Targeting HER2 in Breast Cancer: Latest Developments on Treatment Sequencing and the Introduction of Biosimilars

Megan E. Tesch1 · Karen A. Gelmon1

Abstract

Approximately 20% of all breast cancers overexpress the human epidermal growth factor receptor 2 (HER2). Targeting breast cancer through this vital oncogenic protein has been a major step towards improved patient outcomes. Today, several anti-HER2 agents are in clinical use including: the monoclonal antibodies trastuzumab and pertuzumab; the small molecule inhibitors lapatinib, neratinib, and tucatinib; and the antibody–drug conjugates ado-trastuzumab emtansine and trastuzumab deruxtecan, in some jurisdictions. In addition, several trastuzumab biosimilars have recently been granted regulatory approval in North America and the EU, and are enhancing patient access to HER2-directed therapy. The various agents differ greatly in their side-effect profiles and approved indications, from neoadjuvant and adjuvant use in early disease, to first- and later- line use in metastatic disease. This review discusses the current treatment recommendations for the use of anti-HER2 agents alone and in combination, examines the latest advances in HER2-targeted drugs and how they may be best applied in clini- cal practice, and provides guidance on optimal sequencing of the growing array of therapeutic options for HER2-positive breast cancer.

1 Introduction

Department of Medical Oncology, British Columbia Cancer, 600 W. 10th Avenue, Vancouver, BC V5Z 4E6, Canada Approximately 20% of all breast cancers overexpress the human epidermal growth factor receptor 2 (HER2). HER2- positivity has historically conferred a poor prognosis owing to its association with high-grade histology, lymph node involvement, and higher rates of disease recurrence and mortality [1–5]. Developed over 20 years ago, tras- tuzumab was the first drug to target the oncogenic HER2 protein and represented a revolutionary step in the treat- ment of HER2-positive breast cancer. Anti-HER2 agents have dramatically improved patient outcomes in the neo- adjuvant and adjuvant settings for early disease, and simi- larly in advanced or metastatic disease through first- and later-line application.
Today, several anti-HER2 agents are in clinical use including the monoclonal antibodies trastuzumab and pertuzumab; the small-molecule inhibitors lapatinib, ner- atinib, and tucatinib; and the antibody–drug conjugates ado-trastuzumab emtansine (TDM1) and trastuzumab deruxtecan, in some jurisdictions. In addition, a number of trastuzumab biosimilars have recently been granted regulatory approval in North America and Europe and are enhancing patient access to targeted therapies. Treatment algorithms for HER2-positive breast cancer continue to evolve, and as anti-HER2 agents currently in late-stage clinical development become available we will be faced with an even greater array of options. The aim of this review was to provide a historical background on targeted anti-HER2 agents and summarize key clinical trials, whilst serving as a guide in navigating through current treatment strategies for early and metastatic HER2-positive breast cancer.

2 HER2 Activity and Molecular Targets

HER2 belongs to a family of four receptors (EGFR1, HER2, HER3, HER4) that regulate key cellular processes including proliferation, motility, and survival. These pro- teins contain an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase catalytic domain, which through dimerization activate several downstream signalling pathways such as the PI3K/Akt pathway [6, 7]. Overexpression of HER2 in breast cancer occurs primarily through amplification of the HER2 oncogene on chromosome 17 and results in constitutive signal transduction and promotion of tumori- genesis [7, 8].
HER2 overexpression is a strong predictive factor for response to anti-HER2 therapies, which target different regions of the HER2 protein (Fig. 1). Trastuzumab is a humanized monoclonal antibody that binds to the extracel- lular juxtamembrane domain of HER2 to exert its antitu- mor effects via several mechanisms including inhibition of HER2 shedding, suppression of ligand-independent down- stream signalling, and triggering of antibody-dependent cell-mediated cytotoxicity (ADCC) [9–11]. In contrast, the monoclonal antibody pertuzumab binds to the extracellu- lar dimerization domain of HER2, inhibiting HER2-HER3 heterodimerization [12]. This is a critical antitumor effect, given that the HER2/HER3 heterodimer constitutes the most mitogenic receptor complex within the HER fam- ily [13]. TDM1 is a conjugate of trastuzumab with the microtubule inhibitor DM1, allowing targeted intracel- lular drug delivery to HER2-overexpressing cancer cells [14]. The more recently US Food and Drug Administration (FDA)-approved antibody–drug conjugate trastuzumab deruxtecan is composed of an anti-HER2 monoclonal antibody with the same amino acid sequence as trastu- zumab, a cleavable tetrapeptide-based linker, and a cyto- toxic topoisomerase I inhibitor [15]. The peptide-based linker is selectively cleaved by lysosomal cathepsins, which are upregulated in cancer cells [16], to release the cytotoxic drug. The tyrosine kinase inhibitors (TKIs) act either reversibly or irreversibly to block receptor phospho- rylation and downstream pathways. Lapatinib is a revers- ible inhibitor of the intracellular tyrosine kinase activity of HER2 and EGFR1 [17], while neratinib acts against EGFR1, HER2, and HER4 irreversibly [18]. Tucatinib is a reversible selective inhibitor of HER2 [19].

3 Treatment of Early HER2‑Positive Breast Cancer

3.1 Adjuvant
In the adjuvant setting, trastuzumab is standard of care for stage I–III HER2-positive breast cancer and is universally recommended by current guidelines [20, 21]. This recom- mendation is based on results of four large trials that have demonstrated sustained improvements in disease-free sur- vival (DFS) and overall survival (OS) in updated analyses with 1 year of adjuvant trastuzumab in combination with or after chemotherapy (Table 1).
Not only did these landmark adjuvant trials establish sig- nificant benefit for trastuzumab, but they also drew several other important conclusions. Firstly, it was shown that the addition of trastuzumab to chemotherapy is generally well tolerated. Cardiac dysfunction is the most significant tox- icity associated with trastuzumab, thought to be mediated via inhibition of HER2 signaling in cardiac myocytes [26]. However, overall incidence of cardiac adverse events was low in these trials, ranging from 2 to 4%, and the majority of events were asymptomatic reductions in left ventricular ejection fraction (LVEF). Fortunately, both asymptomatic and symptomatic (including congestive heart failure [CHF]) cardiac side effects are treatable and appear to be largely reversible [27]. Secondly, the NCCTG N9831 trial dem- onstrated that concurrent administration of trastuzumab with taxanes is preferable to sequential administration. This trial comprised two experimental trastuzumab arms; in one arm trastuzumab was delivered concurrently with weekly paclitaxel, and in the other arm trastuzumab was deliv- ered sequentially after paclitaxel [28]. Although DFS was numerically increased in the concurrent arm compared to the sequential arm (84.4% vs. 80.1%), this difference was not statistically significant based on prespecified interim analysis criteria [29]. Nevertheless, in view of the additional practical advantages, guidelines favour concurrent admin- istration of trastuzumab with taxanes [20, 21]. Thirdly, the HERA trial found that 2 years of trastuzumab therapy was not superior to 1 year of treatment and increased the rate of cardiac adverse events [30].
Current areas of interest in the adjuvant treatment of HER2-positive breast cancer lie in de-escalation and esca- lation strategies. One attempt at treatment de-escalation has been to shorten the duration of trastuzumab. Of the five randomized trials addressing this question, only PERSE- PHONE met its noninferiority margin of 3% for 4-year

Fig. 1 Mechanism of action of agents targeting HER2. Trastuzumab and pertuzumab are monoclonal antibodies that bind to the extracel- lular domain of the HER2 receptor to suppress HER2 activity and, in the case of pertuzumab, HER2-HER3 receptor dimerization. The small-molecule inhibitors lapatinib, neratinib, and tucatinib bind to the intracellular tyrosine kinase domains of HER2 and other HER family receptors. Antibody–drug conjugates, such as TDM1 and tras- tuzumab deruxtecan, bind to the extracellular domain of HER2, fol- lowed by internalization of the complex into lysosomes and intracel- lular release of the cytotoxic drug payload. The antitumor effects of these agents are mediated through inhibition of downstream signal- ling pathways responsible for carcinogenesis DFS (Table 2). Despite the increased frequency of cardiac adverse events observed with longer-duration trastuzumab, 12 months of adjuvant trastuzumab remains the standard of care.
A second de-escalation strategy involves the de-inten- sifying of the chemotherapy backbone used with trastu- zumab. Non-anthracycline regimens including the docetaxel, carboplatin, and trastuzumab (TCH) regimen used in the BCIRG006 trial and the weekly paclitaxel with trastuzumab (TH) regimen used in the single-arm phase II APT trial have demonstrated favorable DFS outcomes over long-term fol- low-up and less cardiotoxicity compared to anthracycline regimens [25, 37–39]. The TCH regimen may be considered for patients where avoidance of anthracyclines is desired (e.g. pre-existing cardiac conditions, reduced baseline LVEF), while TH may be considered in small node-negative (≤ 3 cm) tumors where there is proven benefit for trastu- zumab despite being unrepresented in the original landmark trials [40]. The recently reported phase II ATEMPT trial compared TH with a non-chemotherapy regimen, namely TDM1, in patients with stage I HER2-positive breast cancer [41]. Three-year DFS rates were comparable for TH and TDM1 (93.2% vs. 97.5%) and TDM1 had significantly fewer clinically relevant toxicities (25% vs. 36%, p = 0.03). How- ever, further study is required to justify the cost of TDM1.
Dual and sequential HER2 blockade strategies have been explored in attempts to further improve outcomes for patients with early HER2-positive breast cancer. The ALTTO trial compared 12 months of trastuzumab with three experimen- tal regimens: lapatinib for 12 months, lapatinib with tras- tuzumab for 12 months, or sequential therapy with trastu- zumab for 12 weeks followed by lapatinib for 34 weeks [42]. The study failed to show a DFS benefit with any of the lapat- inib approaches over trastuzumab. Lapatinib was also associ- ated with a greater rate of adverse events, including diarrhea, skin rash, and hepatotoxicity. In the ExteNET trial, patients randomly assigned to receive 1 year of neratinib after completing a year of adjuvant trastuzumab had improved 5-year invasive DFS (iDFS) compared to placebo (90.2% vs. 87.7%, hazard ratio (HR) 0.73, 95% confidence interval (CI) 0.57–0.92, p = 0.0083) [43]. Hormone-positive (HR+) tumors derived the most benefit from neratinib (HR 0.60, 95% CI 0.43–0.83), which may stem from the drug’s abil- ity to induce estrogen receptor (ER) function whilst inhibit- ing HER2 signalling [44]. In doing so, neratinib overcomes resistance to hormonal agents that may occur secondary to crosstalk between ER and HER2 pathways [45]. Importantly, diarrhea occurred in over 95% of patients taking neratinib, with approximately 40% of events being grade 3–4 in sever- ity. These results led to FDA approval of neratinib in 2017, with the caveat recommendation for loperamide primary prophylaxis. Finally, the APHINITY trial investigated the addition of pertuzumab to adjuvant chemotherapy with tras- tuzumab [46]. Compared to placebo, pertuzumab conferred a modest improvement in 3-year iDFS (94.1% vs. 93.2%, HR 0.81, 95% CI 0.66–1.00, p = 0.045) that was maintained in an updated analysis (6-year iDFS 90.6% vs. 87.8%, HR 0.76, 95% CI 0.64–0.91) [47]. Both the original and the updated subgroup analyses showed greater magnitude of benefit from pertuzumab in node-positive patients (6-year iDFS 87.9% vs. 83.4%, HR 0.72, 95% CI 0.59–0.87); whereas no benefit was observed in node-negative patients (6-year iDFS HR 1.02, 95% CI 0.69–1.53). Cardiotoxicity was low and no differ- ent between groups, but pertuzumab caused more grade 3 or higher diarrhea (9.8% vs. 3.7%). Interpreting the results of ExteNET and APHINITY, extended therapy with ner- atinib can be considered in the high-risk HR+ population, while dual trastuzumab and pertuzumab can be considered for node-positive patients. However, the small DFS benefit gained with these escalated treatment strategies must be weighed against the added toxicity and cost.

3.2 Neoadjuvant
Compared with other breast cancer subtypes, a higher per- centage of HER2-positive patients achieve a pathologic complete response (pCR) with neoadjuvant therapy. Fur- ther, the pCR rate is markedly increased with the addition of anti-HER2 agents, as was first shown in the NOAH trial. In this study, the addition of trastuzumab to neoadjuvant chemotherapy increased pCR rates (from 19 to 38%) and was associated with a significant improvement in event-free sur- vival in women with locally advanced HER2-positive breast cancer [48]. Benefits of a neoadjuvant approach include post-treatment risk stratification on the basis of pCR, a vali- dated surrogate endpoint for improved DFS and OS [49, 50], and presence of residual disease, with ability to tailor further adjuvant therapies to these higher risk patients. Thus, neoadjuvant treatment is recommended for clinical stage II and III HER2-positive tumors [21, 51].
In an attempt to surpass the efficacy of single-agent trastuzumab in the neoadjuvant setting, several other anti- HER2 agents have been tested against or in combination with trastuzumab, using pCR as the primary endpoint (Table 3). The most efficacious arms in these neoadjuvant trials have been those comprising dual anti-HER2 blockade and neoadjuvant chemotherapy. The combination of lapat- inib and trastuzumab led to numerical increases in pCR in five studies, albeit these differences were only statistically significant in the Neo-ALTTO and CHER-LOB trials [52, 53]. In addition, a common theme of the lapatinib trials was the need for lapatinib dose reductions because of excessive gastrointestinal toxicity and high rates of treatment discon- tinuations. Similarly, co-administration of pertuzumab and trastuzumab achieved high pCR rates in the NeoSphere and TRYPHAENA trials, with considerably less toxicity than the lapatinib and trastuzumab combination. Coupled with the known survival advantage of pertuzumab in the metastatic setting, the FDA granted accelerated approval in 2013 for dual pertuzumab and trastuzumab with neoadjuvant chemo- therapy for stage II-III HER2-positive breast cancer, which was followed by approval by most international regulatory bodies. In contrast, the side-effect profile and unclear benefit of lapatinib has precluded its use in the neoadjuvant setting. Like in the adjuvant setting, de-escalation of the chemo- therapy backbone has been of interest in neoadjuvant man- agement of HER2-positive breast cancer. The TRYPHAENA trial demonstrated safety and efficacy for the non-anthracy- cline-containing regimen of taxane and carboplatin with dual pertuzumab and trastuzumab (TCHP) [65]. Other trials using taxane, carboplatin, and trastuzumab with or without pertuzumab have also shown equivalent pCR rates and lesser cardiac and hematologic toxicity compared to anthracycline- containing regimens; thus, anthracycline may be justly omitted from neoadjuvant chemotherapy when desired [70, 75–77]. TDM1 administered as a single agent or combined with pertu- zumab was capable of inducing pCRs in over 40% of patients in three trials [69, 70, 72]. However, in an updated 3-year analysis of the KRISTINE trial, the TDM1 and pertuzumab arm had a higher risk of an event-free survival event owing to more locoregional progression [71]. HER2 expression was more heterogenous within the tumors from the 15 patients with locoregional disease progression, which suggests that anti-HER2 therapy without chemotherapy may be insufficient in cases of intratumoral HER2 heterogeneity. An additional single-arm phase II trial of neoadjuvant TDM1 plus pertu- zumab showed that pCR rates were substantially inferior in the setting of HER2 heterogeneity [78]. Thus, chemotherapy- free approaches as used in the ATEMPT and KRISTINE trials remain investigational in the curative setting until long-term safety data are available.
Where TDM1 has been practice-changing for early HER2- positive breast cancer has been in the treatment of residual disease following neoadjuvant therapy, a marker of poor prog- nosis when compared to those patients who achieve pCR. The KATHERINE trial randomly assigned 1,486 patients with stage II-III HER2-positive breast cancer to receive 14 cycles of adjuvant TDM1 or trastuzumab, if they did not achieve a pCR after neoadjuvant therapy [79]. It is worth noting only 18.3% of patients received neoadjuvant chemotherapy with trastuzumab plus pertuzumab, and likewise, the control arm comprised trastuzumab alone without pertuzumab. At a EFS event-free survival, DFS disease-free survival, OS overall survival, DDFS distant disease-free survival, RFS relapse-free survival, iDFS invasive disease-free survival, H trastuzumab, P paclitaxel, L lapatinib, T docetaxel, F 5-fluorouracil, E epirubicin, C cyclophosphamide, A doxorubicin, Per pertuzumab, Carbo carboplatin, ET endocrine therapy, N neratinib, HR hormone-receptor apCR defined as ypT0/is ypN0 unless otherwise stated, †pCR defined as ypT0 ypN0 median follow-up of 41.4 months, 3-year iDFS was 88% and 77% in the TDM1 and trastuzumab arms, respectively (HR 0.50, 95% CI 0.39–0.64, p < 0.0001). Importantly, the benefit of TDM1 was observed across all subgroups, regardless of clinicopathologic characteristics like hormone-receptor status or use of dual trastuzumab and pertuzumab in the neoadjuvant regimen (18% of patients). Grade ≥ 3 adverse events including thrombocytopenia and peripheral sensory neuropathy occurred more frequently with TDM1 than trastuzumab (25.7% vs. 15.4%). Adjuvant radiation was delivered concurrently with anti-HER2 therapy when indicated. Although rates of radiation pneumonitis were low overall, they were doubled with TDM1 (1.5% vs. 0.7%), which may influence the timing of adjuvant radiation in clinical practice. The 2019 St. Gallen consensus supports adjuvant TDM1 for all HER2-positive patients with residual cancer after neoadjuvant therapy [51].

4 Treatment of Metastatic HER2‑Positive Breast Cancer

4.1 First‑Line
Single-agent trastuzumab is associated with an over- all response rate (ORR) of 15–26% in advanced HER2- positive breast cancer [80, 81]. However, the addition of standard first-line chemotherapy (either doxorubicin and cyclophosphamide or paclitaxel) to trastuzumab was shown to dramatically increase efficacy, yielding an ORR of 50% and median OS of 25.1 months in a landmark trial by Slamon et al. [82]. Paclitaxel emerged the preferred chemotherapy backbone, given the higher incidence of cardiotoxicity among patients who received concurrent anthracycline, cyclophosphamide, and trastuzumab (27% vs. 13% with paclitaxel and trastuzumab).
Multiple trials were initiated after the Slamon study combining trastuzumab with various other single-agent chemotherapies such as docetaxel, vinorelbine, gemcit- abine, liposomal doxorubicin, capecitabine, and others, all of which were effective albeit not superior to the ini- tial trial regimen [83–86]. In addition, studies were done changing the schedule from once weekly to three weekly dosing of trastuzumab with paclitaxel, reporting equiva- lent efficacy and also defining the pharmacokinetics of trastuzumab [87]. Trials exploring whether doublet-agent chemotherapy was superior to single-agent chemother- apy when combined with trastuzumab had inconsistent results. Improved ORR and PFS were shown when tras- tuzumab was combined with paclitaxel and carboplatin compared to paclitaxel alone [88], as well as with doc- etaxel and capecitabine compared to docetaxel alone [89]. However, the contradictory negative results of two other studies [90, 91], lack of meaningful OS advantage, and increased toxicity of chemotherapy doublet backbones limited their clinical applicability. The HER2-targeted TKIs lapatinib and neratinib were also evaluated in the first-line setting. In the MA.31 trial, PFS was inferior with lapatinib plus paclitaxel compared to trastuzumab plus paclitaxel (9.0 months vs. 11.3 months, HR 1.37, 95% CI 1.13–1.65, p = 0.001) [92]. In the NEfERT-T trial, efficacy of neratinib and paclitaxel was similar to trastuzumab and paclitaxel, with a PFS of 12.9 months in both arms (HR 1.02, 95% CI 0.81–1.27, p = 0.89), but neratinib-treated patients had a significantly lower incidence of brain metas- tases (relative risk: 0.48, 95% CI 0.29–0.79, p = 0.002) and delay in onset of brain metastases (HR 0.45, 95% CI 0.26–0.78, p = 0.004) [93].
The practice-changing CLEOPATRA trial revealed the synergistic effect of dual pertuzumab and trastuzumab for first-line treatment of metastatic HER2-positive breast can- cer [94]. A total of 808 patients with newly diagnosed meta- static disease were randomized to receive 3-weekly trastu- zumab and a recommended minimum six cycles of docetaxel (median, eight cycles) plus either pertuzumab or placebo. The addition of pertuzumab to trastuzumab and docetaxel led to remarkable improvements in progression-free survival (PFS; 18.7 vs. 12.4 months, HR 0.68, 95% CI 0.58–0.80, p < 0.001) and OS (56.5 vs. 40.8 months, HR 0.68, 95% CI 0.56–0.84, p < 0.001), with no additional cardiotoxicity and only small increases in adverse events such as diarrhea, rash, and pruritus that were not detrimental to quality of life [95]. With a recently updated 8-year OS rate of 37% [96], this three-drug regimen has been unanimously established as optimal first-line therapy for advanced HER2-positive breast cancer per clinical guidelines [97, 98]. The other taxanes, paclitaxel and nab-paclitaxel, were shown to be accept- able alternatives to docetaxel in the PERUSE trial [99]. Of note, 29% of patients in the PERUSE study had previously received neoadjuvant or adjuvant trastuzumab compared with 11% in the CLEOPATRA trial, which is slightly more representative of real-world practice. The single-arm phase II VELVET trial enrolled 213 patients with HER2-positive metastatic breast cancer to receive first-line trastuzumab and pertuzumab plus vinorelbine, with the anti-HER2 monoclo- nal antibodies administered sequentially in separate infu- sions in cohort 1 or in a single infusion in cohort 2 [100, 101]. Both cohorts demonstrated favorable ORR (74.2% in cohort 1, 63.7% in cohort 2) and PFS (14.3 months in cohort 1, PFS 11.5 months in cohort 2). Treatment was well toler- ated and consistent with known safety profiles of the individ- ual agents, with less grade ≥ 3 neutropenia (31.1% in cohort 1, 31.8% in cohort 2) and comparable rates of grade ≥ 3 diarrhea (6.6% in cohort 1, 6.5% in cohort 2) to that reported in the CLEOPATRA trial. Thus, vinorelbine represents an alternative chemotherapy backbone for dual trastuzumab and pertuzumab in patients with contraindications to taxanes.

4.2 Second‑Line
Continued HER2 blockade upon progression is critical to improving survival outcomes for metastatic HER2-positive breast cancer. Although shown to be inferior to trastuzumab as first-line therapy [92], lapatinib is effective and approved in the second-line setting in combination with capecitabine. In 399 patients who had progressed on prior trastuzumab- based therapy, lapatinib plus capecitabine significantly prolonged time to progression compared to capecitabine alone (8.4 vs. 4.4 months, HR 0.57, 95% CI 0.43–0.77, p < 0.001), with a trend towards improved OS (HR 0.78, 95% CI 0.55–1.12, p = 0.177) that was likely diluted by early crossover [102, 103].
The EMILIA trial defined a new standard for second-line therapy by demonstrating superiority of TDM1 to lapatinib plus capecitabine. In this phase III study of 978 patients with advanced HER2-positive breast cancer and prior exposure to trastuzumab, TDM1 resulted in significant improvements in PFS (9.6 vs. 6.4 months, HR 0.65, 95% CI 0.55–0.77,p < 0.001) and OS (29.9 vs. 25.9 months, HR 0.75, 95% CI 0.64–0.88) when compared to lapatinib plus capecitabine [104, 105]. Thrombocytopenia and liver enzyme elevations were the main toxicities with TDM1, in comparison to diar- rhea, hand-foot syndrome, and nausea with lapatinib plus capecitabine. TKIs have been hypothesized to penetrate the blood–brain barrier better than larger anti-HER2 monoclo- nal antibodies on the basis of phase II trials [106, 107], an important consideration given that up to 50% of HER2-posi- tive patients will develop brain metastases over the course of their metastatic disease [108–110]. However, a retrospective analysis of the EMILIA study demonstrated similar rates of CNS progression in the lapatinib plus capecitabine and TDM1 arms, irrespective of whether patients had baseline CNS metastases [111]. This echoes the results of the CER- EBEL trial, which demonstrated no significant difference in incidence of CNS metastases for lapatinib plus capecitabine versus trastuzumab plus capecitabine (3% vs. 5%, 95% CI– 2 to 5%, p = 0.360) [112]. Further evidence challenging this assumption comes from a recent post hoc analysis of 398 patients with baseline brain metastases enrolled the sin- gle-arm phase IIIb KAMILLA trial of TDM1 [113]. Among this subset of patients, ORR across all organs was 21.4%, with responses in brain target lesions observed in 49.3% of patients without prior brain radiation therapy (RT) and in 32.7% of patients who received prior brain RT ≥ 30 days before baseline. Patients with baseline brain metastases had a PFS of 5.5 months and OS of 18.9 months, and they expe- rienced similar side effects to patients without baseline brain metastases, except for a slightly higher rate of headache and vomiting.
In summary, the superior efficacy and more favora- ble side-effect profile of TDM1 has pushed lapatinib plus capecitabine to later-line treatment. Studies evaluating the addition of chemotherapies to TDM1, such as capecitabine in the phase 1/2 TRAXHER2 trial, have demonstrated no or modest improvements in efficacy compared to TDM1 alone and increased toxicity [114–116]. TDM1 was also studied in the first-line setting in the MARIANNE trial, alone or in combination with pertuzumab, and found to be equivalent to trastuzumab plus taxane, affirming TDM1 is best suited to second-line therapy [117].

4.3 Third‑Line and Beyond
Multiple HER2-directed therapy options exist in the third-line setting, including the aforementioned lapatinib plus capecitabine, lapatinib plus trastuzumab as per the EGF104900 study, and trastuzumab plus chemotherapy as discussed earlier [118]. The TH3RESA trial compared TDM1 to physician’s choice therapy in patients who had progressed on at least two anti-HER2 agents and demon- strated improved PFS (6.2 vs. 3.3 months, HR 0.53, 95% CI 0.42–0.66, p < 0.001) and OS (22.7 vs. 15.8 months, HR 0.68, 95% CI 0.54–0.85, p = 0.007) with TDM1, proving the drug is also effective in later-line treatment [119, 120].
Over the past year, there has been a wave of new and promising later-line therapeutic options for HER2-positive metastatic breast cancer. In the phase III NALA trial, 621 patients who had received two or more prior lines of HER2- directed therapy for metastatic HER2-positive breast cancer were randomized to neratinib plus capecitabine versus lapa- tinib plus capecitabine [121]. Only asymptomatic and stable brain metastases were included, and 65% of patients were treatment-naïve to pertuzumab and/or TDM1. The 12-month PFS rate doubled from 15% in the lapatinib arm to 29% in the neratinib arm (HR 0.76, 95% CI 0.63–0.93, p = 0.0059) and mean OS at 48 months favored the neratinib arm (24.0 vs. 22.2 months, HR 0.88, 95% CI 0.72–1.07, p = 0.2086).
As anticipated, there was a higher rate of grade 3 diarrhea in neratinib-treated patients (24.4% vs. 12.5%), but both arms had a duration of diarrhea of 4 days and similar rates of treatment discontinuation due to diarrhea (2.6% with ner- atinib vs. 2.3% with lapatinib). In addition, there was no cor- responding significant difference in patient-reported qual- ity of life observed between arms. Of note, fewer patients receiving neratinib required intervention for symptomatic brain metastases (overall cumulative incidence 22.8% vs. 29.2%), suggestive of a delay in central nervous system (CNS) progression and in complement to the findings of the NEfERT-T trial [93].
The phase III HER2CLIMB-01 trial randomized 480 metastatic HER2-positive breast cancer patients previouslytreated with trastuzumab, pertuzumab, and TDM1 (median four prior lines of therapy) to either tucatinib or placebo, in combination with trastuzumab and capecitabine [122]. Tucatinib is a highly selective TKI of HER2, hypothesized to be less toxic given its minimal inhibition of EGFR [19, 123]. In contrast to NALA and most other trials in the advanced setting, HER2CLIMB-01 allowed progressing brain metas- tases not requiring immediate local therapy. It is also worth noting that, given only 6% of enrolled patients had received prior lapatinib, lapatinib may have been a preferable com- parator arm to placebo. Compared to placebo, the addition of tucatinib to trastuzumab and capecitabine significantly pro- longed PFS (7.8 vs. 5.6 months, HR 0.54, 95% CI 0.42–0.71, p < 0.001) and OS (21.9 vs. 17.4 months, HR 0.66, 95% CI0.50–0.88, p = 0.005). The most common grade ≥ 3 adverse events experienced by patients receiving tucatinib versus placebo were diarrhea (13% vs. 9%), hand-foot syndrome (13% vs. 9%), elevated liver enzymes (5% vs. 0.5%), and fatigue (5% vs. 4%). Among the 291 patients (47.5%) with baseline brain metastases, PFS at 1 year was 24.9% with tucatinib and 0% with placebo (HR 0.48, 95% CI 0.34–0.69, p < 0.001), with median PFS of 7.6 months and 5.4 months, respectively. A recent exploratory analysis of this subset of patients demonstrated the addition of tucatinib significantly reduced the risk of intracranial progression or death (HR 0.32, 95% CI 0.22–0.48, p < 0.0001) and death (OS HR 0.58,95% CI 0.40–0.85, p = 0.005) and doubled the intracranial ORR (47.3% vs. 20.0%, p = 0.03) compared to placebo [124]. Median CNS-PFS was 9.9 months in the tucatinib arm versus 4.2 months in the control arm, and median OS was 18.1 months versus 12.0 months.
The SOPHIA study presented at the 2019 San Anto- nio Breast Cancer Symposium compared trastuzumab to the novel monoclonal antibody margetuximab [125, 126]. Margetuximab binds to the same epitope on HER2 as tras- tuzumab, but has an optimized Fc domain with increased affinity for the activating CD16A receptor and decreased affinity for the inhibitory CD32B receptor, resulting in enhanced ADCC [127, 128]. The phase III SOPHIA trial comprised 536 HER2-positive patients with disease progres- sion after ≥ two lines of HER2-directed therapy including pertuzumab (100%) and TDM1 (91%). There was a modest PFS benefit with margetuximab compared to trastuzumab (5.8 vs. 4.9 months, HR 0.76, 95% CI 0.59–0.98, p = 0.033); however, OS data remains immature albeit in favor of mar- getuximab at median follow-up of 15.6 months (21.6 vs. 19.8 months, HR 0.89, 95% CI 0.69–1.13, p = 0.3626). Magetuximab was more effective in carriers of CD16A-158F, a low-affinity allele that has been associated with diminished response to trastuzumab [129, 130], conferring an OS of 23.7 months compared to 19.4 months with trastuzumab (HR 0.79, 95% CI 0.61–1.04, p = 0.087). Infusion-related reactions were more common with margetuximab than trastuzumab (13.3% vs. 3.4%), but safety profiles were oth- erwise comparable between the two drugs.
The DESTINY-Breast01 study explored the efficacy of the novel antibody–drug conjugate trastuzumab deruxtecan. Compared to TDM1, trastuzumab deruxtecan has a higher drug-to-antibody ratio (8% vs. 3–4%) and a membrane-per- meable payload, which can affect neighboring tumor cells irrespective of their HER2 expression status via a bystander effect [16, 131]. In the single-arm phase II DESTINY- Breast01 trial involving 184 heavily pretreated (median six prior lines of therapy) patients with metastatic HER2- positive breast cancer, trastuzumab deruxtecan resulted in an impressive ORR of 60.9% (95% CI 53.4–68.0) and PFS of 16.4 months (95% CI 12.7–NR (not reported)). The most common grade ≥ 3 adverse events were decreased neutro- phil count (20.7%), anemia (8.7%), and nausea (7.6%). Importantly, interstitial lung disease (ILD) occurred in 14% patients, including four patients (2.2%) who died as a result. On the basis of the meaningful results of the DESTINY- Breast01 trial, trastuzumab deruxtecan received accelerated FDA approval in December 2019, with a “black box” warn- ing to monitor closely for signs and symptoms of ILD and treat promptly with steroids when suspected.
In the 50% of HER-positive patients with HR+ disease, consideration can be given to combining anti-HER2 and hormonal agents as a means of overcoming the crosstalk between HER2 and ER pathways that contributes to endo- crine resistance [45]. Three phase III trials confirmed supe- rior PFS and time to progression when aromatase inhibitors (AIs) were combined with either trastuzumab or lapatinib compared to AI monotherapy as first-line treatment for post- menopausal patients with HER2-positive, HR+ metastatic breast cancer [132–134]. The phase II PERTAIN trial ran- domly assigned 258 postmenopausal patients with HER2- positive, HR+ advanced breast cancer to receive first-line pertuzumab plus trastuzumab and AI or trastuzumab plus AI [135]. Dual anti-HER2 blockade with pertuzumab plus trastuzumab conferred an impressive PFS of 18.9 months (vs. 15.8 months in the trastuzumab arm, HR 0.65, 95% CI 0.48–0.89, p = 0.007) comparable to the CLEOPATRA trial, but notably over half of patients received induction chemo- therapy with a taxane prior to initiating endocrine therapy. The ALTERNATIVE trial evaluated the chemotherapy-free regimen of lapatinib plus trastuzumab and AI in metastatic postmenopausal HER2-positive, HR+ patients with disease progression on prior trastuzumab-based chemotherapy in either the (neo)adjuvant or first-line metastatic setting [136]. There was a significant improvement in PFS with this combi- nation compared to trastuzumab plus AI (11 vs. 5.6 months, HR 0.62, 95% CI 0.45–0.88, p = 0.006). Although a chemo- therapy backbone remains the preferred first-line strategy in this patient population given the proven OS benefit, combin- ing anti-HER2 agents with endocrine therapy is an effective and less toxic alternative that may be employed in later-line settings. More recently, CDK 4/6 inhibitors have also shown promise in HER2-positive, HR+ positive patients. The CDK 4/6 inhibitor, abemaciclib, has been shown to enhance activ- ity of anti-HER2 agents and re-sensitize tumors to EGFR/ HER2 blockade in preclinical models [137], which led to the investigation of abemaciclib in the phase II MONARCH- HER trial. In this study, 237 postmenopausaul HER2-pos- itive, HR+ patients who had received two or more lines of prior therapies for advanced breast cancer were randomized to abemaciclib plus trastuzumab and fulvestrant (arm A), abemaciclib plus trastuzumab (arm B), or trastuzumab plus chemotherapy (arm C) [138]. Superior PFS was observed in arm A compared to arm C (8.3 vs 5.7 months, HR 0.67, 95% CI 0.45–1.00, p = 0.025), while there was no difference in PFS between arms B and C. Patients receiving abemaciclib in arm A experienced more grade 3/4 adverse events than arm C (56% vs. 33%), with the most noticeable difference seen in rates of thrombocytopenia (10.3% vs. 2.8%) and diar- rhea (9.0% vs. 2.8%).
Synergistic use of anti-HER2 agents and immunotheapy for HER2-postive breast cancer is an additional area of growing interest for several reasons. Firstly, there is increasing evidence that the innate and adaptive immune systems play a significant role in the therapeutic activity of trastuzumab and other monoclonal anti-HER2 antibod- ies [139]. Secondly, HER2-positive tumours frequently exhibit features of immunogenicity including increased tumour mutational burden (TMB), high tumour-infiltrating lymphocyte (TIL) numbers, and greater programmed cell death-ligand (PD-L1) expression [140–142]. The single-arm phase IB/II PANACEA trial evaluated the efficacy of the PD-1 inhibitor pembrolizumab in combination with trastu- zumab in 58 patients with advanced HER2-positive breast cancer who had previously progressed on trastuzumab- based therapy [143]. The ORR was 15% in PD-L1 positive patients, while there were no responders among the PD-L1 negative cohort. In PD-L1 positive patients with more than 5% TILs, the ORR was higher at 39%, akin to other studies that have demonstrated better outcomes for HER2-positive patients with more TILs in the early and metastatic set- ting [144–147]. Serious adverse events occurred in 50% of patients, the most common being dyspnea (5%), pneumo- nitis (5%), pericardial effusion (3%), and upper respiratory infection (3%). There was one treatment-related death due to the autoimmune Lambert-Eaton myasthenic syndrome. In the phase II KATE2 trial, the PD-L1 inhibitor atezolizumab did not improve PFS, ORR, or OS when added to TDM1 in metastatic HER2-positive patients with progression on prior trastuzumab-based therapy [148, 149]. However, there was a numerical increase in endpoints within the PD-L1 positive and TIL-high (≥ 5%) subgroups. Anti-HER2 com- bination therapies with PD-1/PD-L1 inhibitors, bifunctional antibodies, vaccines, and CAR-T cells are all promising immunomodulatory strategies under current investigation.
Discerning between the emerging array of therapeutic options for metastatic HER2-positive breast cancer in the later-line setting may present a challenge as these agents become more widely available. As of yet, only tucatinib has demonstrated an OS benefit of 4.5 months. In addition, as all patients in the HER2CLIMB-01 trial had prior expo- sure to both TDM1 and pertuzumab, the patient population reflects current clinical cohorts. Finally, the inclusion of both stable and active brain metastases and confirmed intracra- nial efficacy among this subset make tucatinib an appeal- ing choice in this notoriously difficult-to-treat population. While at present HER2-targeted TKIs are best considered for later-line treatment of metastatic HER2-positive breast cancer, the addition of tucatinib or neratinib to TDM1 in the second-line setting is being explored and could have impli- cations for treatment sequencing in future (NCT03975647, NCT02236000). Trastuzumab deruxtecan is a promis- ing option for heavily pretreated patients, and a phase III trial is now underway (NCT03529110). Interestingly, a phase Ib trial utilizing trastuzumab deruxtecan in HER2- low metastatic breast cancer reported an ORR of 37.0% (95% CI 24.3–51.3%) and median duration of response of 10.4 months (95% CI 8.8–NR) in 54 extensively pretreated (median 7.5 prior lines of therapy) patients [150]. Low HER2 expression, defined as IHC 1+/2+ and FISH nega- tive, is present in 40–50% of breast cancers and comprises a heterogenous group including luminal-like HR+ and triple- negative breast cancers [151–153]. The antitumor activity of trastuzumab deruxtecan in HER2-low-expressing tumours may be related to its high drug-to-antibody ratio and potent bystander effect. A phase III trial is currently examining trastuzumab deruxtecan in HER2-low metastatic breast can- cer (NCT03734029), results of which have the potential to invoke a paradigm shift in the definition of HER2 status in breast cancer and expand the subset of patients who may benefit from HER2-directed therapy. Like trastuzumab der- uxtecan, the novel second-generation antibody–drug conju- gate SYD985 has a potent and membrane-permeable duo- carmycin payload, capable of inducing a bystander killing effect [154]. SYD985 has demonstrated favorable ORRs in heavily pretreated HER2-positive and HER2-low metastatic breast cancer patients in a phase I trial [155], and a phase III trial (TULIP) is currently ongoing (NCT03262935).

5 Implementation of Anti‑HER2 Biosimilars
Biosimilar agents are being rapidly developed in oncol- ogy, with the aim of providing cost-effective alternatives to biologics. Unlike small-molecule drugs for which iden- tical generic versions can be manufactured, biologics are larger and structurally complex, such that exact replication is impossible. Biosimilars must therefore undergo abbreviated but rigorous testing to demonstrate bioequivalence in safety, efficacy, and immunogenicity to the reference product.
Currently, there are five trastuzumab biosimilars approved by the FDA and European Medicines Agency (EMA), each having demonstrated equivalent efficacy to trastuzumab in phase III trials using the regulatory recommended equivalence trial design (Table 4). All trials used anthracycline/taxane- based chemotherapy in the neoadjuvant setting and taxane- based chemotherapy in the metastatic setting. Safety analyses of hematological and non-hematological toxicity, cardiotoxic- ity, and immunogenicity revealed no significant differences between any of the biosimilars and the reference product. The LILAC trial was the only study to include a switch from neoadjuvant reference product to adjuvant ABP-980 after surgery [156]. It has been hypothesized that switching from a reference product to its biosimilar could potentially lead to enhanced immunogenicity. However, no increase in frequency and severity of adverse events, nor increased incidence of anti- drug antibodies, was observed in the LILAC switch group. Interestingly, the CI upper boundaries for both ABP-980 and SB3 exceeded the predefined equivalence margin, ruling out non-inferiority but not potential superiority [156, 157]. These findings were considered to have resulted partly from a small downward drift in ADCC activity in the trastuzumab refer- ence product [158], a phenomenon that may occur over time with biologics, and highlights the need for post-marketing pharmacovigilance programs.
Complexities in the evaluation of trastuzumab biosimilars has raised questions about the clinical application of these agents. These include questions regarding the patient population used to establish comparable efficacy, choice of clinical trial endpoints, and extrapolation to other drug com- binations. To mitigate these concerns, there has been a shift towards testing biosimilars in the neoadjuvant setting, as this may represent a more homogenous patient population with fewer confounding characteristics, compared to the heterog- enous group of metastatic cancer patients. Moreover, pCR might be considered a more favorable clinical endpoint than ORR in metastatic breast cancer, given its validated corre- lation with long-term survival. However, it remains uncer- tain whether the equivalence demonstrated in these trials is representative of combination with other anti-HER2 agents, such as pertuzumab in the metastatic setting. To date, phase III trials have only studied trastuzumab biosimilars as single agents in combination with chemotherapy.
An additional factor that could hinder the introduction of biosimilars into clinical practice is the development of sub- cutaneous (SC) trastuzumab and pertuzumab formulations. The short administration times, fixed dosages, and lack of requirement for infusion facilities with SC formulations of anti-HER2 monoclonal antibodies is appealing to patients and healthcare providers alike. The Roche product trastu- zumab hyaluronidase (Herceptin Hylecta) received EMA approval in September 2013 and FDA approval in Febru- ary 2019. The HannaH study was the first phase III trial to evaluate the safety and efficacy of SC trastuzumab [164]. In total, 596 patients with HER2-positive operable or locally advanced breast cancer were randomized to receive eight cycles of either fixed-dose SC trastuzumab or weight-based intravenous (IV) trastuzumab every 3 weeks concurrently with neoadjuvant chemotherapy, followed by surgery and continued adjuvant SC or IV trastuzumab for ten cycles. SC trastuzumab was non-inferior to IV trastuzumab with respect to the proportion of patients achieving pCR (45.4% vs. 40.7%, 95% CI -4.0–13.4), and an updated analysis dem- onstrated similar 6-year event-free survival rates (EFS, 65% vs. 65%, HR 0.98, 95% CI 0.74–1.29) and OS rates (84% vs.
84%, HR 0.94, 95% CI 0.61–1.45) [165]. There were more grade ≥ 3 adverse events with SC compared to IV trastu- zumab (20.9% vs. 10.9%), mainly attributable to infections (8.1% vs. 4.4%). Of note, a pharmacokinetic study has sug- gested that the proportion of patients achieving adequate plasma trough concentrations of trastuzumab is higher with the IV than the SC formulation (93.8% vs. 67.6%, p = 0.042), with even greater differences seen in patients with a body mass index (BMI) > 30 kg/m2 [166]. However, exploratory subgroup analyses in the HannaH trial indicated that 3-year EFS rates were similar irrespective of bodyweight and serum trough trastuzumab concentrations at predose cycle 8 [167]. Other phase III trials have shown comparable efficacy and safety for SC trastuzumab, including the SafeHER study in the adjuvant setting [168], the MetaPHER study of SC tras- tuzumab in combination with IV pertuzumab and docetaxel in the first-line metastatic setting [169], and the PrefHer patient preference study of adjuvant SC trastuzumab fol- lowed by IV trastuzumab or the reverse sequence, in which 88.9% of patients preferred the SC formulation [170]. In June 2020, the FDA approved a fixed-dose combination of trastuzumab and pertuzumab with hyaluronidase (Phesgo) on the basis of the phase III FeDeriCa trial. In this study, 500 patients receiving neoadjuvant chemotherapy for operable or locally advanced HER2-postive breast cancer were randomly assigned to receive either SC or IV trastuzumab and pertu- zumab, with continued adjuvant dual SC or IV trastuzumab and pertuzumab post-surgery to complete a total of 18 cycles [171]. The pCR rate was 60% in both arms and overall toxic- ity, including cardiac safety, was comparable.
An updated analysis at 36 months showed equivalent OS for the trastuzumab biosimilar, MYL-1401O, and trastu- zumab (35.0 vs. 30.2 months, HR 0.9, 95% CI 0.70–1.17) [172]. Emergence of such long-term trial data, as well as real-world efficacy and safety outcomes in previously stud- ied and extrapolated indications, is expected to increase uptake of trastuzumab biosimilars within the oncology
FDA US Food and Drug administration, EMA European Medicines Agency, MBC metastatic breast cancer, EBC early breast cancer, NAT neo- adjuvant therapy, ORR overall response rate, pCR pathologic complete response, RP reference product, RD risk difference, RR risk ratio, Ctrough trough plasma concentration community. It is important that clinicians understand the goal of biosimilar comparability studies are to demonstrate biosimilarity rather than patient benefit, which has already been demonstrated for the reference product. Biosimilars can help alleviate the growing financial burden of can- cer therapies on healthcare budgets, and ideally allow for more spending towards new targeted agents. In addition, in developing countries, where it is estimated less than 10% of patients receive anti-HER2 agents, trastuzumab biosimilars hold the potential to improve patient access to life-saving breast cancer treatment [173]. However, given the superior convenience of SC formulations of anti-HER2 monoclonal antibodies, cost-effectiveness analyses comparing SC tras- tuzumab and biosimilar IV trastuzumab in the treatment of HER2-positive breast cancer would be valuable.

6 Conclusion
The prognosis for patients with HER2-positive breast can- cer has been profoundly changed since the introduction of trastuzumab, the first monoclonal antibody to target the HER2 oncogene. Additional therapeutic advances have been achieved by combining trastuzumab with other anti-HER2 monoclonal antibodies in the neoadjuvant and metastatic set- tings, and using antibody–drug conjugates to treat residual disease after neoadjuvant therapy or when metastatic disease progresses on trastuzumab-based therapy. Newer approaches using more selective and less toxic HER2-targeted TKIs, Fc-engineered monoclonal antibodies, and highly potent antibody–drug conjugates with permeable payloads have demonstrated efficacy in later-line treatment of metastatic HER2-positive cancer.
With multiple approved anti-HER2 agents for advanced disease, possessing different mechanisms of action and safety profiles, identifying the best sequence can be chal- lenging. As drugs such as pertuzumab and TDM1 are increasingly used in the neoadjuvant and adjuvant settings, newer agents may be more effective first-line treatment of recurrent disease, particularly in early relapsers. Patient disease characteristics such as the presence of brain metas- tases or perhaps eventually degree of HER2 expression may help guide optimal therapy choice. Likewise, as uti- lization of TILs and other biomarkers increases, we will be better able to detect which patients may benefit from immune checkpoint inhibitors and additional immuno- therapeutic strategies that have revolutionized the manage- ment of other cancer subtypes. Trastuzumab biosimilars have the potential to not only improve patient access to HER2-directed therapy, but also ease the growing financial burden of anticancer biologics and conceivably open up funding avenues for emerging novel anti-HER2 agents.
In early HER2-positive breast cancer, ongoing research on identifying patient subsets requiring treatment escala- tion and those who can have their treatment safely deesca- lated, perhaps even to chemotherapy-free regimens, will facilitate personalized cancer care approaches. With so many therapeutic options now available in the metastatic setting, future clinical trials will need to consider opti- mal sequencing of HER2-directed therapy and incorpora- tion of biomarkers to better understand patterns of HER2 resistance, as a continued effort to improve outcomes for patients with advanced disease.

Acknowledgements
We would like to thank Dr. Justin Pater for his help in figure preparation.Declarations
Funding No external funding was used in the preparation of this arti- cle.
Conflict of interest M. T. has no disclosures to declare. K. G. reports participation in advisory boards for AstraZeneca, Pfizer, Novartis, Lil- ly, Roche, BMS, Genomic Health, Merck, Mylan, Nanostring, Seattle Genetics, and Knight Pharmaceuticals.
Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable.
Availability of data and material Not applicable.
Code availability Not applicable.
Author contributions MT and KG wrote the manuscript, which was conceived by KG.

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