breast cancer drug trastuzumab induces cardiac toxicity ... · known as cancer (rakoff-nahoum,...

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Breast Cancer Drug Trastuzumab Induces Cardiac Toxicity:

Evaluation of HER2 as a Potential Diagnostic and Prognostic Marker

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2018-0005.R1

Manuscript Type: Review

Date Submitted by the Author: 08-Feb-2018

Complete List of Authors: Johnson, Taylor; University of Central Florida Burnett School of Biomedical

Sciences, Division of Metabolic and Cardiovascular Sciences Singla, Dinender; University of Central Florida,

Is the invited manuscript for consideration in a Special

Issue: IACS Orlando

Keyword: HER2; Breast Cancer; Trastuzumab; Trastuzumab Emtansine; Cardiac Toxicity

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Breast Cancer Drug Trastuzumab Induces Cardiac Toxicity: Evaluation of HER2

as a Potential Diagnostic and Prognostic Marker

Taylor A. Johnson and Dinender K. Singla

Division of Metabolic and Cardiovascular Sciences, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, 32816

Running Title : HER2 Therapy and Cardiovascular Complications Address for Correspondance :

Dinender K. Singla, PhD, FAHA, FIACS Division of Metabolic and Cardiovascular Sciences Burnett School of Biomedical Sciences College of Medicine University of Central Florida 4110 Libra Drive Orlando, FL 32816, USA Email: [email protected] Phone: 407-823-0953 Fax: 407-823-0956

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Abstract

Breast cancer is one of the most prevalent forms of cancer in the United States

and worldwide. Cancer occurs through the uncontrolled development of new abnormal

cell growth. Clinicians and researchers strive to improve diagnostics and treatments in

pursuit of remedying breast cancer, while limiting or removing any potential side effects

that may arise. Unfortunately, traditional treatments, such as anthracyclines (i.e.

Doxorubicin) can damage the cardiovascular system. Recent strategies have utilized

antibody-based compounds as singular treatments, or in conjunction with other

treatments, with the aim to minimize side effects. The Human Epidermal Growth Factor

Receptor 2 (HER2) protein has been the target of numerous antibody-based breast

cancer therapies, such as Trastuzumab (TZM) and Trastuzumab Emtansine (T-DM1).

This review will discuss the HER2 receptor as a diagnostic marker in targeting breast

cancer using the therapeutic agents TZM and T-DM1, as well as discuss the induced

cardiac toxicity following TZM and T-DM1 treatments.

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Keywords:

HER2; Breast Cancer; Trastuzumab; Trastuzumab Emtansine; Cardiac Toxicity

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Abbreviations

ASCO (American Society of Clinical Oncology)

ALT (Alanine Transaminase)

AST (Aspartate Transaminase)

CAP (College of American Pathologists)

CE (Cardiac Event)

Doxo (Doxorubicin)

EGFR (Epidermal Growth Factor Receptor)

FDA (Food and Drug Administration)

HER (Human Epidermal Growth Factor Receptor)

HF (Heart Failure)

IHC (Immunohistochemistry)

ISH (In Situ Hybridization)

LVEF (Left Ventricular Ejection Fraction)

PI3K (Phosphoinositide 3-Kinase)

RT-PCR (Real-time Polymerase Chain Reaction)

SMCC (N-Succinimidyl 4-(Maleimidomethyl) Cyclohexane-1-Carboxylate)

T-DM1 (Trastuzumab Emtansine)

TK (Tyrosine Kinase)

TZM (Trastuzumab)

VEGF (Vascular Endothelial Growth Factor)

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Introduction

Normal tissue function requires that body cells maintain homeostatic conditions

through normal cell division, mediated by apoptosis (Seitz et al., 2000). Unfortunately, in

certain circumstances, such as in genetic disorders, cells can divide in an uncontrolled

manner, leading to new abnormal cell growth (Rakoff-Nahoum, 2006). This group of

new and atypical cells form a mass of tissue called a malignant tumor, more commonly

known as cancer (Rakoff-Nahoum, 2006). Many different types of cancers can be

developed in the body (American Cancer Society & Atlanta, 2017). The most prevalent

types of cancers, according to incidence and mortality statistics reported by the

American Cancer Society, are lung cancer, colon-rectal cancer, pancreatic cancer,

prostate cancer, and breast cancer (specifically in females) (American Cancer Society &

Atlanta, 2017).

Breast cancer is considered to be a heterogeneous disease in which

characterization depends on the cellular involvement, diversity between tumors,

appearance of certain markers, and development of metastasis (Redig & McAllister,

2013). Therefore, the complexity of breast cancer development can influence the

decision of which treatment options are best suitable for an individual patient. The most

common treatment options include surgery (removal of the cancerous tissue), radiation

(killing cancerous cells using x-ray), and chemotherapy (chemical treatment to kill

cancer cells) (Baskar et al., 2014). Unfortunately, these treatments significantly affect

the normal cell populations that reside adjacent to the cancer cells as well as in different

tissues, such as the heart (Baskar et al., 2014). Therefore, recent research efforts have

aimed to identify breast cancer cell specific markers. In this review, we will discuss the

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breast cancer cell marker, human epidermal growth factor receptor 2 (HER2), in

addition to methods of breast cancer diagnosis, treatment options, the mechanism of

anti-breast cancer antibody treatment, and cardiotoxicity that results from anti-breast

cancer treatment.

Human Epidermal Growth Factor Receptor 2

Human Epidermal Growth Factor Receptor 2 (HER2/erbB2), a 185-kD

transmembrane protein in the epidermal growth factor receptor (EGFR) family of

tyrosine kinases (TKs), is a receptor that helps regulate normal cell growth, division, and

repair when needed (Dai et al., 2016). However, when the HER2 gene is not working

correctly and expresses aberrant levels of the HER2 protein, it promotes breast cancer

due to uncontrolled cell growth and division (Dai et al., 2016).

HER2 has also been shown to play a role in cardiovascular development and

regulation. Studies have documented HER2 in the development of the fetal

cardiovascular system, dilated cardiomyopathy prevention, ventricular muscle and valve

development, cardiomyocyte survival, and sympathovagal coordination (Okoshi et al.,

2004;Camenisch et al., 2002;Crone et al., 2002;Ozcelik et al., 2002;Zhao et al., 1998).

Overexpression of HER2 has been shown to lower reactive oxygen species in

cardiomyocytes and H9C2 cardiomyoblast cells, and has been suggested to play a role

in antioxidant defenses (Belmonte et al., 2015). HER-activated cell survival networks

normally protect the heart from agents that would cause cardiovascular damage (De

Keulenaer et al., 2010). In fact, HER2-deficient mice demonstrated left ventricular

dysfunction, chamber dilation, decreased contractility, and increased susceptibility to

drug toxicity (Crone et al., 2002). In addition to cardiac cells, gastric cells (Abrahao-

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Machado & Scapulatempo-Neto, 2016) and esophageal cells (Huang et al., 2013) were

also observed to have the HER2 receptor.

HER2 oncogene expression is amplified in 25-30% of human primary breast

cancers (Slamon et al., 1989) and expresses nearly 1-2 million copies of the receptor

per breast cancer tumor cell (Lewis Phillips et al., 2008). HER2 is commonly linked to

cell survival pathways such as the phosphoinositide-3-kinase (PI3K) pathway (Yuan &

Cantley, 2008). Predominance of the HER2 receptor on cancer cells makes it a strong

candidate to be used as a common diagnostic marker in breast cancer detection assays

(Weigel & Dowsett, 2010). Furthermore, scientists have developed monoclonal

antibodies against the HER2 receptor in order to restrict breast cancer cell growth.

HER2 as a Diagnostic Marker

There have been challenges with proper diagnosis of breast cancer. In 2002, the

National Surgical Adjuvant Breast and Bowel Project (NSABP) generated the B-31

HER2 protocol to screen patients for study eligibility. 18% of the assays used were

inconclusive using HercepTest™ immunohistochemistry (IHC) or fluorescent in situ

hybridization (ISH) (Paik et al., 2002). In addition, smaller laboratories were shown to

produce over-reaching positive results, resulting in inconsistent data (Paik et al., 2002).

To address these inconsistencies in diagnosis, a joint panel, coordinated by the

American Society of Clinical Oncology (ASCO) and the College of American

Pathologists (CAP), constructed guidelines in 2007 that included improved instructions

on performing interpretation of IHC and ISH assays. The guidelines and protocols were

revised in 2013 by ASCO and CAP due to the emergence of new testing methodology,

such as bright-field in situ hybridization and real-time polymerase chain reaction (RT-

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PCR), which warranted an updated review of the diagnostic procedures (Wolff et al.,

2013). While the committee deemed these new DNA and mRNA techniques beneficial

for clinical utility, they required validation with a Food and Drug Administration (FDA)

approved assay as a confirmatory means of diagnosis (Wolff et al., 2013). HER2 testing

is a critical component necessary to guide clinicians in the decision to prescribe

therapeutics in a more personalized way, by targeting HER2 either specifically or non-

specifically. The first method outlined by ASCO and CAP in the selection criterion of

histological diagnostic principles makes use of an immunohistochemistry protocol; a

procedure in which an antibody specific for the HER2 protein is used to identify

expression as shown in Figure 1 (Wolff et al., 2013). In this procedure, tissue isolated

from a patient’s primary tumor or metastatic site is fixed for preservation, and

subsequently stained for HER2 expression. The staining intensity allows for

classification of the patient’s specimen into one of four histological grades (IHC 0, 1+,

2+, 3+) that can be used to establish whether the patient is considered positive or

negative for HER2 expression, and if additional testing is necessary. The process for

this classification system is outlined in Figure 1. In this method, the ASCO and CAP

guidelines define the patient as positive when the biopsy demonstrates intense

circumferential cell staining of HER2 on greater than 10% of the biopsied tumor cells

(Figure 1) (Wolff et al., 2013). Alternatively, when there is no or incomplete

circumferential staining with faint intensity, the patient is considered HER2 negative

(Wolff et al., 2013). This classification system is clinically significant because it provides

a method of analysis that is standardized across all clinical providers; therefore,

decreasing the likelihood of false-positives and administration of non- HER2 specific

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and/or potentially harmful therapies. In addition to IHC, ASCO and CAP have defined

criterion for a second diagnostic test using in situ hybridization. The procedure for this

additional diagnostic differential method is shown in Figure 2. This test can be used

either singularly or in combination with IHC to support a diagnosis. ISH utilizes a DNA

probe that facilitates quantification of the number of HER2 copies present within the cell

nucleus, termed as a single probe for HER2, shown in the bottom left section of Figure

2. The single probe ISH method quantifies the number of cells positive for the HER2

signal in analysis of twenty non-overlapping cells within the patient specimen. If the

biopsy results in an average of ≥6.0 signals/cell among the quantified cells, the patient

is reported as positive and therapies most effective against HER2 cancers are

significantly considered (Figure 2). However, when the average number of signals is <4,

the patient is reported as negative and non- HER2 specific therapies may prove more

effective (Figure 2). The guidelines also outline the use of a dual probe for patient

specimens that fall within the intermediate range of a ≥ 4.0 and <6.0 signal average

(Figure 2). This classification is reported as equivocal, meaning that it requires

additional testing, such as use of a dual probe ISH test. The dual probe targets HER2 in

addition to a centromeric probe for chromosome 17 (CEP17), a HER2 genotypic

abnormality that results in polysomy of chromosome 17 in cancer cells (Page et al.,

2017). The ratio derived from the HER2 probe signal to CEP17 can subsequently

classify a patient sample as HER2 positive and further clarify treatment options (Wolff et

al., 2013).

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HER2 Treatment with Trastuzumab (TZM)

Trastuzumab (Herceptin) (Genentech, Inc.), a monoclonal antibody used to treat

HER2 receptor positive patients, was approved by the United States FDA for cancer

therapy in 1998 (Slamon et al., 2001;Smith et al., 2007). Structurally, TZM contains two

antigen-specific sites that bind to the HER2 extracellular domain (Albanell et al., 1996).

Patients prescribed TZM every 3 weeks for one year following chemotherapy

demonstrated an increase in disease-free survival (Piccart-Gebhart et al., 2005). In

addition, tumor regression of up to 60% was exhibited in patients given weekly

Trastuzumab doses after 3 weeks of single agent treatment (Mohsin et al., 2005).

Mechanism of TZM treatment in Cancer Cells

Recent in vitro and in vivo studies support TZM treatment of cancer cells,

resulting in down modulation of HER2, dimerization prevention, reverse cytokine

resistance, tumor growth inhibition, and impairment of vascular endothelial growth factor

(VEGF) expression (Izumi et al., 2002;Ritter et al., 2007;Sliwkowski et al.,

1999;Valabrega et al., 2007). Moreover, TZM has also been shown to exhibit

multifactorial effects resulting in cancer growth inhibition such as inhibition of ligand-

independent HER2 signaling, activation of antibody dependent cell-mediated

cytotoxicity (ADCC), inhibition of extracellular domain (ECD) (Figure 3) (Clynes et al.,

2000;Cooley et al., 1999), co-expression of EGFR and/or EGFR ligands (Ritter et al.,

2007;Dua et al., 2010;El-Sahwi et al., 2010;Junttila et al., 2009;Tse et al., 2012), and

modification of angiogenesis and properties of vasculature(Izumi et al., 2002;Petit et al.,

1997;Viloria-Petit et al., 2001).

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Mechanisms of TZM Treatment in Cardiac Cells

TZM treatment significantly induces cardiac toxicity as a major side effect. It has

been reported that HER2 receptor is necessary for cardiac development and

cardiomyocyte cell survival, as we mentioned in this paper previously (Crone et al.,

2002). Therefore, treatment with TZM inhibits HER2 receptor function to attenuate

cancer cell growth, but unfortunately also inhibits HER2 receptor presence on healthy

cardiomyocytes in the heart which subsequently induces mitochondrial dysfunction,

oxidative stress and apoptosis (Figure 3) (Cardinale et al., 2010;Gordon et al., 2009).

Development of oxidative stress and apoptosis has been well documented in various

heart diseases in the progression of left ventricular dysfunction, ultimately leading to

heart failure (Hansel et al., 2010;Chien, 2006;Levine, 2005;Singal & Iliskovic, 1998).

Therefore, presence of increased oxidative stress and cardiomyocyte apoptosis with

TZM treatment also leads to heart dysfunction in these cancer patients as a side effect

(Figure 3), as reported elsewhere (ElZarrad et al., 2013;Hahn et al., 2014). Moreover,

the exact mechanisms of cardiotoxicity induced by TZM is complex and not very well

established; however, various other mechanisms leading to cell death has also been

reported such as damage to myofibers and reduction of myofiber thickness (ElZarrad et

al., 2013), hindering neuregulin function (Sandoo et al., 2015), inhibiting autophagy

(Mohan et al., 2016), stimulating oxidative stress (Sandoo et al., 2015;Dirican et al.,

2014;Mohan et al., 2016), inhibiting the PI3K/AKT pathway (Junttila et al., 2009;Mohsin

et al., 2005) and several cytoskeletal proteins (i.e. ALC1, MLCA2a, Connectin) and

transcription factors (i.e. ATF3, DBP) (ElZarrad et al., 2013). TZM treatment develops

into acute and chronic heart dysfunction. Therefore, development of strategies for early

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detection of cardiac dysfunction following TZM treatment would reduce hospitalizations,

and avoid development of chronic heart dysfunction.

Acute Cardiac Dysfunction and Early Detection to Reduce Disease Burden

Cardiotoxicity has been reported to occur in 3-36% of patients that receive TZM-

based chemotherapy (Srikanthan et al., 2017). TZM treatment hinders heart contractility

by interfering with the QT interval and ventricular polarization (Hansel et al., 2010). A

recent study by Dirican et al (2014) observed that patients, post-TZM administration,

had a slower QT interval (.398 seconds pre-TZM vs. .430 seconds post-TZM), which

indicates ventricular depolarization and repolarization dysfunction; however, the QRS

complex duration was unchanged (Dirican et al., 2014). LVEF also decreased from 65%

to 60% upon TZM administration (Dirican et al., 2014). In a separate study, ElZarrad

(2013) revealed that TZM administration in C57BL/6 mice decreased left ventricular

posterior wall thickness, LVEF, and fractional shortening, yet little changes occurred in

the left ventricular diastolic and systolic diameter (ElZarrad et al., 2013). A third study

revealed 28% of TZM administered patients developed cardiotoxicity and registered

significant reductions in LVEF, representative of decreased cardiac function (Baron et

al., 2014). Specifically, patients that registered between a LVEF baseline of 60.8 ±6.9 %

declined over one year to 57.68 ±8.4 % (Baron et al., 2014). Further, TZM

administration has a long serum half-life (Leyland-Jones et al., 2003), which may

contribute to the higher prevalence of cardiotoxicity (2.5 fold) in TZM patients compared

to non-TZM patients (Viani et al., 2007). African-Americans had a higher risk of

developing cardiotoxicity, however more studies are necessary to evaluate susceptibility

in races and genders (Baron et al., 2014). Collectively, it can be inferred that TZM

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treatment promotes the development of cardiac contractility events with high incidence

and reduces LVEF function; however, early detection of TZM induced cardiac

dysfunction can decrease the risk of developing chronic heart dysfunction leading to

heart failure.

Recent studies show that TZM induced left ventricular cardiac dysfunction

leading to chronic heart dysfunction is actually reversible (Zeglinski et al., 2011;Nair &

Gongora, 2016;Wadhwa et al., 2009) if proper management is provided to the patients.

Standard methods to detect chemotherapy related cardiac dysfunction (CRCD) include

the use of echocardiography and presence of clinical symptoms. The use of

echocardiography determines structural damage which may not have been clearly

developed in the early stage of the disease development, and therefore may be

reversible once the TZM regimen is stopped (Onitilo et al., 2014;Jiji et al., 2012).

However, this acute dysfunction may damage the cardiomyocytes in the myocardium

which then start releasing specific proteins and lead to apoptosis. In this case, it is too

late for reversal. Therefore, implementing the use of biomarker panels is becoming a

newly established method for early detection of CRCD (Srikanthan et al., 2017).

Moreover, after TZM treatment cardiac troponin I has been shown to increase in animal

models and humans (ElZarrad et al., 2013;Zeglinski et al., 2011) which could be

harnessed as a future ELISA target to detect cardiac dysfunction developing in the

heart. Identification of the level of cardiac dysfunction following TZM treatment will allow

for optimization of TZM administration to significantly reduce potential of long-term side-

effects and severe consequences.

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Transtuzumab Induced Chronic Heart Dysfunction

Several studies have documented the severity of cardiac events (CE) by

assigning grades and reporting changes in LVEF that occur during treatment. Guarneri

(2006) revealed 28% of patients in a metastatic breast cancer study reported

experiencing a CE; 15.6% experienced a grade 2 CE (asymptomatic with 40-50%

LVEF), and 10.4% experienced a grade 3 CE (congestive heart failure with 40-50%

LVEF) (Guarneri et al., 2006). Furthermore, in a 2 year follow-up to the Herceptin

Adjuvant (HERA) trial, eleven percent of patients prescribed TZM for one year

experienced a grade 3 or 4 cardiac event as defined by the New York Heart Association

as symptomatic congestive heart failure with LVEF below 50% and a 10% decrease

from baseline (Smith et al., 2007). Eight years post-HERA, approximately 1/3 of the

patients that were discontinued from the trial were due to a cardiac-related disorder,

including congestive heart failure and decreased LVEF (de et al., 2014). In addition, 8

years post-HERA significant decreases in LVEF were noted in 4.1% and 7.2% of

patients prescribed 1 and 2 years of TZM treatment, respectively. Interestingly, patients

that previously demonstrated significant reductions of LVEF (less than 50%) showed

recovery of LVEF over time, suggesting some reversibility (de et al., 2014).

Further studies were conducted to evaluate cardiac toxicity in elderly patients. In

a study of 68,536 elderly breast cancer patients, 10% (6,829) had experienced

cardiomyopathy or HF (Tsai et al., 2014). Importantly, use of TZM is associated with a

two-fold increase in cardiac disorders in patients at 66 years of age and older,

regardless of the stage of the disease in the patients’ diagnosis, history of hypertension,

or prior drug treatment (i.e. anthracyclines, taxanes). This study alludes to the potency

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of utilizing TZM in elderly patients. A separate study reports approximately 9% of TZM-

treated patients 70 years of age and older developed symptomatic HF (Serrano et al.,

2012). These sets of data suggest that TZM is an excellent cell specific anti-breast

cancer drug, though it still can cause significant acute and chronic heart dysfunction.

Cardiac Toxicity with Modified TZM Drug Trastuzumab Entansine (T-DM1)

Although TZM is considered the standard therapy for HER2 breast cancer, nearly

40% of patients do not respond to the current regimens and patients develop

cardiotoxicity (Marty et al., 2005). The exact reason for ineffectiveness of TZM and

developed cardiotoxicity is not clearly understood. Recently, a new drug called

Trastuzumab Emtansine (T-DM1) was developed through the combination of two

components: (a) a monoclonal antibody against HER2 receptor and (b) a cytotoxic

agent.

T-DM1 is constructed using the antibody, TZM, and a maytansinoid, DM1. T-

DM1 is a larger, hydrophilic molecule, that requires cells to undergo endocytosis for

drug activity (Dieras & Bachelot, 2014). T-DM1 requires N-Succinimidyl 4-

(Maleimidomethyl) Cyclohexane-1-Carboxylate (SMCC), a cross-linking agent that

creates a thioester bond (Lewis Phillips et al., 2008;Burris, III et al., 2011) allowing the

antibody to release the drug upon tumor site arrival (Xie & Blattler, 2006). TZM acts as

the delivery vehicle for DM1, which acts independently of HER2 signaling to have a

cytotoxic effect (Dieras & Bachelot, 2014). DM1 is a derivative of maytansine, which has

been previously recognized as an anticancer drug that inhibits microtubule construction,

induces cell cycle arrest, and stimulates apoptosis (Dieras & Bachelot, 2014;Cassady et

al., 2004;Chari, 2008). DM1 has been shown to be 25-270 times more potent than

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common chemotherapy agents, such as Paclitaxel, and 180-4000 times stronger than

Doxorubicin (Doxo) (Junttila et al., 2011). In addition, T-DM1 has shown tumor growth

inhibition and regression in the in vivo model 30 days post treatment in mouse

mammary tumor virus (MMTV)-HER2 carrying mice (Lewis Phillips et al., 2008). These

initial results suggest T-DM1 could be a more potent and targeted treatment option in

comparison to TZM alone.

Due to the recent approval of T-DM1 (2013, compared to TZM approval in 1998),

little is known about its adverse effects. However, thus far data shows modified HER2

treatment has reduced cardiac toxicity (Lewis Phillips et al., 2008). Currently, most

studies evaluate T-DM1 in replacement of TZM or in combination with another drug.

Since T-DM1 contains Trastuzumab, the FDA requires patients treated with T-DM1

have baseline and regular assessments during treatment. Current studies have shown

decline in LVEF due to T-DM1 treatment (Callahan, 2014;Dieras et al., 2014).

Additionally, T-DM1 administration has shown to stimulate other complications in

patients. T-DM1 was administered in a clinical trial to 148 early stage breast cancer

(EBC) patients, comprising of many ages, races, and geographical locations (Krop et

al., 2015). Patients were administered T-DM1 approximately 1 year after anthracycline-

based chemotherapy. 31.8% of patients reported epistaxis and 21.6% reported

thrombocytopenia. Hypertension was also reported in 5.4% of patients. Grade 3 events

documented in patients included thrombocytopenia (8.1%), neutropenia (5.4%), and

increases in aspartate and alanine transaminases (AST and ALT) (both at 7.4%) (Krop

et al., 2015).

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Telangiectasia has been documented in multiple clinical studies. One case study

evaluated a 43-year-old woman who developed telangiectasia accompanied with

gingival bleeding, nasal mucosal bleeding, and rectal mucosal bleeding (Kwon et al.,

2016). Echocardiography revealed normal left ventricular function, but right ventricular

dilation and flattening of the interventricular septum. Although catheterization further

confirmed severe pulmonary hypertension, multiple procedures ruled out other causes

of the hypertension. Upon exiting the T-DM1 trial, her telangiectasia significantly

improved; however, she continued to have breathing problems (Kwon et al., 2016). A

separate study documented 5 different female patients, ranging from 43 to 66 in age,

which developed telangiectasia post-initial treatment with T-DM1 (Sibaud et al., 2014).

Additionally, multiple groups prescribed singular treatments or mixed therapies

that included TZM or T-DM1. The combined therapy of TZM, and cyclophosphamide (an

anthracycline) revealed cardiotoxicity in 27% of patients in addition to anemia (35%)

and leukopenia (52%). In contrast, the group that received TZM and Paclitaxel had the

lowest cardiovascular complications (13%, 14%, and 24%, respectively) (Slamon et al.,

2001). A separate study revealed that cardiovascular complications were high in

patients prescribed either Docetaxel or a combined TZM-Docetaxel treatment (Marty et

al., 2005). The percentage of patients that experienced epistaxis was higher by 15% in

patients prescribed TZM and Docetaxel compared to Docetaxel alone. In addition, more

TZM-Docetaxel patients experienced Grade 3 and 4 leukopenia or neutropenia, as well

as reductions in LVEF (Marty et al., 2005).

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These results support the potency and superiority of therapies containing multiple

treatments in reducing HER2 breast cancer; however, the combination of therapies

could result in developing cardiovascular and other complications.

Future Perspectives

In conclusion, Trastuzumab and Trastuzumab Emtansine should be considered a

treatment option for patients with HER2 breast cancer; however, each treatment results

in complications. At this time, TZM has been well documented to hinder contractility of

the heart, in particular reduction LVEF. T-DM1, on the other hand, is relatively new and

requires more clinical and non-clinical studies to further understand its potential side

effects. Although both treatments have been documented to promote hematological

pathologies, such as neutropenia, T-DM1 treatment is strongly correlated with

thrombocytopenia and elevated transaminases, AST and ALT. Further testing is

necessary to evaluate how these antibody-based treatments influence cardiovascular

function, induce these pathologies, and determine whether damage can be minimized

or reversed over time.

Marker based trials have been previously proposed as a method to define

patients that would benefit from dual-therapy treatments (Blank et al., 2010;Bonastre et

al., 2012). To determine cardiotoxicity following TZM treatment, additional biomarkers

specific for heart proteins such as cardiac troponin I (cTnI), cardiac myosin light chain 1

(cMLC1), myeloperoxidase (MPO), placental growth factor (PIGF), and growth

differentiation factor 15 (GDF-15) need to be evaluated in order to understand their role

in pathogenesis prior to the development of cardiac dysfunction and subsequent heart

failure (ElZarrad et al., 2013;Putt et al., 2015). In fact, a recent study revealed 62% of

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patients that developed TZM-induced cardiotoxicity had elevated Troponin I levels

(Cardinale et al., 2010;Fallah-Rad et al., 2011). These biomarkers should be combined

with physiological measurements in order to determine which parameter can first, or

more accurately, predict drug-induced cardiotoxicity. In a recent editorial utilizing the

echocardiography parameter, left atrium global longitudinal strain (LAGLS) was shown

to decrease in both Doxo and TZM induced toxicity prior to a decrease in LVEF (Moreno

et al., 2016).

Exosomes, small 30-100 nm cell-derived vesicles, have recently become the

subject of many research efforts. These vesicles have been utilized as vehicles to

deliver a variety of cargo, including numerous anti-cancer drugs (i.e. Doxo) (Batrakova

& Kim, 2015;Johnsen et al., 2014;Tavakoli et al., 2017). The contents of exosomes are

also of interest, as they contain a variety of miRNAs, siRNAs, and proteins, which could

play a beneficial role in inhibition/reversal of disease progression (Batrakova & Kim,

2015;Johnsen et al., 2014). Collectively, the roles of exosomes provide an exciting area

of further research that warrants investigation into their use as a delivery method to

reduce cardiac toxicity or as a combination therapy approach with TZM or TZM-DM1.

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Acknowledgements

The authors would like to thank Kaley Garner and Zahra Tavakoli Dargani for technical

assistance in preparing the manuscript.

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Disclosures

There are no conflicts of interest to report.

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Reference List

Abrahao-Machado LF & Scapulatempo-Neto C (2016). HER2 testing in gastric cancer: An update. World J Gastroenterol 22, 4619-4625.

Albanell J, Bellmunt J, Molina R, Garcia M, Caragol I, Bermejo B, Ribas A, Carulla J, Gallego OS, Espanol T, & Sole Calvo LA (1996). Node-negative breast cancers with p53(-)/HER2-neu(-) status may identify women with very good prognosis. Anticancer Res 16, 1027-1032.

American Cancer Society & Atlanta G. Cancer Facts & Figure 2017. 2017. Ref Type: Pamphlet

Baron KB, Brown JR, Heiss BL, Marshall J, Tait N, Tkaczuk KH, & Gottlieb SS (2014). Trastuzumab-induced cardiomyopathy: incidence and associated risk factors in an inner-city population. J Card Fail 20, 555-559.

Baskar R, Dai J, Wenlong N, Yeo R, & Yeoh KW (2014). Biological response of cancer cells to radiation treatment. Front Mol Biosci 1, 24.

Batrakova EV & Kim MS (2015). Using exosomes, naturally-equipped nanocarriers, for drug delivery. J Control Release 219, 396-405.

Belmonte F, Das S, Sysa-Shah P, Sivakumaran V, Stanley B, Guo X, Paolocci N, Aon MA, Nagane M, Kuppusamy P, Steenbergen C, & Gabrielson K (2015). ErbB2 overexpression upregulates antioxidant enzymes, reduces basal levels of reactive oxygen species, and protects against doxorubicin cardiotoxicity. Am J Physiol Heart Circ Physiol 309, H1271-H1280.

Blank PR, Dedes KJ, & Szucs TD (2010). Cost effectiveness of cytotoxic and targeted therapy for metastatic breast cancer: a critical and systematic review. Pharmacoeconomics 28, 629-647.

Bonastre J, Jan P, Barthe Y, & Koscielny S (2012). Metastatic breast cancer: we do need primary cost data. Breast 21, 384-388.

Page 22 of 35



Draft

23

Burris HA, III, Tibbitts J, Holden SN, Sliwkowski MX, & Lewis Phillips GD (2011). Trastuzumab emtansine (T-DM1): a novel agent for targeting HER2+ breast cancer. Clin Breast Cancer 11, 275-282.

Callahan R (2014). Ado-Trastuzumab Emtansine in Metastatic HER2-Positive Breast Cancer. J Adv Pract Oncol 5, 134-139.

Camenisch TD, Schroeder JA, Bradley J, Klewer SE, & McDonald JA (2002). Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors. Nat Med 8, 850-855.

Cardinale D, Colombo A, Torrisi R, Sandri MT, Civelli M, Salvatici M, Lamantia G, Colombo N, Cortinovis S, Dessanai MA, Nole F, Veglia F, & Cipolla CM (2010). Trastuzumab-induced cardiotoxicity: clinical and prognostic implications of troponin I evaluation. J Clin Oncol 28, 3910-3916.

Cassady JM, Chan KK, Floss HG, & Leistner E (2004). Recent developments in the maytansinoid antitumor agents. Chem Pharm Bull (Tokyo) 52, 1-26.

Chari RV (2008). Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res 41, 98-107.

Chien KR (2006). Herceptin and the heart--a molecular modifier of cardiac failure. N Engl J Med 354, 789-790.

Clynes RA, Towers TL, Presta LG, & Ravetch JV (2000). Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med 6, 443-446.

Cooley S, Burns LJ, Repka T, & Miller JS (1999). Natural killer cell cytotoxicity of breast cancer targets is enhanced by two distinct mechanisms of antibody-dependent cellular cytotoxicity against LFA-3 and HER2/neu. Exp Hematol 27, 1533-1541.

Crone SA, Zhao YY, Fan L, Gu Y, Minamisawa S, Liu Y, Peterson KL, Chen J, Kahn R, Condorelli G, Ross J, Jr., Chien KR, & Lee KF (2002). ErbB2 is essential in the prevention of dilated cardiomyopathy. Nat Med 8, 459-465.

Dai X, Xiang L, Li T, & Bai Z (2016). Cancer Hallmarks, Biomarkers and Breast Cancer Molecular Subtypes. J Cancer 7, 1281-1294.

Page 23 of 35



Draft

24

De Keulenaer GW, Doggen K, & Lemmens K (2010). The vulnerability of the heart as a pluricellular paracrine organ: lessons from unexpected triggers of heart failure in targeted ErbB2 anticancer therapy. Circ Res 106, 35-46.

de AE, Procter MJ, van Veldhuisen DJ, Agbor-Tarh D, Metzger-Filho O, Steinseifer J, Untch M, Smith IE, Gianni L, Baselga J, Jackisch C, Cameron DA, Bell R, Leyland-Jones B, Dowsett M, Gelber RD, Piccart-Gebhart MJ, & Suter TM (2014). Trastuzumab-associated cardiac events at 8 years of median follow-up in the Herceptin Adjuvant trial (BIG 1-01). J Clin Oncol 32, 2159-2165.

Dieras V & Bachelot T (2014). The success story of trastuzumab emtansine, a targeted therapy in HER2-positive breast cancer. Target Oncol 9, 111-122.

Dieras V, Harbeck N, Budd GT, Greenson JK, Guardino AE, Samant M, Chernyukhin N, Smitt MC, & Krop IE (2014). Trastuzumab emtansine in human epidermal growth factor receptor 2-positive metastatic breast cancer: an integrated safety analysis. J Clin Oncol 32, 2750-2757.

Dirican A, Levent F, Alacacioglu A, Kucukzeybek Y, Varol U, Kocabas U, Senoz O, Emren SV, Demir L, Coban E, Aksun S, Sutcu R, & Tarhan MO (2014). Acute cardiotoxic effects of adjuvant trastuzumab treatment and its relation to oxidative stress. Angiology 65, 944-949.

Dua R, Zhang J, Nhonthachit P, Penuel E, Petropoulos C, & Parry G (2010). EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance. Breast Cancer Res Treat 122, 685-697.

El-Sahwi K, Bellone S, Cocco E, Cargnelutti M, Casagrande F, Bellone M, Abu-Khalaf M, Buza N, Tavassoli FA, Hui P, Silasi DA, Azodi M, Schwartz PE, Rutherford TJ, Pecorelli S, & Santin AD (2010). In vitro activity of pertuzumab in combination with trastuzumab in uterine serous papillary adenocarcinoma. Br J Cancer 102, 134-143.

ElZarrad MK, Mukhopadhyay P, Mohan N, Hao E, Dokmanovic M, Hirsch DS, Shen Y, Pacher P, & Wu WJ (2013). Trastuzumab alters the expression of genes essential for cardiac function and induces ultrastructural changes of cardiomyocytes in mice. PLoS One 8, e79543.

Fallah-Rad N, Walker JR, Wassef A, Lytwyn M, Bohonis S, Fang T, Tian G, Kirkpatrick ID, Singal PK, Krahn M, Grenier D, & Jassal DS (2011). The utility of cardiac biomarkers, tissue velocity and strain imaging, and cardiac magnetic resonance imaging in predicting early left ventricular dysfunction in patients with human epidermal growth

Page 24 of 35



Draft

25

factor receptor II-positive breast cancer treated with adjuvant trastuzumab therapy. J Am Coll Cardiol 57, 2263-2270.

Gordon LI, Burke MA, Singh AT, Prachand S, Lieberman ED, Sun L, Naik TJ, Prasad SV, & Ardehali H (2009). Blockade of the erbB2 receptor induces cardiomyocyte death through mitochondrial and reactive oxygen species-dependent pathways. J Biol Chem 284, 2080-2087.

Guarneri V, Lenihan DJ, Valero V, Durand JB, Broglio K, Hess KR, Michaud LB, Gonzalez-Angulo AM, Hortobagyi GN, & Esteva FJ (2006). Long-term cardiac tolerability of trastuzumab in metastatic breast cancer: the M.D. Anderson Cancer Center experience. J Clin Oncol 24, 4107-4115.

Hahn VS, Lenihan DJ, & Ky B (2014). Cancer therapy-induced cardiotoxicity: basic mechanisms and potential cardioprotective therapies. J Am Heart Assoc 3, e000665.

Hansel TT, Kropshofer H, Singer T, Mitchell JA, & George AJ (2010). The safety and side effects of monoclonal antibodies. Nat Rev Drug Discov 9, 325-338.

Huang JX, Zhao K, Lin M, Wang Q, Xiao W, Lin MS, Yu H, Chen P, & Qian RY (2013). HER2 gene amplification in esophageal squamous cell carcinoma is less than in gastroesophageal junction and gastric adenocarcinoma. Oncol Lett 6, 13-18.

Izumi Y, Xu L, di TE, Fukumura D, & Jain RK (2002). Tumour biology: herceptin acts as an anti-angiogenic cocktail. Nature 416, 279-280.

Jiji RS, Kramer CM, & Salerno M (2012). Non-invasive imaging and monitoring cardiotoxicity of cancer therapeutic drugs. J Nucl Cardiol 19, 377-388.

Johnsen KB, Gudbergsson JM, Skov MN, Pilgaard L, Moos T, & Duroux M (2014). A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy. Biochim Biophys Acta 1846, 75-87.

Junttila TT, Akita RW, Parsons K, Fields C, Lewis Phillips GD, Friedman LS, Sampath D, & Sliwkowski MX (2009). Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 15, 429-440.

Page 25 of 35



Draft

26

Junttila TT, Li G, Parsons K, Phillips GL, & Sliwkowski MX (2011). Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer. Breast Cancer Res Treat 128, 347-356.

Krop IE, Suter TM, Dang CT, Dirix L, Romieu G, Zamagni C, Citron ML, Campone M, Xu N, Smitt M, & Gianni L (2015). Feasibility and cardiac safety of trastuzumab emtansine after anthracycline-based chemotherapy as (neo)adjuvant therapy for human epidermal growth factor receptor 2-positive early-stage breast cancer. J Clin Oncol 33, 1136-1142.

Kwon Y, Gomberg-Maitland M, Pritzker M, & Thenappan T (2016). Telangiectasia and Pulmonary Arterial Hypertension Following Treatment With Trastuzumab Emtansine: A Case Report. Chest 149, e103-e105.

Levine MN (2005). Trastuzumab cardiac side effects: only time will tell. J Clin Oncol 23, 7775-7776.

Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, Blattler WA, Lambert JM, Chari RV, Lutz RJ, Wong WL, Jacobson FS, Koeppen H, Schwall RH, Kenkare-Mitra SR, Spencer SD, & Sliwkowski MX (2008). Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res 68, 9280-9290.

Leyland-Jones B, Gelmon K, Ayoub JP, Arnold A, Verma S, Dias R, & Ghahramani P (2003). Pharmacokinetics, safety, and efficacy of trastuzumab administered every three weeks in combination with paclitaxel. J Clin Oncol 21, 3965-3971.

Marty M, Cognetti F, Maraninchi D, Snyder R, Mauriac L, Tubiana-Hulin M, Chan S, Grimes D, Anton A, Lluch A, Kennedy J, O'Byrne K, Conte P, Green M, Ward C, Mayne K, & Extra JM (2005). Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group. J Clin Oncol 23, 4265-4274.

Mohan N, Shen Y, Endo Y, ElZarrad MK, & Wu WJ (2016). Trastuzumab, but Not Pertuzumab, Dysregulates HER2 Signaling to Mediate Inhibition of Autophagy and Increase in Reactive Oxygen Species Production in Human Cardiomyocytes. Mol Cancer Ther 15, 1321-1331.

Mohsin SK, Weiss HL, Gutierrez MC, Chamness GC, Schiff R, Digiovanna MP, Wang CX, Hilsenbeck SG, Osborne CK, Allred DC, Elledge R, & Chang JC (2005).

Page 26 of 35



Draft

27

Neoadjuvant trastuzumab induces apoptosis in primary breast cancers. J Clin Oncol 23, 2460-2468.

Moreno J, Garcia-Saez JA, Clavero M, Manganaro R, Moreno F, Lopez J, Macaya C, & Perez d, I (2016). Effect of breast cancer cardiotoxic drugs on left atrial myocardium mechanics. Searching for an early cardiotoxicity marker. Int J Cardiol 210, 32-34.

Nair N & Gongora E (2016). Heart failure in chemotherapy-related cardiomyopathy: Can exercise make a difference? BBA Clin 6, 69-75.

Okoshi K, Nakayama M, Yan X, Okoshi MP, Schuldt AJ, Marchionni MA, & Lorell BH (2004). Neuregulins regulate cardiac parasympathetic activity: muscarinic modulation of beta-adrenergic activity in myocytes from mice with neuregulin-1 gene deletion. Circulation 110, 713-717.

Onitilo AA, Engel JM, & Stankowski RV (2014). Cardiovascular toxicity associated with adjuvant trastuzumab therapy: prevalence, patient characteristics, and risk factors. Ther Adv Drug Saf 5, 154-166.

Ozcelik C, Erdmann B, Pilz B, Wettschureck N, Britsch S, Hubner N, Chien KR, Birchmeier C, & Garratt AN (2002). Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy. Proc Natl Acad Sci U S A 99, 8880-8885.

Page DB, Wen H, Brogi E, Dure D, Ross D, Spinelli KJ, Patil S, Norton L, Hudis C, & McArthur HL (2017). Monosomy 17 in potentially curable HER2-amplified breast cancer: prognostic and predictive impact. Breast Cancer Res Treat.

Paik S, Bryant J, Tan-Chiu E, Romond E, Hiller W, Park K, Brown A, Yothers G, Anderson S, Smith R, Wickerham DL, & Wolmark N (2002). Real-world performance of HER2 testing--National Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst 94, 852-854.

Petit AM, Rak J, Hung MC, Rockwell P, Goldstein N, Fendly B, & Kerbel RS (1997). Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol 151, 1523-1530.

Page 27 of 35



Draft

28

Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M, Inbar M, Lichinitser M, Lang I, Nitz U, Iwata H, Thomssen C, Lohrisch C, Suter TM, Ruschoff J, Suto T, Greatorex V, Ward C, Straehle C, McFadden E, Dolci MS, & Gelber RD (2005). Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353, 1659-1672.

Putt M, Hahn VS, Januzzi JL, Sawaya H, Sebag IA, Plana JC, Picard MH, Carver JR, Halpern EF, Kuter I, Passeri J, Cohen V, Banchs J, Martin RP, Gerszten RE, Scherrer-Crosbie M, & Ky B (2015). Longitudinal Changes in Multiple Biomarkers Are Associated with Cardiotoxicity in Breast Cancer Patients Treated with Doxorubicin, Taxanes, and Trastuzumab. Clin Chem 61, 1164-1172.

Rakoff-Nahoum S (2006). Why cancer and inflammation? Yale J Biol Med 79, 123-130.

Redig AJ & McAllister SS (2013). Breast cancer as a systemic disease: a view of metastasis. J Intern Med 274, 113-126.

Ritter CA, Perez-Torres M, Rinehart C, Guix M, Dugger T, Engelman JA, & Arteaga CL (2007). Human breast cancer cells selected for resistance to trastuzumab in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network. Clin Cancer Res 13, 4909-4919.

Sandoo A, Kitas GD, & Carmichael AR (2015). Breast cancer therapy and cardiovascular risk: focus on trastuzumab. Vasc Health Risk Manag 11, 223-228.

Seitz CS, Freiberg RA, Hinata K, & Khavari PA (2000). NF-kappaB determines localization and features of cell death in epidermis. J Clin Invest 105, 253-260.

Serrano C, Cortes J, De Mattos-Arruda L, Bellet M, Gomez P, Saura C, Perez J, Vidal M, Munoz-Couselo E, Carreras MJ, Sanchez-Olle G, Tabernero J, Baselga J, & Di CS (2012). Trastuzumab-related cardiotoxicity in the elderly: a role for cardiovascular risk factors. Ann Oncol 23, 897-902.

Sibaud V, Niec RE, Schindler K, Busam KJ, Roche H, Modi S, Delord JP, & Lacouture ME (2014). Ado-trastuzumab emtansine-associated telangiectasias in metastatic breast cancer: a case series. Breast Cancer Res Treat 146, 451-456.

Singal PK & Iliskovic N (1998). Doxorubicin-induced cardiomyopathy. N Engl J Med 339, 900-905.

Page 28 of 35



Draft

29

Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, & . (1989). Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244, 707-712.

Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, & Norton L (2001). Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344, 783-792.

Sliwkowski MX, Lofgren JA, Lewis GD, Hotaling TE, Fendly BM, & Fox JA (1999). Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). Semin Oncol 26, 60-70.

Smith I, Procter M, Gelber RD, Guillaume S, Feyereislova A, Dowsett M, Goldhirsch A, Untch M, Mariani G, Baselga J, Kaufmann M, Cameron D, Bell R, Bergh J, Coleman R, Wardley A, Harbeck N, Lopez RI, Mallmann P, Gelmon K, Wilcken N, Wist E, Sanchez RP, & Piccart-Gebhart MJ (2007). 2-year follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer: a randomised controlled trial. Lancet 369, 29-36.

Srikanthan K, Klug R, Tirona M, Thompson E, Visweshwar H, Puri N, Shapiro J, & Sodhi K (2017). Creating a Biomarker Panel for Early Detection of Chemotherapy Related Cardiac Dysfunction in Breast Cancer Patients. J Clin Exp Cardiolog 8.

Tavakoli DZ, Singla R, Johnson T, Kukreja R, & Singla DK (2017). Exosomes derived from embryonic stem cells inhibit doxorubicin and inflammation-induced pyroptosis in muscle cells. Can J Physiol Pharmacol 1-4.

Tsai HT, Isaacs C, Fu AZ, Warren JL, Freedman AN, Barac A, Huang CY, & Potosky AL (2014). Risk of cardiovascular adverse events from trastuzumab (Herceptin((R))) in elderly persons with breast cancer: a population-based study. Breast Cancer Res Treat 144, 163-170.

Tse C, Gauchez AS, Jacot W, & Lamy PJ (2012). HER2 shedding and serum HER2 extracellular domain: biology and clinical utility in breast cancer. Cancer Treat Rev 38, 133-142.

Valabrega G, Montemurro F, & Aglietta M (2007). Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol 18, 977-984.

Page 29 of 35



Draft

30

Viani GA, Afonso SL, Stefano EJ, De Fendi LI, & Soares FV (2007). Adjuvant trastuzumab in the treatment of her-2-positive early breast cancer: a meta-analysis of published randomized trials. BMC Cancer 7, 153.

Viloria-Petit A, Crombet T, Jothy S, Hicklin D, Bohlen P, Schlaeppi JM, Rak J, & Kerbel RS (2001). Acquired resistance to the antitumor effect of epidermal growth factor receptor-blocking antibodies in vivo: a role for altered tumor angiogenesis. Cancer Res 61, 5090-5101.

Wadhwa D, Fallah-Rad N, Grenier D, Krahn M, Fang T, Ahmadie R, Walker JR, Lister D, Arora RC, Barac I, Morris A, & Jassal DS (2009). Trastuzumab mediated cardiotoxicity in the setting of adjuvant chemotherapy for breast cancer: a retrospective study. Breast Cancer Res Treat 117, 357-364.

Weigel MT & Dowsett M (2010). Current and emerging biomarkers in breast cancer: prognosis and prediction. Endocr Relat Cancer 17, R245-R262.

Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, Allred DC, Bartlett JM, Bilous M, Fitzgibbons P, Hanna W, Jenkins RB, Mangu PB, Paik S, Perez EA, Press MF, Spears PA, Vance GH, Viale G, & Hayes DF (2013). Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 31, 3997-4013.

Xie H & Blattler WA (2006). In vivo behaviour of antibody-drug conjugates for the targeted treatment of cancer. Expert Opin Biol Ther 6, 281-291.

Yuan TL & Cantley LC (2008). PI3K pathway alterations in cancer: variations on a theme. Oncogene 27, 5497-5510.

Zeglinski M, Ludke A, Jassal DS, & Singal PK (2011). Trastuzumab-induced cardiac dysfunction: A 'dual-hit'. Exp Clin Cardiol 16, 70-74.

Zhao YY, Sawyer DR, Baliga RR, Opel DJ, Han X, Marchionni MA, & Kelly RA (1998). Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes. J Biol Chem 273, 10261-10269.

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Figure Legends

Figure 1: Immunohistochemistry (IHC) based HER2 specific diagnostic procedure

to identify breast cancer patients.

Tissue is initially isolated from a patient that has been clinically diagnosed with breast

cancer using several different methods such as core needle biopsy, extraction of

cytologic specimens, and resection. Upon isolation, the tissue is fixed for 6 to 72 hours

in a 10% neutral buffered formalin solution within 1 hour of isolation. The tissue is then

sectioned and prepared for immunohistochemistry staining using a primary antibody to

the HER2 protein. When the circumferential staining pattern is not observed or is

incomplete with ≤10% of tumor cells positive for HER2, the patient is considered to be

negative and the histological score reported as IHC 0. Similarly, when there is faint or

incomplete staining with HER2 expression in >10% of tumor cells, the patient is

considered to be negative and reported as IHC 1+. Upon faint or moderate staining with

>10% of cells positive for HER2 or complete circumferential staining with ≤10% of cells

positive for HER2, the patient is considered to have equivocal expression which

warrants confirmation by repeating IHC with a new sample or performing an additional

test such as ISH. When circumferential staining is strong and >10% of cells are positive

for HER2, the patient is considered HER2 positive and assigned a histological score of

3+. If the tissue sample has any apparent histopathologic discordance, either IHC or

another HER2 test must be repeated on a new sample.

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Figure 2: In situ Hybridization (ISH) based HER2 Assay to diagnose breast cancer

patients.

In situ hybridization is used to determine the number of HER2 gene signals per cell

nucleus using a DNA probe. Initially, a patient diagnosed clinically with breast cancer

undergoes a procedure to isolate tissue from a primary tumor or metastatic site.

Subsequent to tissue fixation, a single or double probe can be used to determine HER2

signal or HER2 signal in relation to CEP17, respectively. Using the single probe

method, 20 non-overlapping cells are quantified for an average HER2 signal/cell. When

this value is <4 the sample is reported as negative. A value equal to or greater than 6 is

considered positive. When the average HER2 signal is between these values (4 or

greater, but less than 6), it is considered equivocal and a dual probe or IHC test needs

to be performed to confirm. The dual probe method analyzes the presence of HER2 in

addition to CEP17. HER2/C17 ratios ≥2.0 are considered to be positive. HER2/C17

ratios <2 are further classified based on whether HER2 signal is <4. A HER2/C17 ratio

<2 accompanied by a HER2 signal <4 is reported as negative. A HER2/C17 ratio <2

with a HER2 signal >4 and <6 is considered equivocal and requires ISH to be repeated

or confirmed with an additional test.

Figure 3. Flow diagram shows proposed mechanisms of action using Trastuzumab in

the induction of cardiotoxicity (left side) and anti-cancer effects on cancer cells (right

side). (ADCC: Antibody-dependent cell-mediated cytotoxicity, EDC: Extracellular

Domain)

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Equivocal HER2

Expression (IHC 2+)

Negative HER2

Expression (IHC 1+) Negative HER2

Expression (IHC 0)

Positive HER2

Expression (IHC 3+)

No observed staining

OR

Incomplete

circumferential staining;

Faint intensity;

≤ 10% of tumor cells

Incomplete


Faint/Moderate intensity;

> 10% of tumor cells

OR

Complete


Strong intensity;

≤ 10% of tumor cells

Complete


Strong intensity;


Figure 1. Johnson and

Singla 2018

Incomplete


Faint intensity;


Patient Clinically Diagnosed with Breast Cancer

Isolation of tissue from the primary tumor or metastatic site for

HER2 protein expression

(Examples of tissue isolation: Core needle biopsy, cytologic specimens,

resection specimens)

Tissue is fixed within 1 hour of isolation in 10%

neutral buffered formalin for 6 to 72 hours

Immunohistochemistry Staining (IHC)

Use of antibody to analyze HER2 Protein

Expression

Consideration of performing

additional HER2 tests or

repeating IHC on a new sample

Patient is HER2 Negative

(Consideration of Treatment options

non-specific for HER2)

Patient is HER2 Positive

(Consideration of

Treatment options

specific for HER2)

Apparent

histopathologic

discordance

Perform New

IHC or ISH

HER2 test

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Singla 2018 Patient Clinically Diagnosed with Breast Cancer

Isolation of tissue from the primary tumor or metastatic site for

HER2 gene expression

(Examples of Tissue isolation: Core needle biopsy, cytologic specimens,

resection specimens)

In situ Hybridization (ISH)

Determines the number of HER2 copies per

nucleus using a DNA probe

(Examples: Bright-Field ISH and FISH assay)

Tissue is fixed within 1 hour of isolation in 10%

neutral buffered formalin for 6 to 72 hours

Dual Probe for

HER2 and CEP17

Average HER2

Signal/Cell:

< 4.0

Average HER2

Signal/Cell:

≥6.0

HER2/CEP17

Ratio: ≥2.0

or

HER2/CEP17

Ratio: <2.0

with

HER2

Signal/Cell: ≥ 6.0

Genetic

Aneusomy of

Chromosome 17

(CEP17)

Quantification of 20

Non-overlapping cells

Report as Negative

Report as Positive

Quantification of 20

Non-overlapping cells

Report as Positive

Report as Negative

Repeat on new

specimen or

perform IHC

Average HER2

Signal/Cell:

≥ 4.0 and <6.0

Equivocal

Perform Dual Probe

ISH or IHC to Confirm

Single Probe for

HER2 Signal

HER2/C17

Ratio: <2.0

with

HER2 Signal/Cell:

< 4.0

Equivocal

HER2/CEP17

Ratio: <2.0

With HER2

Signal/Cell:≥ 4.0

and <6.0

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Trastuzumab

(HER2 Receptor Antibody)

HER2 Receptor on Cardiomyocytes HER2 Receptor on

Cancer Cells

• Impaired Myocardial Genes

• Impaired Mitochondrial Function

• Damages Myofibers

• Increased Oxidative Stress

• Inhibits Ligand-independent HER2

Signaling

• Activates ADCC

• Prevents HER2 ECD Shedding

Apoptosis of Cardiomyocytes Apoptosis of Cancer Cells

Heart Dysfunction / Cardiotoxicity Anti-Cancer Effects


Singla 2018

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breast cancer drug trastuzumab induces cardiac toxicity ... · known as cancer (rakoff-nahoum,...

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