ado-trastuzumab emtansine targets ...for the treatment of her2-positive metastatic breast cancer in...

Cancer Biology and Signal Transduction

Ado-Trastuzumab Emtansine TargetsHepatocytes Via Human Epidermal Growth FactorReceptor 2 to Induce HepatotoxicityHaoheng Yan1,2, Yukinori Endo1, Yi Shen1, David Rotstein3, Milos Dokmanovic1,Nishant Mohan1, Partha Mukhopadhyay4, Bin Gao5, Pal Pacher4, and Wen Jin Wu1

Abstract

Ado-trastuzumab emtansine (T-DM1) is an antibody–drugconjugate (ADC) approved for the treatment of HER2-positivemetastatic breast cancer. It consists of trastuzumab, a humanizedmAb directed against HER2, and a microtubule inhibitor, DM1,conjugated to trastuzumab via a thioether linker. Hepatotoxicityis one of the serious adverse events associated with T-DM1therapy. Mechanisms underlying T-DM1–induced hepatotoxicityremain elusive. Here, we use hepatocytes and mouse models toinvestigate the mechanisms of T-DM1–induced hepatotoxicity.We show that T-DM1 is internalized upon binding to cell surfaceHER2 and is colocalized with LAMP1, resulting in DM1-associ-ated cytotoxicity, including disorganized microtubules, nuclearfragmentation/multiple nuclei, and cell growth inhibition. Wefurther demonstrate that T-DM1 treatment significantly increasesthe serum levels of aspartate aminotransferase, alanine amino-

transferase, and lactate dehydrogenase in mice and inducesinflammation and necrosis in liver tissues, and that T-DM1–induced hepatotoxicity is dose dependent. Moreover, the geneexpression of TNFa in liver tissues is significantly increased inmice treated with T-DM1 as compared with those treated withtrastuzumab or vehicle. We propose that T-DM1–induced upre-gulation of TNFa enhances the liver injury that may be initiallycaused by DM1-mediated intracellular damage. Our proposal isunderscored by the fact that T-DM1 induces the outer mitochon-drial membrane rupture, a typical morphologic change in themitochondrial-dependent apoptosis, and mitochondrial mem-brane potential dysfunction. Our work provides mechanisticinsights into T-DM1–induced hepatotoxicity, which may yieldnovel strategies tomanage liver injury induced by T-DM1 or otherADCs. Mol Cancer Ther; 15(3); 480–90. �2015 AACR.

IntroductionAntibody–drug conjugates (ADC) are an emerging group of

therapeutic agents that are generated by covalent attachment ofcytotoxic agents to mAbs via linkers (1). They are designed toselectively deliver cytotoxic agents to tumor cells where specifictumor-associated antigens are overexpressed on the cell surface,thereby minimizing systemic toxicity. Ado-trastuzumab emtan-sine (also known as T-DM1) is an ADC approved by the FDAfor the treatment of HER2-positive metastatic breast cancer inpatients previously treated with trastuzumab and taxane (2).T-DM1 consists of trastuzumab, a humanized mAb directed

against HER2, and a microtubule inhibitor, DM1, that is conju-gated to trastuzumab via a thioether linker (3). DM1 is a potentmicrotubule polymerization inhibitor that induces mitotic arrestand kills tumor cells at subnanomolar concentration (4). It is 25-to 270-fold more potent than paclitaxel and 180- to 4,000-foldmore potent than doxorubicin (5, 6). However, its side effects andlack of specificity prevented it from clinical use (7).

HER2 is a member of EGFR/ErbB family of receptor tyrosinekinases with significant roles in breast cancer tumorigenesis andis overexpressed in 15% to 30% of breast cancers (8). Uponbinding to HER2, T-DM1 is internalized and processed inlysosomes to release the active catabolite lysine-N(e)-N-mal-eimidomethyl-cyclohexane-1-carboxylate (MCC)-DM1 (Lys-MCC-DM1; refs. 3, 9–12). The released Lys-MCC-DM1 exertsantimicrotubule functions via microtubule destabilization,which results in mitotic arrest, cell growth inhibition, and celldeath (9–12).

T-DM1 significantly prolongs progression-free and overall sur-vival with less toxicity than lapatinib plus capecitabine in patientswith HER2-positive advanced breast cancer (13, 14). However, T-DM1 therapy is associated with serious grade 3 or greater adverseevents, including hepatotoxicity (13–15). It was reported that allgrade increases in serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) occurred in 208 (23.5%) and139 (15.7%) patients, respectively, out of 884 T-DM1–exposedpatients. Thirty-six patients (4.1%) had grade 3 increase in serumALT (4.1%), 2 patients (0.2%) had grade 4 increase in serum ALT,and 27 patients (3.1%) had grade 3 increase in serum AST (15).Three patients were diagnosed with nodular regenerative

1Division of Biotechnology Review and Research I, Office of Biotech-nology Products, Office of Pharmaceutical Quality, Center for DrugEvaluation and Research, U.S. Food and Drug Administration, SilverSpring, Maryland. 2Interagency Oncology Task Force Fellowship:OncologyProduct Research/ReviewFellow,NCI, Bethesda,Maryland.3Division of Compliance, Office of Surveillance and Compliance, Cen-ter for Veterinary Medicine, U.S. Food and Drug Administration, Der-wood, Maryland. 4Laboratory of Physiologic Studies, National Insti-tute on Alcohol Abuse and Alcoholism, NIH, Bethesda, Maryland.5Laboratory of Liver Diseases, National Institute on Alcohol Abuseand Alcoholism, NIH, Bethesda, Maryland.

H. Yan and Y. Endo contributed equally to this article.

Corresponding Author: Wen Jin Wu, U.S. Food and Drug Administration,Building 52/72, Room 2310, 10903 New Hampshire Avenue, Silver Spring, MD20993. Phone: 240-402-6715; Fax: 240-402-6716; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0580

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(3) March 2016480

on October 4, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 28, 2015; DOI: 10.1158/1535-7163.MCT-15-0580

http://mct.aacrjournals.org/

hyperplasia, which resulted in one death due to liver failure.Hepatotoxicity is one of the black box warnings on the labelingfor the prescription of T-DM1 (http://www.gene.com/download/pdf/kadcyla_prescribing.pdf). However, mechanisms underlyingT-DM1–induced hepatotoxicity remain elusive (15). It has notbeen reported whether T-DM1 directly targets hepatocytes viaHER2 to induce hepatotoxicity.

Gemtuzumab ozogamicin (GO) is an ADC consisting of ananti-CD33 mAb conjugated to calicheamicin (16). GO wasapproved in 2000 for the treatment of acute myeloid leukemia(AML) and was withdrawn from the market in 2010 due toproduct safety and efficacy concerns (17, 18). Among the safetyconcerns were clinical symptoms of hepatotoxicity, includinghepatic veno-occlusive disease, which is a significant GO-relatedtoxicity (19). In addition, 29% of patients had grade 3 or grade 4hyperbilirubinemia and 9% had grade 3 or grade 4 ALT levelabnormalities (20). However, the cause of GO-induced hepato-toxicity remains unproven. It has been reported that CD33 recep-tor, thought to be a specific marker for the cells of the myeloidlineage, is widely distributed in the liver tissue and highlyexpressed on hepatocytes (20, 21). This study suggested that thespecific targeting of hepatocytes by GO resulted in the accumu-lation of antibody–toxin conjugates in hepatocytes causing cali-cheamicin-induced damage (20). Besides GO, signs of liver dys-function were also identified in other ADC therapies in clinicaltesting (22–24). Given the significant clinical potential of ADCtherapies and the substantial increase in the number of ADCs inclinical trials, it is critical to obtain a better understanding ofmechanisms by which ADCs, including T-DM1, inducehepatotoxicity.

Murine model has been widely used to study the mechan-isms of trastuzumab-induced cardiotoxicity (25–29). Riccioand colleagues have shown that trastuzumab binds to mouseHER2 (26) and that mice treated with trastuzumab havereduced left ventricular ejection fraction (25–29). Trastuzumabmay directly block antiapoptotic signaling, leading to prema-ture cardiac dysfunction (28). Although HER2 is present at alow level in mouse cardiomyocytes, the studies from differentlaboratories suggested that trastuzumab-induced cardiotoxicitymay be HER2-dependent (25–29). Furthermore, trastuzumabtreatment induces apoptosis in cardiac sections of heart tissuesfrom mice treated with trastuzumab (25, 28). In this study, weused the cellular and murine models to investigate the mechan-isms by which T-DM1 induces hepatotoxicity. We aimed toevaluate HER2 expression in mouse and human hepatocytesand T-DM1–induced endocytosis. We also examined its asso-ciated intracellular damage and liver injury in mice treated withT-DM1.

Materials and MethodsCell lines and control DM1 ADC

Human hepatocytes (THLE2) and mouse hepatocytes(AML12) were obtained from ATCC and used within six monthsafter cell lines were ordered and cultured in BEGM SingleQuotsmedium containing 10% FBS and DMEM/F-12 (1:1) mixturecontaining 10% FBS, respectively. Human primary hepatocytes(HPH) were obtained from ScienCell and used within 6 monthsafter the cells were ordered. THLE2, AML12, and HPH were notauthenticated in our laboratory.aHFc-NC-DM1 is an anti-humanIgG Fc-specific antibody conjugated to maytansinoid DM1 with a

noncleavable linker. aMFc-NC-DM1 is an anti-mouse IgG Fc-specific antibody conjugated to maytansinoid DM1 with a non-cleavable linker. Both anti-HFc-NC-DM1 and anti-MFc-NC-DM1were purchased from Moradec LLC.

Immunofluorescence microscopyFor LAMP1 and microtubule staining, cells were plated on

fibronectin (10 mg/mL, Sigma-Aldrich) precoated glass cover-slips (12 mm; Fisher Scientific) in 12-well plate (Corning) andcultured overnight. After washing with prewarmed DPBS twice,cells were fixed in 4% paraformaldehyde (PFA, Electron Micros-copy Sciences) in DPBS for 15 minutes and permeabilized with0.2% Triton X-100 in DPBS for 10 minutes. The samples werewashed with DPBS and blocked with 2% BSA in DPBS at roomtemperature for 1 to 2 hours. Anti-a-tubulin (clone DM1A,Sigma-Aldrich) or anti-LAMP1 (BD Biosciences) antibodieswere diluted 1:100 in the blocking solution, and the sampleswere incubated with these antibodies at 4�C overnight. Afterwashing with DPBS three times, the samples were incubatedwith Alexa Fluor 488 or 594-conjugated anti-mouse secondaryantibodies (1:100 dilution; Life Technologies) in the blockingsolution at room temperature for 1 hour. After washing withDPBS for 10 minutes three times, the coverslips were mountedinverted on glass slides with ProLong Gold Antifade Reagentcontaining DAPI (Life Technologies). Stained samples wereimaged on Confocal LSM 510 Meta Microscope (Carl ZeissMicroscopy). For immunostaining using trastuzumab of T-DM1as the primary antibody, cells on fibronectin-precoated coverglass were fixed in 4% PFA in cell culture media, permeabilizedwith 0.5% saponin (EMDMillipore) in TBS at room temperaturefor 15 minutes, and washed with TBS for 5 minutes three times.The samples were blocked with 10% goat serum (JacksonImmunoResearch) in TBS at 37�C for 30 minutes and thenincubated with the purified human IgG (Life Technologies),trastuzumab, or T-DM1 antibodies (each at 50 mg/mL) at 37�Cfor 1 hour. After washing with TBS containing 1% goat serumthree times (10 minutes for each wash), the samples wereincubatedwith Alexa Fluor 488-conjugated anti-human second-ary antibody (1:100 dilution, Life Technologies) diluted in TBSat 37�C for 30 minutes. The samples were washed with TBScontaining 1% goat serum at room temperature three times andthen mounted on glass slides as described above. Images werecaptured on an LSM 510 Meta Confocal Microscope attached toan Axiovert 200 Inverted Microscope (Carl Zeiss).

IHCLivers from healthy C57BL/6 mice were harvested, fixed, and

embedded in paraffin. IHC staining was performed by Histoserv,Inc. According to their IHC protocol, the tissue sections weretreated at 90�C for 20minutes for antigen retrieval and then weretreated with hydrogen peroxide and blocked with BSA. Afterwashing with TBST, the sections were incubated with controlrabbit IgG (Life Technologies) or rabbit anti-HER2 antibody(29D8, 1:400 dilution; Cell Signaling Technology) at room tem-perature overnight. After TBST washing, the sections were incu-bated with secondary antibody for 30 minutes at room temper-ature and then streptavidin–HRP incubation for 30 minutes atroom temperature. After washing with TBST, the slides weredeveloped with diaminobenzidine tetrahydrochloride. Images ofthe immunohistochemical stainingwere collected using Pannora-mic MIDI Digital Slide Scanner (3DHISTECH Ltd).

T-DM1 Induces Hepatotoxicity

www.aacrjournals.org Mol Cancer Ther; 15(3) March 2016 481




Duolink proximity ligation assayDuolink proximity ligation assay (PLA) was performed accord-

ing to the instructions obtained from the manufacturer's website(http://www.olink.com/products/duolink/downloads/duolink-manuals-and-guidelines). Briefly, the cells werefixed andpermea-bilized and incubated with primary antibodies from two differentspecies recognizing either C-terminal region of HER2 (rabbitpolyclonal) or DM1 component of T-DM1 (mousemonoclonal).As a control, cells that were not treated with T-DM1, but stainedforHER2 andDM1,were used. Controls also included no primaryantibodies. After washing and permeabilization step, the cellswere incubated with secondary antibodies with the ligated oli-gonucleotide probes (PLA plus and PLA minus) for 1 hour at37�C, followed by ligation and amplification reactions. In theamplification step, fluorescently labeled oligonucleotides wereadded together with polymerase. The signals from fluorescentlylabeled amplified concatemeric product were visualized by fluo-rescent microscopy. Distinct single fluorescent spots indicateinteraction between HER2 protein and T-DM1.

Animal modelAll animal experiments were approved by and conducted in

accordance with the regulations of the FDA Institutional AnimalCare and Use Committee guidelines. The first cohort of thirtysix C57BL/6 mice, ages 8 to 9 weeks (NCI, Frederick, MD),was randomly assigned into three groups to receive a singletail-vein injection of vehicle (6% sucrose, 0.02% Tween 20, and10 mmol/L sodium succinate), trastuzumab (Genentech, Inc), orT-DM1 (Genentech, Inc). Both trastuzumab and T- DM1 werepurchased from the pharmacy at the NIH (Bethesda,MD). Twelvemice in the T-DM1 group received 30mg/kg of T-DM1, 12mice intrastuzumab group received 29.4 mg/kg trastuzumab (compara-ble mole quantity of trastuzumab to T-DM1), and 12 mice in thesham group received a comparable volume of vehicle. Mice werebled 10 days prior to the drug injection to establish a baseline forserumbiomarkers.Micewere thenbled and sacrificedondays 1, 3,or 7 after the tail-vein injection of vehicle, trastuzumab, and T-DM1. Livers from each mouse were harvested for H&E stainingand electronmicroscopy study. The second cohort of 40 C57BL/6mice was randomly assigned into five groups, including vehicle,trastuzumab (29.4 mg/kg), T-DM1 (3 mg/kg), T-DM1 (10 mg/kg), or T-DM1 (30mg/kg) for the time anddose-dependent study.There were 8 mice in each group. Mice received a single tail-veininjection of vehicle, trastuzumab, or T-DM1. Mice were bled andthen euthanized 12 hours or 72 hours post tail-vein injection.Livers from mice were harvested for H&E staining, TNFa geneexpression and EM study. Livers from healthy C57BL/6mice wereharvested for IHC and Western blot analysis.

Serum hepatic markersBlood collection and serum isolation: Tail vein nick was per-

formed during survival blood collection, and terminal cardiacblood collection was used during euthanasia. ALT, AST, andlactate dehydrogenase (LDH) serum test was conducted in thechemistry laboratory at NIH clinical center (Bethesda, MD) usingthe same produces as those used for human clinical samples.

Reverse transcription and qPCRTotal RNA was isolated from freshly frozen liver tissues and

Reverse Transcriptase Superscript II (Life Technologies) was used

for cDNA synthesis according to the manufacturer's protocol.qPCRwas performed using Taqman Gene Expression PCRMasterMix and commercially available primers for genes with the 7900Fast Real-Time PCR system (Applied Biosystems). Actin was usedas reference to normalize the expression level.

Electron microscopeFresh liver samples were fixed for 1 hour at room temperature

and then overnight at 4�C in TEM/SEM tousimis Glutaraldehyde,2.5% in 0.1 mol/L Na-Cacodylate Buffer (Tousimis), osmicatedfor 1 hour at room temperature in the same buffer, en bloc stainedwith 0.5% uranyl acetate for 1 hour, dehydrated in a gradedethanol series, embedded in Epon 812 substitute, and examinedon a Hitachi H7650 Transmission Electron Microscope.

Mitochondria membrane potential assayCells were plated on fibronectin-precoated glass coverslips in

12-well plate and cultured overnight. Next day, cells weretreated with T-DM1 (0 or 20 mg/mL) and then incubated for2 days. Mitochondrial Transmembrane Potential ApoptosisDetection Kit (Abcam) was used according to the manufac-turer's protocol. Images were captured by EVOS FL system (LifeTechnologies). According to the manufacturer's instructions,the kit utilizes a cationic dye that fluoresces differently inhealthy versus apoptotic cells. In healthy cells, the dye accu-mulates and aggregates in the mitochondria with bright fluo-rescence, whereas in apoptotic cells, the dye cannot aggregate inmitochondria due to the altered mitochondrial transmembranepotential.

Alexa Fluor 488 conjugationAlexa Fluor 488 Protein Labeling Kit (Life Technologies) was

used for the labeling of T-DM1 with Alexa Fluor 488. Briefly, 2.5mgT-DM1(5mg/mL, 0.5mL inDPBS)was conjugatedwithAlexaFluor 488according to themanufacturer's instructions. The kit hasa tetrafluorophenyl ester moiety, and it reacts efficiently withprimary amines of T-DM1 to form stable dye–protein conjugate.The Alexa Fluor 488–labeled T-DM1 was eluted by serum-freeBEGM medium.

Western blotting and immunoprecipitationWhole-cell lysates (WCL) of THLE2 or AML12 cells were used

either for detection of HER2 protein expression or immunopre-cipitation experiments. For immunoprecipitation, WCL wereincubated with control IgG (human IgG, Life Technologies),trastuzumab, T-DM1 or anti-HER2 antibody (29D8, Cell Signal-ing Technology). The immune-precipitated HER2 was detectedusing anti-HER2 antibody (29D8), which recognizes bothmouseand human HER2.

Statistical analysisGraphPad Prism was used for statistical studies. Statistical

significance was determined by Student t test (�, P < 0.05;��, P < 0.01). Data are expressed as mean � SEM.

ResultsHER2 is expressed in mouse and human hepatocytes, andT-DM1 associates with HER2 on the hepatocyte cell surface

To investigate the possibility that specific targeting of hepa-tocytes via HER2 may contribute to hepatotoxicity induced by

Yan et al.

Mol Cancer Ther; 15(3) March 2016 Molecular Cancer Therapeutics482




T-DM1, HER2 expression in human and mouse hepatocytesand mouse liver tissue was analyzed by Western blotanalysis. Figure 1A showed HER2 expression in human hepa-tocyte (THLE2) versus HPH (left) and in THLE2 versus mousehepatocyte (AML12) (right). HER2 was detected in liver tissuesfrom three mice by Western blot analysis (Fig. 1B). HER2expression in HER2-positive breast cancer cells (SKBR3) andAML12 was used as controls (Fig. 1B). Immunoprecipitationstudy (top) showed that both trastuzumab and T-DM1 boundto human HER2 (Fig. 1C), consistent with previous report(30). Figure 1C (bottom) showed that both trastuzumab andT-DM1 were capable of immunoprecipitating HER2 from WCLof mouse hepatocytes, although the binding affinity of trastu-zumab and T-DM1 to mouse HER2 was lower than that ofmouse anti-HER2 antibody (29D8). Using the immunofluo-

rescence approach, we found that HER2 was expressed in bothplasma membrane and inside of cells in HPH (Fig. 1D). Apositive HER2 staining by IHC was observed in mouse livertissue. HER2 expression by IHC in human liver tissues wasreported previously (The Human Protein Atlas: www.proteina-tlas.org).

Wenext examined the interactionof trastuzumaborT-DM1withHER2 in hepatocytes. As shown in Fig. 1F (arrowheads), the strongpositive signal was detected at the cell edges of both human andmouse hepatocytes when either trastuzumab or T-DM1 was usedfor detection of cell surface HER2, suggesting that both trastuzu-mab and T-DM1 bind to HER2 expressed on the cell surface. Incontrast, control IgG did not give rise to any positive signal on thecell surface (Fig. 1F).UsingDuolinkPLA,we further confirmed thatT-DM1 bound to HER2 expressed in hepatocytes (Fig. 1G).

CA

IP:THLE2

AML12

HER2

HER2

HER2

Actin

D

THLE2 AML12

IgG

T-D

M1

Tras

tuzu

mab

F

IgG

B

HER

2

G

HPH 2REHGgIE

T-DM1

IgG(Anti-HER2/DM1)

(No antibodies)

(Anti-HER2/DM1)

T-DM1

50 μg/mL 200 μg/mL

HER2

Figure 1.HER2 is expressed in human andmousehepatocytes and mouse liver tissues,and both trastuzumab and T-DM1 bindto HER2 on the cell surface ofhepatocytes. A, levels of endogenousHER2 in WCL of human and mousehepatocytes (THLE2 and AML12,respectively) and HPH weredetermined by Western blot analysisusing anti-HER2 antibody (29D8). B,levels of endogenous HER2 in WCL ofAML12 (20 mg) and mouse liver tissue(20 mg) were determined by Westernblot analysis using anti-HER2 antibody(29D8). C, endogenous HER2 in WCLcollected from THLE2 and AML12 wasimmunoprecipitated (IP) using humancontrol IgG, trastuzumab, T-DM1, andanti-HER2 antibody (29D8).Immunoprecipitated HER2 protein wasdetected using anti-HER2 antibody(29D8). D, endogenous HER2 in HPHwas detected by anti-HER2 antibody(29D8) using immune-fluorescenceapproach. Bar, 50 mm. E, endogenousHER2 in mouse liver tissues wasdetected by immunohistochemicalstaining. Bar, 50 mm. F, binding oftrastuzumab and T-DM1 to the cellsurface HER2 of THLE2 and AML12 wasdetected using immunofluorescenceapproach. Human IgG was used as anegative control. Bar, 20 mm. G, THLE2cells were plated overnight, and theneither control treated with IgG (50 or200 mg/mL) or treated with T-DM1 (50or 200 mg/mL) for 1 hour. Followingtreatment, the cells were fixed andsubjected to Duolink PLA according tothe manufacturer's instructions (seeMaterials and Methods section); top,cells that were not treated with T-DM1but stained with both primary and bothsecondary antibodies (right);middle, T-DM1–treated cells, which were notstained with either primary antibodiesbut stained with both secondaryantibodies; bottom, T-DM1–treatedcells, which were stained with bothprimary and both secondaryantibodies. Bar, 50 mm.






T-DM1 is internalized into the human hepatocytes, resulting indisorganized microtubule networks, nucleus fragmentation/multiple nuclei, and cell growth inhibition

Wenext addressed the question of whether T-DM1was capableof inducing endocytosis upon binding to HER2 expressed onhepatocyte cell surface. Human hepatocytes (THLE2) were incu-bated with Alexa Fluor 488–conjugated T-DM1 for 1 hour. Thecolocalization of the Alexa Fluor 488–conjugated T-DM1with the

lysosomal marker LAMP1 was examined. As shown in Fig. 2A,Alexa Fluor 488–conjugated T-DM1 was internalized into THLE2cells and colocalized with LAMP1 (arrows), indicating that T-DM1has the ability to induce endocytosis when it binds toHER2.

After internalization of the receptor T-DM1 complex, intracel-lular release of DM1-containing moieties from T-DM1 occursfollowing lysosomal degradation of trastuzumab component.Wenext tested whether T-DM1 induces microtubule destabilization

**

0.00

Con

trol

T-D

M1

α-tubulin/nucleus

Con

trol

T-D

M1

NucleusLAMP1 T-DM1A

THLE

2

AM

L12

α-tubulin/nucleus

α-tubulin/nucleus

α-tubulin/nucleus

CB

Merge

Cel

l num

ber (

x105

)

1

0 µg/mL T-DM1

0 1 2 3 4

5 µg/mL T-DM110 µg/mL T-DM1

0 µg/mL T-DM15 µg/mL T-DM1

10 µg/mL T-DM1

0

Day

Cel

l num

ber (

x105

)

1

2

00 1 2 3 4

Day

0.5

D

Cel

l num

ber (

x105

) 2

00 1 2 3 4

Day

1

No treatmentT-DM1αHFc-NC-DM1αMFc-NC-DM1

No treatmentT-DM1αHFc-NC-DM1αMFc-NC-DM1

0 1 2 3 4Day

Cel

l num

ber (

x105

) 2

0

1

E

**

** **

(5 µg ADC/mL) (10 µg ADC/mL)

Figure 2.T-DM1 is internalized, leading to thedisorganized microtubules, multipleand fragmented nuclei, and cell growthinhibition in THLE2 and AML12 cells. A,human hepatocytes (THLE2) wereseeded on fibronectin-precoated glasscoverslips overnight and then wereincubated with Alexa Fluor 488–conjugated T-DM1 for 1 hour. Cells werethen fixed, permeabilized, and stainedfor LAMP1 (red). Merge (red arrows),Alexa Fluor 488–conjugated T-DM1(green) and lysosomal marker LAMP1colocalized. Nuclei were stained withDAPI (blue). Bar, 20 mm. B, THLE2 cellswere plated on fibronectin-precoatedglass coverslips and treated with T-DM1overnight or left untreated. Cells werethen fixed, permeabilized, and stainedfor microtubules (green). Nuclei werestained with DAPI (blue).White arrows,disorganized microtubules. Redarrows, nucleus fragmentation/multiple nuclei. Graph, growth profilesof cells treated with indicatedconcentrations of T-DM1. Cells wereseeded at 0.5 � 105, harvested at theindicated times, and counted. Dataare the mean � SEM of threeindependent experiments in triplicate.Bar, 50mm. C, experimental procedureswere essentially the same as describedin Fig. 2B except AML12 cells wereseeded at 0.25� 105. Bar, 50 mm. D andE, graph, growth profiles of THLE2 cellstreated with either 5 mg/mL (D) or10 mg/mL (E) of T-DM1, aHFc-NC-DM1or aMFc-NC-DM1. The THLE2 cells wereseeded at 0.5 � 105, harvested at theindicated times, and counted. Data arethe mean � SEM (�� , P < 0.01).

Yan et al.





and the mitotic arrest leading to growth inhibition. As shownin Fig. 2B, after cells were incubated with T-DM1 for 48 hours,microtubule structures in THLE2 cells were disorganized (whitearrow) as compared with those in the untreated cells (orangearrow). Furthermore, multiple nuclei and fragmented nucleuswere also observed in the cells treated with T-DM1 (Fig. 2B; redarrows), indicating that the internalized T-DM1preventedmitosisof THLE2 cells and induced apoptosis. Figure 2B (graph) showedgrowth profiles of THLE2 cells treated with T-DM1 or left untreat-ed and demonstrated that the growth of hepatocyte was inhibitedby T-DM1 in a dose-dependent manner. It should be notedthat the inhibition of cell growth by T-DM1 shown in Fig. 2Bwas not due to possible residual-free DM1 that may be present inthe T-DM1 solution since after immunodepletion of T-DM1

from the solution using Protein A/G agarose, the supernatantdid not exhibit any inhibitory effect on cell growth (data notshown). Figure 2C also demonstrated that T-DM1 was capable ofdestabilizing microtubule structures (white arrow), inducingmultinuclei in mouse hepatocytes (red arrows), and inhibitingAML12 cell growth (Fig. 2C; graph). Furthermore, as shownin Fig. 2D and E, the ability to inhibit THLE2 cell growth bynon-HER2 targeting control ADCs (aHFc-NC–DM1 and aMFc-NC-DM1) was significantly reduced as compared with T-DM1,indicating that the T-DM1–induced cytotoxicity is mainly HER2-dependent in this in vitro toxicology study. Taken together, thesedata suggest that T-DM1 can specifically target both human andmouse hepatocytes via HER2, resulting in DM1-associated intra-cellular damages.

Trastuzumab 29.4 mg/kg

Trastuzumab 29.4 mg/kg

0

1

2

3

4

5

**

**

**

**

**

**

**

0

1

2

3

4

5

0

1

2

3

4

5

0

2

4

6

A

C

B

D

**

**

** *

*

TNFα7 Days

Fold

diff

eren

ces

Control T-DM1 30 mg/kg

LDHASTALT

2

3

4

5

1

0

Fold

diff

eren

ces

LDHASTALT

2

3

4

5

1

0

3 Days1 Day

Fold

diff

eren

ces

LDHASTALT

2

3

4

5

1

0

0

1

2

3

0

1

2

3

FE

* * *

*

**

*

*

Control T-DM1 3 mg/kg

T-DM1 10 mg/kg T-DM1 30 mg/kg

72 h12 h

Fold

diff

eren

ces

LDHASTALT

2

3

1

0

Fold

diff

eren

ces

LDHASTALT

2

3

1

0

Fold

diff

eren

ces

Day 7Day 3Day 1

4

6

2

0

Figure 3.T-DM1 treatment increases the serumlevels of AST, ALT, and LDH and thegene expression of TNFa in livertissues. A–C, C57BL/6 mice wereadministered a single tail-veininjection of vehicle, trastuzumab (29.4mg/kg), or T-DM1 (30mg/kg) and thenbled and sacrificed on day 1, 3, or 7. Theserum levels of ALT, AST, and LDHwere determined. Data are the means� SEM of 4 mice (� , P < 0.05). D, TNFagene expression in the freshly frozenliver tissues was determined usingTaqman Gene Expression PCR MasterMix. Actin was used as reference tonormalize the expression level. Dataare the means � SEM of 4 mice(� , P < 0.05). E and F, C57/BL6 micewere administered a single tail-veininjection of vehicle, trastuzumab (29.4mg/kg), or T-DM1 (3, 10, or 30 mg/kg).At the indicated times, mice were bledand then euthanized. The serum levelsof ALT, AST, and LDH weredetermined. Data are the means �SEM of 4 mice (�, P < 0.05).






T-DM1 treatment increases the serum levels of AST, ALT, andLDH, and TNFa gene expression in liver tissue

Data presented in Figs. 1 and 2 suggest that mouse can be usedas an animal model to study the mechanisms of T-DM1–inducedhepatotoxicity. We next investigated whether T-DM1 treatmentinduced acute liver injury in mice. T-DM1 was administered in asingle tail-vein injection, and time-dependent effects of T-DM1onliver function were evaluated bymeasuring AST, ALT, and LDH inthe serum. Consistent with the previous report (30), a single doseof T-DM1 at 30 mg/kg was tolerated in mice (data not shown).While mice administered control vehicle or trastuzumab at 29.4mg/kg showed no significant increases in ALT, AST, and LDH atthree different time points (day 1, 3, and 7), mice administered T-DM1 showed significant increases in serum levels of ALT, AST, andLDH compared with that of control or trastuzumab-treated mice(Fig. 3A–C). It has been shown that tissue macrophages such asKupffer cells respond to the liver injury and secret preinflamma-tory cytokines such as TNFa (31–33). The cytokines can furtherpromote the accumulation of neutrophils in liver, leading toserious hepatic damage (32, 33). Thus, we measured TNFa geneexpression in liver tissues from mice treated with control vehicle,trastuzumab, or T-DM1at different timepoints and found that theTNFa gene expression was significantly elevated in mice treatedwith T-DM1 as compared with those treated with control vehicleor trastuzumab (Fig. 3D). We observed a downward trend in theserum levels of TNFa from day 1 to day 7 (Fig. 3D).

We next addressed whether T-DM1 induced the elevation ofserumALT, AST, and LDH in a dose-dependentmanner. As shownin Fig. 3E, the significant increases in serum levels of ALT and AST,but not LDH, were observed inmice treated with the highest doseof T-DM1 (30 mg/kg) at the 12-hour post T-DM1 injection. At 72hours posttreatment of T-DM1, the serum levels of ALT, AST, andLDH were significantly increased at the dose of 30 mg/kg of T-DM1 (Fig. 3F), consistent with data shown in Fig. 3B. The serumlevels of ALT and LDH, but not AST, were also significantly

increased at the dose of 10 mg/kg of T-DM1, and there were nosignificant increases in ALT, AST, and LDH at the dose of 3 mg/kgof T-DM1 (Fig. 3F). These data indicated that T-DM1–inducedliver injure was in a dose-dependent manner. Surprisingly, theserum levels of ALT were also increased in mice treated withtrastuzumab at 72 hours postinjection as compared with controlmice (Fig. 3F).

T-DM1 treatment induces inflammation and necrosis in livertissues

The hallmarks of acute hepatocellular injury are inflammationand/or necrosis (34). Both inflammation (green arrows) andnecrosis (yellow arrows) were found in liver tissues from themice treated with T-DM1 at the dose of 30 mg/kg, whereasinflammation and necrosis were not observed in liver tissuesfrom control mice (Table 1; Fig. 4A, C, and D). Inflammation,but not necrosis, was noticed in liver tissue in one mouse treatedwith trastuzumab at the dose of 29.4 mg/kg on day 7 (Table 1;Fig. 4B, green arrow). However, the reason that caused inflam-mation remains unclear. T-DM1–induced necrosis in liver tissueswas observed as early as 12 hours post–T-DM1 treatment atthe doses of 10 mg/kg and 30 mg/kg, but not at 3 mg/kg. At72 hours post–T-DM1 treatment, necrosis was found in all tissuesamples obtained from themice treated with three different dosesof T-DM1 (Table 2). No necrosis was found in liver tissuesobtained from the mice treated with vehicle control or trastuzu-mab (Table 2).

T-DM1 induces the outer mitochondrial membrane ruptureand mitochondrial membrane potential dysfunction

The outer mitochondrial membrane rupture (OMR), whichreleases mitochondrial proteins of the intermembrane space intothe cytoplasm, has been implicated as the central event of themitochondrial-dependent apoptosis (35). These mitochondrialproteins initiate and execute the apoptotic processes (35). Electron

Table 1. T-DM1 causes hepatocellular injury in mouse liver tissues (the first cohort of mice)

Hepatocellular injuryDay 1 Day 3 Day 7

Controla No histopathologic changes No histopathologic changes No histopathologic changesTrastuzumabb No histopathologic changes No histopathologic changes 1 mouse with liver inflammationT-DM1c 2 mice with liver necrosis 2 mice with liver necrosis 2 mice with liver necrosis

NOTE: The first cohort of 36 C57/BL6 mice was randomly assigned into three groups to receive a single tail-vein injection of vehicle (6% sucrose, 0.02% Tween 20,and 10 mmol/L Sodium succinate), trastuzumab (29.4 mg/kg), or T-DM1 (30 mg/kg). At the indicated times, mice were sacrificed and livers were harvested forH&E staining.aLiver tissue from 1 mouse was randomly tested for H&E staining at each time point.bLiver tissues from 2 mice were randomly tested for H&E staining at each time point.cLiver tissues from 2 mice were randomly tested for H&E staining at each time point.

Table 2. T-DM1 causes hepatocellular injury in mouse liver tissues (the second cohort of mice)

Hepatocellular injury12 hours 72 hours

Control 1 of 4 mice with liver inflammation 1 of 4 mice with liver inflammationTrastuzumab 4 mice with liver inflammation No histopathologic changesT-DM1, 3 mg/kg 3 of 4 mice with liver inflammation 4 mice with liver necrosisT-DM1, 10 mg/kg 4 mice with liver necrosis 4 mice with liver necrosisT-DM1, 30 mg/kg 2 of 4 mice with liver

necrosis, 2 of 4 mice with liveinflammation and necrosis

4 mice with liver necrosis

NOTE: The second cohort of 40 C57/BL6mice was randomly assigned into five groups to receive a single tail-vein injection of vehicle, trastuzumab (29.4 mg/kg), T-DM1 (3mg/kg), T-DM1 (10mg/kg), or T-DM1 (30mg/kg). At the indicated times,micewere sacrificed, and liverswere harvested for H&E staining. All groups contained4 mice tested for H&E staining at each time point, except trastuzumab 72-hour group in which 3 mice were tested for H&E staining.

Yan et al.





microscopy study demonstrated that OMR was frequentlyobserved in T-DM1–treated liver cells, whereas control- and tras-tuzumab-treated liver cells showed no damages in cell organellestructures (Fig. 5A, red arrows). To confirm the observation fromthe mice, we also examined whether mitochondrial dysfunctioncould be found in hepatocytes treated with T-DM1. As shownin Fig. 5B, using Mitochondrial Transmembrane Potential Apo-ptosis Detection Kit, the cationic dye (fluorescence dots) accumu-lated in the cells that were not treatedwith T-DM1,whereas the dyeaccumulation in mitochondria was inhibited in cells treated withT-DM1, suggesting that T-DM1 inducedmitochondrialmembranedysfunction inhepatocytes. Taken together, thesedata indicate thatT-DM1 induced apoptosis of hepatocytes in vivo and in vitro.

DiscussionHepatotoxicity is widely regarded as the leading cause of failure

of drug development programs and a serious safety concern inclinical studies and postmarketing surveillance (36, 37). In theanalysis of drugs withdrawn for toxicity reasons in the periodbetween 1992 and 2002, hepatotoxicity was identified in 27%of cases (36). Although mechanisms underlying most instancesof drug-induced hepatotoxicity are still not well understood,different studies implicated factors such as host metabolism,detoxification, liver-regeneration, and immune response path-ways (38, 39).

Hepatotoxicity has been reported in several other ADC thera-pies currently undergoing different stages of clinical testing (22–24). In a phase I study of cantuzumab mertansine, hepatotoxicity(reversible elevations of hepatic transaminases and occasionally

alkaline phosphatase and bilirubin) was identified as a principaltoxic side effect of the therapy (22). In a phase I study ofinotuzumab ozogamicine, increased ASTs and bilirubinemiawere reported for 18.4% and 22.4% of study participants, respec-tively (23). In a phase I study of MLN2704, which is an ADCdesigned to deliver DM1 to prostate-specific membrane antigen–expressing cells, in patients with progressive metastatic castra-tion–resistant prostate cancer, abnormal hepatic functions wereidentified inmore than 20% of study participants (24). Given thesignificant clinical promise of ADC therapies, better understand-ing of the molecular basis for hepatotoxicity induced by ADC,including T-DM1, will benefit ADC clinical development pro-grams, as well as postmarketing surveillance.

Thrombocytopenia is the dose-limiting toxicity induced by T-DM1 (13, 40). A study conducted by Uppal and colleaguessuggests that human megakaryocytes (MKs) internalize T-DM1in a HER2-independent, FcgRIIa-dependent manner, resulting inintracellular release of DM1 (40). However, the mechanism ofDM1 release remains elusive as the authors were unable to detectcolocalization between the internalized T-DM1 and lysosomalLAMP1 by immunofluorescence. Thon and colleagues reportedthat T-DM1-induced thrombocytopenia occurred via a mecha-nism that is both HER2- and FcgRIIa-independent due to the factthat mouse MKs and platelets do not express HER2 and FcgRIIaand that both takeupT-DM1(41). This study suggests that T-DM1is endocytosed through an alternative pathway (41). Although itis still a matter of debate whether trastuzumab associates withmouseHER2,many laboratories have beenusingmousemodel toinvestigate the mechanisms of trastuzumab-induced cardiotoxi-city, and data from these studies suggest that trastuzumab-

Control Trastuzumab

T-DM1 T-DM1

A B

DC

Figure 4.T-DM1 treatment inducesinflammation and necrosis in mouseliver. Liver tissues obtained from themice (first cohort) treated withvehicle control (A), trastuzumab (B),and T-DM1 (C and D) were collectedfor H&E staining. Green arrows,inflammation; yellow arrows, necrosis.Bar, 50 mm.






induced cardiotoxicity isHER2dependent (25–29).Weusedbothcellular and mouse model systems to investigate a clinicallyrelevant case of drug-induced hepatotoxicity and to elucidate thepossible mechanisms of T-DM1–induced hepatotoxicity. Wefound thatHER2was expressed inmouse and humanhepatocytesas well as mouse liver and that T-DM1 was able to associate withmouse and human HER2, although binding affinity of trastuzu-mabor T-DM1 tomouseHER2 is lower than that of humanHER2.

We also demonstrated that T-DM1 was capable of mediatingendocytosis and that the internalized T-DM1 colocalized withlysosomal LAMP1, induced destabilization of microtubules, andinhibited hepatocyte growth, indicating that DM1was released inlysosomes and that the released T-DM1 induced cytotoxicity inhepatocytes. According to the public resource of data (www.proteinatlas.org), FcgRIIa, FcgRIIb, and FcgRIIIa are not expressedin hepatocytes (42), suggesting that T-DM1 internalization is

B

mt

mtmt

mt

A TrastuzumabControl

T-DM1

mt

mt

mt

Enlarged

T-DM1Control

Figure 5.T-DM1 induces the OMR andmitochondrial membrane (mt)potential dysfunction. A, electronmicroscopy images of liver tissuesfrom mice treated with control,trastuzumab, (29.4 mg/kg), or T-DM1(30 mg/kg). Red arrows, OMR. Bar,1 mm. B, images of a cationic dyeaccumulated in cells (punctatedfluorescence dots); left, image fromthe cells (THLE2) left untreated; right,image from the cells treated withT-DM1 (20 mg/mL). Bar, 50 mm.

Yan et al.





unlikely mediated by Fc receptors. On the basis of the data wehave, we propose a novel mechanism by which T-DM1 directlytargets hepatocytes via HER2 contributing to T-DM1–inducedhepatotoxicity.

Results from our cellular model provide scientific rationaleto use mouse as a relevant animal model to further investigatemechanisms of T-DM1–induced hepatotoxicity. Our animaldata reveal some important findings. First of all, T-DM1–induced hepatotoxicity is dose dependent. Second, trastuzu-mab was found to induce inflammation, but not necrosis, inliver tissue. Trastuzumab-induced inflammation was recoveredwithin three days, except one mouse in the first cohort (Table1B). Third, T-DM1–induced hepatocellular injury containsboth inflammation and necrosis (H&E staining) with signifi-cant liver function damage, including increase in serum levelsof ALT, AST, and LDH. Fourth, TNFa gene expression in livertissue is significantly increased in mice treated with T-DM1 ascompared with those treated with trastuzumab or vehicle.TNFa is a proinflammatory cytokine capable of inducing apo-ptosis. Cytokine-mediated proapoptotic signaling is an impor-tant component in the pathophysiology of drug-induced liverinjury (43, 44). It has been reported that TNFa severelyenhances liver damage caused by various xenobiotics(43, 45–47) and is the major cytokine to be excreted by theliver stationary macrophages (Kupffer cells) in response tohepatocyte damage (33). It is possible that T-DM1–mediatedsecretion of TNFa, which activates proapoptotic signalingpathway, significantly enhances the liver damage that is ini-tially caused by DM1-mediated intracellular stress. Themechanisms by which T-DM1 induces TNFa-mediated apopto-sis are further supported by our EM study in that T-DM1induces the OMR, an indication of mitochondrial-dependentapoptosis. Our work sheds new light on the mechanism bywhich T-DM1 induces hepatotoxicity, which may yield novelstrategies to manage T-DM1, as well as other ADCs-inducedliver toxicity.

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

DisclaimerThis article reflects the views of the author and should not be construed to

represent FDA's views or policies. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Authors' ContributionsConception and design:H. Yan, P. Mukhopadhyay, B. Gao, P. Pacher, W.J. WuDevelopment of methodology: H. Yan, Y. Endo, M. Dokmanovic, W.J. WuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):H. Yan, Y. Endo, Y. Shen, M. Dokmanovic, N. Mohan,W.J. WuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Yan, Y. Endo, Y. Shen, D. Rotstein, M. Dokma-novic, B. Gao, W.J. WuWriting, review, and/or revision of the manuscript: H. Yan, Y. Endo,D. Rotstein, M. Dokmanovic, N. Mohan, B. Gao, P. Pacher, W.J. WuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. YanStudy supervision: P. Mukhopadhyay, P. Pacher, W.J. Wu

AcknowledgmentsThe authors thank Drs. Tao Wang and Eric Hales of FDA for critical internal

review of this article. The authors also thank Dr. George Kunos, the scientificdirector of NIAAA and NIH for his support.

Grant SupportThis study is supported by the Interagency Oncology Task Force Joint

Fellowship Program sponsored by the FDA and NCI. This work was alsosupported by the Food and Drug Administration Office of Women's Healthand in part by an appointment to the ORISE Research Participation Program atthe Center for Drug Evaluation and Research, FDA, administered by the OakRidge Institute for Science and Education through an interagency agreementbetween the U.S. Department of Energy and FDA. Dr. Nishant Mohan is anORISE research fellow supported by Food and Drug Administration Office ofWomen's Health.

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 July 13, 2015; revised December 2, 2015; accepted December 4,2015; published OnlineFirst December 28, 2015.

References1. Ducry L, Stump B. Antibody-drug conjugates: linking cytotoxic payloads to

monoclonal antibodies. Bioconjug Chem 2010;21:5–13.2. Giordano SH, Temin S, Kirshner JJ, Chandarlapaty S, Crews JR, David-

son NE, et al. Systemic therapy for patients with advanced humanepidermal growth factor receptor 2-positive breast cancer: AmericanSociety of Clinical Oncology clinical practice guideline. J Clin Oncol2014;32:2078–99.

3. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, et al.Targeting HER2-positive breast cancer with trastuzumab-DM1, an anti-body-cytotoxic drug conjugate. Cancer Res 2008;68:9280–90.

4. Remillard S, Rebhun LI,HowieGA, Kupchan SM. Antimitotic activity of thepotent tumor inhibitor maytansine. Science 1975;189:1002–05.

5. Junttila TT, Li G, Parsons K, Phillips GL, Sliwkowski MX. Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab andefficiently inhibits growth of lapatinib insensitive breast cancer. BreastCancer Res Treat 2011;128:347–56.

6. Krop I, Winer EP. Trastuzumab emtansine: a novel antibody-drug conju-gate for HER2-positive breast cancer. Clin Cancer Res 2014;20:15–20.

7. Cassady JM, Chan KK, Floss HG, Leistner E. Recent developments in themaytansinoid antitumor agents. Chem Pharm Bull 2004;52:1–26.

8. Hudis CA. Trastuzumab–mechanism of action and use in clinical practice.N Engl J Med 2007;357:39–51.

9. Erickson HK, Lewis Phillips GD, Leipold DD, Provenzano CA, Mai E,Johnson HA, et al. The effect of different linkers on target cell catabolismand pharmacokinetics/pharmacodynamics of trastuzumab maytansinoidconjugates. Mol Cancer Ther 2012;11:1133–42.

10. Dokmanovic M, ElZarrad KM, Hirsch DS, Wu WJ. Antibody-drug con-jugates as therapeutic agents in oncology: overview and perspectives.In: Atta-ur-Rahman, M. Iqbal Choudhary, editors. Frontiers in anti-cancer drug discovery. Oak Park, IL: Bentham Science Publishers; 2013.p.139–89.

11. Senter PD. Potent antibody drug conjugates for cancer therapy. Curr OpinChem Biol 2009;13:235–44.

12. Boyraz B, Sendur MA, Aksoy S, Babacan T, Roach EC, Kizilarslanoglu MC,et al. Trastuzumab emtansine (T-DM1) for HER2-positive breast cancer.Curr Med Res Opin 2013;29:405–14.

13. Krop IE, LoRusso P, Miller KD, Modi S, Yardley D, Rodriguez G, et al. Aphase II study of trastuzumab emtansine in patients with human epider-mal growth factor receptor 2-positive metastatic breast cancer who werepreviously treated with trastuzumab, lapatinib, an anthracycline, a taxane,and capecitabine. J Clin Oncol 2012;30:3234–41.

14. Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, et al. Trastu-zumab emtansine for HER2-positive advanced breast cancer. N Engl J Med2012;367:1783–91.






15. Di�eras V,HarbeckN, BuddGT, Greenson JK, Guardino AE, SamantM, et al.Trastuzumab emtansine in human epidermal growth factor receptor 2-positivemetastatic breast cancer: an integrated safety analysis. J ClinOncol2014;32:2750–7.

16. Voutsadakis IA. Gemtuzumab Ozogamicin (CMA-676, Mylotarg) for thetreatment of CD33þ acute myeloid leukemia. Anticancer Drugs 2002;13:685–92.

17. Tanimoto T, TsubokuraM,Mori J, PietrekM,OnoS, KamiM.Differences indrug approval processes of 3 regulatory agencies: a case study of gemtu-zumab ozogamicin. Invest New Drugs 2013;31:473–8.

18. Rowe JM, L€owenberg B. Gemtuzumab ozogamicin in acute myeloidleukemia: a remarkable saga about an active drug. Blood 2013;121:4838–41.

19. McKoy JM, Angelotta C, Bennett CL, Tallman MS, Wadleigh M, Evens AM,et al. Gemtuzumab ozogamicin-associated sinusoidal obstructive syn-drome (SOS): an overview from the research on adverse drug events andreports (RADAR) project. Leuk Res 2007;31:599–604.

20. Maniecki MB, Hasle H, Bendix K, Møller HJ. Is hepatotoxicity in patientstreated with gemtuzumabozogamicine due to specific targeting ofhepatocytes? Leuk Res 2011;35:e84–6.

21. Larson RA, Sievers EL, Stadtmauer EA, L€owenberg B, Estey EH, Dombret H,et al. Final report of the efficacy and safety of gemtuzumab ozogamicin(Mylotarg) in patients with CD33-positive acute myeloid leukemia in firstrecurrence. Cancer 2005;104:1442–52.

22. Tolcher AW, Ochoa L, Hammond LA, Patnaik A, Edwards T, Takimoto C,et al. CantuzumabMertansine, amaytansinoid immunoconjugate directedto the CanAg antigen: a Phase I, pharmacokinetic, and biologic correlativestudy. J Clin Oncol 2003;21:211–22.

23. KantarjianH, ThomasD, Jorgensen J, Kebriaei P, Jabbour E, RyttingM, et al.Results of inotuzumab ozogamicin, a CD22 monoclonal antibody, inrefractory and relapsed acute lymphocytic leukemia. Cancer 2013;119:2728–36.

24. Galsky MD, Eisenberger M, Moore-Cooper S, Kelly WK, Slovin SF, DeLa-Cruz A, et al. Phase I trial of the prostate-specific membrane antigen-directed immunoconjugate MLN2704 in patients with progressive castra-tion-resistant prostate cancer. J Clin Oncol 2008;26:2147–54.

25. Jassal DS, Han SY, Hans C, Sharma A, Fang T, Ahmadie R, et al. Utility oftissueDoppler and strain rate imaging in the early detection of trastuzumaband anthracycline mediated cardiomyopathy. J Am Soc Echocardiogr2009;22:418–24.

26. RiccioG, EspositoG, Leoncini E, Contu R, Condorelli G, ChiarielloM, et al.Cardiotoxic effects, or lack thereof, of anti-ErbB2 immunoagents. FASEB J2009;23:3171–8.

27. Walker JR, Sharma A, LytwynM, Bohonis S, Thliveris J, Singal PK, et al. Thecardioprotective role of probucol against anthracycline and trastuzumab-mediated cardiotoxicity. J Am Soc Echocardiogr 2011;24:699–705.

28. Milano G, Raucci A, Scopece A, Daniele R, Guerrini U, Sironi L, et al.Doxorubicin and trastuzumab regimen induces biventricular failure inmice. J Am Soc Echocardiogr 2014;27:568–79.

29. ElZarradMK, Mukhopadhyay P, Mohan N, Hao E, Dokmanovic M, HirschDS, et al. Trastuzumab alters the expression of genes essential for cardiacfunction and induces ultrastructural changes of cardiomyocytes in mice.PLoS One 2014;8:e79543.

30. Poon KA, Flagella K, Beyer J, Tibbitts J, Kaur S, Saad O, et al. Preclinicalsafety profile of trastuzumab emtansine (T-DM1): mechanism of action of

its cytotoxic component retained with improved tolerability. Toxicol ApplPharmacol 2013;273:298–313.

31. BilzerM, Roggel F, Gerbes AL. Role of Kupffer cells in host defense and liverdisease. Liver Int 2006;26:1175–86.

32. Adams DH, Ju C, Ramaiah SK, Uetrecht J, Jaeschke H. Mechanisms ofimmune-mediated liver injury. Toxicol Sci 2010;115:307–21.

33. Roberts RA, Ganey PE, Ju C, Kamendulis LM, Rusyn I, Klauning JE. Role ofthe Kupffer cell in mediating hepatic toxicity and carcinogenesis. ToxicolSci 2007;96:2–15.

34. Ramachandran R, Kakar S. Histological patterns in drug-induced liverdisease. J Clin Pathol 2014;62:481–92.

35. Sesso A, Beliz�ario JE, Marques MM, Higuchi ML, Schumacher RI,Colquhoun A, et al. Mitochondrial swelling and incipient outer mem-brane rupture in preapoptotic and apoptotic cells. Anat Rec 2012;295:1647–59.

36. Schuster D, Laggner C, Langer T. Why drugs fail-a study on side effects innew chemical entities. Curr Pharm Des 2005;11:3545–59.

37. Hewitt M, Enoch SJ,Madden JC, Przybylak KR, CroninMT.Hepatotoxicity:a scheme for generating chemical categories for read-across, structural alertsand insights into mechanism(s) of action. Crit Rev Toxicol 2013;43:537–58.

38. Erickson HK, Park PU, WiddisonWC, Kovtun YV, Garrett LM, Hoffman K ,et al. Antibody-maytansinoid conjugates are activated in targeted cancercells by lysosomal degradation and linker-dependent intracellular proces-sing. Cancer Res 2006;66:4426–33.

39. Tujios S, Fontana RJ. Mechanisms of drug-induced liver injury: frombedside to bench. Nat Rev Gastroenterol Hepatol 2011;8:202–11.

40. Uppal H, Doudement E, Mahapatra K, Darbonne WC, Bumbaca D,Shen BQ , et al. Potential mechanisms for thrombocytopenia devel-opment with trastuzumab emtansine (T-DM1). Clin Cancer Res 2015;21:123–33.

41. Thon JN,DevineMT, JurakBegonja A, Tibbitts J, Italiano JE Jr.High-contentlive-cell imaging assay used to establish mechanism of trastuzumabemtansine (T-DM1)–mediated inhibition of platelet production. Blood2012;120:1975–84.

42. Uhl�en M, Bj€orling E, Agaton C, Szigyarto CA, Amini B, Andersen E, et al. Ahuman protein atlas for normal and cancer tissues based on antibodyproteomics. Mol Cell Proteomics 2005;4:1920–32.

43. Fredriksson L, Herpers B, Benedetti G, Matadin Q, Puigvert JC, deBont H,et al. Diclofenac inhibits tumor necrosis factor-a-induced nuclear factor-kBactivation causing synergistic hepatocyte apoptosis. Hepatology 2011;53:2027–41.

44. Roth RA, Ganey PE. Animal models of idiosyncratic drug induced liverinjury-current status. Crit Rev Toxicol 2011;41:723–39.

45. Cosgrove BD, Alexopoulos LG, Hang TC, Hendriks BS, Sorger PK, GriffithLG, et al. Cytokine-associated drug toxicity in human hepatocytes isassociated with signaling network dysregulation Mol Biosyst 2010;6:1195–206.

46. Lu J, Jones AD, Harkema JR, Roth RA, Ganey PE. Amiodaroneexposure during modest inflammation induces idiosyncrasy-like liverinjury in rats: role of tumor necrosis factor-alpha. Toxicol Sci 2012;125:126–33.

47. Shaw PJ, Hopfensperger MJ, Ganey PE, Roth RA. Lipopolysaccharide andtrovafloxacin coexposure in mice causes idiosyncrasy-like liver injurydependent on tumor necrosis factor-alpha. Toxicol Sci 2007;100:259–66.


Yan et al.




2016;15:480-490. Published OnlineFirst December 28, 2015.Mol Cancer Ther Haoheng Yan, Yukinori Endo, Yi Shen, et al. Epidermal Growth Factor Receptor 2 to Induce HepatotoxicityAdo-Trastuzumab Emtansine Targets Hepatocytes Via Human

Updated version

10.1158/1535-7163.MCT-15-0580doi:

Access the most recent version of this article at:

Cited articles

http://mct.aacrjournals.org/content/15/3/480.full#ref-list-1

This article cites 46 articles, 14 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/15/3/480.full#related-urls

This article has been cited by 3 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/15/3/480To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-15-0580

http://mct.aacrjournals.org/content/15/3/480.full#ref-list-1

http://mct.aacrjournals.org/content/15/3/480.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/15/3/480


ado-trastuzumab emtansine targets ...for the treatment of her2-positive metastatic breast cancer in...

Documents