswith endocrine resistance and/or established coexpression ofboth receptors; the promising strategy of co-blockade hasbeen delivered in the clinic with the recent approval of thecombination of lapatinib with letrozole in HER2-positiveMBC patients. Table 1 summarizes major clinical trials withanti-HER2 agents.

2.2 Phosphatidylinositol 3-kinase/AKT/mTOR

signaling pathwaysThe phosphoinositide-3 kinase (PI3K) pathway has beenidentified as an important target in breast cancer research.PI3K pathway is frequently aberrantly activated in breast can-cer with mutations occurring in up to one quarter ofendocrine-resistant breast cancer [39]. Several agents targetingthe PI3K pathway are currently under development includingmonoclonal antibodies, TKIs, PI3K inhibitors, Akt inhibi-tors, rapamycin analogs and mammalian target of rapamycin(mTOR) inhibitors. Their development is based on the strat-egy of co-blockade; multiple signaling inhibition is manda-tory since anti-mTOR agents and PI3K inhibitors mayresult in the activation of compensatory feedback loops thatwould result in reduced activity.

2.2.1 mTOR inhibitorsThe first agents against the pathway that were studied in theclinic were rapamycin analogs. Clinical data suggest thatmTOR inhibition may play a role in the therapy ofendocrine-resistant breast cancer. In the neoadjuvant setting,a randomized Phase II study patients with early ER-positivebreast cancer were randomized to receive either letrozoleplus placebo for 16 weeks or letrozole plus daily everolimus

Anti-IGR-1R Ab and

TKIs

Anti-EGFR

Dasatinib

PI3K and mTORInhibitors

T-DM1, pertuzumab

Neratinib, lapatinib

Olaparib

Neratinib

Veliparib

Luminal A

Luminal B

HER2-enriched

Basal-like

UB

E2C

PT

TG

1M

YB

L2C

CN

B1

BIR

C5

HS

PC

150T

YM

S

KN

TC

2C

EP

55C

DC

6R

RM

2O

RC

6LA

NLN

KIF

2CE

XO

1C

EN

PF

CD

CA

1C

DC

20M

KI67

CC

NE

1G

RB

7T

ME

M45B

ER

BB

2B

LVR

AG

PR

160F

OX

A1

MM

P11

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XC

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6P

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T14

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L2P

HG

DH

CD

H3

EG

FR

FG

FR

4M

DM

2M

LPH

MA

PT

ME

LK

Normal-like

Figure 1. Molecular subtype of breast cancer and new potential agents to be explored in the neoadjuvant setting.EGFR: Epidermal growth factor receptor; mTOR: Mammalian target of rapamycin; PI3K: Phosphoinositide-3 kinase; TKI: Tyrosine kinase inhibitor.

Curigliano, Locatelli, Fumagalli, et al.

Expert Opin. Investig. Drugs (2012) 21(2) 193

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(RAD001), a rapamycin analog. The primary endpoint of thetrial was response rate to the combination therapy [40].Response rate by clinical palpation in the everolimus armwas higher than that with letrozole alone (i.e., placebo;68.1 vs 59.1%). An antiproliferative response, as defined bya reduction in Ki-67 expression to natural logarithm ofpercentage positive Ki-67 of less than 1 at day 15, occurredin 52 (57%) of 91 patients in the everolimus arm and in25 (30%) of 82 patients in the placebo arm (p <. 01) [40]. Inanother trial in the metastatic setting, patients (n = 109)were randomly assigned to receive 75 or 250 mg of temsiroli-mus (monoclonal antibody against mTOR) weekly [41].Patients were evaluated for tumor response, time to tumorprogression, adverse events (AEs) and pharmacokinetics oftemsirolimus. Temsirolimus produced an objective responserate of 9.2% (10 partial responses) in the intent-to-treat pop-ulation. Median time to tumor progression was 12.0 weeks.Efficacy was similar for both dose levels but toxicity wasmore common with the higher dose level [41]. Ridaforolimus(MK-8669) in combination to exemestane is actually underinvestigation in early clinical trials for ER-positive breastcancer progressing to nonsteroidal aromatase inhibitors.

2.2.2 PI3K inhibitorsPan-PI3K inhibitors include GDC-0941 (Genentech, Inc.),which is under investigation in a Phase I -- II clinical trial,in combination with endocrine therapy and mTOR inhibi-tors. The XL147 agent is a potent inhibitor of the class IPI3K family. A Phase I/II randomized study of letrozole andXL147 versus letrozole and XL765 (Exelixis/Sanofi-Aventis),a dual mTOR and PI3K inhibitors, is also planned.

2.3 Histone deacetylaseThe use of demethylating agents or histone deacetylase(HDAC) inhibitors can reactivate expression of a functionalER in cell lines in which ER silencing exists because of pro-moter methylation [42]. HDACs are crucial components ofthe ER transcriptional complex. Preclinically, HDACinhibitors can reverse tamoxifen/aromatase inhibitor resis-tance in hormone receptor-positive breast cancer [42]. In aPhase II trial, patients with ER-positive MBC progressingon endocrine therapy were treated with 400 mg of vorino-stat (Merck & Co., Inc., White House Station, New Jersey)daily for 3 of 4 weeks and 20 mg/day tamoxifen, continu-ously. The objective response rate was 19% and the CBRwas 40%. The median response duration was 10.3 months(CI: 8.1 -- 12.4) [43].

2.4 Insulin-like growth factor receptorInsulin-like growth factor-1 receptor (IGF-1R) is a homodi-meric receptor tyrosine kinase activated by IGF I/II ligand bind-ing, which results in tumor growth and apoptosis blockade [44].IGF-1R antagonists have been shown to interact with both ERpathways. This cross talk between ER suggests that IGF-1Rmay be an attractive treatment target especially for the ‘luminalT

able

1.Endocrinetherapiesco

mbinedwithbiologicaltargetedagents

inestrogenreceptor(ER)-positivebreast

cancer.

Clinicalsetting

Trialphase

No.ofpatients

Intervention

Clinicalendpoints

Ref.

Metastaticbreast

cancer

Phase

IIn=15

Anastrozole

andgefitinib

Response

rate

Noresponse

Nostable

disease

MitaM

etal.

[29]

Phase

IIn=150

Letrozole

andgefitinib

Clinicalbenefit11/20patients

MayerIetal.

[30]

Earlybreast

cancer

Phase

IIrandomized

n=206

Anastrozole

vsgefitinib

plus

anastrozole

Response

rate

61%

anastrozole

vs45%

(combinationarm

)p=0.067

Smithetal.

[31]

Metastaticbreast

cancer

Phase

IIIrandomized

n=207

Anastrozole

vstrastuzumabplus

anastrozole

PFS

=2.4

(anastrozole)vs

4.8

months

(anastrozole

plustrastuzumab)p=0.0016

Kaufm

anBetal.

[37]

Metastaticbreast

cancer

Phase

IIIrandomized

n=219

Letrozole

vsletrozole

pluslapatinib

PFS

=3.0

(letrozole)vs

8.2

months(letrozole

pluslapatinib)

JohnsonSetal.

[38]

Metastaticbreast

cancer

Randomizedphase

IIn=150

Tamoxifenvs

tamoxifenplusgefitinib

PFS

=8.8

(tamoxifen)vs

10.9

months

(tamoxifenplusgefitinib)

OsborneCK

[33]

Randomizedphase

IIn=206

Anastrozole

plusgefitinib

vsanastrozole

PFS

=14.6

(anastrozole

plusgefitinib)vs

8.2

months(anastrozole)

CristofanilliM

[34]



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B’ breast cancers. This is supported by in vitro experimentsshowing a synergistic effect when co-targeting the IGF-1Rreceptor along with antiestrogen agent [45]. Moreover, growthof tamoxifen-resistant MCF-7 cells declines when anti-IGF-1Rantibody is added to the cells [46]. Several monoclonal antibodiesand TKIs are in early clinical development in the treatment ofbreast cancer. Phase II randomized trials are currently ongoingfor patients progressing to nonsteroidal aromatase inhibitorsand randomized to exemestane versus exemestane with figitu-mumab (Pfizer, Inc.), a fully humanized anti-IGF-1R antibody.In a recent randomized Phase II study, the investigational agentAMG 479 (Amgen, Inc.), a fully human monoclonal antibodyagainst the IGF-1R, failed to revert resistance to hormonal ther-apy in patients with endocrine therapy-resistant, ER-positiveMBC [47]. Indeed, the drug showed a trend toward worse PFSand objective response in a Phase II trial [47]. When AMG479 was paired with exemestane or fulvestrant, patients in theexperimental arm had a median PFS of 3.9 months, comparedwith 5.7 months for patients on exemestane or fulvestrant alone(hazard ratio, 1.17; p =. 435). In this study, AMG 479 in com-bination with either fulvestrant or exemestane does not appear todelay or reverse resistance to hormonal therapy in this popula-tion of patients with prior endocrine therapy-resistant hormonereceptor-positive MBC. Other trials are currently ongoing inhormone-resistant breast cancer patents using TKIs targetingthe IGF-1R pathway.

2.5 Src family tyrosine kinaseSrc is specifically involved in coordinating signaling from the ste-roid receptors, including the ER and androgen receptor (AR).Multiple studies have shown cross talk between ER/AR andSrc, with ER/AR activation leading to activation of Src, and sub-sequent Src-mediated cell proliferation [48]. Blocking the interac-tion between ER/AR and Src leads to inhibition of downstreamcellular pathways and cessation of cell growth [48]. Several studieshave shown associations between resistance to endocrine therapyand both increased levels of Src activity and an increasingly inva-sive and aggressive tumor phenotype [49,50]. Given this data, spe-cifically targeting Src may overcome endocrine resistance inhormonally driven cancers. Several inhibitors of Src have beendeveloped. One of the best studied is dasatinib (Sprycel,BMS354825; Bristol-Myers Squibb Oncology). Dasatinib is apotent oral small-molecule inhibitor of the Src tyrosine kinase.Another agent, bosutinib (SKI-606, Wyeth), is an oral dual-selective competitive inhibitor of both Src (IC50 = 1.0 nmol/l)and Abl tyrosine kinases, with moderate inhibition of the Axltyrosine kinase, Eph receptors and Ste20 family kinases [51].Mul-tiple other agents with activity against Src, including saracatinib(AZD0530; AstraZeneca) and XL999 (Exelixis), are in preclini-cal or early-phase clinical development. A Phase II monotherapystudy was open to patients with both ER-positive and/or HER2-positive disease. Of the response-evaluable population from bothsubtypes, a response rate of 4% was seen, with a CBR of 8% inthe HER2+ cohort, and 16% in the ER+ cohort. Interestingly,all benefit was seen in patients with ER+ tumors [52]. Another

Phase II randomized trial was designed for patients withER-positive MBC progressing to nonsteroidal aromatase inhibi-tors. Patients are randomized to exemestane plus dasatinib versusexemestane plus placebo. The accrual to this trial has just beencompleted. Progesterone receptors also interact with c-Src. PRcontains an SH3 domain interaction motif in the N-terminusthat mediates interaction with Src tyrosine kinases and othersignaling molecules. This interaction mediates rapid progester-one activation of Src/MAP K signaling pathways and defines amolecularmechanism for some of the rapid non-genomic actionsof progesterone. Interactions of PR with c-Src or proteinkinases downstream of c-Src are required for breast cancer cellproliferation [53].

3. New targeted therapies for the treatmentof HER2-positive breast cancer

Many breast cancer patients with HER2 overexpression donot respond to initial therapy with trastuzumab (Herceptin,Roche), and a vast majority of these develop resistance tothis monoclonal antibody. Several molecular mechanismsleading to the development of trastuzumab resistance havebeen described, including circulating HER2 extracellulardomain [54], loss of PTEN [55], activation of alternative path-ways (e.g., IGFR) [56], receptor--antibody interaction block [57]

or innate modulation of the immunological response [58].Identification of upregulated pathways may lead to develop-ment on new therapeutic targets that potentially overcomeresistance to trastuzumab. Several agents are currently underdevelopment to overcome trastuzumab resistance.

3.1 Trastuzumab-DM1Genentech, Inc., in collaboration with Roche, has recentlydeveloped trastuzumab-DM1 (a maytansine conjugated to tras-tuzumab, RG-3502, T-DM1), which is active on HER2 overex-pressing breast cancer and also on trastuzumab-refractorytumors. Maytansinoids are very potent anticancer agents origi-nally isolated from plant families: Celastraceae, Rhamnaceaeand Euphorbiaceae and later from microorganism producingantibiotics (Actinosynnema pretiosum) [59,60]. They are 19-mem-bered microcyclic lactams related to amsamycin. The maytansi-noid DM1 is 100- to 1000-fold more potent that anticanceragents in clinical use [59,60]. The maytansine DM1 bindsto microtubules in a manner similar to Vinca alkaloids, but is20- to 100-fold more potent than vincristine in blockingmitosis [59]. Therefore, the maytansinoid DM1 was conjugatedto the humanized HER2 antibody trastuzumab (Tmab, whichis a protein) using --S-S- (disulfide)-containing linkers(Tmab-SPDT-DM1, Tmab-SPP-DM1, Tmab-SSNPPDM1,Tmab-SSNPP-DM4) (Figure 2).

The first-in-human Phase I, multicenter, open-label, dose-escalation study of single-agent T-DM1 in patients withHER2-positive MBC, who had previously received atrastuzumab-containing chemotherapy regimen, demonstratedthat, at the maximum-tolerated dose (MTD) of 3.6 mg/kg every



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3 weeks, T-DM1 was safe and had considerable clinicalactivity. The CBR (RR plus stable disease (SD) at 6 months)among 15 patients treated at MTD was 73%, including fiveobjective responses [61]. Phase II studies of T-DM1 in patientswith HER2-positive MBC who progressed while receivingHER2--directed therapy (trastuzumab or lapatinib), or whowere previously treated with several lines of chemotherapy,have demonstrated an objective response rate, by independentassessment, of 25.9% (95% CI 18.4 -- 34.4%). Median dura-tion of response was not reached as a result of insufficient events(lower limit of 95% CI 6.2 months), and median PFS time was4.6 months (95% CI 3.9 -- 8.6 months) [62]. Several randomizedclinical trials are actually ongoing in metastatic HER2-positivebreast cancer patients. An open-label, Phase III trial (EMILIA)will compare the safety and efficacy of T-DM1 with that ofcapecitabine in combination with lapatinib in patients withHER2-positive MBC previously treated with a trastuzumab-based therapy [63]. Another first-line trial (MARIANNE) is cur-rently ongoing for the treatment of MBC [64]. This randomized,three-arm, multicenter study will evaluate the efficacy and safetyof trastuzumab-DM1 with pertuzumab or T-DM1 with pertu-zumab--placebo, versus the combination of trastuzumab plustaxane (docetaxel or paclitaxel) in patients with HER2-positiveprogressive or recurrent locally advanced or previously untreatedMBC. Patients will be randomized to one of three treatmentarms (arms A, B or C). Arm A will be open-label, whereasarms B and C will be blinded [64].

3.2 PertuzumabPertuzumab is a novel recombinant humanized monoclonalantibody directed against the highly conserved dimerizationdomain of HER2, and as such, it inhibits HER2 homo- andheterodimerization. Pertuzumab-mediated blockage of HER2dimerization inhibits HER family downstream signaling (i.e.,the Akt cell survival pathway and the mitogen-activated proteinkinase pathway) [65]. In a Phase II randomized trial

investigating the efficacy and safety of pertuzumab in patientswith HER2-positive MBC, the only measurable therapeuticbenefit observed was a stable disease of a relatively short dura-tion [66]. The idea that the combination of pertuzumab andtrastuzumab might be a clinically meaningful therapy inMBC came from the single-arm Phase II trial of trastuzumabplus pertuzumab, which demonstrated that the combinationwas well tolerated and active in patients with HER2-positiveMBC who had progressed during trastuzumab therapy [67]. Inthis trial, the objective response rate was 24.2%, and theCBR was 50%. Cardiac dysfunction was minimal, and nopatients withdrew as a result of cardiac-related AEs. Recentlythe combination of pertuzumab and trastuzumab has beentested in patients with HER2-positive first diagnosed earlybreast cancer. The NEOSPHERE study (Neoadjuvant Studyof Pertuzumab and Herceptin in an Early Regimen Evaluation)is a randomized multicenter Phase III study, which was con-ducted in 417 women with newly diagnosed HER2-positiveearly, inflammatory or locally advanced breast cancer whonever received trastuzumab. Prior to surgery (neoadjuvant treat-ment), these women were randomized to four study arms. Theprimary endpoint was pathological complete response (pCR)and the results were i) Arm A: pCR of 29% for trastuzumaband docetaxel; ii) arm B: pCR of 45.8% for trastuzumab, per-tuzumab and docetaxel; iii) arm C: pCR of 16.8% for trastuzu-mab and pertuzumab; and iv) arm D: pCR of 24 % forpertuzumab and docetaxel [68]. The findings of the NEO-SPHERE study suggested that this new approach was effectivefor early HER2-positive breast cancer and suggest a potentialapplication of the double targeting approach.

3.3 LapatinibIntegration of lapatinib in early breast cancer has been investi-gated in the Neoadjuvant Lapatinib and/or Trastuzumab Treat-ment Optimisation Study (Neo-ALTTO study). This is arandomized, open-label multicenter Phase III study comparingthe efficacy of neoadjuvant lapatinib plus paclitaxel, versus trastu-zumab plus paclitaxel, versus concomitant lapatinib and trastuzu-mab plus paclitaxel given as neoadjuvant treatment in HER2/ErbB2 overexpressing and/or amplified primary breast cancer.Patients have been randomized to receive either lapatinib1500 mg/day, trastuzumab 4 mg/kg i.v. load followed by2 mg/kg i.v. weekly or lapatinib 1000 mg/day with trastuzumab4 mg/kg i.v. load followed by 2 mg/kg i.v. weekly for a total of6 weeks. After this biological window, patients on monotherapyarms continued on the same targeted therapy plus weekly pacli-taxel 80 mg/m2 for a further 12 weeks, up to definitive surgery.In the combination arm, patients received lapatinib 750 mg/day in combination with trastuzumab 2 mg/kg i.v. plus weeklypaclitaxel 80 mg/m2 i.v. for a further 12 weeks, up to definitivesurgery. After surgery, patients received three courses of adjuvantchemotherapy with fluorouracil, epirubicin and cyclophospha-mide followed by the same targeted therapy as in the biologicalwindow of the neoadjuvant setting for a further 34 weeks (inthe combination arm, lapatinib dose will be 1000 mg/day in

Trastuzumab

DM1MCC

Figure 2. T-DM1: maytansinoid DM1 was conjugated to the

humanized HER2 antibody trastuzumab.



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combination with trastuzumab). The planned total duration ofthe anti-HER2 therapy was 1 year. Results of this study havebeen presented during the SanAntonio 2010Breast Cancer Sym-posium [69]. In this study 455 patients have been randomized.The primary endpoint of pCR at the time of surgery in theITT population was statistically significantly higher in subjectsreceiving lapatinib plus trastuzumab with paclitaxel comparedwith those receiving trastuzumab with paclitaxel. The pCR inthe lapatinib plus trastuzumab group was 51.32% comparedwith 29.53% in the trastuzumab group; unstratified binomialp-value was p < 0.0001. No difference in pCR was observedbetween lapatinib with paclitaxel and trastuzumabwith paclitaxel(24.68 vs 29.53%). When adjusting for the stratification factors(tumor size, clinical nodal status, ER/PgR status, candidate forconservative surgery), the pCR remained higher in the lapatinibplus trastuzumab group compared with trastuzumab. The ORwas 2.62 (97.5% CI 1.50, 4.58; stratified log rank p < 0.001).When considering the secondary endpoint of locoregional pCR(extended definition of pCR including regional lymph nodestatus of pN0), the rate was also higher in the lapatinib plus tras-tuzumab group compared with trastuzumab (43.66 vs 26.53%).No difference in locoregional pCR was observed between lapati-nib and trastuzumab (18.12 vs 26.53%). In the ITT population,94.2, 90.8 and 85.9% of subjects reported AEs in the lapatinib,lapatinib plus trastuzumab and trastuzumab groups, respectively.More subjects in the lapatinib and lapatinib plus trastuzumabgroups reported serious adverse events (SAEs) compared withtrastuzumab (39 (25.3%) and 34 (22.4%) subjects, respectively,vs 9 (6.0%)). The most frequently reported SAEs in thelapatinib-containing treatment groups were hypertransaminase-mia, diarrhea and hyperbilirubinemia. No clinically significantcardiac dysfunction occurred and there were no deaths duringthe neoadjuvant treatment phase.

3.4 NeratinibNeratinib, is an orally available pan-ErbB TKI, differing inthat it inhibits HER4 as well as HER1/EGFR andHER2 [70]. The efficacy and safety of neratinib were evaluatedin a trial including two cohorts of patients with advancedErbB2-positive breast cancer, and those with and those with-out prior trastuzumab treatment, in an open-label, multicen-ter, Phase II trial. The 16-week PFS rates were 59% forpatients with prior trastuzumab treatment and 78% forpatients with no prior trastuzumab treatment. Median PFSwas 22.3 and 39.6 weeks, respectively. Objective responserates were 24% among patients with prior trastuzumab treat-ment and 56% in the trastuzumab-naive cohort [70]. Emergingagents for treatment of HER2-resistant tumors are reportedin Table 2.

4. Ductal triple-negative breast cancer

Numerous transcriptional pathways are under investigation todetermine how best to target therapies to specific mutations ormolecular events in basal-like breast cancers. Each one of these

pathways will require careful investigation to assess howimportant therapeutic interventions along this pathway willbe. Table 3 summarizes clinical data of new agents in ductaltriple-negative breast cancer (TNBC).

4.1 Poly-(adenosine diphosphate (ADP)--ribose)

polymerases pathwayDNA lesions such as single-strand breaks (SSBs) and double-strand breaks (DSBs) are common by products of normal cellularmetabolism and may also result from exposure to harmful envi-ronmental agents. Briefly four DNA repair mechanisms areresponsible for repairing these lesions: i) base-excision repair(BER), ii) nucleotide-excision repair (NER), iii) mismatch repair(MMR) and iv) recombinational repair (with homologousrecombination and nonhomologous end joining (NHEJ)) [71].When SSBs occur, they are repaired using the intact complemen-tary strand as a template by BER, NER and MMR. A key com-ponent of the BER pathway, PARP1, is the most importantmember of the poly-(adenosine diphosphate (ADP)--ribose)polymerase (PARP) family of enzymes [72,73]. PARP inhibitionleads to accumulation of DNA SSBs and subsequent DNADSBs at replication forks. These breaks normally are repairedvia the homologous recombination double-strandedDNA repairpathway, major components of which are the tumor-suppressorproteins BRCA1 and BRCA2 [73]. PARP1 is upregulated differ-entially in primary breast cancers, including ERnegative, PRneg-ative, HER2 negative (ductal triple negative). Preclinical studiesof in vitro activity of PARP inhibitors demonstrated inhibitionof tumor cell growth only if the cell was BRCA deficient [73].Inhibition of PARP to kill tumor cells selectively, therefore, is anovel approach to cancer therapy.

4.1.1 OlaparibOlaparib, a novel, orally active PARP inhibitor, induced syn-thetic lethality in BRCA-deficient cells. A proof-on-concept trialin BRCA-mutated patients assessed the efficacy, safety and toler-ability of olaparib alone in women with advanced breast can-cer [74]. Patients had been given a median of three previouschemotherapy regimens (range 1 -- 5 in cohort 1, and 2 -- 4 incohort 2). Response rate was 11 (41%) of 27 patients (95% CI25 -- 59) in the cohort assigned to 400 mg twice daily, and six(22%) of 27 (11 -- 41) in the cohort assigned to 100 mg twicedaily [74]. The results of this study provide positive proof of con-cept for PARP inhibition in BRCA-deficient breast cancers andshow a favorable therapeutic index for a novel targeted treatmentstrategy in patients with tumors that have genetic loss of functionof BRCA1-associated or BRCA2-associated DNA repair.Phase I studies are currently ongoing combining cisplatin andolaparib. Agents such as platinum salts bind to DNA directlyand result in the formation of DNA-platinum adducts and, con-sequently, intrastrand and interstrand DNA cross-links thatimpede cell division. As a consequence, cisplatinmay be an effec-tive treatment for patients with hereditary BRCA1-mutatedbreast cancers. Because sporadic TNBC and BRCA1-associatedbreast cancer share features suggesting common pathogenesis, a



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Table

2.HER2-targetedagents

inearlyandmetastaticbreast

cancer.

Clinicalsetting

Trialphase

No.ofpatients

Intervention

Clinicalendpoints

Ref.

Metastaticbreast

cancer

Phase

In=24

Trastuzumab-DM1

Clinicalbenefitrate

at3.6

mg/kgwas73%

including

five

partialresponse

KropIetal.

[61]

Phase

IIn=112

Trastuzumab-DM13.6

mg/kg

Partialresponse

25.9%

MedianPFS

=4.6

mo

BurrisHA

etal.

[62]

Metastaticbreast

cancer

Phase

IIrandomized

n=79

Pertuzumab420mg(arm

A)

Pertuzumab1050mg(arm

B)

Arm

A:2patients

PR

18patients

SD

Arm

B:SD14patients

GianniLetal.

[66]

Metastaticbreast

cancer

Phase

IIsingle

arm

Trastuzumabpluspertuzumab

PFS

=5.5

months

PR=24.2%

Clinicalbenefitrate:50%

BaselgaJetal.

[67]

Metastaticbreast

cancer

Phase

IIIsingle

arm

n=136

Neratinib

240mg/day

Cohort1:66patients

priortrastuzumab

Cohort2:70patients

firstline

Cohort1:16weeksPFS

=59%

(22.3

weeks).PR:24%

Cohort2:16weeksPFS

=78%

(39.6

weeks);PR:56%

Burstein

etal.

[70]

EarlyBreast

cancer

Phase

IIIfourarm

sn=417

i)Arm

A:trastuzumabanddocetaxel;

ii)arm

B:trastuzumab,pertuzumaband

docetaxel;iii)arm

C:trastuzumaband

pertuzumab;iv)arm

D:pertuzumaband

docetaxel

Arm

A:

pCR29%

Arm

B:

pCR45.8

%Arm

C:

pCR16.8%

;Arm

D:

pCR24%

GianniL.

etal.

[68]

Earlybreast

cancer

Phase

IIIthreearm

sn=455

Arm

A:lapatinib

pluspaclitaxel

Arm

B:trastuzumabpluspaclitaxel

Arm

C:concomitantlapatinib

and

trastuzumabpluspaclitaxel

Arm

A:

pCR24.68%

Arm

B:

pCR29.53%

Arm

C:pCR51.32%

BaselgaJ.etal.

[69]



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Table

3.New

agents

inductaltriple-negativebreast

cancer.

Clinicalsetting

Trialphase

No.ofpatients

Intervention

Clinicalendpoints

Ref.

MetastaticBRCA1andBRCA2

mutatedbreast

cancerpatients

Phase

In=60

22mutationcarrier

Olaparib200mgtw

icedaily

inmutation

carried

Objectiveantitumoractivitywasreported

onlyin

mutationcarriers

FongPCetal.

[72]

Phase

IIn=54

Cohort

1:27

Cohort

2:27

Olaparib400mgtw

icedaily:Cohort1

Olaparib100mgtw

icedaily:Cohort2

Cohort1:Overallresponse

rate:41%

Cohort2:Overallresponse

rate

22%

TuttA

etal.

[74]

Metastatictriple-negative

breast

cancerpatients

Phase

IIrandomized

n=123

Iniparib5.6

mg/kgpluscarboplatin/

gemcitabinevs

Carboplatin/gemcitabine

Overallresponse

rate

52%

ininiparibarm

vs32in

chemotherapyarm

(p=0.01).

MedianPFS:5.9

vs3.6

months(p

=0.01);

OS:12.3

vs7.7

months(p

=0.01)

O’Shaughnessyetal.

[76]

Metastaticbreast

cancer

Phase

IIsingle

arm

n=41

Veliparib40mgtw

icedaily

plus

temozolomide

Overallresponse

rate

=7%

PRin

BRCA1/2

mutated:37.5%

IsakoffSJetal.

[79]

Metastaticbreast

cancer

Phase

IIItw

oarm

n=519

Iniparib5.6

mg/kgpluscarboplatin/

gemcitabinevs

Placebo/carboplatin/

gemcitabine

Iniparibarm

MedianPFS

5.1

months

comparedwith4.1

monthsin

theplacebo

group(p

=0.027)

MedianOS11.8

monthswithinipariband

11.1

monthsin

theplaceboarm

(p=0.28)

O’Shaughnessyetal.

[78]



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neoadjuvant trial of cisplatin in TNBC was conducted [75]. Six(22%) of twenty-eight patients achieved pathologic completeresponses, including both patients with BRCA1 germline muta-tions; 18 (64%) patients had a clinical complete or partialresponse in the BRCA1 mutation group. These backgrounddata suggest that combination of PARP inhibitors and cisplatincan be potentially very active.

4.1.2 IniparibIniparib (previously BSI 201) (4-iodo-3-nitrobenzamide) is adrug that acts as an irreversible inhibitor of PARP1 (hence, itis a PARP inhibitor) and possibly other enzymes throughcovalent modification [76]. An open-label, Phase II study tocompare the efficacy and safety of gemcitabine and carbopla-tin with or without iniparib, a small molecule with PARP-inhibitory activity, in patients with metastatic TNBC, wasconducted. A total of 123 patients were randomly assignedto receive gemcitabine (1000 mg/m2 of body surface area)and carboplatin (at a dose equivalent to an area under theconcentration-time curve of 2) on days 1 and 8 -- with orwithout iniparib (at a dose of 5.6 mg/kg of body weight) ondays 1, 4, 8 and 11 -- every 21 days. Primary endpoints werethe rate of clinical benefit (i.e., the rate of objective response(complete or partial response) plus the rate of stable diseasefor ‡ 6 months) and safety. Additional endpoints includedthe rate of objective response, PFS and overall survival [76].The addition of iniparib to gemcitabine and carboplatinimproved the rate of clinical benefit from 34 to 56%(p = 0.01) and the rate of overall response from 32 to 52%(p = 0.02). The addition of iniparib also prolonged themedian PFS from 3.6 to 5.9 months (hazard ratio for progres-sion, 0.59; p = 0.01) and the median overall survival from7.7 to 12.3 months (hazard ratio for death, 0.57;p = 0.01) [76]. Another large randomized trial included519 women with metastatic TNBC. Patients were random-ized to receive a standard chemotherapy regimen (gemcitabineand carboplatin) on days 1 and 8 of each 21-day cycle, with orwithout iniparib 5.6 mg/kg, which was administered on days1, 4, 8 and 11 of each 21-day cycle [77]. Patients in the studyhad received up to two previous lines of chemotherapy in ametastatic setting. The co-primary endpoints were overall sur-vival and PFS. Recently final data of this study have been pre-sented at ASCO 2011 meeting. Authors announced that thetrial did not meet the prespecified criteria for significancefor co-primary endpoints of overall survival and PFS [78].The Phase III trial involved 519 women with metastaticTNBC, treated with two or fewer metastatic regimens. Allpatients received gemcitabine and carboplatin and were ran-domized to iniparib or placebo. Patients who progressed onplacebo could cross over to the iniparib arm, and 96% ofpatients in the placebo arm subsequently crossed over. Thetrial had co-primary endpoints of overall survival and PFS,and the study would be considered positive if either endpointfavored iniparib. Secondary endpoints included objectiveresponse rate, safety and tolerability. The study population

had a median age of 53 -- 54, and all had good performancestatus. O’Shaughnessy reported that 56 -- 57% of patientswere receiving first-line therapy for metastatic disease, and43 -- 44% were being treated in second or third line. Patientsin the placebo group had a median disease-free interval of15 months compared with 12 months for the iniparib group.The rates and types of treatment-emergent AEs were similarbetween the two groups, suggesting that iniparib did notadd appreciably to the anticipated toxicity of the chemother-apy. When the trial ended, the iniparib arm had a medianPFS of 5.1 months compared with 4.1 months in the placebogroup. The associated probability value (p = 0.027) did notmeet the prespecified definition of p = 0.01. Median overallsurvival was 11.8 months with iniparib and 11.1 months inthe placebo arm (p = 0.28). Analysis of secondary endpointsshowed an objective response rate of 34% in the iniparibgroup and 30% in the placebo arm. The two groups had sim-ilar rates of complete and partial responses. An exploratoryanalysis of the primary endpoints by treatment history showedthat patients receiving first-line metastatic therapy had similaroverall survival and PFS. By contrast, patients in second- andthird-line therapy had a median PFS of 4.2 months with ini-parib and 2.5 months with placebo, a difference that trans-lated into a hazard ratio of 0.57 in favor of iniparib. Medianoverall survival was 10.8 months in the iniparib arm and8.1 months in the placebo arm, representing a 35% reductionin the hazard [78].

4.1.3 VeliparibVeliparib (ABT-888) is a potent inhibitor of both PARP-1and PARP-2 [78]. Preclinical studies showed that temozolo-mide potentiation by PARP inhibition occurs in TNBC [79].A single-arm Phase II trial of veliparib in combination withtemozolomide was conducted in patients who had receivedat least one prior regimen for MBC [79]. Patients received veli-parib (40 mg twice daily on days 1 -- 7) and oral temozolo-mide (150 mg/m2/day on days 1 -- 5) every 28 days;temozolomide was increased to 200 mg/m2 as tolerated. Theprimary endpoint was objective response rate and secondaryendpoints were PFS, CBR and safety and tolerability. Ofthe 41 patients, 23 had TNBC. Objective response was 7%(one complete response and two partial responses; 95% CI2 -- 20%). The rate of stable disease at 16 weeks was 10%(four patients). When response among all BRCA1/BRCA2 mutation carriers was determined, objective responsewas 37.5% (one complete response and two partial responses)and CBR was 62.5% (n = 5) [79].

5. Conclusion

The ‘wiring diagrams’ of breast cancer subtypes define thatthe signaling circuitry describing the intercommunicationbetween various pathways should be charted in far greaterdetail and clarity, in order to better understand ‘drivers’ and‘passengers.’ We continue to foresee breast cancer research as



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an increasingly ‘computational’ science, in which in silicomodels should predict underlying pathways that sustain can-cer progression and proliferation. The selection of patientsfor targeted therapy remains a challenge, because we lack reli-able biomarkers to predict activity for most of the targetedagents. Traditional methodologies applied for drug develop-ment may be inappropriate for new targeted agents. Resis-tance to many of traditional and new drugs is a majorclinical challenge. The use of high-throughput technologieswill help us to understand the molecular biology of signalingpathways as the roads of the ‘genomic landscape’ of breastcancer. The number of potential driver genes is large, even ifmore limited is the number of ‘driver’ pathways. Patientselection, rational combination therapies, surrogate markersidentification and tumor tissue banking will be key areasof research.

6. Expert opinion

Clinical research in the field of breast cancer should be appliedfor the future to specific subtypes of breast cancer. Trials of thepast did not follow such concept of heterogeneity, using extrap-olation of data from retrospective analyses, frequently difficultand imprecise. Future research should achieve the goal to recog-nizing the diversity of targets in each subtype of breast cancer,taking advantage from molecular characterization tools. Newprospective trials will specifically address the questions of tar-geting multiple pathways in each breast cancer subtype, to max-imize response to treatment and minimize the toxicity. Recentlarge-scale tumor sequencing studies, including wide genomeanalysis studies, have identified a number of mutations thatmight be involved in breast cancer tumorigenesis. Analysis ofthe frequency of specific mutations across different tumorshas been able to identify some, but not all of the mutated genesthat contribute to tumor initiation and progression. One reasonfor this is that other functionally important genes are likely tobe mutated more rarely and only in specific contexts. Thus,for example, mutation in one member of a collection of func-tionally related genes may result in the same net effect, and/or mutations in certain genes may be observed less frequentlyif they play functional roles in later stages of tumor develop-ment, such as metastasis. The biggest challenge for the futurewill be to apply a network reconstruction and coexpressionmodule identification-based approach to identify functionally

related gene modules targeted by somatic mutations in cancer.The ultimate goal of this approach is to identify network ofpathways and potential cross talks within pathways. Dual ormultiple targeting in order to shut down ‘driver’ pathwayswill be the future of breast cancer treatment within several sub-types. This method was applied to available breast cancersequence data and identified several pathways as targets ofrare driver mutations in breast. These mutations do not appearto alter genes that play a central role in these pathways, butrather contribute to a more refined shaping or ‘tuning’ of thefunctioning of these pathways in such a way as to result inthe inhibition of their tumor-suppressive signaling arms andthereby conserve or enhance tumor-promoting processes. Webelieve a gene network reconstruction strategy-based approachcan successfully identify cancer driver mutations throughenrichment of mutations within modules. We should high-light a few important caveats in the field. Next-generationsequencing technologies used to reconstruct genetic networkscan be altered to generate networks of different sizes, or toreflect different coexpression relationships, depending on theinvestigator requirements and/or sample size and likelihoodthat module enrichment will be observed in different-sizedmodules. Specifically genome remodeling during cancerprogression or upon resistance to therapies can upregulatepathways due to downregulation of current driver path-ways. Additionally, this approach probably does not captureall of the secondary driver mutations, which may requireeither additional complementary systems biology appro-aches or larger sample sizes to capture other mutation-enriched coexpression modules. Overall, we believe that thisapproach shows tremendous promise for the identification ofrare tumorigenic driver mutations, which is a crucial task forthe upcoming large-scale cancer resequencing projects, as it isthese more private mutations that may be driving intratumorheterogeneity, inter-patient heterogeneity and ultimately alter-ing response to therapeutic intervention. The future of manyinvestigational therapeutics in breast cancer is, therefore,linked to our ability to identify the most druggable target ineach subtype.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.



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AffiliationGiuseppe Curigliano† MD PhD,

Marzia Locatelli, Luca Fumagalli, Janaina Brollo,

Elisabetta Munzone, Franco Nole,

Carmen Criscitiello & Aron Goldhirsch†Author for correspondence

Division of Medical Oncology,

Istituto Europeo di Oncologia,

Via Ripamonti 435,

20141 Milano, Italia

Tel: +39 02 57489788; Fax: +39 02 57489581;

E-mail: [email protected]



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http://clinicaltrials.gov/ct2/show/NCT01130259






targeting the subtypes of breast cancer: rethinking investigational drugs

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