	Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is an aggressive type of breast cancer with an inferior prognosis. Trastuzumab, a human monoclonal anti-HER2 antibody, was the first targeted agent for this breast cancer subtype. However, de novo and acquired trastuzumab resistance does occur, resulting in the need for other HER2-directed therapies. One approach has been the use of antibody drug conjugates (ADCs), which are cytotoxic drugs connected by chemical linkers to monoclonal antibodies that are specific for a tumor-associated antigen. This results in the preferential delivery of a drug to cancer cells while minimizing exposure to normal tissues. Ado-trastuzumab emtansine (T-DM1) is a novel ADC that consists of trastuzumab bound via a thioether linker to the cytotoxic agent, DM1, a highly potent antimitotic drug. Currently, T-DM1 is approved for patients with HER2-positive metastatic breast cancer who have previously received trastuzumab and a taxane, progressed on their most recent treatment in the locally advanced or metastatic setting, or are within 6 months of adjuvant trastuzumab for early-stage disease. It is currently being investigated in the neoadjuvant and adjuvant settings. This article provides a concise review of the clinical development, use, safety, and pharmacokinetics of T-DM1, as well as future research directions of T-DM1 in the field of HER2-directed therapies.

Human epidermal growth factor receptor 2 (HER2)-positive disease accounts for 20% to 25% of breast cancers, as represented by amplification of the HER2 gene and/or HER2 protein overexpression. This breast cancer subtype has an aggressive clinical course and inferior outcome.

 Trastuzumab is a humanized monoclonal anti-HER2 antibody that binds to the extracellular domain of HER2 and has significantly improved outcomes for patients with both early-stage and metastatic HER2-positive disease.

 Despite these significant advances, both de novo and acquired trastuzumab resistance occurs. Approximately 15% of women develop metastatic breast cancer (MBC) despite trastuzumab-based adjuvant therapy.

 This has led to the development of other HER2-directed therapies. Lapatinib, a potent small molecule dual tyrosine kinase inhibitor of HER2 and epidermal growth factor receptor (EGFR), was approved by the FDA in 2007 either in combination with capecitabine or trastuzumab for patients with HER2-positive MBC following disease progression on trastuzumab.

 In 2012, pertuzumab, a monoclonal antibody that binds subdomain II of the HER2 extracellular domain and blocks HER2 dimerization and signaling, was approved in combination with trastuzumab and docetaxel in the first-line setting for HER2-positive MBC (

 Most recently, the FDA provided accelerated drug approval for dual HER2 blockade in the neoadjuvant setting using pertuzumab and trastuzumab in combination with chemotherapy.

		Figure. Mechanisms of Action of HER2-Directed Agents for Breast Cancer

		Human epidermal growth factor (EGF) receptors HER1, HER2, HER3, and HER4 are receptor tyrosine kinases that are involved in the signal transduction pathways that lead to cell survival and differentiation. Each of these receptors consists of three components: an extracellular binding domain, a transmembrane lipophilic segment, and a functional intracellular tyrosine kinase domain (with the exception of HER3). When a ligand of an EGF receptor binds to the extracellular binding domain, the tyrosine kinase domains are activated by both homodimerization and heterodimerization. The HER2 receptor is an orphan receptor and does not require the binding of a ligand for activation. HER2 is also the preferential dimerization partner of other members of the EGF family, and dimerization can be induced by receptor overexpression and mutation. Homo- and heterodimerization of the HER family leads to phosphorylation of the tyrosine kinase domain and activation of downstream signaling pathway, including the PI3K-AKT-mTOR and the RAF-MEK-MAPK pathways. Downstream effects include survival, proliferation, and increased vascular endothelial growth factor (VEGF). Anti-HER2 strategies include: (A) Trastuzumab is a monoclonal antibody that binds to the extracellular domain IV of the HER2 receptor. (B) Lapatinib is a dual tyrosine kinase inhibitor against HER2 and EGFR (HER1). (C) Pertuzumab is a monoclonal antibody that binds to extracellular domain II of HER2 and inhibits the dimerization of HER2 with other HER family receptors, especially HER3. The HER2-HER3 heterodimer is a strong activator of signaling through the PI3K-AKT-mTOR pathway. (D) T-DM1 is a novel antibody-drug conjugate of trastuzumab and DM1, a derivative of maytansine and a highly potent antimitotic drug. The drug binds to the extracellular domain of HER2, allowing for the selective delivery of DM-1 to HER2-overexpressing cells.

One novel approach in the development of new HER2-targeted therapies has been the development of HER2-directed antibody-drug conjugates (ADCs).

 ADCs are cytotoxic drugs that are connected by chemical linkers to monoclonal antibodies specific for a tumor-associated antigen. ADCs are designed to preferentially deliver a cytotoxic drug to cancer cells while minimizing exposure to normal tissues, thus improving the therapeutic index.

 The use of ADCs in oncology first began with the development of gemtuzumab ozogamicin, an anti-CD33 antibody linked to the cytotoxic antibiotic, calicheamicin, for CD33-positive acute myelogenous leukemia (AML).

 Subsequently, brentuximab vedotin, a CD30-specific ADC, was developed and is approved for use in Hodgkin lymphoma and anaplastic large cell lymphoma.

Ado-trastuzumab emtansine (T-DM1) is an ADC that was developed to treat HER2-positive MBC. T-DM1 consists of trastuzumab covalently bound via a thioether linker, (N-maleimidomethyl) cyclohexane-1-carboxylate (MCC), to the cytotoxic agent, DM1, a derivative of maytansine. DM1 is a highly potent antimitotic drug that binds to microtubules in a similar way to that of vinca alkaloids.

 Vinca alkaloids have demonstrated synergy with trastuzumab.

 T-DM1 binds to the extracellular domain of HER2, and the complex is then internalized into the cell. The antibody is degraded by proteases and the active metabolite, lysine-Nε—MCC-DM1, is released into the cytoplasm (

 This metabolite is a charged molecule and is relatively membrane impermeable. This reduces the chance that DM1 can enter a neighboring cell and limits the possibility of nonspecific toxicity.19 T-DM1 also retains the effector functions of trastuzumab: inhibition of HER2-mediated signal transfusion and activation of antibody-dependent cellular cytotoxicity.

T-DM1 was initially evaluated as a single agent in a phase I dose escalation study in 24 patients with trastuzumab-refractory HER2-positive MBC (TDM3569g).

 Weekly and every-3-week dosing schedules were tested. The maximum tolerated dose (MTD) for the 3-week regimen was determined to be 3.6 mg/ kg. T-DM1 was well tolerated and the most common adverse events (AEs) were thrombocytopenia, elevated transaminases, fatigue, anemia, and nausea.

 In the 3-week regimen, the objective response rate (ORR) was 44% in patients treated at the MTD and was comparable to patients treated on the weekly regimen (40%). Subsequently, the every-3-week schedule has been utilized in the majority of subsequent studies, largely due to the convenience for patients.

T-DM1 monotherapy was then studied in two single arm phase II trials (TDM4258g and TDM4374g). TDM4258g evaluated T-DM1 at 3.6 mg/kg every 3 weeks for patients with HER2-positive MBC who had progressed on at least one line of HER2-directed therapy. A total of 112 patients with a median of 8 (range 2-19) prior anticancer agents in all settings were enrolled. The primary endpoint, ORR, was 25.9% (95% confidence interval [CI], 18.4% to 34.4%). Median progression free survival (PFS) was 4.6 months (95% CI, 3.9 to 8.6 months), and median duration of response (DOR) was not reached. In a post hoc exploratory analysis, the ORR in patients who had received prior lapatinib and trastuzumab was not significantly different than the overall ORR, suggesting that T-DM1 has activity after progression on prior HER2-directed therapies.

TDM4374g enrolled 110 patients who had previously received trastuzumab, lapatinib, capecitabine, a taxane, and an anthracycline for HER2-positive breast cancer and had progressed on at least two HER2-directed agents in the metastatic or locally advanced setting.

 This was in contrast to TDM4258g, which only required that patients had received and progressed on at least one prior HER2-directed therapy and had received at least one chemotherapy regimen in the metastatic setting.

 In TDM4374g, all patients were heavily pretreated with a median of 7 (range 3-17) anticancer agents in the metastatic setting. The ORR was 34.5% (95% CI, 26.1% to 43.9%) with a median PFS of 6.9 months (95% CI, 4.2 to 8.4 months) and a clinical benefit rate (CBR) of 48.2% (95% CI, 38.8% to 57.9%). This confirmed the single agent activity of T-DM1 in the metastatic setting.

T-DM1 was subsequently evaluated in several large, randomized studies. The pivotal EMILIA study was a phase III, international trial in patients with locally advanced or metastatic, centrally confirmed, HER2-positive breast cancer. Patients had previously received a taxane and trastuzumab and had progressed on their most recent treatment in the locally advanced or metastatic setting or within 6 months of completion of adjuvant trastuzumab for early-stage disease. A total of 991 patients were randomized 1:1 to T-DM1 (3.6 mg/kg every 3 weeks) or to lapatinib (1250 mg daily) and capecitabine (2000 mg/m

 days 1-14). The primary endpoints were PFS and overall survival (OS). Treatment with T-DM1 significantly improved median PFS compared with lapatinib plus capecitabine (9.6 vs 6.4 months; hazard ratio [HR] 0.65; 95% CI, 0.55 to 0.77; 

<.001). OS also favored T-DM1 (30.9 vs 25.1 months, HR 0.68; 95% CI, 0.55 to 0.85; 

<.001). ORR and DOR were superior in patients treated with T-DM1. Serious AEs were reported for 18% of patients in the lapatinib-capecitabine group versus 15.5% in the T-DM1 group. There were more grade ≥3 AEs in the lapatinib-capecitabine group versus in the T-DM1 group (57% vs 40.8%). The most commonly reported grade 3 or 4 AEs were diarrhea and palmar-plantar erythrodysesthesia in the lapatinib-capecitabine group and thrombocytopenia and elevated transaminases in the T-DM1 group.

 The results of the EMILIA study led to the FDA approval of T-DM1 in February 2013 for patients with HER2-positive MBC.

The TH3RESA trial is another phase III trial, which evaluated the efficacy and safety of T-DM1 in comparison with treatment of physician’s choice (TPC) in 602 patients with MBC or unresectable locally advanced/recurrent HER2-positive breast cancer who had received at least two prior HER2-directed therapies, including trastuzumab and lapatinib (NCT01419197).

 The first results from this trial, presented at the 2013 European Cancer Congress, showed that median PFS increased by nearly 3 months (6.2 vs 3.3 months; HR 0.528; 95% CI, 0.422-0.661; 

<.0001) in patients receiving T-DM1 compared with patients receiving TPC.

T-DM1 has also been evaluated in the first-line metastatic setting. The TDM4450g study was a phase II trial in which 137 patients with previously untreated HER2-positive metastatic or locally advanced breast cancer were randomized to T-DM1 or trastuzumab plus docetaxel. The primary endpoints were PFS and safety. PFS was significantly longer in patients receiving T-DM1 compared with those receiving trastuzumab plus docetaxel (14.2 months vs 9.2 months, HR 0.59; 95% CI, 0.36 to 0.97; 

=.035). Patients receiving T-DM1 had fewer grade ≥3 AEs compared with the trastuzumab plus docetaxel group (46.4% vs 90%). Of note, only 27.1% of the patients in the trastuzumab plus docetaxel group and 17.9% of the patients in the T-DM1 group had received trastuzumab in the neoadjuvant or adjuvant setting.

Encouraged by the synergistic activity noted in xenograft models, the combination of T-DM1 plus pertuzumab was then evaluated in a phase Ib/II clinical trial (TDM4373g). The safety and efficacy of T-DM1 plus pertuzumab in 67 patients with HER2-positive locally advanced or MBC was assessed. Patients received the combination either in the first-line or relapsed setting (defined as progression on prior HER2 therapy for MBC). All patients in the relapsed setting (n=46) had received prior trastuzumab and 75% had received prior lapatinib; of these 46 patients, 16 had confirmed responses. Interim results showed ORRs of 34.8% and 57.1% in the relapsed and first-line settings, respectively. The combination was deemed to be safe and tolerable.

Notably, the combination regimen did result in a 24% incidence of peripheral sensory neuropathy, and one patient was discontinued from the trial due to a decrease in left ventricular ejection fraction. However, the peripheral neuropathy was mild and it is not known if it was noted specifically in the patients treated with T-DM1 in the first-line setting versus those treated in the relapsed setting. In the 4374g phase II trial of single agent T-DM1, there was 18% incidence of peripheral neuropathy. However, these patients were heavily pretreated. The incidence of neuropathy will perhaps be further elucidated in the MARIANNE study.

The MARIANNE study is a randomized, 3-arm, multicenter, phase III study that is evaluating the efficacy and safety of T-DM1 with pertuzumab in patients with HER2-positive progressive or recurrent locally advanced or previously untreated MBC (NCT01120184). Patients were randomized to one of the three cohorts: T-DM1 with pertuzumab, T-DM1 with pertuzumab-placebo (blinded for pertuzumab), or trastuzumab combined with a taxane. This trial has completed enrollment and results are eagerly awaited later in 2014.

An additional phase 1b study of T-DM1 and paclitaxel with or without pertuzumab showed that the combination was feasible, and updated results regarding dosing schedules and safety are awaited.

					T-DM1 + capecitabine in HER2+ MBC and HER2+ locally advanced or metastatic gastric cancer (followed by phase II component)

					T-DM1 in HER2+ MBC and normal or reduced hepatic function

					Neoadjuvant T-DM1 with or without standard endocrine therapy vs trastuzumab with standard endocrine therapy for 12 weeks in patients with operable HER2+/HR+ breast cancer

					Pertuzumab + trastuzumab with or without chemotherapy both followed by T-DM1 in case of progression in patients with HER2+ MBC

					Pertuzumab + trastuzumab vs pertuzumab + trastuzumab + metronomic chemotherapy in the elderly HER2+ MBC population who may continue on T-DM1 alone following disease progression

					T-DM1 + pertuzumab + standard chemotherapy in women with HER2+ locally advanced breast cancer

					Assessing cardiac safety of trastuzumab vs pertuzumab vs T-DM1 in adjuvant or metastatic setting in patients with LVEF between 40% and 50%

					Cardiac events or asymptomatic worsening of cardiac function

					T-DM1 vs paclitaxel + trastuzumab for stage I HER2+ breast cancer

					T-DM1 in HER2+ locally advanced or MBC patients who have received prior anti-HER2 and chemotherapy-based treatment

					Adjuvant T-DM1 vs trastuzumab in HER2+ breast cancer with residual tumor in breast or axillary lymph nodes following neoadjuvant therapy

		DFS, disease-free survival; HR, hormone receptors; LVEF, left ventricular ejection fraction; MTD, maximum tolerated dose; MBC, metastatic breast cancer; ORR, overall response rate; OS, overall survival; pCR, pathologic complete response; PFS, progression free survival.

In addition to evaluating T-DM1 in the metastatic setting, T-DM1 is also being studied in the neoadjuvant and adjuvant settings. The I-SPY 2 study is evaluating T-DM1 and pertuzumab in the neoadjuvant setting.

 The KATHERINE trial is an adaptive trial design wherein patients with HER2-positive primary breast cancer with residual disease in the breast or axillary lymph nodes following neoadjuvant therapy are being randomized to T-DM1 versus trastuzumab.

Overall, T-DM1 is well tolerated with a relatively low rate of clinically significant AEs (

 A pooled analysis of toxicity data from 882 patients treated in seven clinical trials with single agent T-DM1 at 3.6 mg/kg every 3 weeks found that the most common AEs of any grade were fatigue (45.4%), nausea (42.3%), headache (28.7%), thrombocytopenia (28.7%), and constipation (25.5%).

 Grade ≥3 AEs occurring in ≥2% of patients were thrombocytopenia, increased transaminases, fatigue, hypokalemia, and anemia.

 It is important to note that up to two dose modifications (3 mg/kg and 2.4 mg/kg) are allowed with T-DM1 treatment. After two dose reductions, any further requirements for dose reductions should result in discontinuation of T-DM1.

The most common AEs of special interest are thrombocytopenia, hepatic toxicity, and cardiotoxicity. Thrombocytopenia is typically grade 1 or 2 and rapidly reversible, and has generally not been associated with clinically significant bleeding.

Platelet counts can start to decline as soon as 24 hours after dosing, nadir around day 8, and recover by day 15.20 In approximately 20% of patients, platelet counts do not completely recover to baseline after repeated dosing.

 Hepatic toxicity typically presents as transient and asymptomatic transaminase elevations.

 Serious hepatobiliary disorders, including two fatal cases of severe drug-induced liver injury and associated hepatic encephalopathy, have been reported; as such, the prescribing information for T-DM1 includes a boxed warning for hepatotoxicity.

 Rare cases of nodular regenerative hyperplasia (NRH) have also been described (3 cases out of 882 treated patients).

 NRH is a rare liver disorder characterized by a widespread transformation of the hepatic parenchyma into small regenerative nodules and can result in noncirrhotic portal hypertension.

NRH can only be diagnosed by a liver biopsy, which should be considered in any patient receiving T-DM1 who develops signs and/or symptoms of portal hypertension.

		Table 2. Most Common Adverse Events (>20% incidence in at least one single agent trial) Reported in Clinical Trials of T-DM1

Patients were treated with 0.3-4.8 mg/kg every 3 weeks. This trial also included a separate cohort of a weekly regimen, which is not reported here.

 TDM4450g compared T-DM1 (n=67) versus trastuzumab plus docetaxel (HT) (n=70). Only the T-DM1 arm is reported here. 67 patients were enrolled in the T-DM1 arm but two patients in the HT arm mistakenly received a dose of T-DM1 and were included in the T-DM1 safety analyses.

 TDM4373g compared T-DM1 with pertuzumab. The AEs reported here are of the combination regimen.

EMILIA compared T-DM1 (n=490) versus lapatinib plus capecitabine (n=488). Only the T-DM1 arm is reported here. NR, not reported.

 One patient required a dose reduction for neuropathy but exact grade was not recorded.

T-DM1 should be discontinued if NRH develops.

 For both thrombocytopenia and hepatic toxicity, patients can typically continue on T-DM1 with dose modifications.

 Cardiotoxicity has been carefully studied in patients receiving T-DM1 because of the cardiac toxicity of trastuzumab, and a boxed warning for left ventricular dysfunction exists for T-DM1.

 In the pooled safety analysis, there were 13 cardiac AEs (1.5%) and all but 2 of the events were grade 1 or 2.

 Rare AEs of special interest include interstitial lung disease and pneumonitis, infusion-related reactions and hypersensitivity reactions, and peripheral neuropathy. T-DM1 is pregnancy category D and carries a boxed warning for embryo-fetal toxicity

The half-life of T-DM1 is approximately 3.5-4 days and there is no significant accumulation of T-DM1 when given every 3 weeks.

 The level of free DM1 in the plasma is very low (average of approximately 5 ng/mL) throughout the treatment cycle, which is likely related to the favorable toxicity profile of T-DM1. Pretreatment residual levels of trastuzumab have not been shown to influence the pharmacokinetics or clinical efficacy of T-DM1.

As the therapeutic armamentarium of HER2-directed therapies has expanded in the past decade, it has become imperative to identify patients who are most likely to respond to a specific agent or be resistant to it. The mechanisms that contribute to the frequent development of resistance to trastuzumab are only beginning to be understood.

 One mechanism of resistance to trastuzumab is the development of mutations in the phosphoinositide 3-kinase/protein kinase B /mammalian target of rapamycin (PI3K/AKT/mTOR) pathway.

 Preclinical studies have shown potent T-DM1 activity in breast cancer cells with PIK3CA mutations.

 T-DM1 inhibits the growth of lapatinib-resistant breast cancer cells with an activated PI3K pathway.

 A recent analysis performed on archival tumor tissue from the EMILIA study showed that patients with PIK3CA mutations who were assigned to the lapatinib-capecitabine arm had a shorter PFS and OS compared with patients with wild-type cancers in the lapatinib-capecitabine arm. However, patients treated with T-DM1 had a similar PFS and OS, regardless of the presence of a PIK3CA mutation.

 These results need further validation but highlight the need for identification of biomarkers of response to help identify which patients are specifically likely to benefit from T-DM1.

 Future steps also include identifying mechanisms of resistance to T-DM1. Currently, potential mechanisms include downregulating HER2 expression and upregulating expression of drug efflux pumps, such as multidrug resistance 1 (MDR1), and thus, reduction of intracellular exposure to DM1.

Overall, T-DM1, a novel ADC, is an attractive HER2-directed therapy given its favorable toxicity profile and clinical efficacy in the locally advanced and metastatic settings. Ongoing and future trials, as demonstrated in 

, will help elucidate the efficacy in the neoadjuvant and early disease settings. Research efforts are also focused on best selecting patients who are most likely to benefit from this therapy.

Given that there are now four approved agents in the field of HER2-directed therapy with various others under investigation, identifying biomarkers of response and/or resistance to these agents and determining the exact sequence of these promising therapies to best optimize the outcomes of patients is critical. Cost-effectiveness and toxicity profiles of these agents will come to play a significant role. Nevertheless, the future is bright and exciting, and evolving data bear further hope for the improvement of outcomes in this patient population.

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