Layer-specific distribution of myocardial deformation from anthracycline-induced cardiotoxicity in patients with breast cancer—From bedside to bench

IJCA-28292; No of Pages 7 International Journal of Cardiology xxx (xxxx) xxx

Contents lists available at ScienceDirect

International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Layer-specific distribution of myocardial deformation from anthracycline-induced cardiotoxicity in patients with breast cancer— From bedside to bench Wei-Ting Chang a,b,c, Yin-Hsun Feng d, Yu Hsuan Kuo d, Wei-Yu Chen d, Hong-Chang Wu d, Chien-Tai Huang d, Tzu-Ling Huang a, Zhih-Cherng Chen a,e,⁎ a

Department of Cardiology, Chi Mei Medical Center, Tainan, Taiwan Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan c Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan d Division of Oncology, Department of Internal Medicine, Chi-Mei Medical Center, Tainan, Taiwan e Department of Pharmacy, Chia Nan University of Pharmacy & Science, Tainan, Taiwan b

a r t i c l e

i n f o

Article history: Received 28 July 2019 Received in revised form 9 October 2019 Accepted 15 January 2020 Available online xxxx Keywords: Cancer therapy-related cardiac dysfunction Sub-endocardium Strain Breast cancer

a b s t r a c t Background: Anthracycline anticancer drugs such as epirubicin and doxorubicin may induce myocardial dysfunction, leading to poor prognosis. Early detection of minor left ventricular (LV) myocardial dysfunction is important for the prevention of anthracylcine-induced cardiotoxicity. Using layer-specific speckle tracking echocardiography (STE), we investigated the progressive distribution of myocardial dysfunction in both breast cancer patients and an animal toxicity model. Methods: Patients with preserved LV ejection fraction (LVEF) preparing for epirubicin chemotherapy (N = 125) were prospectively enrolled. Layer-specific STE, including LV longitudinal and circumferential strains on subepicardium and subendocardium, were evaluated at baseline and after the first cycle, third cycle and six months of epirubicin therapy. A decline of LVEF above 10% to b55% at six months was defined as cardiotoxicity. These same strain measures were obtained in doxorubicin-treated rats and the distribution of myocardial fibrosis evaluated. Results: In patients developing cardiotoxicity, LV longitudinal strain on subendocardium (LVLSendo) was significantly reduced after three cycles of therapy despite no significant changes in conventional LV systolic, diastolic parameters as well as LV circumferential strains at that moment. Compared to conventional echocardiographic parameters, LVLSendo was significantly predictive of cardiotoxicity. Declines in LVLSendo were also observed in doxorubicin-treated rats at an early stage. These reductions also predicted significant fibrosis in the subendocardial layer. Conclusion: LVLSendo is useful for the early detection of minor cardiac dysfunction during chemotherapy, thereby implicating endocardial involvement in the development of cardiotoxicity. © 2020 Elsevier B.V. All rights reserved.

1. Introduction With the advances in cancer therapies, the number of cancer survivors continues to increase. As many anticancer agents can induce cardiotoxicity, there has been a parallel increase in the number of patients with cardiovascular complications related to cancer therapy [1]. Although recurrence of cancer may be the eventual cause of death, cardiovascular disease, particularly myocardial dysfunction, may also lead to subsequent morbidity and mortality [1]. Currently, cancer therapyrelated cardiac dysfunction (CTRCD) is defined as a decline in left ventricular ejection fraction (LVEF) of at least 10% to b55% [2]. Therefore, ⁎ Corresponding author at: 901, Zhonghua Road, Yongkang District, Tainan, Taiwan. E-mail address: [email protected] (Z.-C. Chen).

most international consensus statements recommend frequent monitoring of patients receiving potentially cardiotoxic regimens for the early detection of myocardial dysfunction [1]. Compared to traditional echocardiographic parameters, speckle tracking echocardiography (STE) is superior for detecting subtle myocardial dysfunction even in the context of normal LVEF [3,4]. Among chemotherapy regimens for breast cancer, the widely used anthracyclines, including doxorubicin and Epirubicin, are potentially cardiotoxic [5]. Nevertheless, the natural course of cardiac remodeling during and after anthracycline therapy is still largely unknown, including how regional myocardial degeneration is associated with the deterioration of LVEF. Thus, upon the hypothesis that layer-specific STE could be an early detector of CTRCD, this study investigated the value of layer-specific STE in both patients and animals receiving anthracycline treatment. This insight is fundamental to

https://doi.org/10.1016/j.ijcard.2020.01.036 0167-5273/© 2020 Elsevier B.V. All rights reserved.

Please cite this article as: W.-T. Chang, Y.-H. Feng, Y.H. Kuo, et al., Layer-specific distribution of myocardial deformation from anthracyclineinduced cardiotoxicity in p..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2020.01.036

2

W.-T. Chang et al. / International Journal of Cardiology xxx (xxxx) xxx

advance our understanding regarding the pathophysiology and management of cardiac dysfunction in this population. 2. Materials and methods 2.1. Objectives The study was conducted in strict accordance with the Declaration of Helsinki on Biomedical Research involving human subjects and was approved by the local ethics committee (IRB: 10307–003). Written informed consent was obtained from each participant. One hundred and forty-two patients newly diagnosed with early-stage breast cancer at Chi-Mei Medical Center from June 2014 to March 2017 were prospectively enrolled for this study. We excluded 37 patients while 3 of them underwent or were undergoing chemo- or radiotherapy, 4 had valvular heart disease more severe than mild, 10 exhibited impaired systolic function (LVEF b50%) at baseline, 20 did not provide an adequate echocardiographic view or lost follow-up. Ultimately, 125 patients were enrolled in this study (Fig. S1). Participants received or planned to receive six to eight cycles of adjuvant Epirubicin therapy, with each cycle 21 days apart. All participants received echocardiographic evaluation before the onset of the chemotherapeutic regimen (T1), after the first cycle (T2), after three cycles (T3) and after 6 months of the regimen (T4). CTRCD was defined as a decline in LVEF of at least 10% to b55%. The end point was CTRCD at six months (T4). 2.2. Echocardiography in patients with breast cancer Standard echocardiography was performed with a 3.5-MHz multiphase-array probe and Vivid E9;GE system (Vingmed Ultrasound AS, Horten, Norway) in accordance with the recommendations of the American Society of Echocardiography [6]. In addition to LV dimension, LVEF was measured using the biplane Simpson's method from the apical 4-chamber view. Measured LV diastolic function-associated parameters included isovolumic relaxation time (IVRT), ejection time (ET), deceleration time (DT) and trans-mitral early filling velocity (E) to atrial velocity (A) ratio. Early (e’) and late (a’) annular diastolic velocities were also measured. In addition, myocardial performance index, also known as the Tei index, was calculated by the equation (IVCT+IVRT)/ET. 2.3. Multi-layer speckle tracking echocardiography analysis Standard apical 4-, 2-, and 3-chamber views were recorded in digital loops for the longitudinal strain analysis of the LV. The images were acquired at frame rates of 70–90/s. The images were analyzed off-line using computer software (EchoPAC, GE-Vingmed Ultrasound AS, Horten, Norway). As described previously [7], the endocardial border was manually traced at end-diastole. The region of interest (ROI) for strain analysis was adjusted manually. The locations of the tracking points were adjusted when necessary so that the ROI extended from endocardial to epicardial borders to encompass the myocardium, which was automatically divided into subendocardial and subepicardial layers by the software. Peak systolic longitudinal strain of subendocardial myocardium and subepicardial myocardium was derived. In addition to LV global longitudinal (LVLS) and circumferential strains (LVCS), we also focused compared subendocardial (LVLSendo; LVCSendo) and subepicardial (LVLSepi; LVCSepi) layer strains between groups. 2.4. Animal echocardiography The measurement of layer specific strains in both of humans and rats has been validated in previous literature [7]. In brief, echocardiography was performed under anesthesia with 3% isoflurane to minimize the effects on heart rate. Throughout the procedure, heart rate was maintained above 200 beats/min and recorded at a frame rate of 300–350/s using a pediatric transducer with a transmission frequency of 10 MHz

and S6 ultrasound cardiovascular system (GE-Vingmed Ultrasound AS, Horten, Norway). During scans, rats were placed in a left lateral decubitus position. Measurements included long- and short-axis views with ECG gating. Long-axis views were used for fractional shortening (FS) measurements while axial views at the papillary muscle level were used for STE analysis. In brief, end-systolic and end-diastolic LV dimensions were measured by marking the endocardial borders and measuring the area of the LV cavity. FS was defined as the difference between end-diastolic dimension and end-systolic dimension divided by end-diastolic dimension. The endocardial border was tracked and followed by defining the width of myocardium using EchoPAC software (GE-Vingmed Ultrasound AS, Horten, Norway) as described above. The myocardium was divided into subendocardial, middle, and subepicardial myocardium layers automatically and the global and layer-specific strains were subsequently displayed. Echocardiography including STE was performed at baseline and weekly. Also, body weight and survival were recorded and compared to sham (N = 4) and normal controls (N = 4). 2.5. Chronic doxorubicin rat model of cardiotoxicity Male Sprague–Dawley rats weighing 200–250 g were obtained from the Animal Center of National Cheng Kung University Medical College. The animal experiments were approved and conducted according to guidelines for the care and use of laboratory animals of Chi-Mei Medical Center (No. 100052307) and conformed to the Guide for the Care and Use of Laboratory Animals. To mimic the slowly progressive heart failure in patients developing CTRCD, according to previous report we established the chronic doxorubicin (cDOX) model by weekly intraperitoneal injection for four weeks to reach a cumulative dose of 20 mg/kg (N = 8) (Fig. S2). Echocardiography including STE was performed at baseline and weekly. Also, body weight and survival were recorded and compared to sham (N = 4) and normal controls (N = 4). 2.6. Masson's trichrome and Tunel immunohistochemistry staining After eight weeks, the rats were euthanized. The hearts were harvested, fixed overnight in 4% paraformaldehyde, and then embedded in paraffin. Serial sections were prepared at 5 μm sections and stained with Masson's trichrome for the quantification of myocardial fibrosis. Image acquisition and analysis were performed in a blinded fashion. We randomly selected ten areas to measure the ratio of fibrotic to the whole tissue areas. Also, the tissue was stained with terminal deoxynucleotidyl transferase dUTP nick end labelling (Tunel) for the detection of apoptotic cells. 2.7. Reproducibility To assess intra-observer and inter-observer variability, strain parameters were reevaluated using Bland-Altman limits of agreement and intraclass correlation coefficients. Images from 10 randomly selected patients were analyzed by two experienced readers who were blinded to previous measurements. The average value was used to test reproducibility. Intra-observer measurements yielded generally high ICC values (0.98 for LVLSendo, 0.96 for LVLSepi). Similarly, inter-observer measurements yielded high ICC values (0.96 for LVLSendo, 0.94 for LVLSepi) (Table S1). These results indicate satisfactory reproducibility of layer-specific STE for measurement of longitudinal strain value. 2.8. Statistical analysis Differences among normal control subjects and breast cancer patients before and after Epirubicin therapy were compared using Student's t-tests for normally distributed continuous variables or nonparametric tests for non-normally distributed continuous variables.

Please cite this article as: W.-T. Chang, Y.-H. Feng, Y.H. Kuo, et al., Layer-specific distribution of myocardial deformation from anthracyclineinduced cardiotoxicity in p..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2020.01.036

W.-T. Chang et al. / International Journal of Cardiology xxx (xxxx) xxx

Categorical variable were compared by χ2 tests. These same tests were used for comparing corresponding physiological and echocardiographic parameters among experimental rats (control, sham, doxorubicin groups). A p b 0.05 was considered significant for all tests. Factors with p b 0.1 were included in the univariate logistic regression analyses. Differences among layer-specific STE measurements at baseline, after one Epirubicin cycle, after three cycles, and after six months (T1–T4) were compared by one-way repeated measures ANOVA and Tukey post hoc tests for pair-wise comparisons. All analyses were performed using SPSS version 18 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Changes in clinical parameters during and following chemotherapy There was no significant difference in age, body weight/height, heart rate, blood pressure, blood glucose, lipid profile or renal function at baseline between breast cancer patients with non-cardiotoxicity and cardiotoxicity (Table 1). Among patients with breast cancer, the single

3

and cumulative doses of Epirubicin were within the recommended safety thresholds according to the current international consensus. The use of Trastuzumab and radiotherapy were also similar between the two groups. Post three cycles of Epirubicin therapies, despite slight increases of patients with hypertension, diabetes and hyperlipidemia, the changes of clinical characteristics were not significant. 3.2. Changes in echocardiographic parameters during and following chemotherapy There were no significant differences in chamber dimensions, left ventricular mass index, and LVEF between patient with noncardiotoxicity and cardiotoxicity at baseline p. The diastolic parameters E/A ratio, e’, E/e’, IVRT, and DT were also similar (Table 1). Post Epirubicin therapy for three cycles (T3), though there was slight declines of LVEF in both patients with non-cardiotoxicity (from 68.5 ± 5.7% to 64.5 ± 7.7%) and cardiotoxicity (from 69.2 ± 6.5% to 63.8 ± 8.7%, p = 0.12), the changes were not significant (Fig. 1A). Conversely, as we focused on the15 patients who developed CTRCD, the average

Table 1 The baseline clinical characteristics, conventional and layer specific speckle tracking echocardiography of breast cancer patients developing and free from cardiotoxicity at baseline (T1) and post three cycles of Epirubicin (T3). Non-Cardiotoxicity (n = 110) Baseline Clinical parameters Age (years) Body height (cm) Body weight (kg) Heart rate (bpm) Hypertension Diabetes Coronary artery disease Hyperlipidemia Smoking Renal failure ALT The use of Trastuzumab Concomitant radiotherapy

53 ± 9.6 155.2 ± 13.3 60.3 ± 12.4 84.2 ± 8.3 15 (13.6) 6 (5.4) 6 (5.4) 15 (13.6) 7 (6.3) 2 (1.8) 21.4 ± 16.6 32 (29) 32 (29.1)

Echocardiographic parameters Left atrium (cm) LVMI (g/ m2) LVIDd (cm) LVIDs (cm) LVEF (%) Changes of LVEF (%) E (cm/s) E/A e' (cm/s) E/e' IVRT (ms) DT (ms) MPI LVLS (%) Changes of LVLS (%) LVLSendo(%) Changes of LVLSendo (%) LVLSepi(%) Changes of LVLSepi (%) LVCS (%) Changes of LVCS (%) LVCSendo(%) Changes of LVCSendo (%) LVCSepi(%) Changes of LVCSepi (%)

3.5 ± 1.3 83.3 ± 29.2 4.3 ± 0.9 2.4 ± 0.9 68.5 ± 5.7 −2.3 ± 6.6 70.8 ± 18.9 0.7 ± 0.3 9.3 ± 2.2 8.6 ± 3.1 93.8 ± 20.4 201.7 ± 43.8 0.4 ± 0.2 −22.9 ± 5.1 1.7 ± 3.4 −24.2 ± 8.0 2.1 ± 6.1 −20.6 ± 8.9 2.1 ± 4.2 −25.6 ± 5.5 0.4 ± 7.1 −26.2 ± 5.7 1.6 ± 7.4 −25.0 ± 5.7 −0.2 ± 7.9

Cardiotoxicity (n = 15) Post 3 cycles of Epirubicin

155.8 ± 4.05 60.6 ± 12.8 80.6 ± 10.1 19 (17.2) 10 (9.1) 7 (6.3) 18 (16.3) 4 (3.6) 20.8 ± 12.6

3.4 ± 1.1 84.2 ± 19.7 4.0 ± 1.2 2.5 ± 1.1 64.5 ± 7.7 72.4 ± 16.8 1.0 ± 0.4 8.4 ± 3.2 8.6 ± 3.4 93.8 ± 23.1 198.3 ± 57.8 0.4 ± 0.1 −20.7 ± 7.4 −22.1 ± 8.4 −19.6 ± 7.9 −25.1 ± 6.7 −24.5 ± 5.7 −25.6 ± 5.2

Baseline 55.3 ± 12.1 155.2 ± 13.3 59.6 ± 10.1 85.7 ± 9.1 1 (6.7) 2 (13.3) 0 (0) 2 (13.3) 2 (13.3) 0 (0) 17.6 ± 4.2 4(26.6) 5 (33.3)

3.4 ± 1.2 85.8 ± 39.8 4.1 ± 0.8 2.6 ± 0.4 69.2 ± 6.5 −5.4 ± 6.8 67.3 ± 12.7 0.9 ± 0.3 8.8 ± 2.7 9.5 ± 1.4 86.7 ± 13.2 154.2 ± 42.2 0.5 ± 0.1 −20.9 ± 2.3 4.5 ± 4.1 −25.8 ± 7.3 7.6 ± 9.6⁎ −16.3 ± 6.4 2.4 ± 3.2 −26.3 ± 2.2 1.4 ± 2.7 −26. 9 ± 3.8 1.4 ± 5 −26.1 ± 4.4 1.5 ± 5.9

Post 3 cycles of Epirubicin

155.0 ± 13.5 58.7 ± 11.9 83.4 ± 13.8 3 (20) 4 (26.6) 0 (0) 3 (20) 0 (0) 22.4 ± 7.2

3.3 ± 1.5 85.7 ± 40.2 4.1 ± 1.1 2.6 ± 0.7 63.8 ± 8.7 78.7 ± 21.3 0.9 ± 0.7 8.2 ± 1.4 10.8 ± 2.8 80.7 ± 21.5 198.4 ± 43.7 0.4 ± 0.1 −15.2 ± 7.7# −16.8 ± 4.7# −14.8 ± 5.3 −25.1 ± 2.9 −25.4 ± 2.9 −24.6 ± 2.8

Data are expressed as mean ± SD or number (%). ALT = alanine aminotransferase, LVMI = left ventricular mass index, LVIDd = Left ventricular internal dimension at end-diastole, LVIDs = Left ventricular internal dimension at end-systole, LVEF = left ventricular ejection fraction, E/A = early to late transmitral velocity ratio, E/e’ = early transmitral velocity to tissue Doppler mitral annular early diastolic velocity ratio, IVRT = Isovolumic relaxation time, DT = deceleration time, MPI = myocardial performance index, LVLS = left ventricular global longitudinal strain, LVLSendo = left ventricular longitudinal strain at sub-endocardial layer, LVLSepi = left ventricular longitudinal strain at sub-epicardial layer. # P b 0.05, compared with the baseline (T1) at each group. ⁎ P b 0.05, compared with the changes of non-cardiotoxicity.

Please cite this article as: W.-T. Chang, Y.-H. Feng, Y.H. Kuo, et al., Layer-specific distribution of myocardial deformation from anthracyclineinduced cardiotoxicity in p..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2020.01.036

4

W.-T. Chang et al. / International Journal of Cardiology xxx (xxxx) xxx

LVEF significantly dropped from 69.2 ± 6.5% to 53.6 ± 5.8% (p = 0.005) after six months of therapy (T3 to T4) (Table S2). However, though LVEF did decrease by T4, it failed to reflect the early decline of myocardial function during Epirubicin therapy (from T1 to T3) (Fig. 1B). Alternatively, global LVLS (from −20.9 ± 2.3% to −15.2 ± 7.7%, p = 0.02) declined in patients post three cycles of therapies (from T1 to T3) and it distinguished patients who developed CTRCD from patients that did not (Table 1). However, global LVCS failed to show a significant change in patients subsequently developing CTRCD. Despite the feasibility of strain imaging for detecting CTRCD, how and where chemotherapy causes myocardial dysfunction remains uncertain. To further investigate the pathophysiology, we applied layer-specific STE (Fig. S3). Though among all enrolled patients with breast cancer, there was no significant change in LVLSendo during the course of Epirubicin therapy (Fig. 1C), in patients reaching CTRCD criteria, LVLSendo was impaired as early as after the first cycle of chemotherapy (from T1 to T2) and the changes persisted over the next several months (Fig. 1D). Conversely, there were no significant changes of LVLSepi during the course of Epirubicin therapy, even in patients reaching CTRCD (Fig. 1E, F). Differently, as we divided LVCS to the subendocardial and subepicardial layers, there were no significant changes of LVCSendo and LVCSepi in patients developing cardiotoxicity. Using univariate logistic regression, we further investigated the efficacy of conventional echocardiographic parameters and layer-specific STE for predicting the development of CTRCD. LVLSendo predicted CTRCD (Odd ratio: 2.14, CI: 1.01–5.82, p = 0.005), while LVEF, global LVLS and LVLSepi at baseline and after three cycles (T3, which is the international consensus recommended timing for cardiac function monitoring), were not associated with CTRCD (Table 2). 3.3. Predictive values of layer-specific STE in the doxorubicin rat model To further study layer-specific chemotherapy-induced myocardial injury, we established a chronic doxorubicin (cDox) rat model mimicking CTRCD in humans. In this cDox model, rats could survive for longer than fifty days, adequate for observing changes in cardiac structure and function (Fig. S4A). Seven days after the final doxorubicin administration, there was a significant decline in body weight compared to control

Table 2 Univariate logistic regression of echocardiographic parameters in predicting the development of cardiotoxicity in breast patients receiving Epirubicin therapy. Parameter

Univariate model Odds ratio (95% CI)

P-value

Age

1.02 (0.95–1.1)

0.52

Baseline characteristics (T1) LVEF LVLS LVLSepi

1.01 (0.88–1.15) 1.05 (0.87–1.72) 0.95 (0.77–1.17)

0.88 0.64 0.72

Post three cycles of Epirubicin (T3) LVEF 1.04 (0.94–1.68) LVLS 1.28 (1.01–1.62) LVLSendo 2.14 (1.01–5.82)⁎

0.08 0.06 0.005

The changes of strains LVLSendo

0.81

1(0.93–1.09)

⁎ P b 0.05 with significance. Abbreviation as Table 2.

and sham groups (Fig. S4B). Sequential echocardiographic measurements revealed no significant change in heart rate after doxorubicin treatment (Fig. S5A), and all heart rates were maintained with the normal physiological range (about 200 beats/min). In doxorubicin-treated rats, FS did not decrease until measurement 5 (Day 22) and was generally stable in the control and sham groups (Fig. S5B). In addition, there were no significant changes in end-diastolic volume and LV mass following doxorubicin treatment and the values remained similar among cDox, control and sham groups (Fig. S5C, D). Alternatively, there were significant changes in LVLSend following doxorubicin treatment, suggesting layer-specific damage (Fig. 2). Thus, layer-specific STE is a feasible method for the sequential monitoring of cardiac remodeling. Specifically, we found a significant decline of LVLSendo starting on measurement 3 (Day 8) post-doxorubicin treatment, about two weeks earlier than any detectable changes in conventional FS (Fig. 2A). Conversely, there were no significant changes in LVLSepi throughout treatment (Fig. 2B). Collectively, our findings suggest that anthracyclines including doxorubicin and Epirubicin induce cardiotoxicity starting in the

Fig. 1. During the course of Epirubicin therapy (A) no significant change in left ventricular ejection fraction (LVEF) for the whole patient group; (B) LVEF did not decrease until six months of therapy (T4) among 15 patients developing cancer therapy-related cardiac dysfunction (CTRCD); (C) no significant change in subendocardial left ventricular longitudinal strain (LVLSendo) for the whole patient group; (D) LVLSendo declined after the first cycle of chemotherapy (T2) in those with CTRCD (E,F) No significant changes of subepicardial left ventricular longitudinal strain (LVLSepi).

Please cite this article as: W.-T. Chang, Y.-H. Feng, Y.H. Kuo, et al., Layer-specific distribution of myocardial deformation from anthracyclineinduced cardiotoxicity in p..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2020.01.036

W.-T. Chang et al. / International Journal of Cardiology xxx (xxxx) xxx

5

Fig. 2. Significant changes in LVLSendo and LVLSepi following doxorubicin treatment; (A) A significant decline of LVLSendo starting on measurement 3 (Day 8) post-doxorubicin treatment (N = 8); (B) conversely, there were no significant changes in LVLSepi throughout treatment; (C) In Masson's trichrome staining, more severe myocardial fibrosis in the subendocardial layer compared to the subepicardial layer post-doxorubicin treatment. (D) In the Tunel assay, there were more apoptotic cells in the subendocardial layer compared with that in the subepicardial layer.

subendocardial layer and progressing to full myocardial dysfunction. Masson's trichrome staining indicated more severe myocardial fibrosis in the subendocardial layer compared to the subepicardial layer postdoxorubicin treatment (Fig. 2C), a result in accord with our imaging observations in patients. To differentiate the etiologies of declined strains mainly in the subendocardial layer, the Tunel assay also indicated a higher percentage of apoptotic cells in the subendocardial layer compared with that in the subepicardial layer (Fig. 2D). 4. Discussion Anthracyclines including doxorubicin and Epirubicin are highly effective treatments for breast cancer; however, an estimated 9% to 26% of patients receiving doxorubicin develop myocardial dysfunction,

mainly through oxidative stress [1,5,8]. Although breast cancer survival continues to improve, permanent injury to myocardium resulting from anthracycline toxicity may lead to higher non-cancer-related mortality and morbidity [1]. Therefore, early detection and prevention of CTRCD is crucial. Current treatment consensus suggests an interval of three to six months before monitoring of cardiac function [1,2,9]. However, conventional LVEF lacks sensitivity for detecting subtle myocardial dysfunction and thus speckle tracking has been introduced [3,4]. A number of studies have demonstrated the diagnostic and predictive utility of longitudinal strain measures following anthracycline therapy [1–4]. In the ESC position paper, a reduction of LVLS N15% implied the myocardial dysfunction post anti-cancer therapies [1]. However, the cut-off of 15% is a huge change of LVLS and may not provide satisfactory sensitivity for the early detection of CTRCD.

Please cite this article as: W.-T. Chang, Y.-H. Feng, Y.H. Kuo, et al., Layer-specific distribution of myocardial deformation from anthracyclineinduced cardiotoxicity in p..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2020.01.036

6

W.-T. Chang et al. / International Journal of Cardiology xxx (xxxx) xxx

Our results add to these findings by highlighting the potential mechanism of myocardial involvement in anthracycline-induced cardiotoxicity and also the applicability of layer-specific speckle tracing for early detection of CTRCD. The sentinel decline of LVLSendo prior to LVEF changes during and following suggest overt myocardial dysfunction initiated from the subendocardial layer. Given the different orientations of myocardial fibers, the amplitude of myocardial contraction varies between the subendocardial and subepicardial layers [3,4]. Generally, contractile force is greater in the subendocardial layer than in the subepicardial layer [4], but it is also more susceptible to injury given its higher energy requirements [10]. Previous studies have demonstrated that layerspecific strain is more accurate for discriminating the various intensities of transmural scarring compared to cardiac magnetic resonance imaging (MRI) [11]. In particular, layer-specific strain analysis provides additional information on disease progression compared to global strain under different physiological conditions, including heart failure, hypertensive cardiomyopathy, aortic stenosis, and ischemic dysfunction [4,12–15]. Luo and colleagues found that breast cancer patients receiving higher doses of Epirubicin (≥360 mg/m2) exhibited greater left ventricular longitudinal systolic dysfunction, especially of the endocardium, compared to patients receiving lower cumulative doses [16]. Kang et al. also reported subendocardial deformation in long-term lymphoma survivors after exposure to anthracycline [4]. Notably, in the former study, echocardiography was not performed until six months after treatment initiation while in the latter study, the interval from the last dose of anthracycline to echocardiography was longer than 50 months [16–19]. Conversely, in our study, echocardiography was conducted after only one cycle of chemotherapy in order to detect early myocardial dysfunction. In addition, this is the first study assessing the impact of layer-specific strain between patients developing and free from CTRCD, while previous studies did not include subjects reaching the criteria of CTRCD. In the present study, we adopted a multi-layer strain approach in analysing layer-specific ventricular deformation and observed the decrease of subendocardial strain values and transmural gradient in long-term survivors exposed to anthracycline. It has been proved in animal models of anthracycline cardiotoxicity that severe myocytolysis mainly involved the subendocardium of the ventricle [20]. Moreover, Perel et al. [21] observed a regional and diffuse pattern of subendocardial enhancement using cardiac magnetic resonance imaging in patients with anthracycline-induced cardiomyopathy. Notably, despite an early reduction of LVLS in patients developing CTRCD, there were no significant changes of either LVEF or LVCS in patients developing cardiotoxicity. In our opinion, although the increasing fibrosis of the subendocardial layer was represented in the declined LVLS, LVEF could be spare by the preserved circumferential myocardial fibers and torsional forces [22]. It is supported that most progressive myocardial diseases mainly result in subendocardial dysfunction in an early stages and lead to a reduction in longitudinal myofilaments [23]. Nevertheless, since the epicardialfibers remain preserved, circumferential strain could be normal or even increased, compensating for the longitudinal mechanical dysfunction. Hence, the findings in our study of reduction of subendocardial strain values and transmural gradient but preserved subepicardial strain was consistent with the same hypothesis of subendocardial injury induced by anthracycline. Moreover, it has been proved [24] that in patients with chronic ischemic cardiomyopathy, subendocardial strain was a powerful predictor of cardiac events and appeared to be a better parameter than LVEF and other strain variables analyzed by echocardiography. Also, in Cadeddu and colleagues' work, in patients with cardiac syndrome X the subendocardial contractile dysfunction during exercise indicated that layer-specific echocardiography could be a sensitive diagnostic modality in the assessment of cardiac syndrome X [25]. Therefore, we believed that further importance may need to be attached to the changes of subendocardial strain. Animal models play a pivotal role in pre-clinical studies but the difference between animals and human is always a concern. Using a chronic murine model of LV dysfunction, the accuracy and applicability of strain parameters have been validated by standard conductance

catheter measurements [19]. Layer-specific speckle tracking has also been applied in various animal models such as diabetic cardiomyopathy and myocardial infarction models [11,20–24]. Bachner-Hinenzon and colleagues report that despite heterogeneous segmental strains, layer longitudinal strain was equal in rats and humans [7]. Our findings in the cDox animal model also reflected our clinical observations. Masson's trichrome staining showed a greater extent of myocardial fibrosis in the subendocardial layer compared to the subepicardial layer following doxorubicin treatment. The findings of Tunel assay also supported that more apoptotic cardiac cells located in the sub-endocardial layer that in the sub-epicardial layer. In a previous study of autopsy cases with a history of anthracycline administration, inflammatory cell infiltration was recognized not only in the myocardium but in the endocardium, where atrophy, cell loss, and hypereosinophilia were observed [26]. There were evidences that anthracycline induced endothelial cells apoptosis, [27] senescence [28] and also disruption of cardiac microvascular homeostasis [29]. These mechanisms may explain a subendocardium to sub-epicardium progression of the carediotoxicity. Therefore, our findings are in accord with both experimental and clinical results. We acknowledge some limitations to our study. First, as an observational study we are only able to assess correlations among parameters instead of causal relationships during a limited amount of time of the follow-up. Second, despite excellent measurement reliability, the normal range of strain values in humans and animals remains uncertain. Also, there is no established optimal cut-off value to differentiate subjects with and without cardiotoxicity. Third, in this study we focused on the cardiotoxicity of anthracycline therapy, but these drugs may be used in combination with others. Although combined agents such as trastuzumab may also induce myocardial suppression, this is generally reversible and has only minor effects on heart function. Last, given the stress, relatively dehydration and systemic conditions, the baseline LVEFs in some patients were higher than 75%. The hyperdynamic status may interfere the accuracy of strain analysis. 5. Conclusion Layer-specific STE measures, especially LSendo, detected CTRCD earlier than conventional echocardiographic parameters during Epirubicin treatment. Further, given the correspondence between LSendo changes with subendocardial fibrosis, endocardial involvement in the development of cardiotoxicity could be a sentinel for early intervention to prevent subsequent cardiovascular complications. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijcard.2020.01.036. Declaration of competing interest None. Acknowledgments This study is supported by Ministry of Science and Technology (MOST 105-2628-B-384-001-MY3), National Health Research Institute, Taiwan (NHRI-EX106-10618SC) and Chi-Mei Medical Canter. References [1] J.L. Zamorano, P. Lancellotti, D. Rodriguez Muñoz, V. Aboyans, R. Asteggiano, M. Galderisi, G. Habib, D.J. Lenihan, G.Y.H. Lip, A.R. Lyon, T. Lopez Fernandez, D. Mohty, M.F. Piepoli, J. Tamargo, A. Torbicki, T.M. Suter, 2016 ESC position paper on cancer treatments and cardiovascular toxicity developed under the auspices of the ESC committee for practice guidelines: the task force for cancer treatments and cardiovascular toxicity of the European society of cardiology, Eur. Heart J. 37 (2016) 2768–2801. [2] A. Seidman, C. Hudis, M.K. Pierri, S. Shak, V. Paton, M. Ashby, M. Murphy, S.J. Stewart, D. Keefe, Cardiac dysfunction in the trastuzumab clinical trials experience, J. Clin. Oncol. 20 (2002) 1215–1221.

Please cite this article as: W.-T. Chang, Y.-H. Feng, Y.H. Kuo, et al., Layer-specific distribution of myocardial deformation from anthracyclineinduced cardiotoxicity in p..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2020.01.036

W.-T. Chang et al. / International Journal of Cardiology xxx (xxxx) xxx [3] A.F. Yu, J. Raikhelkar, E.C. Zabor, E.S. Tonorezos, C.S. Moskowitz, R. Adsuar, E. Mara, K. Huie, K.C. Oeffinger, R.M. Steingart, J.E. Liu, Two-dimensional speckle tracking echocardiography detects subclinical left ventricular systolic dysfunction among adult survivors of childhood, adolescent, and young adult cancer, Biomed. Res. Int. 2016 (2016) 9363951. [4] Y. Kang, F. Xiao, H. Chen, W. Wang, L. Shen, H. Zhao, X. Shen, F. Chen, B. He, Subclinical anthracycline-induced cardiotoxicity in the long - term follow-up of lymphoma survivors: a multi-layer speckle tracking analysis, Arq. Bras. Cardiol. 110 (2018) 219–228. [5] H.K. Narayan, B. Finkelman, B. French, T. Plappert, D. Hyman, A.M. Smith, K.B. Margulies, B. Ky, Detailed echocardiographic phenotyping in breast cancer patients: associations with ejection fraction decline, recovery, and heart failure symptoms over 3 years of follow-up, Circulation 135 (2017) 1397–1412. [6] R.M. Lang, L.P. Badano, V. Mor-Avi, J. Afilalo, A. Armstrong, L. Ernande, F.A. Flachskampf, E. Foster, S.A. Goldstein, T. Kuznetsova, P. Lancellotti, D. Muraru, M.H. Picard, E.R. Rietzschel, L. Rudski, K.T. Spencer, W. Tsang, J.U. Voigt, Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American society of echocardiography and the European association of, cardiovascular imaging, Eur. Heart J. Cardiovasc. Imaging 16 (2015) 233–270. [7] N. Bachner-Hinenzon, O. Ertracht, M. Leitman, Z. Vered, S. Shimoni, R. Beeri, O. Binah, D. Adam, Layer-specific strain analysis by speckle tracking echocardiography reveals differences in left ventricular function between rats and humans, Am. J. Physiol. Heart Circ. Physiol. 299 (2010) H664–H672. [8] D. Cappetta, A. De Angelis, L. Sapio, L. Prezioso, M. Illiano, F. Quaini, F. Rossi, L. Berrino, S. Naviglio, K. Urbanek, Oxidative stress and cellular response to doxorubicin: a common factor in the complex milieu of anthracycline cardiotoxicity, Oxidative Med. Cell. Longev. 2017 (2017) 1521020. [9] D. Cardinale, A. Colombo, G. Bacchiani, I. Tedeschi, C.A. Meroni, F. Veglia, M. Civelli, G. Lamantia, N. Colombo, G. Curigliano, C. Fiorentini, C.M. Cipolla, Early detection of anthracycline cardiotoxicity and improvement with heart failure therapy, Circulation 131 (2015) 1981–1988. [10] D. Algranati, G.S. Kassab, Y. Lanir, Why is the subendocardium more vulnerable to ischemia? A new paradigm, Am. J. Physiol. Heart Circ. Physiol. 300 (2011) H1090–H1100. [11] E. Altiok, M. Neizel, S. Tiemann, V. Krass, M. Becker, C. Zwicker, R. Koos, M. Kelm, N. Kraemer, F. Schoth, N. Marx, R. Hoffmann, Layer-specific analysis of myocardial deformation for assessment of infarct transmurality: comparison of strain-encoded cardiovascular magnetic resonance with 2d speckle tracking echocardiography, Eur. Heart J. Cardiovasc. Imaging 14 (2013) 570–578. [12] L. Zhang, W.C. Wu, H. Ma, H. Wang, Usefulness of layer-specific strain for identifying complex cad and predicting the severity of coronary lesions in patients with non-stsegment elevation acute coronary syndrome: compared with syntax score, Int. J. Cardiol. 223 (2016) 1045–1052. [13] K. Kimura, K. Takenaka, A. Ebihara, K. Uno, H. Iwata, M. Sata, T. Kohro, H. Morita, Y. Yatomi, R. Nagai, Reproducibility and diagnostic accuracy of three-layer speckle tracking echocardiography in a swine chronic ischemia model, Echocardiography 28 (2011) 1148–1155. [14] S. Hamada, J. Schroeder, R. Hoffmann, E. Altiok, A. Keszei, M. Almalla, A. Napp, N. Marx, M. Becker, Prediction of outcomes in patients with chronic ischemic cardiomyopathy by layer-specific strain echocardiography: a proof of concept, J. Am. Soc. Echocardiogr. 29 (2016) 412–420.

7

[15] K. Ozawa, N. Funabashi, T. Kamata, Y. Kobayashi, Inter- and intraobserver consistency in lv myocardial strain measurement using a novel multi-layer technique in patients with severe aortic stenosis and preserved lv ejection fraction, Int. J. Cardiol. 228 (2017) 687–693. [16] R. Luo, H. Cui, D. Huang, G. Li, Early assessment of the left ventricular function by Epirubicin-induced cardiotoxicity in postoperative breast cancer patients, Echocardiography 34 (2017) 1601–1609. [17] J. Bogaert, F.E. Rademakers, Regional nonuniformity of normal adult human left ventricle, Am. J. Physiol. Heart Circ. Physiol. 280 (2001) H610–H620. [18] K. Howard-Quijano, M. McCabe, A. Cheng, W. Zhou, K. Yamakawa, E. Mazor, J.C. Scovotti, A. Mahajan, Left ventricular endocardial and epicardial strain changes with apical myocardial ischemia in an open-chest porcine model, Physiol Rep 4 (2016). [19] V. Ferferieva, A. Van den Bergh, P. Claus, R. Jasaityte, A. La Gerche, F. Rademakers, P. Herijgers, J. D’hooge, Assessment of strain and strain rate by two-dimensional speckle tracking in mice: comparison with tissue doppler echocardiography and conductance catheter measurements, Eur. Heart J. Cardiovasc. Imaging 14 (2013) 765–773. [20] M. Becker, E. Altiok, C. Lente, S. Otten, Z. Friedman, D. Adam, R. Hoffmann, R. Koos, G. Krombach, N. Marx, R. Hoffmann, Layer-specific analysis of myocardial function for accurate prediction of reversible ischaemic dysfunction in intermediate viability defined by contrast-enhanced MRI, Heart 97 (2011) 748–756. [21] S.I. Sarvari, K.H. Haugaa, W. Zahid, B. Bendz, S. Aakhus, L. Aaberge, T. Edvardsen, Layer-specific quantification of myocardial deformation by strain echocardiography may reveal significant cad in patients with non-st-segment elevation acute coronary syndrome, JACC Cardiovasc. Imaging 6 (2013) 535–544. [22] P. Claus, A.M.S. Omar, G. Pedrizzetti, P.P. Sengupta, E. Nagel, Tissue tracking technology for assessing cardiac mechanics: principles, normal values, and clinical applications, JACC Cardiovasc. Imaging 8 (12) (2015 Dec) 1444–1460. [23] D. Vinereanu, P.O. Lim, M.P. Frenneaux, A.G. Fraser, Reduced myocardial velocities of left ventricular long-axis contraction identify both systolic and diastolic heart failure —a comparison with brain natriuretic peptide, Eur. J. Heart Fail. 7 (2005) 512–519. [24] Enomoto M, Ishizu T, Seo Y, Yamamoto M, Suzuki H, Shimano H, Kawakami Y, Aonuma K.. Subendocardial systolic dysfunction in asymptomatic normotensive diabetic patients. Circ J79 (2015)1749–1755. [25] C. Cadeddu, S. Nocco, M. Deidda, F. Pau, P. Column, G. Mercuro, Altered transmural contractility in postmenopausal women with cardiac syndrome X, J. Am. Soc. Echocardiogr. 27 (February 2014) 208–214. [26] H. Kajihara, H. Yokozaki, M. Yamahara, Y. Kadomoto, E. Tahara, Anthracycline induced myocardial damage. An analysis of 16 autopsy cases, Pathol. Res. Pract. 181 (1986) 434–44124. [27] S. Wu, Y.S. Ko, M.S. Teng, Y.L. Ko, L.A. Hsu, C. Hsueh, Y.Y. Chou, C.C. Liew, Y.S. Lee, Adriamycin-induced cardiomyocyte and endothelial cell apoptosis: in vitro and in vivo studies, J. Mol. Cell. Cardiol. 34 (2002) 1595–1607. [28] E.H. Bent, L.A. Gilbert, M.T. Hemann, A senescence secretory switch mediated by PI3K/AKT/mTOR activation controls chemoprotective endothelial secretory responses, Genes Dev. 30 (2016) 1811–1821. [29] D.M. Eckman, R.B. Stacey, R. Rowe, R. D’Agostino Jr., N.D. Kock, D.C. Sane, F.M. Torti, J. Yeboah, S. Workman, K.S. Lane, W.G. Hundley, Weekly doxorubicin increases coronary arteriolar wall and adventitial thickness, PLoS One 8 (2013) e57554.

Please cite this article as: W.-T. Chang, Y.-H. Feng, Y.H. Kuo, et al., Layer-specific distribution of myocardial deformation from anthracyclineinduced cardiotoxicity in p..., International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2020.01.036