Dual HER-2 blockade therapy increases the risk of developing cardiac toxicities in HER-2 positive breast cancer: an up-to-date comprehensive meta-analysis

Background To investigate the incidence and risk of cardiac toxicities between dual HER-2 blockade and anti-HER-2 monotherapy. Materials and Methods We searched PubMed, EMBASE and Cochrane library databases to identify relevant trials between January 1 1990 and October 31 2019. Statistical analyses were conducted to calculate the summary incidence, Petro odds radio (Peto ORs) and 95% confidence intervals (CIs) by using either random-effects or fixed-effects models. Results A total of 16,375 patients from 15 randomized controlled trials were included for analysis; the pooled incidence of LVEF decline and CHF in dual HER-2 blocked were 4.6% and 0.9%, which was higher than that in anti-HER-2 monotherapy (3.2% and 0.7%, respectively). Dual HER-2 blockade therapy in breast cancer patients significantly increased the risk of developing LVEF decline (OR:1.19, 95%CI: 1.02-1.40, p =0.031) and CHF (OR:1.45, 95%CI: 1.00-2.11, p =0.049) when compared to anti-HER2 monotherapy. Sub-group analysis showed that addition of dual HER-2 blockade to adjuvant treatment for breast cancer significantly increased the risk of developing LVEF decline ( p =0.048) and CHF ( p= 0.005). In addition, dual HER-2 blockade in breast cancer patients significantly increased the risk of developing LVEF decline ( p =0.004) when compared to lapatinib alone, but not for CHF ( p =0.11, respectively). Conclusion Dual HER-2 targeted therapy in HER-2 positive breast cancer significantly increase the risk of developing LVEF and CHF when compared to anti-HER-2 alone, though the overall incidence of cardiac toxicities is very low. Physicians should be aware of this risk and provide close monitoring during the administration of dual HER-2 targeted therapy.


Introduction
During the past decades, the increased knowledge of biological mechanisms involving tumor proliferation and progression has led to the introduction of novel approaches for the treatment of cancer. The epidermal growth factor receptor (EGFR) family of transmembrane receptor tyrosine kinases is one such therapeutic target, which is overexpressed or mutated in multiple cancers [1,2].
Unlike other members of EGFR family, HER2/ErbB2 is a non-ligand-binding member of this family and exerts its activity through heterodimerization with other EGFR family members(EGFR, HER3,HER4) [3,4]. And HER2 overexpression could also lead to HER2 dimerization and constitutive activation in the absence of ligand. Initially, HER2 overexpression is found in approximately 15-20% of breast cancer [5], then its overexpression was also detected in a subsets of many other cancer types, such as gastric [6], lung [7] or colorectal cancer [8]. In the last two decades, the introduction of monoclonal antibodies (MoAbs), TKIs or antibody-drug conjugates, which directly targets HER2 gene, has impressively improved the outcomes of HER-2 positive breast cancer patient in all disease stages [9][10][11]. Subsequently, the addition of anti-HER2 MoAbs to chemotherapy in HER-2 positive locally advanced gastric or gastro-oesophageal junction (GEJ) cancer also significantly improve overall survival. Currently, routine assessment of HER2 status in these tumors is mandatory, and the use of anti-HER2 agents have become a standard treatment for in HER-2 positive breast or gastric or GEJ cancer.
However, nearly all patients with metastatic HER2-positive breast cancer or gastric or GEJ cancer would eventually progress on anti-HER2 therapy due to de novo or acquired resistance, potentially due to incomplete blockade of HER2 pathway. Therefore, inhibiting the HER2 signaling pathway more effectively with dual blockade approaches by using improved anti-HER2 therapies has been extensively investigated in both breast cancer and gastric or GEJ cancer, and dual HER2 blockade shows promising results in HER-2 positive breast cancer. Nevertheless, with the increasing use of dual HER2 blockade, particularly in the curative adjuvant setting, concerns regarding cardiac toxicities associated with dual anti-HER2 treatment in breast cancer patients has been increased. Therefore, there is an urgent need to clearly determine the overall incidence and risk of cardiac toxicities associated with dual HER2 blockade. Prior to the present study, Valachis A [12]. performed a metaanalysis to investigate the risk of cardiac toxicities related to dual HER2 blockade in breast cancer, and found that cardiac toxicities in anti-HER2 combination therapy was comparable to that of anti-HER2 monotherapy [12]. But the sample size in that study are relative small, and most of the included studies have updated cardiac toxicities data. In addition, more randomized controlled trials assessing efficacy and toxicities of dual HER2 blockade in breast cancer have been conducted since then. As a result, we perform the present study to comprehensively investigate the incidence and risk of cardiac toxicities associated with dual HER-2 blockade in breast cancer when compared to anti-HER2 monotherapy by using a meta-analysis.

Study design
Based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement guidelines 2009, the authors conducted the present systematic review and meta-analysis [13].

Data source
From January 1 1990 to October 31 2019, we searched related citations in PubMed by using the following keywords: trastuzumab, lapatinib, pertuzumab, T-DM1, neratinib, pyrotinib, anti-HER2 agents, HER2 blockade, breast cancer, breast carcinoma, clinical trial and randomized. We also performed independent searches in EMBASE and Cochrane library databases to identify relevant trials. Moreover, abstracts of dual HER-2 blockade therapy in breast cancer presented at the American

Outcome definition
Incidence and risk of left ventricular ejection fraction (LVEF) decline and congestive heart failure (CHF) were the primary endpoints for the present study. According to the CTC-AE grading system, LVEF decline was defined as LVEF decline less than 50% or a decrease of more than 10% from baseline, while CHF defined as New York Heart Association (NYHA) class III or IV or cardiac death. To avoid loss of information, trials reported LVEF decline more than 15% or 20% from baseline was also regarded as LVEF decline in the present analysis.

Data extraction
Two investigators (WXQ and JYC) initially performed the data extraction including the followings: Trial name, treatment setting, anti-HER2 regimen and dosage, duration of anti-HER2 therapy, concomitant therapy. Data related to the primary outcomes were also extracted including No. of LVEF decline, No. of CHF and No. of patients for safety analysis. The extracted data were cross-checked by another two investigators (CX and UC), and any discrepancies were resolved by consensus.

Study selection
Prospective randomized, controlled phase 2 or 3 trials investigating the cardiac safety of dual HER2 blockade versus anti-HER2 monotherapy (lapatinib or trastuzumab or pertuzumab or T-DM1) in breast cancer were included for analysis. For trials with multiple intervention arms (for example, three-arms trials with two anti-HER2 monotherapy arms and one combined anti-HER2 therapy arm), we merged the two relevant (anti-HER2 monotherapy) arms into one group, then compared the merged group with dual HER-2 blockade group as previously reported [12]. Additionally, we used the five-item Jadad scale including randomization, double-blinding, and withdrawals as previously described to assess the quality of included trials. the final score was reported between 0 and 5 [14].

Statistical analysis
Events of cardiac toxicities (LVEF decline or CHF) and total number of breast cancer patients treated with anti-HER2 agents or dual HER-2 blockade were extracted to calculate the overall incidence and corresponding 95% confidence intervals (CIs). An appropriate statistical model (fixed or random-effect model) was used to pool the overall incidence and corresponding 95% CIs based on the heterogeneity results. Binary data of cardiac toxicities associated with dual HER-2 blockade versus anti-HER-2 monotherapy were meta-analyzed with the Peto method, because this method provided the best CI coverage and was more appropriate method for meta-analysis when dealing with low event rates(<5%) [15]. Additionally, we conducted the following prespecified subgroup analyses: the treatment setting (neoadjuvant, adjuvant and metastatic), the type of anti-HER2 therapies (dual HER2 blockade vs. trastuzumab; dual HER2 blockade vs. lapatinib), concomitant treatment (none, chemotherapy and hormonal therapy) to investigate the risk of cardiac toxicities associated with dual anti-HER2 blockade within particular groups. A statistical test with a p-value less than 0.05 was considered significant. The overall heterogeneity was assessed by using the I 2 statistic, and I 2 greater than 50% indicated high heterogeneity. Sensitivity analysis using different statistical models was performed to assess the stability of results. Potential publication bias was checked by visual inspection of the symmetry of funnel plots and the Egger regression asymmetry test. All statistical analyses were performed by using Version 2 of the Comprehensive MetaAnalysis program (Biostat, Englewood, NJ) and Open Meta-Analyst software version 4. 16.12 (Tufts University).

Search results
Based on the electronic search protocol, a total of 470 studies were initially identified. of these studies, 338 did not meet the inclusion criteria and were therefore excluded. Among the remaining 132 studies, a total of 22 trials met the inclusion criteria . Of these 22 included trials, six trials were update reports of published trials [17,25,26,28,29, 33], one trial involving gastric or GEJ carcinoma [18]. Thus finally 15 randomized controlled trials were included in this meta-analysis (figure 1). Table 1 listed the baseline characteristics of patients and studies. The quality of each included study was roughly assessed according to Jadad scale, four trials [16,21,34,37] were double-blind, placebo-controlled trials, thus had a Jadad score of 5, and the remaining eleven trials were open-label controlled trials, thus had a Jadad score of 3.

Incidence of LVEF decline
A total of 7,184 patients receiving dual HER-2 blockade in 15 RCTs were available for LVEF decline analysis. There were 302 LVEF decline events among these patients. The highest incidence (8.7%; 95% CI 5.3% to 13.9%) was observed in a phase III breast cancer neoadjuvant trial of trastuzumab plus lapatinib when concomitant with paclitaxel [22], while the lowest incidence was observed in four trials in which no events of LVEF decline occurred [16,27,31,37]. Using a random-effects model (χ2based Q statistic test: Q= 31.75; p=0.004; I 2 = 55%), the summary incidence of LVEF decline in cancer patients treated with dual HER2 blockade was 4.6% (95% CI, 3.7% to 5.7%, figure 2A).

Incidence of CHF
A total of 6,818 patients receiving dual HER-2 blockade in 14 RCTs were available for CHF analysis.
There were 56 total CHF events among these patients. The highest incidence (2.0%; 95% CI 0.7 to 6.1%) was observed in a phase III breast cancer trial of trastuzumab plus lapatinib after prior trastuzumab-based therapies[33], while no events of CHF occurred in five trials. Using a fixed-effects model (χ2-based Q statistic test: Q=8.26; p=0.82; I 2 =0%), the summary incidence of CHF in cancer patients treated with dual HER2 blockade was 0.9% (95% CI, 0.7% to 1.2%, figure 2C). As for CHF associated with anti-HER2 monotherapy, a total of 9,283 patients were included for analysis. Using a random-effects model (χ2-based Q statistic test: Q=52.77; p<0.001; I 2 =73.42%), the summary incidence of CHF in cancer patients treated with anti-HER2 monotherapy was 0.7% (95% CI, 0.4% to 1.3%, figure 2D).

Risk of LVEF decline and CHF associated with dual HER2 blockade
All of the 15 included trials reported the LVEF decline data, thus included for calculating the OR of LVEF decline associated with dual HER2 blockade. A total of 284 LVEF decline events were observed in dual HER2 blockade versus 357 LVEF decline events in anti-HER2 monotherapy. The pooled results demonstrated that the dual HER-2 blockade in cancer patients significantly increased the risk of developing LVEF decline with an OR of 1.20 (95% CI 1.02-1.41, p=0.031, Figure 3A) using a fixedeffects model (χ2-based Q statistic test: Q=22.58; p=0.091, I 2 =32.6).
A total of 14 randomized trials reported CHF data. A total of 55 CHF events were observed in dual HER2 blockade versus 57 CHF events in anti-HER2 monotherapy. The pooled results showed that the dual HER-2 blockade in cancer patients significantly increased risk of developing CHF with an OR of 1.45 (95% CI 1.00-2.11, p=0.049, Figure 3B)  We also did sub-group analysis according to anti-HER-2 monotherapy. Our result showed that dual HER-2 blockade significantly increased the risk of developing LVEF decline (OR 1.49, 95%CI: 1.14-1.96, p=0.004) when compared to lapatinib, but not for CHF (OR 2.62, 95%CI: 0.90-2.94, p=0.11). In comparison with trastuzumab alone, no significantly increased risk of developing LVEF decline(p=0.37) and CHF (p=0.24, table 2) was observed in dual HER-2 blockade group.

Publication Bias
We performed Begg's funnel plot and Egger's test to detect the publication bias of literatures. No significant evidence of publication bias for LVEF decline and CHF was detected by using funnel plots (supplemental figure 1 for LVEF decline; and supplemental figure 2 for CHF), Begg's test (LVEF decline, p=0.62; CHF, p=0.53) and Egger's test (LVEF decline, p=0.38; CHF, p=0.93).

Discussion
To our best knowledge, this meta-analysis is the largest and most comprehensive meta-analysis to assess the risk of cardiac toxicities associated with dual HER2 blockade versus anti-HER2 monotherapy. A total of 16,375 patients from 15 randomized controlled trials were included for analysis. Our pooled results indicate that the overall incidence rate of LVEF decline and CHF in dual HER-2 blockade are 4.6% and 0.9%, which is higher than that of anti-HER-2 monotherapy (3.2% and 0.7%, respectively). Importantly, the present study for the first time demonstrates that dual HER-2 blockade therapy in breast cancer patients increases 19% risk of developing LVEF decline and 45% risk of developing CHF when compared to anti-HER2 monotherapy. It should be noted that patients with inadequate cardiac function would be excluded from treatment and close cardiac monitoring has been performed during the administration of anti-HER-2 agents, both of them would significantly reduced the incidence of cardiac toxicities. Although the overall incidence of cardiac toxicities is very low, a slight but significant risk of developing cardiac toxicities has been observed in dual HER-2 blockade when compared to anti-HER-2 monotherapy.
Sub-group analysis showed that addition of dual HER-2 blockade to adjuvant treatment in breast cancer significantly increased the risk of developing LVEF decline (p = 0.031) and CHF (p = 0.049), but not for neoadjuvant or metastatic settings. Additionally, cardiac toxicities associated with specific anti-HER-2 agents might be difference, which might be attributable to the unique epitopes of HER2 recognized by each antibody and differential effects on downstream signaling pathways [38]. The cardiac toxicities associated with trastuzumab seems higher than that of lapatinib. In a previous publication based on 29,000 breast cancer, the authors found that incidence severe cardiac toxicities with trastuzumab was 3.0%(95%CI: 2.41-3.64)[39], while the overall cardiac toxicities associated with lapatinib was 3.0% [40]. We therefore perform sub-group analysis based on anti-HER-2 agents, and find that dual HER-2 blockade in breast cancer patients significantly increased the risk of developing LVEF decline (p = 0.004) when compared to lapatinib alone, but not for CHF (p = 0.11, respectively).
No significant difference of cardiac toxicities is found between dual HER-2 blockade and trastuzumab.
Additionally, the concomitant hormonal treatment with dual HER-2 blockade in breast cancer significantly increases the risk of developing LVEF decline in comparison with hormonal therapy plus anti-HER2 monotherapy (p = 0.022), while concomitant chemotherapy with dual HER2 blockade treatment also significantly increases the risk of developing CHF when compared to anti-HER2 monotherapy plus chemotherapy (p = 0.052). Based on our finding, dual HER-2 blockade therapy in breast cancer is associated with a small but statistically significant risk of developing LVEF decline and CHF when compared with anti-HER2 monotherapy. In comparison with anti-HER-2 monotherapy, addition of dual HER-2 blockade to neoadjuvant or metastatic settings for breast cancer is safe in terms of cardiac toxicities, but not for adjuvant setting. Physicians should pay more attention to cardiac toxicities during the administration of dual HER-2 treatment when concomitant hormonal/chemotherapy treatment.
The molecular mechanisms related to cardiac toxicities induced by anti-HER2 agents, either alone or in combination, remains unknown. One potential explanation for the mechanism is the inhibition of

Consent for publication
Not applicable.

Availability of supporting data
All data included in this study are available upon request by contact with the corresponding author.

Competing interests
The authors declare that they have no competing interests.