Breast cancers over-express the human epidermal growth factor receptor 2 (HER2) in about 15% of patients. This transmembrane tyrosine kinase receptor activates downstream signaling pathways and leads to proliferation of cancer cells. Trastuzumab, an anti-HER2 monoclonal antibody, improves outcome in women with early and metastatic breast cancer. Resistance to trastuzumab involves the phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway, truncation of the Her2 receptor or lack of immune response. The last decade has seen major advances in strategies to overcome resistance to trastuzumab. This includes the development of antibody-drug conjugates, dual HER2 inhibition strategies, inhibition of PI3K/mTOR pathway and development of modulators of immune checkpoints. About 15% of breast cancer over-expresses the human epidermal growth factor receptor 2 (HER2) . Over-exTrastuzumab is a humanized recombinant monoclonal antibody that targets the HER2 extracellular domain. The use of trastuzumab is considered as standard of care both in early and metastatic HER2 over-expressing breast cancer. Numerous clinical trials have confirmed that trastuzumab improves overall survival (OS) in metastatic breast cancers –13. In evia the PI3K and MAPK pathways, and activation of the immune response via antibody dependent cell-mediated cytotoxicity (ADCC) and eventually adaptive immune response [Its antitumor activity is hypothesized to be related to two different mechanisms of action: downregulation of the intracellular signaling pathway response –22.Unfortunately, resistances to trastuzumab occur, mainly in the metastatic setting, where most of the patients treated with trastuzumab have a disease progression within one year . MoleculIn the present review, we will present clinical data on the main strategies that aimed at overcoming trastuzumab resistance. The targets and drug family under investigation are reported in Figure Lapatinib is a small molecule, dual tyrosine kinase inhibitor (TKI) of EGFR and HER2. It inhibits the intracellular kinase domain of HER2 contrary to trastuzumab that inhibits the extracellular domain and blocks ligand-induced heterodimer signaling. It could prevent signaling related to truncated HER2 receptor or enhance trastuzumab-dependant ADCC, thanks to an accumulation of HER2 at the cell surface.P <0.001). In the same trial, the median overall survival times were 75 weeks for the combination arm and 64.7 weeks for the capecitabine arm  [P = 0.0124) [It has shown its efficacy when combined with capecitabine, in terms of time to progression (hazard ratio (HR): 0.57; 95% confidence interval (CI) 0.43 to 0.77; = 0.210) –38. Lapa 0.0124) .P = 0.011) and OS  [The efficacy of dual HER2 blockade with trastuzumab and lapatinib was investigated in the phase III study EGF104900. Patients with HER2-positive metastatic breast cancer (MBC) who progressed during trastuzumab treatment were randomly assigned to receive lapatinib in monotherapy or lapatinib in combination with trastuzumab. The combination was associated with better outcome both in progression free survival (PFS)  .P = 0.98). The dual HER2 blockade was associated with a higher pCR  as compared to single-agent HER2 therapy but the difference was not statistically significant (P = 0.095) [Following the results of these randomized trials in the metastatic setting, the efficacy of lapatinib has been investigated in early breast cancer, mainly in the context of trials testing dual inhibition of HER2. In the phase III trial of NSABP (protocol B-41), 529 patients were randomized to receive weekly paclitaxel with either trastuzumab weekly, lapatinib daily or the association trastuzumab plus lapatinib before undergoing surgery. Lapatinib alone had a similar percentage of pCR as trastuzumab . There was no significant difference in pCR between the lapatinib and the trastuzumab group [In another randomized trial performed in the neo-adjuvant setting (Neo ALTTO), patients were randomized among lapatinib, trastuzumab or lapatinib plus trastuzumab, all in combination with paclitaxel after six weeks of targeted therapy alone. The pCR rate was significantly higher in the group treated with dual inhibition (51.3%) as compared to trastuzumab alone (29.5%) . Nevertheless, this lack of benefit could be related to the low number of events.First results from the phase III ALLTO trial comparing one year of lapatinib alone, trastuzumab alone, their sequence or their combination in an adjuvant setting in 8,381 HER2 positive breast cancers have been reported . AccordiNeratinib is an oral irreversible pan-HER inhibitor. Preclinical data on breast cancer cell lines suggest that it could overcome both primary and acquired trastuzumab resistance in HER2 positive breast cancer cell lines .Neratinib has shown antitumor activity among both pretreated and trastuzumab naive patients. In a phase II trial, the median PFS observed with neratinib was 22.3 weeks among patients with prior trastuzumab treatment and 39.6 weeks with trastuzumab naive patients. Objective response rates were 24% and 56%, respectively .P = 0.231 and P = 0.280, respectively) [Neratinib single agent has been compared with the association of lapatinib plus capecitabine. Both median PFS (4.5 months) and OS (19.7 months) for neratinib single agent were found to be numerically inferior to that of the combination therapy, although not statistically significant  .Neratinib is currently developed in combination with paclitaxel, vinorelbine, capecitabine and temsirolimus –49. The The most common adverse event of this treatment is diarrhea in more than 90% of the cases, and then neutropenia (50% of the cases) ,48.The vascular endothelial growth factor (VEGF) plays a key role in progression of this cancer by promoting tumor angiogenesis . BevacizP = 0.0162). The median PFS was 16.8 months in the bevacizumab arm versus 13.9 months [In the AVEREL study, the efficacy of bevacizumab was evaluated in first line therapy for locally recurrent or metastatic HER2 positive breast cancer. In this phase III study, the HR for progression was 0.72 . In this trial, patients were randomly assigned to receive chemotherapy, trastuzumab plus bevacizumab, or chemotherapy and trastuzumab alone. No difference of efficacy was observed between the two arms  .Other antiangiogenic agents that can target VEGFR, such as multitargeted anti-angiogenic TKIs  have shown promising results .Resistance to trastuzumab can be explained by signaling through other HER dimerization . Pertuzuet al. have evaluated whether adding pertuzumab to trastuzumab could reverse trastuzumab resistance. The objective response rate and clinical benefit rate (CBR) were 3.4% and 10.3% in patients who received pertuzumab monotherapy after trastuzumab. At the opposite end, objective response rates and CBR were 17.6% and 41.2% in patients who received the combination after progression on trastuzumab [Cortes stuzumab .Based on these data, the efficacy of adding pertuzumab to trastuzumab has been investigated.P <0.001). The pertuzumab arm was also associated with an OS improvement. The median OS in the placebo group was 37.6 months (95% CI 34.3 to NE (not estimable)) and had been not reached in the pertuzumab group (95% CI 42.4 to NE)  [In the CLEOPATRA study, patients were randomized between placebo plus trastuzumab plus docetaxel or pertuzumab plus trastuzumab plus docetaxel. The median PFS was 12.4 months in the placebo group versus 18.7 months in the pertuzumab group  .The combination of chemotherapy with trastuzumab and pertuzumab has also shown interesting results in the neo-adjuvant setting.P = 0.0141). Interestingly, pertuzumab and trastuzumab without chemotherapy was associated with a 16.8% pCR [The NeoSphere study (multicenter phase II study) evaluated the efficacy of dual inhibition using pertuzumab. Patients who received pertuzumab and trastuzumab plus docetaxel had a significantly higher pCR rate  compared to patients treated with trastuzumab and docetaxel  then three cycles of docetaxel, or three FEC then three docetaxel with the combination of trastuzumab and pertuzumab or docetaxel plus carboplatin plus the combination during six cycles. The primary endpoint was to assess the cardiac safety. The pCR rates were quite similar in the three arms  .In the early stage of breast cancer, the efficacy of pertuzumab is currently being investigated in the adjuvant setting .Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate and is composed of trastuzumab covalently linked to maytansine, a cytotoxic agent .P <0.001). The median OS at the second interim analysis was 30.9 months in the T-DM1 arm versus 25.1 months in the lapatinib arm  [The EMILIA study, a phase III registration trial compared T-DM1 to lapatinib and capecitabine in patients with HER2 positive advanced breast cancer previously treated with trastuzumab and a taxane. The median PFS was 9.6 months with T-DM1 versus 6.4 months with lapatinib plus capecitabine  .P <0.0001). Final overall survival analysis is still awaited but interim analysis showed a trend favoring T-DM1 with a lower incidence of grade 3 or worse adverse events [The phase III TH3RESA trial compared third line treatment (including trastuzumab and lapatinib) of metastatic or unresectable locally advanced or recurrent HER2 positive breast cancer with T-DM1 to the treatment of the physician’s choice. T-DM1 treatment significantly improved PFS compared with physician’s choice , compares single-agent T-DM1 to T-DM1 combined with pertuzumab to trastuzumab plus a taxane in first line treatment of metastatic breast cancer.These studies will provide more information about the indications of T-DM1 in the treatment algorithms for HER2-positive disease.The mammalian target of rapamycin (mTOR) is a serine-threonine protein kinase that mediates mRNA translation and protein synthesis. Activation of this pathway is known as a mechanism of trastuzumab resistance ,64. PrecIn a phase I/II study, patients with HER2-positive metastatic breast cancer received trastuzumab combined with everolimus, after resistance to trastuzumab. Fifteen percent of patients had a partial response and 19% had a long stable disease (≥6 months). The clinical benefit rate was 34% .A phase II study evaluated the efficacy of everolimus combined with trastuzumab and paclitaxel in patients who were resistant to trastuzumab and taxane therapy. The median PFS was 5.5 months and the median OS was 18.1 months . This coP = 0.0067). In this study, several biomarkers  were analyzed to find some subpopulation for whom the benefit of everolimus was higher. Patients with a low PTEN and high pS6 level seemed to derive more benefit from addition of everolimus. Median PFS gain was 12 weeks for the high pS6 level subgroup  and 18 weeks for the low PTEN subgroup . Unfortunately, there was no marker-treatment interaction with PIK3CA mutation. These promising results deserve additional research.The BOLERO-3 study compared the combination of everolimus, trastuzumab plus vinorelbine to trastuzumab and vinorelbine. The association of the mTOR inhibitor with vinorelbine significantly improved PFS  is a chaperone which stabilizes oncogenic proteins. Inhibition of HSP90 leads to the degradation of these proteins involved in cancer biology . HSP90 i17-Demethoxygeldanamycin (17-AAG) inhibits the activity of HSP90, thereby inducing the degradation of many oncogenic proteins. In a phase II study, 17-AAG (tanespimycin) was given in combination with trastuzumab in patients who previously failed to trastuzumab. The overall response rate (ORR) was 22%, the CBR was 59%, the median PFS was six months and the median OS was seventeen months .In another phase II study, retaspimycin (IPI-504) given with trastuzumab showed modest clinical activity, but it is possible that under-dosing limited efficacy . Other sThese findings are promising and other studies are expected to develop these new targeted therapies.Programmed death 1 (PD-1) is a co-inhibitory receptor and acts as a negative regulator of the immune system. It is overexpressed on tumor-infiltrating lymphocytes (TIL). The PD-1 ligand, PD-L1, is expressed by multiple carcinoma, including breast cancers. This suggests that the PD-1/PD-L1 signaling pathway could be a candidate target in breast and other cancers.T cell infiltration is predictive for the efficacy of trastuzumab –76. BiomPreclinical studies have shown a synergism between trastuzumab and anti-PD1 antibodies .Other monoclonal antibody (mAb)-based therapies are being investigated including anti CD73. Pre-clinical data have suggested that it can delay tumor growth and inhibit the development of metastases .de novo resistance is a serious concern. The understanding of resistance mechanisms could allow developing strategies to prevent or overcome this resistance. The development of novel targeted therapies has changed the practices in metastatic settings.Although trastuzumab remains the standard treatment in patients with HER2 overexpressing breast cancer in neoadjuvant, adjuvant and metastatic settings, the presence of acquired and New standards of care include trastuzumab plus pertuzumab plus docetaxel in first line treatment and TDM-1 for trastuzumab-resistant patients. In early breast cancer, dual HER2 blockade has shown promising results in the neoadjuvant setting. This strategy is being evaluated in the adjuvant setting in several randomized trials.Since several different targets are under investigation, there is a need to identify predictive biomarkers to optimize combination strategies for suitable patients. Loss of PTEN and a high level of pS6 could facilitate the selection of appropriate patients who can benefit from personalized targeted therapy.


Breast cancer is the most common cancer in US women, affecting approximately 1 in 8 women over the course of alifetime. Nearly 40,000 women die of breast cancer every year . In the past decade,we have come to understand that breast cancer is a heterogeneous disease, comprising subtypes that are defined bygene expression profiling . Treatment choices such as chemotherapy, endocrine therapy, andtherapies targeted toward specific genetic alterations have resulted in improved survival.Approximately 25% of all breast cancers are classified as HER2 positive, in which there is overexpression of the HER2protein (as measured by immunohistochemistry) and/or amplification of the HER2 gene (determined by fluorescence insitu hybridization). HER2 activation triggers an intracellular signaling cascade, which results in cell proliferation, survival,invasion, and angiogenesis. HER2-positive breast cancers have an aggressive phenotype. However, with the advent ofHER2-targeted therapy, the natural history of HER2-positive breast cancer has been dramatically improved .Trastuzumab (Herceptin) is a monoclonal antibody that targets the HER2 receptor tyrosine kinase. In a study of 469patients with meta-static breast cancer randomized to receive standard chemotherapy vs. chemotherapy plustrastuzumab, the group receiving trastuzumab demonstrated response rates of 50% vs. 32% and median survival timesof 25.1 vs. 20.3 months, respectively . Trastuzumab was approved by the US Food and DrugAdministration (FDA) for use in patients with metastatic breast cancer in 1998. It was subsequently evaluated in thenonmetastatic setting and showed striking benefits in relapse-free survival and overall survival (OS), leading to itsapproved use in patients with early-stage HER2-positive breast cancer in 2006 .Despite the benefits of adjuvant trastuzumab for HER2-positive breast cancer, recurrences do occur. For thesepatients, resistance to trastuzumab ultimately develops, and they eventually succumb to their disease. Therefore, therewas a need to develop other drugs for patients who develop trastuzumab resistance.Lapatinib (Tykerb) was the second HER2-directed agent approved by the FDA for use in metastatic breast cancer. It isa small-molecule tyrosine kinase inhibitor that targets the intracellular portion of the HER2 receptor. Its approval waslargely based on a study of patients with advanced breast cancer whose disease had progressed on an anthracycline, ataxane, and trastuzumab; patients were randomized to receive either capecitabine or capecitabine with lapatinib. Theaddition of lapatinib improved median time to progression from 4.4 to 8.4 months, and the combination was approvedby the FDA in March 2007 . Since that time, studies evaluating the use of trastuzumab with varyingchemotherapeutic agents have been performed, and it has been incorporated into widespread use . The use of lapatinib in combination with endocrine therapy has also been evaluated and is approved for use inmetastatic HER2-positive breast cancer.In June 2012, pertuzumab (Perjeta), a new monoclonal antibody that binds and blocks the action of the HER2receptor in a manner complementary to that of trastuzumab, was approved for use in first-line metastatic HER2-positive breast cancer in combination with taxane chemotherapy and trastuzumab. This approval was based on ademonstrable benefit in progression-free survival  when pertuzumab was added to docetaxeland trastuzumab .The newest agent in the armamentarium against HER2-positive breast cancer is ado-trastuzumab emtansine(Kadcyla), also known as T-DM1, which earned FDA approval in February 2013 for metastatic HER2-positive breastcancer that has progressed on a trastuzumab-containing regimen . See the Table on the next page fora summary of key information.Ado-trastuzumab emtansine is described as an antibody drug conjugate (ADC). It contains trastuzumab attached toa chemotherapy molecule (DM1) by a stable linker molecule. Trastuzumab is a humanized, anti-HER2 IgG1 antibody.The DM1 chemotherapy molecule is a maytansine derivative that was studied in the 1970s at the National CancerInstitute. Development was halted at that time due to unacceptable toxicity in human trials. However, in ado-trastuzumab emtansine, a thioether linker (MCC) stabilizes the compound so that the DM1 chemotherapy molecule istargeted to the HER2+ cancer cell. The chemotherapy is internalized and released into that cell only after it is attachedto the HER2+ cell. Therefore, the cancer cells are destroyed, and surrounding normal tissue is spared the toxicity of thechemotherapy.Ado-trastuzumab emtansine demonstrates mechanisms of action of both trastuzumab and DM1. In vitro, similar totrastuzumab, it inhibits HER2 receptor signaling and enhances antibody-dependent cell-mediated cytotoxicity. Ado-trastuzumab emtansine, like trastuzumab, inhibits shedding of the HER2 extracellular domain, which is a knownmechanism of resistance to trastuzumab. The DM1 chemotherapy molecule, upon internalization and release into thecancer cell, inhibits microtubule function, thereby interfering with progression through the cell cycle .A phase I, multicenter, open-label, dose-escalation study of single-agent ado-trastuzumab emtansine was initiatedto assess its safety, tolerability, and pharmacokinetics in patients with HER2-positive metastatic breast cancer who hadpreviously received trastuzumab .Twenty-four patients were treated at doses ranging from 0.3 to 4.8 mg/kg. In this study, 3.6 mg/kg was identified asthe maximum tolerated dose (MTD). The dose-limiting toxicity was thrombocytopenia, yet it was usually mild (grade 1or 2) and rapidly reversible upon discontinuation of therapy. Platelet count usually dropped starting 1 day after ado-trastuzumab emtansine administration, with a nadir around day 8 and recovery by day 15. There were no bleedingevents that required transfusion or any other intervention.Overall, the most commonly reported adverse events (AEs) were thrombocytopenia (54.2%), elevatedtransaminases (41.7%), fatigue (37.5%), anemia (29.2%), and nausea (25.0%). No patients experienced grade > 1nausea, vomiting, alopecia, or neuropathy. The overall response rate (ORR) was 44% in the 9 patients with measurabledisease treated at the MTD. The clinical benefit rate (CBR) was 73% (ORR + stable disease) among the 15 patientstreated at the MTD. The median duration of treatment was 238 days for these 15 patients.Based on the favorable phase I safety and efficacy profile, the clinical development of ado-trastuzumab emtansinecontinued. A phase II, open-label, single-arm study of ado-trastuzumab emtansine (TDM4374g) included 110 patientswho had previously been treated with trastuzumab, lapatinib, an anthracycline, a taxane, and capecitabine. Theprimary objectives were ORR and safety. On average, patients had been treated for metastatic breast cancer with sevenprior agents. The ORR was 34.5% and the CBR was 48.2%. The median PFS was 6.9 months in this heavily pretreatedgroup of patients . In another phase II, open-label, single-arm study of ado-trastuzumab emtansine(TDM4258g) that included 112 patients, ORR was 26% and median PFS was 4.6 months .2 twice daily for 14 of every 21 days. The primary endpointswere PFS and OS. A total of 88% of the patients had received prior systemic treatment in the metastatic setting; 12% ofthe patients had prior treatment only in the neoadjuvant or adjuvant setting with disease relapse within 6 months.Patients received a median of three systemic agents for metastatic disease.The EMILIA study was a phase III, randomized, multicenter, open-label trial of ado-trastuzumab emtansine vs.capecitabine plus lapatinib in 991 women with metastatic HER2-positive breast cancer who progressed on or within 6months of having received trastuzumab and who had received taxane chemotherapy . Patients wererandomly allocated (1:1) to receive ado-trastuzumab emtansine 3.6 mg/kg once every 21 days or oral lapatinib 1,250mg/day once daily plus oral capecitabine 1,000 mg/mMedian PFS was 9.6 months in the ado-trastuzumab emtansine group vs. 6.4 months in the lapatinib pluscapecitabine group. The hazard ratio for PFS was 0.65 with ado-trastuzumab emtansine vs. capecitabine plus lapatinib.Median duration of survival was 30.9 months in the ado-trastuzumab emtansine group vs. 25.1 months in the lapatinibplus capecitabine group .2 (HT) at the investigator’s discretion, once every 3 weeks. The primary endpoint was PFSand safety. Secondary endpoints included OS, ORR, duration of response (DOR), CBR, and quality of life (QOL). Of note,only 27.1% and 17.9% of patients in the ado-trastuzumab emtansine and HT groups, respectively, had received priortrastuzumab in the adjuvant or neoadjuvant setting. The ado-trastuzumab emtansine group demonstrated a medianPFS of 14.2 months compared with 9.2 months in the HT group. The hazard ratio for PFS was 0.59 with ado-trastuzumab emtansine vs. HT. The ORR and CBR in the ado-trastuzumab emtansine group was 64.2% vs. 58% in the HTgroup .More recently, results from TDM4450g, a phase II, multicenter, open-label study of ado-trastuzumab emtansine inthe first-line setting, were published. This was the first trial to directly compare ado-trastuzumab emtansine with astandard trastuzumab plus chemotherapy regimen in patients who had not received treatment for metastatic disease.In this trial, 137 patients with advanced HER2-positive breast cancer were randomized in a 1:1 ratio to receive ado-trastuzumab emtansine 3.6 mg/kg IV once every 3 weeks vs. trastuzumab 8 mg/kg loading dose then 6 mg/kg IV anddocetaxel 75 or 100 mg/mIn the single-arm, phase II study TDM4374g, fatigue (61.8%), nausea (37.3%), and thrombocytopenia (38.2%) wereamong the most common adverse events (AEs) seen. Thrombocytopenia (9.1%), fatigue (4.5%), and cellulitis (3.6%)were the most common grade 3 adverse events. In TDM4258g, the other single-arm phase II study, the most frequentgrade 3 AEs were hypokalemia (8.9%), thrombocytopenia (8.0%), and fatigue .In the EMILIA study, grade 3 adverse events were more common with capecitabine plus lapatinib vs. ado-trastuzumab emtansine (57% vs. 40.8%). The most commonly reported grade 3 AEs in the capecitabine plus lapatinibgroup were diarrhea (20.7%) and palmar-plantar erythrodysesthesia (16.4%). Thrombocytopenia and elevated serumconcentrations of aspartate transaminase (AST) and alanine transaminase (ALT) were the most commonly reportedgrade 3 adverse events in the ado-trastuzumab emtansine group, affecting 12.9%, 4.3%, and 2.9% of patients,respectively.While thrombocytopenia is a common side effect of ado-trastuzumab emtansine, it led to discontinuation oftherapy in only 2% of patients in the EMILIA study. Thrombocytopenia was usually seen in the first two cycles oftherapy, responded to dose reductions, and was rarely associated with significant bleeding. The rate of grade 3 or 4bleeding events was low overall in the ado-trastuzumab emtansine group .In the phase II TDM4450g trial, treatment with ado-trastuzumab emtansine also resulted in fewer grade ≥ 3 adverseevents than with the comparator—in that case, HT (AEs: 46.4% vs. 90.9%). Grade 4 AEs occurred in 57.6% of patientsreceiving HT vs. 5.8% in patients receiving ado-trastuzumab emtansine. A total of 40.9% of patients in the HT groupexperienced AEs leading to treatment discontinuation (of either drug) vs. 7.2% in the ado-trastuzumab emtansinegroup. In the HT group, the most common AEs of any grade were alopecia, neutropenia, diarrhea, and fatigue. The mostcommon AEs in the ado-trastuzumab emtansine group were fatigue, nausea, increase in serum AST, fever, andheadache. A notable difference in side-effect profile between ado-trastuzumab emtansine and taxane-basedchemotherapy with trastuzumab is the lack of alopecia seen with ado-trastuzumab emtansine. Consistent with the AEprofile of both study arms, QOL assessments in this study favored ado-trastuzumab emtansine over HT .Studies of therapy with ado-trastuzumab emtansine took special note of cardiac events due to the known potentialfor trastuzumab-associated cardiotoxicity. With trastuzumab, this cardiotoxicity, manifest by decreases in leftventricular ejection fraction (LVEF), is usually asymptomatic and readily reversible. Additionally, trastuzumab-associatedcardiotoxicity is worsened in patients receiving concurrent or prior anthracycline therapy .In a single-arm phase II study (TDM4374g), at a median follow-up of 17.4 months, no left ventricular ejectionfraction decline to ≤ 45% or symptomatic congestive heart failure was observed, and no patients discontinuedtreatment because of cardiotoxicity. Similarly, in TDM4258g, there was no dose-limiting cardiotoxicity observed . In the randomized TDM4450g trial, there were no clinically significant cardiac events in either group. At amedian follow-up of 23 months, 3 patients—1 in the ado-trastuzumab emtansine group and 2 in the HT group—had anasymptomatic decline in LVEF to ≤ 40% .Primarily because it contains trastuzumab, the FDA-approved prescribing information recommends that all patientstreated with ado-trastuzumab emtansine have a baseline LVEF assessment at baseline and at regular intervals  during treatment . At least temporary discontinuation of therapy is recommended ifthe LVEF falls to < 40% or is 40% to 45% with a 10% absolute decrease below the pretreatment value. In this case, LVEFshould be rechecked 3 weeks later. If it has not improved or worsened, then permanent discontinuation isrecommended. However, if there is improvement, it is acceptable to restart ado-trastuzumab emtansine with closecardiac monitoring.In a phase III study of patients with HER2-positive metastatic breast cancer previously treated with trastuzumab,ado-trastuzumab emtansine resulted in a 35% reduction in the risk of progressive disease compared with standardtreatment . In the phase II trial of ado-trastuzumab emtansine used in the first-line setting, therewas a 41% reduction in risk of progressive disease compared with a standard therapy . Remarkably,these improvements in efficacy came with a decrease in toxicity vs. the comparator arm.Ado-trastuzumab emtansine is an effective, tolerable, IV, "smart" therapy, now approved for use in patients withHER2-positive breast cancer who have progressed on or within 6 months of a trastuzumab-containing treatmentregimen. Ongoing studies are evaluating its use as first-line therapy for metastatic disease, in combination with otherHER2-directed therapies and in the neoadjuvant setting. The use of ado-trastuzumab emtansine may be expanded ifthese trials demonstrate safety and efficacy in these studies. Therefore, the use of ado-trastuzumab emtansine isexpected to increase dramatically. In light of this, it is essential for the oncology advanced practitioner to understand itsmechanism of action, efficacy, and adverse event profile.


The improvement in cancer therapy and the increasing number of long term survivors unearth the issue of cardiovascular side effects of anticancer treatments. As a paradox in cancer survivors, delayed cardiotoxicity has emerged as a significant problem. Two categories of cardiotoxic side effects of antineoplastic drugs have been previously proposed: Type I cardiotoxicity, defined as permanent cardiotoxicity, is usually caused by anthracyclines; Type II cardiotoxicity, considered as reversible cardiotoxicity, has been mainly related to monoclonal antibodies. The cardiotoxicity of antibodies has been associated to trastuzumab, a humanized anti-ErbB2 monoclonal antibody currently in clinical use for the therapy of breast carcinomas, which induces cardiac dysfunction when used in monotherapy, or in combination with anthracyclines. Furthermore, recent retrospective studies have shown an increased incidence of heart failure and/or cardiomyopathy in patients treated with trastuzumab, that can persist many years after the conclusion of the therapy, thus suggesting that the side toxic effects are not always reversible as it was initially proposed. On the other hand, early detection and prompt therapy of anthracycline associated cardiotoxicity can lead to substantial recovery of cardiac function. On the basis of these observations, we propose to find a new different classification for cardiotoxic side effects of drugs used in cancer therapy. BCbreast carcinomasCHFcongestive heart failureLVEFleft ventricular ejection fractionHFheart failureCMcardiomyopathyLVESVleft ventricular end systolic volumeLVEDDleft ventricular end diastolic dimension; RS, radial strainIt has been estimated that in January 2014 in USA about 14,5 million people with a history of cancer were still alive,The improvement in cancer therapy and the increasing number of long-term survivors, unearth the relevant issue of cardiovascular side effects of anticancer treatment. Type I cardiotoxicity, defined as permanent cardiotoxicity, is usually caused by anthracyclines. Type II cardiotoxicity, considered as reversible cardiotoxicity, has been mainly related to monoclonal antibodies or tyrosine kinase inhibitors.The mechanism underlying anthracycline cardiotoxicity is well described in literature and includes cell damage due to radical free formation and oxidative stress.The cardiotoxicity of anti-ErbB2 antibodies has been associated to trastuzumab, a humanized anti-ErbB2 monoclonal antibody, currently in clinical use for the therapy of BC.However, cardiac toxicity was recognized as an important side effect at an early stage in the development of Trastuzumab, manifested as symptomatic CHF or asymptomatic LVEF decline,Large-scale clinical studies with Trastuzumab, have shown that up to 7% or 28% of patients suffer from cardiac dysfunction when Trastuzumab is used in monotherapy, or in combination with anthracyclines, respectively.Recently independent studies have shown an increased incidence of HF and/or CM in patients treated with trastuzumab in monotherapy (up to 30%) or in combination with anthracyclines (up to 40%), particularly in elderly breast cancer patients with a history of other diseases.In these studies an interesting aspect has been evidenced: the trastuzumab related cardiotoxic side effects progressively increased during 3–5 years after the end of treatment with trastuzumab, thus suggesting that the cardiotoxicity onset could occur even post treatment and the patients were not appropriately subjected to follow up inspections.In agreement with these observations, ICARO Network showed an increased trastuzumab-related cardiotoxicity (up to 38%) in elder patients (age > 60 years) according with data previously described.Cardiotoxicity induced by Trastuzumab had long been considered reversible as it has been supposed that the withdrawal of the antibody allows for the return of function of the ErbB2 cardiomyocyte survival pathway and reversal of EF decline, in contrast to the permanent myocyte dysfunction induced by anthracyclines.The mechanism of cardiotoxic effects of trastuzumab is not completely clarified, but it has been attributed to blockade of HER-2 signaling in cardiac myocytes as trastuzumab interferes with ErbB2/ErbB4 heterodimerization induced by Neuregulin1in vitro and in vivo studies in human cardiac cell cultures and in mice, respectively, clearly indicate that Trastuzumab induces apoptosis of cardiomyocytes thus leading to irreversible cell death.However recent retrospective studies suggest that the cardiotoxic side effects of Trastuzumab should be carefully reconsidered as they can persist many years after the conclusion of the therapy, thus strongly suggesting that they are not always reversible as it was initially proposed.Several studies report on risk prediction model based on echocardiography imaging analyses in patients treated with anthracyclines or trastuzumab,Chen J et al. observed, in a cohort of 45537 women with early stage breast cancer, that the cumulative incidence of HF or CM was higher for patients treated over three years with trastuzumab alone (32.1%) and trastuzumab with anthracyclines (A+T) (41.9%) compared with patients who received no adjuvant therapy (18.1%). In secondary analyses carried out by evaluating HF and CM separately, the combined treatment of anthracyclines and trastuzumab was associated with HF alone and CM alone compared with no adjuvant therapy in their models. Trastuzumab in the absence of anthracycline therapy was borderline significantly associated with HF alone, but was clearly associated with CM alone compared with no adjuvant therapy. Furthermore, compared with patients who received no adjuvant chemotherapy or trastuzumab, the use of trastuzumab was associated with an absolute 14% higher incidence rate for HF or CM.Another study reports on a retrospective analysis of a cohort of 12500 women diagnosed with breast cancer and treated by adjuvant therapy.On the contrary, the hazard ratio for HF and CM (the incidence of HF and CM in treated patients with respect to untreated control population) associated with anthracyclines seems to be higher in younger patients as it is statistically significant among women younger than 55 years but not among older women. Similarly the hazard ratio associated with trastuzumab treatment is higher in younger women (< 64 years) with respect to older women (> 74 years). Bowles et al. report an hazard ratio of 15.46 or 10.16 in women younger than 55 years and women aged 55–64 years respectively, 5 years since start of treatment. In elder women a hazard ratio of 2.57 was reported. These hazard ratios suggest a fourfold increase in the risk of HF or CM among women treated with trastuzumab.Trastuzumab toxicity is not only directly related to the presence of other risk factors, as in a more accurate analysis Bowles et al. reported the hazard ratio corrected by excluding women with comorbidities, and the results did not change significantly. The overall risk of incidence of HF or CM was increased in women treated with anthracyclines, but it was even greater in women treated with trastuzumab.Moreover some authors discuss how trastuzumab, classified as type II cardiotoxic agent, can also trigger irreversible cardiotoxicity,Altogether these are important observations because they strongly indicate that side effects of trastuzumab not only affect women during treatment, but they also occur many years after the treatment as a delayed cardiotoxicity. The mechanisms of cardiotoxicity induced by anthracycline or by trastuzumab may explain the difference in the risk of HF or CM observed after cancer therapy with these antineoplastic drugs.Several studies indicate that ErbB2 has an important role in heart development and function. In cardiac tissues ErbB2 works as a co-receptor for another ErbB receptor tyrosine kinase family member, ErbB4 and its peptide ligand neuregulin 1 (NRG1).Even though trastuzumab prolongs the survival of women with Her2 positive breast cancer, its cardiotoxic side effects are important. The initial hypothesis has been made that trastuzumab-associated cardiotoxicity is related to the inhibition of the neuregulin-1 activated pathway.L/BCL-XS ratio, which likely leads to loss of mitochondrial membrane integrity with multiple major deleterious intracellular consequences including loss of electron transport, generation of free radicals, uncoupling of oxidative phosphorylation which leads to ATP reduction, release of pro-apoptotic proteins such as cytochrome c.Another possible mechanism for trastuzumab-associated cardiotoxicity is represented by mithocondrial dysfunction and distruption of cellular energetic due to the altered BCL-Xin vitro on cultures of cardiac cells and in vivo on animal models. In vivo studies in mice have shown that trastuzumab treatment significantly affects both functional and structural properties of the heart. It altered the expression of genes involved in cardiac functions, adaptability to pressure, vasodilatation and contractility ,Trastuzumab-associated cardiotoxicity was confirmed by findings obtained Cardiac damage has been observed also by electron microscopy imaging. Trastuzumab treatment causes cardiomyocyte ultrastructure alteration, associated with ultrastuctural damages of heart tissues in mice. In particular, alterations in intermitochondrial distance, thickness of myofibers and number of mitochondria have been observed.L ratio.Moreover, in trastuzumab-treated mice it has been shown an activation of apoptotic pathways, such as PARP cleavage, caspase 3/7 activation and increase of Bax/BCL-XFunctional alterations of heart in treated mice were also detected as trastuzumab was found to significantly reduce fractional shortening by increasing left ventricular end-diastolic dimension (LVEDD).in vitro assays Trastuzumab was found to significantly reduce cell viability of cardiomyocytes.L in cardiomyocytes, thus inducing apoptosis.In in vitro studies it has been also confirmed that the cardiotoxic effects of trastuzumab are also based on its ability to prevent the assembly of NRG-1/ErbB2/ErbB4 complex, required for cardiomyocyte survival. Indeed trastuzumab treatment was capable of inhibiting ligand-induced cell proliferation, thus affecting not only the basal cardiomyocyte survival but also the ligand-induced ErbB2/ErbB4 heterodimerization signaling pathway downstream In these Doxorubicin is a highly effective anthracycline but its clinical use is compromised by the development of a severe form of cardiomyopathy and heart failure. The mechanisms of the doxorubicin-related cardiotoxicity have been extensively studied and two main pathways have been proposed. First, doxorubicin has been supposed to induce cardiotoxicity by ROS formation,In the past few decades, novel anticancer drugs have been developed that effectively induce tumor regression, thus prolonging patient's survival. However, cardiovascular side effects of these new drugs can lead to therapy related heart failure. Cardiac side effects of anti-Her2 anticancer agents, such as trastuzumab or tyrosine kinase inhibitors, were totally unexpected.Thus, the initial hypothesis has been made that trastuzumab-associated cardiotoxicity is related to the inhibition of the NRG1 activated pathway,in vitro and in vivo.Our studies have demonstrated that trastuzumab directly induces damage in cardiomyocytes both  Systolic and diastolic dysfunctions, asymptomatic myocardial ischemia or undiagnosed myocarditis are more frequently in the elderly. Thus it is likely that ErbB2/ErbB4 pathway is chronically activated in these conditions and its blockage by trastuzumab treatment may empathize trastuzumab-related cardiotoxicity in elder patients with undiagnosed heart diseases.This may explain the increased trastuzumab-associated cardiotoxicity in elderly or in patients with other risk factors related to old age such as the higher incidence of systo-diastolic left ventricular dysfunction or chronic myocardial ischemia in diabetic patients.Some data suggest that cardiotoxicity in patients treated with anthracyclines is not necessarily irreversible but it may become reversible if early diagnosed.These findings indicate that a different approach to antitumor treatment-related cardiotoxicity is needed and we cannot anymore consider trastuzumab-related cardiotoxicity less important than anthracycline-related cardiotoxicity or only as a consequence of previous anthracycline treatment as it occurs also in trastuzumab monotherapy. Thus it becomes essential to identify patients with high risk factors or asymptomatic heart dysfunction before starting the treatment with trastuzumab, in order to treat them appropriately or strongly reduce the risk factors.Considering these important observations, the type I and type II classification, based on reversible or not-reversible cardiotoxicity, has become inappropriate as the reversibility concept is ambiguous. It has been demonstrated that ErbB2 inhibitors can be toxic for cardiomyocytes when used in monotherapy in a fashion similar to doxorubicin.The incidence of cardiotoxicity in the real-world population of cancer patients treated with trastuzumab monotherapy is completely different with respect to that observed in clinical trials, as adjuvant clinical trials of trastuzumab have typically enrolled younger women without cardiac comorbidities, excluding older people or people affected by cardiovascular diseases,Furthermore, the incidence of Trastuzumab cardiotoxicity after ten years follow up is underrated, because it does not represent the incidence of real-world population of cancer patients treated with trastuzumab alone.Since older patients are increasingly treated with these anti-ErbB2 drugs, and trastuzumab is widely used for its efficient therapeutic effects on metastatic cancer thus prolonging even more patient's survival in last decades, a different approach is needed to guarantee the survival of treated patients to avoid that women recovered from cancer paradoxically die for heart failure.Also other immunotherapies and targeted drugs have been included in the type II cardiotoxicity such as angiogenesis inhibitors Bevacizumab (anti-VEGF humanized mAb), and TK inhibitors On the basis of these considerations may we still consider Type I and Type II cardiotoxicities clearly different? We propose to find a new different classification for cardiotoxic side effects mainly based only on the types of drugs used in cancer therapy, so that we can discriminate the anthracycline-related cardiotoxicity triggered by anthracyclines or anthracycline-like drugs, ErbB2-related cardiotoxicity, due to trastuzumab or other ErbB2 inhibitors and anti-angiogenic – related cardiotoxicity .


The discovery of human epidermal growth factor receptor 2 (HER2) and its role in the biology of breast cancer and the subsequent development of HER2-targeted therapies, have dramatically improved clinical outcomes for women with early-stage and advanced HER2-positive breast cancer (BC).HER-2 targeted therapies represent a major step forward in achieving the goal of delivering individualized targeted therapy for BC, and trastuzumab was the first anti-HER-2 strategy to be approved for treatment of HER-2 positive BC.This review discusses the treatment of metastatic HER2-positive BC and describes efficacy and safety of novel anti-HER2 target therapies in first-line metastatic settings and the future challenges include refining such treatments, reducing toxicity and simultaneously developing innovative therapies. Furthermore, combinations of trastuzumab and drugs targeting the downstream pathway are described.In the next future will be possible to use an ample armamentarium of combination therapies directed against HER2 and key signaling components integrated in the HER network. This approach will allow clinicians to tailor the management of the individual patient on the basis of tumor- specific biomarker profiles.There is an urgent need for prospective biomarker-driven trials to identify patients for whom targeting is cost-effective. About 20 % of invasive breast cancers (BC) are HER2-positive and characterized by amplification and/or overexpression of HER2, a transmembrane receptor with tyrosine kinase (TK) activity, resulting in HER2 gene amplification on chromosome 17. This subtype of BC shows an intrinsic malignancy and has a poor prognosis in the absence of specific treatment. About 10 % of the cases will be diagnosed as metastatic disease and the 5-year overall survival (OS) in these patients achieves 20 % , of which 2–5 % are long-term survivors –11.Trastuzumab, a humanized monoclonal antibody that selectively binds to the HER2 on the surface of tumour cells, when added to chemotherapy in the first-line advanced treatment of HER2-positive BC is associated with a significantly reduced risk of progression and death compared to chemotherapy alone –16.However, despite these deep therapeutic advances, the progression free survival (PFS), and OS of patients treated with trastuzumab and chemotherapy-based regimens for metastatic BC (MBC) is about 7 months (vs about 5 months without trastuzumab) and 25 months (in contrast to 20 months without trastuzumab) respectively , 18.In the last 5 years we are witnessing an exponential development of new anti HER2 molecules and results of their activity in terms of outcome for the advance tumor are very exciting.In this article, we will analyze the most relevant aspects related to new developments with anti-HER2 therapies and the more recent clinical evidences and therapeutic strategies with the modern use of biological anti HER2 drugs at the first progression of BC patients.With regard to blocking gene expression, the most significant strategy in HER2-targeted therapies have been made using monoclonal antibodies directed against the extracellular domain of the HER2 protein, named trastuzumab. Binding with high affinity to the extracellular domain of HER2, trastuzumab inhibits the proliferation of tumour cells that overexpress HER2. It demonstrated antitumour effects when administered as a single agent, and additive and synergistic effects when administered in combination with a range of antineoplastic agents (1).vs. 32 %, p < 0.001)], duration of response [DR ], time to progression [TTP ], time to failure [TTF ]. The addition of trastuzumab to paclitaxel increased the ORr from 17 to 41 % (p < 0.001), the DR from 4.5 to 10.5 months (p < 0.01), the TTP from 3.0 to 6.9 months (p < 0.001) and the TTF from 2.9 to 5.8 months (p < 0.001). The addition of trastuzumab to adriamycin plus cyclophosphamide (AC) increased the RR from 42 to 56 % (p = 0.02), the DR from 6.7 to 9.1 months (p = 0.005), the TTP from 6.1 to 7.8 months (p < 0.001) and the TTF from 5.6 to 7.2 months (p < 0.001). The median OS for patients who received trastuzumab and chemotherapy combination was 25 months vs. 20 months for those who received chemotherapy alone (p = 0.046). This result is even more significant in light of the fact that two-thirds of the women who are progressive on chemotherapy alone arm went on to receive open-label trastuzumab on a compassionate-use protocol .Also the visceral disease seems to get a better PFS, but the number of patients with non-visceral disease was very limited, and therfore no defined considerations it is possible to drive.The safety profile of pertuzumab, trastuzumab and taxane was consistent with the known safety of patients with long-term exposure to dual targeting. It means that we now have a treatment that improves both PFS and OS without affecting the QoL of patients in terms of haematological, not haematological and cardiac safety. This information is very important for the long duration of exposure to the two biological molecules which the patient can support.Constitutive activation of PI3K/AKT/mTOR signalling due to PTEN loss can lead to trastuzumab resistance. mTOR inhibition sensitises PTEN-deficient tumours to trastuzumab, thereby suggesting that the combination of everolimus, the mTOR inhibitor, and trastuzumab have a role in the treatment of HER2-overexpressing BC , 36.The addition of the mTOR inhibitor, everolimus, to trastuzumab and chemotherapy showed clinical benefit in heavily pretreated patients with HER2-positive MBC progressing on previous trastuzumab and taxane therapy .No so high enthusiasm was declared when the two target combination was tested in first-line MBC. In Bolero 1 trial, patients were randomly assigned to receive either everolimus 10 mg once a day orally or placebo plus weekly trastuzumab intravenously (4 mg/kg loading dose on day 1 with subsequent weekly doses of 2 mg/kg of each 4 week cycle) and paclitaxel intravenously  .p = 0. 11). In the HRs negative subpopulation, median PFS with everolimus arm was 20 months (95 % CI: 14.9-24.0) versus 13 months (10.0-16.5) with placebo arm ; There was a clinically relevant prolongation of PFS with the addition of everolimus in HRs negative patients (7 months), but the p value did not meet prespecified criteria for significance by a small margin. However, based on the statistical design, the threshold for statistical significance in the HRs-negative subpopulation was rather stringent (p = 0.0044) .In the recent years, a new group of compounds that bind irreversibly to the adenosine triphosphate binding pocket of HER receptors have been developed. One of these compounds, neratinib, has passed preclinical phases and is currently undergoing various clinical trials. Importantly, pan-HER TKI are very attractive strategies for this purpose. Among them, JNJ-28871063 has been shown preclinically to penetrate the blood barrier brain (BBB) more effectively than lapatinib, being also effective in improving survival of xenograft mouse models with HER2 positive intracranial metastases . NeratinHormonal therapy and trastuzumab represent one of the oldest and one of the newest treatment modalities for BC, respectively. Recent data have suggested that HER2 overexpression is associated with resistance to hormonal therapy and there is considerable preclinical evidence to support the existence of interaction or cross talk between HER2 and estrogen-receptor signalling pathways in BC. Preclinical data also demonstrate that adding trastuzumab to hormonal therapy results in greater antitumour activity than either agent alone. The existence of an inverse relationship between estrogen receptors ER expression and HER2 overexpression has also been well established clinically. Thus, a range of clinical trials are now ongoing to determine whether the addition of trastuzumab to hormonal therapy will provide BC patients with benefits in clinical practice. For patients with bone-only spreading disease and indolent disease progression, the combination of anti-HER2 and endocrine therapy as first-line treatment represents a valid therapeutic option. Three trials have examined the addition of HER2-targeted agents to aromatase inhibitors (AI) in postmenopausal women with a first recurrence Table .Table 1Svs. 2.4 months; HR 0.63) and an irrelevant improvement in OS (28.5 months vs. 24 months). Also the response rate was in favour of the combination therapy (20 vs. 7 %). The most common toxicities seen in the combination arm were fatigue (21 %), vomiting (21 %), and diarrhea (20 %); however, the vast majority of events were grades 1 and 2.In the TANDEM study, 207 patients were randomized to anastrozole (1 mg daily) plus trastuzumab  or to anastrozole alone . The comvs. 3 months; HR: 0.67), the duration of which was similar to the duration achieved in the HER2-negative group (15 months). The rates of response and clinical benefit were 27 vs 13 % and 65 vs 39 %, respectively, in favour of both trastuzumab-containing arm.The combination of letrozole plus trastuzumab was compared with letrozole alone in the Electra study ; fifty-sA third study, EGF30008, compared the all-oral combination of letrozole (2.5 mg daily) and lapatinib (1500 mg daily) with letrozole alone . Of the On Table The DETECT V/CHEVENDO trial is a randomized phase III study which aims to compare the combination of trastuzumab, pertuzumab and a chemotherapy drug  with the combination of trastuzumab, pertuzumab and hormonal therapy . It is an ongoing trial currently recruiting participants [NCT02344472]. Finally, the phase II 1303GCC trial will compare trastuzumab in combination with pertuzumab alone vs trastuzumab, pertuzumab and eribuline vs trastuzumab, pertuzumab and hormonal therapy (anastrozole or fulvestrant) in locally advanced or metastatic BC affecting patients aged 60 or more [NCT02000596].Although for patients with HRs/HER2 positive disease improvements in TTP or PFS were seen with the addition of anti-HER2 molecules to endocrine therapy, the gains are modest, and no study has demonstrated an improvement in OS. Hence, the use of such approaches has to be weighted against the significant benefit in PFS and OS seen when anti-HER2 molecules are combined with chemotherapy as outlined earlier.CNS progression is a frequent phenomenon in trastuzumab-treated patients. Whether or not it reflects the lack of penetration of trastuzumab through the blood brain barrier or a higher propensity of HER-2 positive disease to spread into the CNS, it is reasonable to continue trastuzumab in the subgroup of patients who develop CNS metastases as the only site of disease progression, since increasing evidence suggests a benefit in terms of OS for patients continuing trastuzumab after receiving radiotherapy for intracranial disease , 56. On An important challenge is the prevention of CNS metastases in HER2 positive metastatic disease For the first time, in the recent CEREBEL study  patientsPatients enrolled onto CEREBEL had similar population characteristics to those reported in other large prospective clinical trials of HER2-positive MBC that used a capecitabine combination , 59, 60 Recent findings, revealed intersting activity of T-DM1 in HER2 postive patients with brain metastasis, also in that case without any loco-regional treatment for CNS recurrences. Probably T-DM1 therapy might be able to prevent out-growth to macro-metastases. In addition, T-DM1 could be useful for patients with established brain metastases that qualify for primary systemic therapy, although WBRT is likely to become necessary eventually , 62.Still, these results suggest that ongoing investigation of T-DM1 in brain metastases is warranted.p = 0.002), as well as a follow up of two years relapse of disease at CNS was much lower  [Recently an interesting finding has been highlighted with the use of neratinib. The phase 2 study NeferTT compared paclitaxel associated with trastuzumab or neratinib in a cohort of 479 HER2 positive patients. Despite the result in terms of PFS was not in favour of either treatment arms, in the group treated with Neratinib the incidence of symptomatic brain metastases was significantly lower  . NeratinLimitations of the research include the lack of specific data on patients with HER2-positive brain disease, how to measure efficacy of various chemotherapy agents and of anti-HER2 molecules. When there is a lack of multiple robust comparative studies, this precludes recommendations on the basis of high-quality evidence .In Table Recently, preliminary results on safety of IIIb PERUSE trial (NCT01572038)  were shoPERTAIN trial (NCT01491737) is looking at combining pertuzumab with trastuzumab and an aromatase inhibitor to treat HER2/HR positive advanced BC; The trial want to find out how safe the combination of pertuzumab, trastuzumab and an aromatase inhibitor is and the activity of the double anti HER2 block with endocrine therapy in postmenopausal patients.In order to test the introduction of different chemotherapic agents from taxane therapy in association with pertuzumab and trastuzumab, phase II VELVET study (NCT01565083) is enrolling patients receiving or the two anti-HER2 molecule as separate infusion or receiving pertzumab and trastuzumab in single saline infusion bag after the first cycle.To date is not investigated the subcutaneous trastuzumab administretion in association with pertuzumab. METAPHER trial (NCT02019277) is a multicenter, international, single-arm post licence investigation that was designed in order to evaluate the safety and efficacy of the combination with pertuzumab plus trastuzumab subcutaneously associated to taxane.In HER2 positive disease the use of trastuzumab is well established. The strong recent data clearly recommend the early use of pertuzumab in combination with trastuzumab and paclitaxel or docetaxel in MBC and ongoing trial will define the integration in the adjuvant setting of these double HER2 inhibition. The mandatory use of this combination in first-line metastatic setting is regardless of the site of the disease concerned. Although we do not have today any results regarding the activity of pertuzumab in population that develops early brain metastases, there is prelimnary evidence of a delayed onset of the CNS spreading with the inclusion of pertuzumab treatment to the standard HER2 double biological therapy. An attrattive field of research would be the response of double block on CNS metastases at the first recurrence of disease.Positive data support the combination of trastuzumab and endocrine agents in that case in which is not well indicated chemotherapy incuding agents, such as elderly patients, indolent progressive disease, absence of visceral crisis. Actually, we have no data to support the use of HER2 double block plus hormonal therapy in selective cases with first recurrence of disease.Unfortunately, all the HER-2 positive MBC patients will eventually develop resistance to pertuzumab plus trastuzumab and taxanes, or to trastuzumab combined with endocrine therapy, even those who initially benefit from the treatment.No other anti HER2 biological agents showed better efficacy than pertuzumab and trastuzumab in first-line therapy. T-DM1 seems to offer improved safety and efficacy over two widely used treatments for HER2-positive MBC. T-DM1-associated adverse reactions were generally manageable with appropriate dose modification and supportive care. The safety profile in first-line MBC or early BC is expected to be similar, and clinical trials are under way to evaluate T-DM1 in these settings.There is an urgent need for prospective biomarker-driven trials to identify patients for whom dual HER2 targeting is cost-effective –68.In conclusion, pertuzumab associated to trastuzumab represents now the milestone of first therapeutic approach in HER2 postive subtypes tumor, and the benefit in terms of outcome disease achieved with “Cleopatra treatment” is one of the more deep enthusiastic result obtained in the story of MBC cure.Finally, the research has focused on molecular mechanisms underlying resistance to trastuzumab and pertuzumab. However, there is still much that we need to lern, in particular to better define activity correlated with biomarkers that could be define resistence or sensitivity to the specific target molecule.The future is one in which it will be possible to use an ample armamentarium of combination therapies directed against HER2 and key signaling components integrated in the HER network. This therapeutic approach will allow clinicians to tailor the clinical management of the individual patient on the basis of tumor-specific biomarker profiles, as captured at the time of diagnosis and in the course of treatment.BBB, blood barrier brain; BC, breast cancer; CNS, central nervous system; EMA, European Medicines Agency; FDA, Food and Drug Administration; HER2, human epidermal growth factor receptor 2; MBC, metastatic breast cancer; ORr, overall response rate; OS, overall survival; PFS, progression free survival; TK, tyrosine kinase; TTP, time to progression


By increasing the understanding of the molecular mechanisms of combined HER2-targeted therapies, we aim to be better able to select patients who would respond to these treatments and understand some of the mechanisms of resistance to HER2-targeted treatments. Recent studies have demonstrated an increased effectiveness of dual targeted HER2 therapies against HER2-amplified breast cancer as compared with single blockade. These studies have resulted in the recent US Food and Drug Administration approval of the combination of taxane chemotherapy with pertuzumab and trastuzumab in the first-line metastatic setting as well as an accelerated approval in the neoadjuvant setting. Another mechanism for overcoming resistance to HER2 targeted therapies is the antibody-drug conjugate trastuzumab-emtansine, which targets the HER2 receptor conjugated to the potent antimicrotubule agent mertansine, allowing for intracellular release of the cytotoxic drug. Studies evaluating the efficacy of dual blockade with antibody-drug conjugate are currently ongoing. This article reviews recent data on different combinations of anti-HER2 treatments as well as ongoing and future research in this area.The estrogen receptor and human epidermal growth factor receptor (HER) signaling pathways are the dominant drivers of cell proliferation and survival in the majority of human breast cancers. Not surprisingly, targeting these pathways provides the most effective therapies in appropriately selected patients. However, The online version of this article (doi:10.1186/s13058-014-0419-5) contains supplementary material, which is available to authorized users. Recognition of the impact of human epidermal growth factor receptor (HER)-2 overexpression or amplification in approximately 15 to 20% of all cases of invasive breast cancer has resulted in the development of multiple drugs that inhibit the proliferative signal pathway associated with this molecular alteration. The incorporation of HER2-directed therapy has improved the overall survival (OS) of metastatic breast cancer (MBC) patients by greater than 20% and has increased the cure rate of breast cancer in the adjuvant setting by approximately 30 to 40% ..14].Table Despite the success of these agents that target the HER family as single agents, there are a number of escape mechanisms from HER-targeted therapies. Clinically, a more complete blockade of the HER receptor layer has been shown to be therapeutically meaningful in prolonging survival in patients. With incomplete blockade of the receptor input layer, proliferative and survival signals can be generated from several different dimer pairs. The idea that the redundancy in the input layer of the network might provide an escape mechanism around a single-agent block has been explored in preclinical trials and neoadjuvant trials as well as in adjuvant trials. Dual HER2 blockade is defined as a more complete blockade of the HER2 and HER signaling pathway by combining two inhibitors with complementary mechanisms of action. In this article, we will review the data supporting these findings and the plans for further evaluation of dual HER2 blockade.de novo and acquired mechanisms of drug resistance exist. Several possible causes of resistance to both trastuzumab and lapatinib have been identified in preclinical studies. Few of these have been prospectively validated in clinical trials. There is enough indication to suggest that some of them do limit the effectiveness of HER2-directed therapy, particularly when these agents are used as single agents.Despite the success in MBC, responses to single-agent trastuzumab are limited and cancer will eventually progress. Many patients treated with adjuvant trastuzumab will be cured of the disease, but disease will recur in some of them. This suggests that both n = 1,082) did not find an impact on DFS . Ba. Ba31]. P = 0.08) and a trend towards improved OS  in patients receiving the combination . A . A 42].  0.0008) .P = 0.0141) as well as in the breast and lymph node pCR (39.3% vs. 21.5%). Consistent with previous studies, a lower pCR rate was noted for hormone receptor-positive patients compared with hormone receptor-negative tumors (26% vs. 63%). The combination targeted therapies only arm also had a 16.8% breast-only pCR rate and an 11.2% breast and lymph node pCR rate ,,[In MDA-175 cells, the combination of T-DM1 and pertuzumab showed enhanced antiproliferative activity and induction of apoptosis compared with either agent alone . In Caluin vivo in the KPL-4 breast tumor xenografts resulted in statistically significant inhibition of tumor growth as compared with the single-agent treatment group. Sustained tumor growth inhibition was also seen for the duration of the study (88 days) compared with 40 days with T-DM1 alone [The combination of T-DM1 and pertuzumab M1 alone .A global phase Ib/II study was conducted to investigate the safety and efficacy of T-DM1 and pertuzumab. The phase Ib results demonstrated acceptable tolerability and promising efficacy (response rate 44.4%) in heavily pretreated MBC patients . The MARin vivo in the BT474-me breast cancer cell line . Tumors treated with single-agent T-DM1 and those treated with the combination of trastuzumab plus lapatinib showed a similar reduction in tumor size. The most significant tumor size reduction was observed in the group treated with the combination of TDM-1 plus lapatinib, where tumor regression was observed in the first 2 days and was significantly superior to the regression observed in the tumors treated with trastuzumab plus lapatinib combination . Proliferation was reduced by lapatinib and lapatinib combination in both cell lines  was observed with afatinib, demonstrating the activity of this agent in this refractory metastatic setting .Neratinib is an oral irreversible inhibitor of endothelial growth factor receptor and HER2 tyrosine kinases. A phase II trial of single-agent neratinib showed a 24% response rate in MBC patients who had progression following trastuzumab and a 56% response rate as first-line therapy . DiarrheSeveral completed neoadjuvant trials have demonstrated significant differences in the rates of pCR among hormone receptor-positive and hormone receptor-negative subsets, with higher pCR rates (at least twice as high) in the hormone receptor-negative subsets Table . The higde novo and acquired resistance exist. With the help of a neoadjuvant model, many studies have demonstrated that single-agent HER2-targeted therapies are efficacious but response is incomplete. Large randomized clinical trials have also demonstrated that dual HER2 targeted combinations with trastuzumab/lapatinib and trastuzumab/pertuzumab are synergistic. Recently, the ADC T-DM1 has been approved for the treatment of HER2-overexpressing MBC. Combination of dual HER2-targeted treatments with T-DM1 and lapatinib or pertuzumab may demonstrate improved efficacy in patients. The results of the ALTTO trial highlight the challenge of determining which patients need multiple targeted therapies. In the future, correlative science embedded within the clinical trials will be invaluable in developing personalized therapy. Studies aimed at characterizing the different genetic alterations associated with resistance to HER2-directed therapies may lead to the discovery of new targets that may overcome resistance.Therapies directed at HER2 establish a successful treatment paradigm, but `Recent advances in breast cancer treatment-, edited by Jenny Chang. Other articles in this series can be found at http://breast-cancer-research.com/series/treatment.This article is part of a series on


Preoperative therapy with chemotherapy and the HER2-targeted monoclonal antibody trastuzumab is valuable for patients with large or locally advanced HER2-positive (HER2+) breast cancers but traditional methods of measuring HER2 expression do not accurately stratify patients for likelihood of response. Quantitative immunofluorescent approaches have the potential to provide a mathematically continuous measure of HER2. Here we seek to determine whether quantitative measurement of HER2 or phospho-HER2 correlates with likelihood of response to trastuzumab- containing neoadjuvant therapy.in situ protein expression. Patients then received 18 weeks of treatment, followed by surgery to assess pathologic response to the neoadjuvant regimen.We evaluated core biopsy samples from 27 HER2+ breast cancer patients enrolled in a preoperative clinical trial using trastuzumab, nab-paclitaxel and carboplatin combination therapy (BrUOG BR-211B (NCT00617942)). Tumor core biopsies were taken before initiation of treatment and 9–13 days after patients received "run-in" doses of either single agent trastuzumab or nab-paclitaxel. The AQUA method of quantitative immunofluorescence was used for analysis of A HER2 score of 2111 by AQUA analysis has been shown to be equivalent to HER2 3+ by immunohistochemical staining in previous studies. Of 20 evaluable patients, 10 cases who achieved a pathologic complete response (pathCR) with neoadjuvant treatment had a mean HER2 level of 10251 compared with 4766 in the patients without pathCR (p = 0.0021). Measurement of phospho-HER2 showed no difference in pathCR vs non-pathCR groups. In 9 patients who had HER2 levels repeated after a single treatment with trastuzumab there was no evidence of a reduction in the HER2 or phospho-HER2 levels following that exposure.High levels of HER2 are associated with achievement of a pathCR in the preoperative setting, while levels of Phospho-HER2 were not predictive of response. This data suggests that accurate measurement of HER2 may help determine the likelihood of response in the pre-surgical setting. Further validation in larger cohorts is required, but this pilot data shows the feasibility of this approach. Human epidermal growth factor receptor 2 (HER2) is amplified or over-expressed in around 20% of breast cancer cases, and the amplification of HER2 is usually associated with worse prognosis-3. TrastPre-surgical or neoadjuvant chemotherapy is standard therapy for inflammatory and locally advanced breast cancer. The addition of trastuzumab to chemotherapy in the pre-surgical setting in HER2+ patients has been tested in several phase II studies-14, withAlthough the use of trastuzumab as part of the pre-surgical regimen for breast cancer has increased, a uniform clinical benefit of trastuzumab in combination with chemotherapy is not observed. The likelihood of achieving a pathCR in this cohort is higher in trastuzumab treated patients with hormone receptor- negative HER2+ cancers compared to hormone receptor-positive HER2+ cancers. Other gBrUOG BR-211B (NCT00617942) was a pre-surgical trial for stage II-III HER2+ breast cancer patients led by the Brown University Oncology Group (BrUOG) and the Yale Cancer Center. Patients were treated with q3week carboplatin, weekly nab-paclitaxel and trastuzumab for 18 weeks. In this trial, research biopsies were collected before initiation of treatment and again after a brief "run-in" exposure to either trastuzumab or nab-paclitaxel before patients received their first doses of the entire treatment regimen, allowing the testing of two hypotheses: 1) that the level of HER2 or pHER2 is associated with the likelihood of achieving a pathological complete response; and 2) that short exposure "run-in" treatment with trastuzumab alters the expression of HER2 or pHER2. In this paper, we quantitatively measured the level of HER2 and pHER2 (pY1248) to address these two issues.2 (determined by the institution at which the patient enrolled on the study). After 9 – 13 days biopsies were repeated. Patients then received trastuzumab 2 mg/kg weekly (patients who received "run-in" nab-paclitaxel were treated with 4 mg/kg for the first week), weekly nab-paclitaxel 100 mg/m2 and carboplatin AUC 6 every 3 weeks for 18 weeks. Pathologic complete response  was then assessed at definitive surgery within 6 weeks from the last dose of pre-surgical therapy. Pairs of biopsies (before and after "run-in" treatment) from twenty-seven patients were obtained for biomarker studies , was designed to determine the clinical and pathologic response rates of treatment with q3week carboplatin, weekly nab-paclitaxel and weekly trastuzumab in resectable and unresectable locally advanced breast cancer. Eligibility for the trial included histologically documented adenocarcinoma, female age greater than 18, stage IIA-IIIC disease, no evidence of metastatic disease, no prior systemic therapy, not pregnant or lactating, no baseline neuropathy greater than or equal to grade 2, and HER2 positive defined by IHC 3+ or FISH ratio greater than or equal to 2.0. Baseline biopsies were obtained before the treatment. Randomized "run-in" treatment was given to patients with either a single dose of trastuzumab 6 mg/kg or two weekly doses of nab-paclitaxel 100 mg/mFormalin fixed paraffin embedded tissue biopsies from twenty-seven patients were collected and immediately fixed in formalin to prevent artifact associated with delayed time to fixation. Specimens were then processed, sectioned and deparaffinized in xylene, followed by antigen retrieval at 97°C in pH 6 sodium citric buffer for 20 minutes. Slides were incubated with 0.3% bovine serum albumin in 0.1 M tris-buffered saline with 0.05% Tween-20 to block non-specific binding, then incubated with primary antibodies at 4C overnight. The primary antibodies to HER2  and to phospho-HER2 pY1248  have been previously extensively validated and were only tested for reproducibility before use in our studies. Antibodies diluted 1:1000 and 1:100 respectively showed uniform membrane staining as has been previously described for each . Pathological complete response was used to stratify patients, Fischer’s PLSD was used to compare the mean difference of target biomarkers within each groups. Ordinary least square (OLS) method and paired t-test were used to test if run-in treatment alternated the expression of biomarkers.Among the 27 patients, assessment of pathologic response was available for 23. Of these patients, 20 pretreatment biopsies were evaluable, as were 12 of the repeat biopsies after ‘run-in’ treatment. Among the 20 patients with evaluable tissue and known response, 50% had pathSince there is a known inverse association between estrogen receptor (ER) and response in the pre-surgical setting, we evaluated the mean levels of HER2 protein as a function of clinical ER status. The mean AQUA score in ER negative patients was 8615 compared to the 5498 in ER positive patients. This difference is not statistically significant in a two sample mean test . This slope is not statistically significantly different from 1, suggesting that HER2 level is not affected by a single dose of trastuzumab treatment. The result is confirmed by the paired t-test of these 9 pairs of baseline and post-treatment biopsies – the mean of post baseline difference is 449.4 AQUA score units (p = 0.4181) . The mean of pHER2 level measured by the PN2A antibody in the pathological complete response group was 2768.0 AQUA score units, compared with 2183.7 in the no PathCR group (p = 0.4689) . The slope is not statistically significantly different from 1 (p = 0.212), suggesting that pHER2 level is not altered by the single dose of trastuzumab treatment. This result is confirmed by the paired t-test of the 9 pairs of baseline and post-treatment biopsies – the mean of post baseline difference is 61.3 AQUA score units (p = 0.8781)  levels. Among baseline biopsies, 21 samples had both evaluable tissues for HER2 and pHER2, and among post-treatment biopsies, 15 samples had evaluable tissues for both HER2 and pHER2. We did not observe an association between HER2 and pHER2 in either baseline biopsies or in post-treatment biopsies . M. Abu-Khalaf, L. Harris, N. Sinclair, H. Cheng, Y. Bai, and V. Bossuyt have no competing interests.HC did the quantitative measurements in the lab and the data analysis and wrote the first draft of the manuscript. YB assisted in the quantification of the specimens. WS designed the trial, dealt with clinical issues and problems during the conduct of the trial, oversaw clinical data collection and analysis, and assisted in manuscript preparation. NS assisted with the trial execution at the Brown site, overall clinical data collection and analysis, and assisted in manuscript preparation. VB read the pathology at Yale and did all aspects of the work related to determination of pathologic complete response. MK assisted with the trial execution at the Yale site. LH ran the trial at the Yale site, assisted with data analysis and assisted in manuscript preparation. DR runs the lab in which the work was done, reviewed all of the data collection and analysis and assisted in manuscript preparation. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/326/prepub


ERBB2 analogous to those detected in EGFR and never coexisting with EGFR, KRAS, or ALK alterations  or of HER2 gene amplification by in situ techniques. Interpretation and reporting are crucial moments, especially in in situ hybridization, where amplification of the chromosome 17 centromeric region (CEP17) has been shownto account for misleading HER2 FISH results, precluding anti-HER2-based therapy in some patients. In addition, heterogeneity of HER2 amplification must be considered in clinical therapeutic decisions, despite pilot results suggesting that a similar trastuzumab benefit for patients with HER2 amplification, either diffuse or focal  to block their aggressive signal underscores the need for in-depth analysis of diagnostic properties specific for these alterations as part of clinical HER2 status evaluation before choosing among HER2-directed therapeutic options. In this context, Sapino and coworkers  present or focal . Anotheror focal .2-terminal residues into the rat EC5-TM cut-down plasmid to regenerate the whole extracellular domain (HuRT) or by cloning the first 410 NH2-terminal rat residues into the remaining residues from human HER2 (RhuT) showed superior performance in breaking tolerance to the HER2 self-antigen (Molecular alterations of HER2 and its presence on the tumor cell membrane endow this oncoprotein with relevant immunological properties, making it an ideal target antigen for long-term cancer immunoprevention. As thoroughly described by Lollini et al. , HER2 is-antigen , 18. PreOverall, while the benefit of anti-HER2 therapies demonstrated in clinical trials indicates that HER2 is, to date, one of the most useful molecules for targeted therapy, optimization of these therapies for HER2-positive cancer patients awaits further studies. The issues considered in this Research Topic point to the need to better define the role of activating molecular alterations of HER2; refine HER2 clinical testing to design the most tailored treatment for breast cancer patients; and tailor anti-HER2 vaccines to prevent relapse in high-risk breast cancer patients or progression in patients with HER2-overexpressing minimal residual disease.Such knowledge will also be highly relevant in considering the ability of new HER2-targeted drugs, i.e., the monoclonal antibody pertuzumab, the tyrosine kinase inhibitor lapatinib, and the antibody–drug conjugated trastuzumab-DM1, to improve the therapeutic effects of trastuzumab. While these new drugs also target HER2, it is not yet known whether activating HER2 mutations are sensitive to novel strategies. In view of recent results obtained in different clinical trials indicating that combining two anti-HER2 drugs with chemotherapy is the most effective treatment modality for HER2-positive patients , HER2 clThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HER2 drives tumorigenesis and cancer progression in a subset of patients with gastric cancer (GC), and treatment with trastuzumab, a humanized HER2-neutralizing antibody, improves the overall survival rate of HER2-positive patients. However, a considerable portion of the patients does not respond to trastuzumab and the molecular mechanisms underlying the intrinsic resistance to anti-HER2 therapy in GC is not fully understood.Genetic amplification of HER2 fusion to discover the DNA integration structure. A multicolor FISH assay for HER2 split screening was conducted to confirm HER2 fusion and IHC (HercepTest™) was used to detect the membranous expression of HER2. Fusion cDNA were transfected into NIH/3T3 cells and generate stable cell line by lentivirus. The expression of exogenous HER2 fusion proteins and pHER2 were examined by western blot analysis. In vitro efficacy studies were also conducted by PD assay and softagar assay in cell line expression wild type and fusion HER2. T-DM1 was used to assess its binding to NIH/3T3 cells ectopically expressing wild-type and fusion HER2. Finally, the anti-tumor efficacy of trastuzumab was tested in NIH/3 T3 xenografts expressing the HER2 fusion variants.We performed whole-transcriptome sequencing on 21 HER2-positive tumor specimens from Chinese GC patients. Whole genome sequencing was performed on the three samples with HER2 fusions with ZNF207, MDK, or NOS2 in 21 HER2-amplified GC samples . Two of the fusions, ZNF207-HER2, and MDK-HER2, which are oncogenic, lead to aberrant activation of HER2 kinase. Treatment with trastuzumab inhibited tumor growth significantly in xenografts expressing MDK-HER2 fusion. In contrast, trastuzumab had no effect on the growth of xenografts expressing ZNF207-HER2 fusion, due to its inability to bind to trastuzumab.We identified three new Our results provide the molecular basis of a novel resistance mechanism to trastuzumab-based anti-HER2 therapy, supporting additional molecule stratification within HER2-positive GC patients for more effective therapy options.The online version of this article (doi:10.1186/s12967-015-0476-2) contains supplementary material, which is available to authorized users. Gastric cancer (GC), as the second leading cause of cancer deaths worldwide, accounted for 989,600 new cases and 738,000 deaths globally in 2011 . TrastuzHER2 gene amplification was initially discovered as an oncogene in breast cancer (BC), which led to the development of HER2-targeted therapeutics for treating HER2-positive BC  × 100, and was used to evaluate anti-tumor efficacy. Student’s t tests were used to compare the TGI of the treatment group with that of the control group. Statistical tests were two sided, with P < 0.05 considered significant.6- to 8-week-old female nude (HER2 (chr17q12) in-frame fusion transcripts with 5’ partners of ZNF207 (chr17q11.2), MDK (chr11p11.2), or NOS2 (chr17q11.2) in three HER2-positive GC samples of GC196, 431-9540474 T, and GC334, respectively  on 21 HER2-positive tumor specimens from Chinese GC patients whose tumors were surgically removed and who were treatment naïve, using the HiSeq2000 system (Illumina). A number of candidate fusion transcripts were identified with more than three chimerical reads, which were subsequently followed by RT-PCR/Sanger sequencing confirmation. This led to the identification of three HER2 fusion genes—ZNF207 (exon 1–2)/HER2 (exon 18–30), MDK (exon 1–4)/HER2 (exon 11–30), and NOS2 (exon 1–2)/HER2 (exon 2–30)—were further confirmed in these tumor DNA samples  were captured for the genomic sequences outside the fusion gene. In the GC334 specimen, in addition to the high reads (>1800) of the fusion partners of NOS2-HER2 fusion that were detected, an average of 500 reads was also captured for the entire genomic sequence of wild-type HER2 gene.In the GC196 sample, over 100 reads were detected for -fusion-positive tumor specimens . In the tumor harboring ZNF207-HER2, the increased expression of HER2 mRNA was observed only after the fusion site, whereas the increased expression of ZNF207 mRNA was detected only before the fusion site, further confirming that the amplification of the ZNF207-HER2 fusion was a homogeneous event in the sample  assay to assess the genetic amplification status of the ZNF207-HER2 and NOS2-HER2 fusion genes, as well as to dissect their relationship to the wild-type HER2 gene in the GC196 and GC334 samples, respectively. Based on hematoxylin and eosin staining, both primary tumors with the two HER2 fusions were defined as adenocarcinoma by pathologists  sample of 431-9540474 T was unavailable for the multicolor FISH analysis, the whole genome sequencing data suggested a mixture of tumor cell populations harboring gene amplifications of wild-type HER2, MDK-HER2, and wild-type MDK  with a HercepTest™ in FFPE samples of GC196 and GC334. Strong staining (IHC 3+) was detected in both samples , which are common genetic alterations identified in GC [HER2 fusions from these known oncogenic alterations suggests that the HER2 fusions are oncogenic drivers. In addition, sequence analysis revealed that proteins encoded by ZNF207-HER2 and MDK-HER2 fusion variants contained a partial extracellular domain, a transmembrane domain, and a full kinase domain of HER2   Figure C. TogethMDK-HER2 or ZNF207-HER2, similar to the results observed in the original primary tumor samples  : a fusion between CDK12 exon 12 and HER2 intron 4, and a second fusion between NEUROD2 exon 1 and HER2 exon 8 [HER2 fusions. In the current study, we performed a whole-transcriptome sequencing of 21 HER2-positive GC tumor samples taken from Chinese patients, and discovered three HER2 fusion transcripts due to HER2 gene fusions. Two of them, ZNF207-HER2 and MDK-HER2 were truncated in the N-terminal extracellular domains, but they remained intact in the kinase and transmembrane domains of HER2. The amplification and overexpression of the three HER2 gene fusions were detected in the primary GC samples by multicolor FISH and RNAseq or IHC-based HercepTest™ analysis. The ectopic expression of ZNF207-HER2 and MDK-HER2 in the NIH/3T3 cells led to a constitutive activation of HER2 and downstream signaling, and thus, cell transformation in vitro and tumorigenesis in vivo, demonstrating the oncogenic driver of the fusions. Furthermore, the xenografts ectopically expressing MDK-HER2 but not ZNF207-HER2 were sensitive to trastuzumab. Interestingly, the HER2 fusions were found to be mutually exclusive with mutations of PI3KCA, BRAF, KRAS, and HER3 and amplifications of FGFR2 and c-MET. Collectively, our data confirmed for the first time the presence of oncogenic HER2 arrangements in patients with GC. The ZNF207-HER2 fusion represents a novel intrinsic resistance mechanism to trastuzumab-based anti-HER2 therapy, due to the loss of binding ability to trastuzumab.Several studies have recently used NGS to understand the molecular basis of GC, and a number of previously unknown genetic alterations have been reported , includi2 exon 8 . HoweverHER2 amplification, a significant portion of the patients do not respond clinically to the treatment, and there is little understanding of the molecular mechanism underlying this intrinsic resistance. In contrast, the understanding of the mechanisms of both intrinsic and acquired resistance to HER2 inhibitors in HER2-positive BC is far more advanced [HER2-Δ16) in the extracellular domain of the HER2 receptor, preventing disruption of HER2 homodimers upon binding by trastuzumab [HER2-Δ16 in a GC tumor sample, which naturally harbored HER2-Δ16. Surprisingly, its corresponding patient-derived gastric cancer xenograft (PDGCX), which retains HER2-Δ16 (data not shown), responded well to trastuzumab treatment, with a significant tumor regression being observed (data not shown). Given the lack of clinical evidence of association between HER2-Δ16 and resistance to trastuzumab in BC, as well as the lack of preclinical data on the anti-tumor efficacy of trastuzumab in xenografts carrying HER2-Δ16 [HER2-Δ16 was not a resistance mechanism to trastuzumab in GC when tested in the PDGCX model. Additional HER2-Δ16 positive PDGCX models are warranted to confirm this observation further.Despite the overall survival benefit achieved with trastuzumab in GC patients carrying advanced ,23. For advanced ,25; a spstuzumab ; In our HER2-Δ16 , our datHER2 fusions, we also found two recurrent in-frame BRAF fusion transcripts, BAIAP2L1-BRAF (data not shown), in another cohort of HER2-amplified GC patients. Although further work is needed to demonstrate its oncogenic activity and sensitivity to trastuzumab or BRAF inhibitor, the findings suggest that BAIAP2L1-BRAF could be a potential resistance mechanism to trastuzumab, as it maintains an intact BRAF kinase domain [Surprisingly, in addition to the e domain .HER2 gene amplification is determined by the ratio between the numbers of signals from the hybridization of the HER2 gene probe (covers the whole HER2 gene) and the number of signals from the hybridization of the reference chromosome 17 centromere probe [HER2 fusions  score of 3+ (HercepTest™ score of 2+ needs further FISH confirmation). re probe . The antHER2 fusions. This notion is further supported by our observations that NIH/3T3 cells expressing either ZNF207-HER2 or MDK-HER2 showed to be sensitivity to lapatinib, afatinib, and neratinib in vitro (data not shown).In addition to the antibody, a number of small-molecule HER2 kinase inhibitors are available: lapatinib, an approved agent for HER2-positive BC patients, and afatinib and neratinib, two irreversible kinase inhibitors, currently in phase III clinical trials for HER2-positive BC. The modulation of AKT signaling by lapatinib in the cells expressing the HER2 fusions Figure A suggestHER2 amplification in 41% (21/51) of HER2-amplified tumors. The mutually exclusive co-amplification of HER2 with EGFR, FGFR2, FGFR2 and MET was also observed respectively in some of the tumors, suggesting the potential challenges for design of targeted-therapy approaches [MDK-HER2 and NOS2-HER2 with wild HER2 (intra-tumor heterogeneity) is yet to be uncovered. Further studies to explore the responsiveness of the HER2 fusions to combination of trastuzumab with pertuzumab or chemotherapies may lead to additional insights into the impact of the HER2 fusions to anti-HER2 therapies.Besides the functional and phonotypical heterogeneity, emerging evidences indicate that genetic heterogeneity among tumor cells contributes to the advantages for survival, proliferation, metastasis and resistance to anti-cancer therapies. Recently, Tajiri et al. examined 475 GC samples using multiple ligation-dependent probe amplification (MLPA) and FISH analysis and revealed intratumoral heterogeneity of proaches . The homIt is noteworthy that the HER2 positive GC samples used for this study were collected prior the introduction of trastuzumab to China, thus we lack evidence for clinical response of the tumors harboring HER2 fusions to trastuzuamb. Further studies on GC samples from patients treated with trastuzumab at different stages will help to confirm the effect of the HER2 fusions to trastuzmab therapy and their oncogenic property.ZNF207-HER2, along with the discovery of two recurrent BAIAP2L1-BRAF, strongly indicate a large degree of molecular heterogeneity, even in the well-defined HER2-positive segment, representing potential de novo resistance to trastuzumab-based GC therapies. In addition, whether a similar mechanism would exist in BC also needs to be exploited.Although further large-scale clinical investigations are needed to understand the clinical prevalence in GC patients, our data on HER2 fusion genes in GC patients whose tumors were clinically classified as HER2-positive. Our results suggest that these HER2 fusions are genetically amplified driver oncogenes that respond differently to the HER2-neutralizing antibody trastuzumab. The resistance of ZNF207-HER2 to trastuzumab and the existence of the recurrent BRAF fusion variants warrant molecular subtype diagnoses of HER2-positive GC patients for more effective personalized trastuzumab therapies and for future treatment options.In summary, we uncovered three previously unidentified


The evolving field of HER2-targeted therapy has significantly improved the outcome of women diagnosed with HER2-positive invasive breast cancer. In this review, we sought to summarise the efficacy of trastuzumab-based regimens in the adjuvant and neoadjuvant setting with a special emphasis on relevant clinical questions: treatment duration, sequence of trastuzumab administration, toxicity, the role of anthracycline-based regimens, and optimal management of small HER2+ tumours. Controversial topics are discussed taking into consideration the development of modern anti-HER2 agents. Breast cancer (BC) is a heterogeneous disease with several biological subtypes. Human epidermal growth factor receptor 2 (HER2) is a strong phenotype determinant present in approximately 20%  of all BIn the adjuvant setting, anti-HER2-targeted therapy with trastuzumab greatly contributed to improvement of clinical outcomes, as measured by disease-free survival (DFS) and overall survival (OS). Several multicentre international trials were designed to assess the role of trastuzumab treatment in high-risk early-stage BC patients, defined as node positive or node negative with tumours larger than 1 cm  or 2 cm These findings were further scrutinised in a meta-analysis that gathered the results of eight trials  testing the benefit of trastuzumab when added to adjuvant chemotherapy versus chemotherapy alone . With a Recently, trastuzumab was also prospectively tested in a cohort of patients mainly with stage I HER2-positive BC . The antFinally, population-based studies reported reassuring patient safety outcomes and treatment compliance –13.Neoadjuvant therapy is the standard approach for treating locally advanced and inflammatory BCs; however, it can be also considered an equally valid strategy for the treatment of early-stage BCs . In HER2In the neoadjuvant setting, trastuzumab has been tested in combination with chemotherapy and other anti-HER2 agents . The ‘fiSubsequently, the ‘second generation’ trials compared trastuzumab to other anti-HER2 agents or the added benefit of other anti-HER2 agents to trastuzumab plus chemotherapy. The GeparQuinto trial  performeper se effective in achieving pCR in a subset of patients without any additional chemotherapy.Besides lapatinib, trastuzumab was also tested in combination with pertuzumab. The NeoSphere trial  was a muOnly recently was the treatment duration of trastuzumab more clearly established. The pivotal trials studying the role of trastuzumab in early BC used an empirical reference therapy duration of one year –4. HowevThe FinHER trial  tested aMoreover, the phase III study PHARE  randomlyIn search of improved efficacy, the HERA trial, an international, multicentre, open-label phase III trial, besides comparing one year of adjuvant trastuzumab with observation after standard adjuvant chemotherapy, also enrolled patients to a third arm treated with two years of trastuzumab . This trGrounded in the current evidence, one year of adjuvant trastuzumab remains the standard treatment duration.Anthracycline-based chemotherapy regimens were used in the majority of the trials evaluating trastuzumab in the adjuvant setting . As prevWhereas some of the pivotal trials opted to give trastuzumab after chemotherapy , others preferred to administer trastuzumab concurrent with paclitaxel . The NCCFinally, some studies also assessed the sequence of chemotherapies (i.e. anthracycline and taxane) given in combination with trastuzumab. A randomised phase III neoadjuvant clinical trial  tested aThe studies leading to the approval of trastuzumab used an intravenous (IV) formulation of the drug. While IV trastuzumab has already been shown to be a cost-effective therapy , its admHER2 overexpression remains the only validated predictive marker of response to anti-HER2 therapy, even though the response to anti-HER2 directed therapies based on this marker is not homogeneous. The development of markers that would allow selecting patients with the highest likelihood to respond would be of much benefit to maximise benefit and decrease harm.18F-FDG positron emission tomography (PET)/computed tomography (CT) imaging is being tested as a predictor of the likelihood of obtaining a pCR. The NeoALTTO trial . It. It41]. Trastuzumab emtansine (T-DM1) is a conjugate between trastuzumab antibody and emtansine (DM1), a cytotoxic drug  45]. In. In45]. a-b) were considered to be ‘low risk’ tumours and often considered not to benefit from anti-HER2 directed therapy. However, some retrospective studies [As previously discussed, trastuzumab significantly enhanced the clinical outcomes in patients with ‘high risk’ HER2-positive BCs, i.e. tumours larger than 1 cm  or 2 cm  studies –48 and a studies . Yet, st studies .The NeoSphere trial  (previouHER2 blockade has brought significant clinical benefit with acceptable toxicity, leading to a paradigm shift in the management of this population with hitherto poor prognosis. The preliminary data from combining new anti-HER2 therapies with trastuzumab are exciting, and several large studies are underway to validate these in the neoadjuvant and adjuvant settings.The authors declare no conflicts of interest.