We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients.A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan – Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates.HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3  and with FISH HER2 . All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS , and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP  and OS .The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment. HER2, a proto-oncogene encoding HER2 tyrosine kinase receptor, is amplified in 10 to 20% of breast cancers, leading to HER2 protein overexpression and an aggressive tumor phenotype associated with reduced survival and high metastatic potential. The advent of molecular targeting of HER2 receptor with trastuzumab has substantially improved the outcome of breast cancer patients. Although single-agent trastuzumab exerts some antitumor activity, the highest clinical benefit is derived when trastuzumab is combined with chemotherapy In recent years, targeted therapies such as the anti-HER2 humanized monoclonal antibody trastuzumab, have changed the therapeutic landscape in breast cancer. Accurate assessment of HER2 status is necessary to recommend therapy for patients who are most likely to benefit from the treatment and minimize unnecessary overtreatment in the setting where potential side effects may occur Despite the reported and proven benefits of trastuzumab in HER2-overexpressing metastatic breast cancer patients, approximately 50% of them The assessment of HER2 overexpression by two immunohistochemical (IHC) assays and three fluorescent in-situ hybridization (FISH) assays have been approved by the US Food and Drug Administration (FDA) http://clinicaltrials.gov/ct2/show/NCT00005970?term=N9831&rank=2. Accessed April 18, 2011), showed decrease in the number of patients eligible for trastuzumab therapy when the ASCO/CAP criteria were applied To minimize discrepancies, the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), developed guidelines for optimal laboratory evaluation of HER2 status by modifying the FDA criteria, which had been used in pivotal trastuzumab trials type DX RT-PCR for HER2 assessment was reported. This high false-negative rate of RT-PCR assay highlights the shortcomings of this non-morphologic assay and the importance of standard immunohistological methods in HER2 testing. This result could open the door for a new method of in situ quantitative analysis of HER2 mRNA levels that would reflect more precisely HER2 status by combining quantitative determination of gene expression levels and morphologic assessment.In spite of these changes, efforts to optimize testing methods are ongoing. It has been suggested that HER2 status can be assessed with different approaches as a continuous variable and can be assessed on mRNA level The aim of this study was to assess HER2- mRNA as a potential predictor of benefit from trastuzumab-based chemotherapy and to correlate it to HER2 protein levels and to FISH by using a combination of automated-quantitative immunofluorescence and a new method of mRNA in situ hybridization marketed as RNAscope.st line (n = 130) were included in the analysis. The translational research protocol was approved by the Bioethics Committee of the Aristotle University of Thessaloniki School of Medicine  under the general title “Investigation of major mechanisms of resistance to treatment with trastuzumab in patients with metastatic breast cancer”. All patients included in the study after 2005 provided written informed consent for the provision of biological material for future research studies, before receiving any treatment. Waiver of consent was obtained from the Bioethics Committee for patients included in the study before 2005.The breast cancer cohort used for this study consists of 149 patients classified as HER2-overexpressing by locally performed IHC at the time of pathological assessment, who were diagnosed in Greece from 1999 through 2006 with metastatic breast cancer. Patients were treated with various Trastuzumab-based combinations for metastatic disease in situ hybridization (ISH) formalin-fixed paraffin embedded (FFPE) tissue has been previously described The RNAscope  technique of mRNA Briefly, the assay uses a pool of up to 20 probe pair sets for each mRNA target of interest. Probe pairs bind along an mRNA region and create a unique 28 base-pair sequence recognized by the preAMP which then allows for binding during the subsequent amplification steps and finally the amplified target is detected by cy5 tyramide & AQUA .ERBB2 gene (by ACD), the housekeeping gene ubiquitin C (UbC) as a positive control or the bacterial gene DapB as a negative control. ERBB2 gene or UbC specific hybridization signals were detected with Cy5-tyramide. Sections were then incubated with 0.3% bovine serum albumin (BSA) in 0.1 mol/L of Tris-buffered saline  for 30 minutes at room temperature followed by incubation with a wide-spectrum rabbit anti-cow cytokeratin antibody  in BSA/tris-buffered saline for 1 hour at room temperature. The cytokeratin signal was detected with Alexa 546 conjugated goat anti-rabbit  incubated for 1 hour at room temperature. Slides were then mounted using ProlongGold plus 4,6-diamidino-2-phenylindole (DAPI).HER2 mRNA status was assessed by in situ hybridization using the RNAscope FFPE assay kit according to the manufacturer’s instructions modified for fluorescence detection of transcripts using Cy5-tyramide. In brief, slides with TMA sections were treated with heat and protease digestion followed by hybridization with target probes to In Situ quantitative measurement of biomarkers was done by using the following:anti-HER2 mouse monoclonal antibody, clone CB11 (by Biocare) Epitope: Intracellular domain of human HER2 receptor.anti-HER2 mouse monoclonal antibody, clone SP3 (by Thermo Fischer) Epitope: Extracellular domain of human c-erbB2.ERRB2 probe, for HER2 mRNA (RNAscope assay by ACD), according to the manufacturer’s protocol modified for detection with Cy-5 Tyramide.Each antibody was validated by performing 1) titering, 2) reproducibility assessment on index arrays, and 3) verification of linearity with expression on cell line series, according to a previously described protocol The AQUA method of QIF has been described elsewhere Correlation of each biomarker with immunohistochemistry/FISH was assessed by using the Pearson and Spearman’s rank correlation coefficient. All biomarkers were treated as binary variables. Dichotomization of CB11 and SP3 was based on the corresponding median AQUA scores (620.41 and 99.42 respectively), whilst the signal-to-noise threshold was used as a cut point for HER2mRNA (<100: negative vs. ≥100 positive). Overall survival (OS) and time to progression (TTP) were the primary endpoints of interest. TTP survival times were calculated in months, from Trastuzumab initiation to the date of disease progression, censoring or last follow-up exam. Survival curves were calculated using the Kaplan-Meier method and differences in survival times between groups were assessed by using the log-rank test. Multivariate Cox regression models were used to assess the performance of each marker and FISH HER2 after adjusting for other important predictors, namely age group (<50 vs. ≥50 years), disease grade (I & II vs. III), status of distant metastasis and ER status. For a simultaneous assessment of all HER2 biomarkers and FISH in the presence of the aforementioned pre-specified set of prognostic factors, multivariate Cox regression models, with backward selection, were also fitted. During the backward elimination process, the possibility of significant interactions between any pair of biomarkers and each biomarker by ER status was also tested. None of these candidates was found statistically significant and hence no interaction terms are included in the final multivariate Cox proportional hazards (PH) models.http://www.r-project.org/).Statistical analysis was performed using R Statistical Software  and with FISH HER2  for CB11, HR = 0.46  for SP3 and HR = 1.68  for HER2 mRNA. In a similar model with all pre-specified prognostic factors present, TTP survival was also more favorable for patients with amplified HER2 gene status .The multivariate TTP analysis included all biomarkers and FISH HER2 as well as the aforementioned prognostic factors. Applying a backward elimination process, it was found that ECD HER2, as measured by SP3, is the only biomarker that retains its prognostic ability for TTP survival . FISH HER2 was also marginally significant at α = 10% .OS was found significantly longer for patients with high HER2 ICD , high HER2 ECD , high HER2 mRNA  and amplified HER2 gene status .Multivariate analysis showed that after adjustment for the pre-specified set of prognostic factors, high HER2 ECD protein levels, as measured by SP3, predict better overall survival , Herceptest and FISH and showed a higher discrimination power compared to HercepTest One possible explanation of our findings may reside in the molecular target of trastuzumab which is the extracellular domain of HER-2 Our study also shows a significant correlation of HER2-mRNA levels with conventional immunohistochemistry and in situ-hybridization methods as well as FISH. We found that HER2 status by FISH, AQUA HER2-proteins and HER2-mRNA levels are all independent predictor factors for TTP after Trastuzumab-containing chemotherapy in this metastatic cohort. In our study, HER2 mRNA status was assessed by in situ hybridization using the RNAscope FFPE assay in combination with the AQUA method of automated quantitative immunofluorescence. In situ quantitative measurement of both HER2-mRNA and protein; is reproducible, automated method that reduces intra-observer variability. Since the interpretation of HER2 immunostaining and in situ-hybridization may be influenced by laboratory and observer variability, the use of the AQUA automated method in measurement of HER2 protein along with the HER2-mRNA level could improve the diagnostic accuracy of HER2 status,. QISH enables a relatively easy, fast and reproducible quantification of HER2-mRNA expression feasible in routine FFPE tissue.In summary, our study demonstrates that measurement of HER2-m RNA levels with this novel method has a predictive value for response to Herceptin-based chemotherapy in metastatic breast cancer. Additional studies in prospective cohorts are required to validate these findings with the ultimate goal to build a potential predictive model of trastuzumab therapy and complete the puzzle of HER2 testing optimization.Figure S1He2mRNA hybridization.(TIF)Click here for additional data file.


Breast cancer is the second leading cause of cancer death among women. Human epidermal receptor 2 (HER2) positive breast cancer (HER2+ BC) is the most aggressive subtype of breast cancer, with poor prognosis and a high rate of recurrence. About one third of breast cancer is HER2+ BC with significantly high expression level of HER2 protein compared to other subtypes. Therefore, HER2 is an important biomarker and an ideal target for developing therapeutic strategies for the treatment HER2+ BC. In this review, HER2 structure and physiological and pathological roles in HER2+ BC are discussed. Two diagnostic tests, immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH), for evaluating HER2 expression levels are briefly introduced. The current mainstay targeted therapies for HER2+ BC include monoclonal antibodies, small molecule tyrosine kinase inhibitors, antibody–drug conjugates (ADC) and other emerging anti-HER2 agents. In clinical practice, combination therapies are commonly adopted in order to achieve synergistic drug response. This review will help to better understand the molecular mechanism of HER2+ BC and further facilitate the development of more effective therapeutic strategies against HER2+ BC. Breast cancer is one of the most common cancers and the second leading cause of cancer death among women of all races . AccordiHER2 gene amplification and HER2 protein overexpression, accounts for about 25%–30% of all breast cancers . In. In51]. On the other hand, IHC testing has some limitations, for example the effect caused by the pre-analytic, analytic and post-analytic processes. There is no uniform control for time, type of tissue fixation, and temperature of paraffin embedding, and there is no standard for processing the tissue samples, all of which may result in fluctuation of the IHC results of the same specimens. Moreover, reduced immuno-reactivity for HER2 proteins to their corresponding antibodies may happen due to prolonged storage of unstained slides, causing the IHC scores to be negatively affected. Most importantly, IHC test is a semi-quantitative method, as it is based on a subjective determination of the intensity of the color reaction .FISH is a more reliable, sensitive and accurate testing tool with less influence by pre-analytic and analytic variable factors. A major advantage of FISH compared to IHC is that the evaluation of HER2 status is much more quantitative and includes internal control cells which are located adjacent to tumor cells in the same tissue section for parallel comparison . This meHER2/neu gene [HER2/neu gene copies can be estimated. The ratio of HER2/neu gene to chromosome 17q centromere (CEP 17) will then be calculated [Three types of FISH assays are approved by the FDA, including PathVysion (Abbott Laboratories) and Dako PharmDx (Dako Corporation) FISH test which two use dual probes (one for HER2 and the other for the centromere 17) and the Ventana Inform  which uses a single probe only for HER2. These testing methods share a similar testing procedure, which utilizes fluorescently labeled probes that are partly complementary to the neu gene . After blculated . Accordilculated .However, FISH also has its limitations. It usually takes about two days and requires the use of expensive fluorescence microscopy. In addition, it requires extensive training and expertise to distinguish between normal cells and malignant cells from the FISH results . FurtherOverexpression of HER2 is closely related to the development and progression of breast cancer. Therefore, HER2 becomes a critical target for developing therapeutic drugs against HER2+ BC. The development of anti-HER2 therapies has significantly improved the clinical outcome for patients with HER2+ BC . In this®, Genetech) is the first humanized monoclonal antibody approved for the treatment of HER2+ BC by the FDA in 1998. With the advent of trastuzumab, the prognosis of patients with HER2+ BC both in metastatic and adjuvant settings has been dramatically improved. Several clinical trials have shown that trastuzumab improves overall survival (OS) in metastatic breast cancer [Trastuzumab , disruption of downstream signaling pathways, inhibition of cleavage of HER2, and promoting endocytosis of HER2 receptors ,60. ThesThough trastuzumab exerts its effect quite successfully in the treatment of early and advanced HER2+ BC, a proportion of patients have intrinsic or acquired resistance to it. In general, there are four proposed mechanisms for resistance to trastuzumab ,62,63,64First, the interaction between receptor and antibody is blocked. Obstacles preventing trastuzumab binding to HER2 also explain the resistance to some extent. Several studies reported that MUC4, also a transmembrane protein, may mask the epitope on HER2. Hence, trastuzumab has difficulty to access and bind to HER2. Therefore, the efficacy of trastuzumab may be significantly reduced ,66.Second, the immune system fails to respond. As is mentioned above, ADCC may take a non-negligible role in the antitumor action of trastuzumab. If polymorphisms and other dysfunctions of Fc receptor make it fail to trigger an immune-mediated mechanism, this would reduce the ADCC response to trastuzumab, leading to resistance to trastuzumab .p < 0.0001) [Third, downstream signaling pathways are upregulated. Phosphatase and tensin homolog (PTEN) loss, presence of p95 (extracellular receptor cleavage forming truncated form of HER2 p95) ,68 and p 0.0001) . Presenc 0.0001) .Fourth, alternative signaling pathways are activated. Even though trastuzumab successfully recognizes and acts on HER2 preventing the signaling transduction pathways induced by HER2-containing dimers, alternative pathways unrelated with HER2 can be activated by other factors, such as insulin-like growth factor-I receptor (IGF-IR) .®, Genetech) is the second humanized monoclonal antibody for anti-HER2 therapy approved by the FDA in 2013. Like trastuzumab, it exerts its function by binding with an epitope on the extracellular domain of HER2 receptors, therefore preventing the formation of HER2-containing heterodimers. However, pertuzumab could form a complex with HER2 on the heterodimerization interface, which has different epitopes from trastuzumab [Pertuzumab  is one of them, showing great promise. It is an Fc-optimized chimeric monoclonal antibody with enhanced ADCC . CurrentMonoclonal antibodies exert their functions by binding to the extracellular domain of HER2 receptor, which prevents the dimerization of HER2 and other EGF receptors, while tyrosine kinase inhibitors (TKIs) bind to the intracellular tyrosine kinase domain of HER2 to prevent the phosphorylation of tyrosine kinase and thus inhibit the activation of downstream signaling pathways.®, GlaxoSmithKline) is the second anti-HER2 targeting agent, approved by the FDA in 2005. It is an orally active, reversible and dual small molecular inhibitor of HER2 and HER1 with prolonged inhibition of tyrosine phosphorylation in tumor cells [n = 138) who received treatment of lapatinib for 17.6 weeks demonstrated an overall response (OR) of 31%, and progression-free survival (PFS) rate of 63% at Month 4 and 43% at Month 6. The most common adverse events caused by lapatinib were diarrhea, rash, pruritus, and nausea. However, these events were primarily graded at mild level as 1 or 2 [Lapatinib  and stable disease was 37% (n = 37) [Afatinib (BIBW-2992) (Boehringer Ingelheim) is an orally active irreversible dual inhibitor of EGFR and HER2. Its chemical structure is presented in (n = 37) . Though n = 66) and the other was not pretreated (n = 70). The overall response rate (ORR) was 24% and 56%, respectively. Efficacy results were better in the non-pretreated group, with PFS rate 78% in the trastuzumab native group and 59% in the trastuzumab pretreated group. Moreover, the study also reported that a majority of the recruited patients showed reduction in their tumor sizes [Neratinib  is also an orally taken and active irreversible inhibitor of EGFR, HER2 and HER4 receptors. or sizes . TherefoAdo–trastuzumab emtansine  is an ADC which was approved by the FDA in 2013 . The strThe potential acting mechanism is shown in Recently, a study constructed a novel HER2 directed ADC of a biparatopic antibody linked with a tubulysin-based microtubule inhibitor. It was found that this novel ADC showed a greater efficacy for HER2+ BC than T-DM1. The reported biparatopic antibody can recognize and bind to two distinct epitopes on the HER2 receptor. This interaction could induce clustering of HER2 which could then facilitate the internalization, lysosomal trafficking and degradation of the clustered HER2. Through thoughtful experimental design, the study also indicated that a broader group of patients (eligible or ineligible and relapsed/refractory for T-DM1 treatment) could benefit from this biparatopic ADC .Heat shock protein 90 (HSP90) inhibitors are novel therapeutic approach for HER2+ BC. HSP90 is a molecular chaperone which is necessary for maintaining the stability and function of proteins. When HSP90 is inhibited by its corresponding inhibitors, its client protein will be unstable and eventually undergo degradation by proteinases. HER2 is one of the most sensitive client proteins of HSP90 . High leInhibitors of downstream signaling pathways are also a class of emerging agents against HER2+ BC. These inhibitors, which include mammalian target of rapamycin (mTOR) inhibitors and PI3K inhibitors, act directly on downstream signaling pathways to inhibit tumor cell proliferation and promote apoptosis ,14,68. PStrategies of combining HER2 targeted agents with each other and combining anti-HER2 drugs with chemotherapy are usually carried out for the treatment of HER2+ BC. Various therapeutic approaches have been developed . CombinaRecent studies reported that activating mutations of HER2 in patients without HER2 amplification might occur in 2%–4% of breast cancers . These HHER2 is one of the best characterized molecular biomarkers and an ideal target for developing therapeutic strategy for the treatment of HER2+ BC. Clinically, methods for evaluating HER2 status are generally utilized to determine the subtype of breast cancers, and then identify the appropriate molecular therapy for corresponding breast cancer. Based on the understanding of the HER2 biology and its role in breast cancer, a variety of HER2 targeted agents have been developed. Among those agents, monoclonal antibody trastuzumab as the prior choice for the treatment of HER2+ BC benefits patients significantly in clinical practice. However, intrinsic or acquired drug resistance to trastuzumab may be engendered in many patients with HER2+ BC. Therefore, clinically, combination therapies of trastuzumab and other anti-HER2 agents and combination therapies of anti-HER2 agents and chemotherapy are commonly conducted to optimize their therapeutic efficacy. However, these combination therapies still have some limitations, such as side effects. The economic costs of combination of HER2 targeted therapies may be prohibitive for patients. This means that only a minority of patients can gain benefits from the combination. The ultimate purpose is to be able to customize or personalize drug therapy with high efficacy and low toxicity. Hence, a great deal of effort is still needed to seek better therapeutic strategies to combat HER2+ BC.


The purpose of this study was to retrospectively review the pathologic complete response (pCR) rate from patients (n=86) with stage II and III HER2-positive breast cancer treated with neoadjuvant chemotherapy at our institution from 2008 to 2013 and to determine possible predictive and prognostic factors. Immunohistochemistry for hormone receptors and Ki-67 was carried out. Clinical and pathological features were analyzed as predictive factors of response to therapy. For survival analysis, we used Kaplan-Meier curves to estimate 5-year survival rates and the log-rank test to compare the curves. The addition of trastuzumab to neoadjuvant chemotherapy significantly improved pCR rate from 4.8 to 46.8%, regardless of the number of preoperative trastuzumab cycles (P=0.0012). Stage II patients achieved a higher response rate compared to stage III (P=0.03). The disease-free and overall survivals were not significantly different between the group of patients that received trastuzumab in the neoadjuvant setting  and the group that initiated it post-operatively . Axillary pCR post neoadjuvant chemotherapy with trastuzumab was associated with reduced risk of recurrence  and death . In conclusion, we confirmed that trastuzumab improves pCR rates and verified that this improvement occurs even with less than four cycles of the drug. Hormone receptors and Ki-67 expressions were not predictive of response in this subset of patients. Axillary pCR clearly denotes prognosis after neoadjuvant target therapy and should be considered to be a marker of resistance, providing an opportunity to investigate new strategies for HER2-positive treatment. Breast cancer is the most common malignancy among women all over the world, and it is considered a heterogeneous disease. Around 25% of all breast cancer overexpress the human epidermal growth factor receptor 2 (HER2), and is traditionally associated with worse prognosis . The actin vivo assessment of tumor response to chemotherapy  is usually defined as absence of residual invasive cancer in breast and lymph nodes in surgical specimens after neoadjuvant therapy. It is considered an important prognostic factor and is associated with long-term survival. pCR has been adopted as the primary end point for neoadjuvant trials and reaches up to 78% in patients treated with trastuzumab ,7. HowevIn this article, we reviewed the rate of pCR from patients with HER2-positive breast cancer treated with neoadjuvant chemotherapy at our institution and investigated possible clinical, histological and immunohistochemical predictive and prognostic factors. This knowledge would allow a better selection of patients that could benefit from therapy with trastuzumab. In addition, costs and adverse events could be minimized, leading to an individualized cancer treatment .This cohort study with retrospective data collection evaluated clinical, histological, and immunohistochemical characteristics of all patients with histologically confirmed HER2-positive, stage II and III breast cancer, treated with neoadjuvant chemotherapy between 2008 and 2013 at our institution. The Institutional Review Board of the Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, SP, Brazil, approved this study (#497.095).in situ hybridization (CISH).The HER2 status was assessed by immunohistochemistry (IHC) and tumors were considered HER2-positive when IHC staining was 3+  and 2+ , in which the HER2 gene amplification was confirmed by chromogenic 2 in one recipient paraffin block.We used microscopically selected representative samples from breast tissues stored in our Pathology department. A paraffin tissue microarray (TMA) block was built to minimize experimental variability and reduce costs. We used the Tissue Microarray Builder Kit  to arrange 24 cylinders of 2 mm).Immunohistochemical staining for estrogen receptor, progesterone receptor and Ki-67 was performed in the TMA sections using mouse monoclonal antibody at 1:200, 1:200 and 1:100 dilutions, respectively , and used the software ImageScope (USA) for cell count. A 14% cut-off was used to define a high expression of Ki-67. At least 500 contiguous tumor cells were counted to establish the index.In order to confirm the amplification of the HER2 gene we performed a dual color chromogenic (CISH) assay using the Kit CISH™ HER2 SPOT-Light¯, Zymed  in the TMA sections. The interpretation followed the College of American Pathologists guidelines. The amplification was confirmed in all 41 tumors tested.Patients were treated with neoadjuvant chemotherapy with anthracyclines and/or taxanes, for at least 4 cycles, according to the current protocol. The 86 patients included in the study were divided into two groups: 65 patients received neoadjuvant chemotherapy with trastuzumab followed by adjuvant trastuzumab while 21 patients received neoadjuvant chemotherapy alone followed by adjuvant trastuzumab.2 epirubicin + 500–600 mg/m2 cyclophosphamide followed by 4 cycles of 100 mg/m2 docetaxel every 21 days +/- trastuzumab; 2) 4 to 6 cycles of 80–100 mg/m2 docetaxel + trastuzumab every 21 days; 3) 6 cycles of 500 mg/m2 fluorouracil + 75 mg/m2 epirubicin + 500 mg/m2 cyclophosphamide every 21 days; 4) 4 cycles of 75 mg/m2 docetaxel + 60 mg/m2 epirubicin every 21 days. All patients completed 1 year of treatment with adjuvant trastuzumab, except those that developed severe toxicity.The chemotherapy regimens used were: 1) 4 cycles of 75 mg/mDisease-free survival was considered as the length of time between the surgery and the first recurrence event . Overall survival was considered as the length of time between the cancer diagnosis and death. Patients that were lost to follow-up or did not reach any event (recurrence or death) were censored in the analysis.Statistical analyses were done with GraphPad Prism 6 software (USA). To analyze the categorical variables, we used Pearson chi-square test or Fisher's exact test. For survival analysis, we used Kaplan-Meier curves to estimate 5-year survival rates and the log-rank test to compare the curves. Statistical significance was assumed at a 0.05 level (P<0.05).All of the 86 patients were treated with adjuvant trastuzumab, 66 completed 1 year of adjuvant treatment, 8 developed cardiac toxicity severe enough to interrupt the drug (9.5%), while 11 were still under treatment with trastuzumab at the time of the study. One patient from the first group died during the neoadjuvant treatment from febrile neutropenia and, therefore, did not receive surgical or adjuvant treatment.The addition of trastuzumab to the neoadjuvant chemotherapy significantly improved the pCR rate. Of the 64 patients that received at least one cycle of trastuzumab in the neoadjuvant setting, 30 achieved pCR (46.8%), while in the group of 21 patients treated with neoadjuvant chemotherapy without trastuzumab only 1 achieved pCR (4.8%).In a subgroup analysis, we evaluated if the number of trastuzumab cycles received preoperatively influenced the pCR rate. Of the 65 patients treated with neoadjuvant chemotherapy with trastuzumab, 46 received at least 4 cycles of trastuzumab (T group) and 19 received only 1 to 3 cycles of the drug together with neoadjuvant chemotherapy (Chemo/T group). As can be seen in =0.1). In the group of patients that achieved pCR, 35% underwent breast-conserving surgery, while in the group with residual tumor in the breast this rate was 39% (P=0.8).The rates of breast-conserving surgery were not significantly influenced by the addition of trastuzumab nor was pCR. Of patients treated with neoadjuvant chemotherapy with trastuzumab, 33% had conservative surgery, compared with 52% from the chemotherapy alone .The disease-free and overall survival  were notWe investigated clinical, histological and immunohistochemical predictive factors for response to neoadjuvant chemotherapy with trastuzumab in the 65 patients treated with at least one cycle of the drug preoperatively . The onlWe evaluated possible prognostic factors in our population of HER2-positive breast cancer patients treated with neoadjuvant chemotherapy associated with trastuzumab. pCR is considered an important prognostic factor and is associated with better disease-free and overall survival . pCR no. The 5-yOur findings confirmed that the addition of trastuzumab to neoadjuvant chemotherapy significantly improves pCR rates. This is consistent with other trials that demonstrated that 1 year of trastuzumab starting as neoadjuvant almost doubled pCR rates . The ideIn a meta-analysis published in 2011, even though the probability to achieve pCR was higher in the trastuzumab plus chemotherapy group , breast-conserving surgery rates were similar to the group that used chemotherapy alone . Our stuThe 3 major trials that evaluated neoadjuvant treatment with trastuzumab had a limitation in the survival analyses, because they compared it with patients that did not receive adjuvant trastuzumab and patients with HER2 negative tumors ,14,15. OEven though trastuzumab presented a great improvement in the treatment of HER2-positive breast cancer, it is associated with relevant adverse cardiac events and with significantly elevated cost of treatment ,18,19. IOur findings reinforce the importance of early diagnoses in some aspects. Only 22 of the 86 patients were at stage II disease, but they had significantly greater chance of achieving pCR and having conservative surgery when compared to the patients at stage III. The relevance of the TNM staging has been questioned in the last few years . A recenWe evaluated possible prognostic factors in our population of HER2-positive breast cancer patients treated with neoadjuvant chemotherapy associated with trastuzumab. Several studies have demonstrated a relation between the number of positive nodes in the axilla after neoadjuvant treatment and survival –25. Ou


NeoSphere showed significantly higher pathologic complete response (pCR) with neoadjuvant pertuzumab, trastuzumab, and docetaxel compared with trastuzumab plus docetaxel, pertuzumab plus trastuzumab, or pertuzumab plus docetaxel. We assessed associations between human epidermal growth factor receptor 2 (HER2) pathway-related biomarkers and clinical outcome in response to these regimens.Tumor, serum, and whole blood samples were collected at baseline and post neoadjuvant treatment before surgery. Associations between biomarkers and pCR, and between biomarkers and clinical variables were assessed in the overall and estrogen receptor (ER)-positive and ER-negative populations. Changes in serum marker levels between baseline and post-neoadjuvant treatment were examined.P = 0.001) and a significant treatment interaction (P = 0.0236) with pertuzumab, trastuzumab, and docetaxel . Low serum transforming growth factor alpha (TGFα) was associated with higher pCR rates with pertuzumab plus trastuzumab (P = 0.04) without a significant treatment interaction. Presence of truncated HER2 did not affect pCR. A non-significant decreased pCR benefit was observed consistently across groups in patients with mutated PIK3CA while the treatment benefit from pertuzumab was maintained when comparing the trastuzumab plus docetaxel and pertuzumab, trastuzumab, and docetaxel groups. Notably, PIK3CA exon 9 mutations were associated with residual disease (pooled groups), which was not found for exon 20 mutations. Serum HER2 extracellular domain levels were significantly increased between baseline and post-neoadjuvant treatment in the non-trastuzumab-treated group, and decreased in the trastuzumab-containing groups (likely due to trastuzumab’s mechanism of action). Differences in biomarker profiles according to ER status were observed.No markers were associated with pCR across all groups; however, significant associations were observed for two markers in individual groups. High HER2 was significantly associated with higher pCR rates  contains supplementary material, which is available to authorized users. Hoffmann-La Roche Ltd., Basel, Switzerland) is directed at the dimerization domain of HER2 and inhibits dimerization with other HER receptors while stimulating antibody-dependent cell-mediated cytotoxicity (ADCC) [®, F. Hoffmann-La Roche Ltd.) binds to the transmembrane domain to inhibit mitogenic signaling, block HER2 cleavage, and stimulate ADCC [Human epidermal growth factor receptor 2 (HER2) is the clinically validated biomarker for HER2-targeted therapies, several of which are approved in the neoadjuvant, adjuvant, and metastatic settings for HER2-positive breast cancer. Pertuzumab (PERJETAy (ADCC) –3. Trastate ADCC –6. Due tate ADCC .®, Sanofi-Aventis, Paris, France) (group B) had a significantly higher pathologic complete response (pCR) rate compared with those treated with trastuzumab plus docetaxel (group A), pertuzumab plus trastuzumab (group C), or pertuzumab plus docetaxel (group D)  [In the NeoSphere study, patients treated with neoadjuvant pertuzumab, trastuzumab, and docetaxel (Taxoterectively) . The comHER2-positive/estrogen receptor (ER)-positive and HER2-positive/ER-negative breast cancer are known to have different patterns of gene expression, and treatment outcome seems to be driven by different biologic pathways , 11–14. HER2, with regards to pCR, as p95 was previously reported to predict worse outcomes in patients with HER2-positive breast cancer [We assessed a panel of biomarkers, in tumor specimens and in sera, to characterize the molecular profile of the biomarker population of NeoSphere, and to explore biomarkers correlated with different treatments and/or different subsets of patients in this trial. The objective of this exploratory analysis was to identify those biomarkers (or combinations of biomarkers) with the best association (positive or negative) with pCR in response to the treatment regimens used in the NeoSphere study. In particular, we attempted to identify biomarkers that would predict a benefit from the addition of pertuzumab to a trastuzumab-based regimen. The markers tested included those that are involved in downstream signaling of HER2, belong to a group of related receptor tyrosine kinases that could serve as salvage routes for an inhibited HER2 pathway, or are ligands of HER family proteins that induce activation of the HER2 pathway. We also assessed the presence of truncated forms of HER2, including p95t cancer .in situ lesions were allowed [The study design and patient characteristics have been reported previously . Briefly allowed .Collection of core biopsies, sera, and whole blood from all patients was mandatory at baseline. Tumor samples were obtained as formalin-fixed, paraffin-embedded tissue. Tissue obtained after the neoadjuvant treatment period was derived from resection specimens.in situ hybridization (FISH), RNA extraction, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and DNA isolation were performed centrally by Targos Molecular Pathology GmbH, Kassel, Germany. Targos followed the protocols and processing instructions developed by Roche Diagnostics GmbH, Penzberg, Germany. Commercially available assays or kits were used where specified, and performed according to the manufacturer’s instructions. All other assays were developed by Roche Diagnostics for exploratory research purposes only.Tissue processing, immunohistochemistry (IHC), fluorescence Expression of HER2 , HER3 , insulin-like growth factor 1 receptor , phosphatase and tensin homolog , and pAKT  were assessed by IHC, and a modified H-score  was deriTherefore the modified H-score has a maximum value of 400 instead of the standard H-score of 300. The percentage of cells stained with an intensity of 0 was used only for quality control. Cases with no staining on the tissue section were assigned a score of 0. Modified H-scores were calculated for subcellular compartments for which specific staining was identified and for which there was a biologic rationale for the subcellular location of the respective marker (e.g. for PTEN and AKT nuclear staining).Image acquisition and analysis of HER2 staining intensity took place at the Royal Marsden Hospital  using the Ariol image analysis system (Leica Microsystems (Gateshead) Ltd., UK) equipped with a BX61 microscope  and a black and white MegaPlus ES 4.0/E camera . Slides were scanned and analyzed as previously described , except HER1, HER2, HER3, Amphiregulin (AREG) and Betacellulin mRNA levels in tumor tissue were assessed relative to the G6PD gene by qRT-PCR . c-Myc amplification was assessed by FISH .PIK3CA) on tumor DNA were performed using TaqMan-PCR  at Roche Translational Research Sciences . This led to a technical success rate of approximately 60%. To increase the sample size, a different method requiring less DNA input was applied at Genentech Laboratories, San Francisco, CA, USA. DNA was extracted from IHC-stained slides, amplified, and subsequently assessed using the DxS assay , which detects mutations E542K, E545K/D, and H1047R in exons 9 and 20. Mutational analysis per exon is presented to allow for pooling of the data on 328 cases overall. PIK3CA subgroups were defined based on the presence or absence of any mutation (mutant versus wild-type). “Wild-type” was only assigned to samples in which all reactions gave a valid result for “no mutation.” If one or more reactions failed, that sample was classified as “not assessable.”Mutational analyses of eight mutations at four hot spots in exons 7, 9, and 20 within the gene encoding the catalytic subunit of phosphoinositide 3-kinase (Enzyme-linked immunosorbent assay (ELISA) was used to detect AREG, EGF, serum HER2 extracellular domain (sHER2), and transforming growth factor alpha (TGFα) in serum using an immunologic multiparametric chip technique (Roche Diagnostics). Levels of each serum marker were compared at baseline and at surgery in matched pairs from the same patients. Truncated forms of HER2 (the intracellular domain (ICD) and HER2 extracellular domain (ECD), where a ratio <1 indicates the presence of truncated forms), were measured in formalin-fixed, paraffin-embedded breast cancer tissue as described by Krüger et al. .PIK3CA mutation and c-Myc amplification, where cutoffs were applied following a biologic rationale (wild-type versus mutant PIK3CA and c-Myc:centromere ratio ≥2). Instead of a data-driven cutoff, we used a prespecified median cutoff to reduce the number of false-positive findings due to the concomitant multiple testing and optimal cutoff selection. Box and whisker plots and summary statistics were used for initial data exploration and selection of relevant biomarkers. Spearman’s correlation was performed to identify the correlated biomarkers. Biomarkers with measurements near the limit of quantification were excluded from analyses.The patient population used (the biomarker population) was a subset of the intent-to-treat (ITT) population that contributed meaningful data to the analysis  and biomarkers were assessed in the overall biomarker population using the Chi-square test.The association between biomarkers and pCR by treatment group were assessed by the Cochran–Mantel–Haenszel Chi-square test, stratified by hormone receptor status and breast cancer type. The treatment interaction test was performed for groups A and B to analyze the interaction between biomarkers and treatment in the association with pCR.Analyses of the association between biomarkers and clinical variables, and of the association between biomarkers and pCR by treatment group, were repeated in ER-positive and ER-negative subgroups. These analyses were exploratory and secondary in nature, and therefore were not taken into account when adjusting for multiple comparisons.AREG and Betacellulin measured by qRT-PCR were generally very low (less than the limits of variability for these assays); hence, detailed interpretation of the data was not considered meaningful. The baseline levels of all biomarkers were well-balanced across all four treatment groups   while the remaining biomarkers showed no significant difference when adjusted for ER status and breast cancer type  Fig. . These f02) Fig. . In grouIn an exploratory analysis performed on truncated forms of HER2 without using high/low cutoffs, there was no association observed between pCR and these truncated proteins via the HER2 ECD/ICD ratio Fig. .Fig. 3HuP = 0.0236) with a large benefit from the combination of pertuzumab, trastuzumab, and docetaxel in the group with expression of HER2 above the median (odds ratio (OR) 2.07 ) and no significant benefit for low HER2 expression. However, clustering of H-scores within a small dynamic range .A comparison of baseline levels of the serum markers AREG, EGF, TGFα, and sHER2 between patients achieving pCR and those with residual disease did not predict pCR , EGFR mRNA expression, sHER2, HER2 mRNA expression, and membrane HER2 protein expression were higher in ER-negative samples  with ER-negative tumors (P = 0.05) but not ER-positive tumors (P = 0.21). Detailed analyses of biomarker levels by ER status are available online  but not the ER-positive subgroup (P = 0.54)  versus group A (trastuzumab plus docetaxel) in the ER-negative (54) Fig. .Fig. 6InEGFR mRNA, HER2 mRNA, and HER2/HER3 mRNA were associated with ER-negative disease, while higher levels of HER3 protein, IGF1R protein, serum AREG, AREG mRNA, and HER3 mRNA were associated with ER-positive disease. Other clinical variables were associated with higher levels of certain biomarkers; however, no clear biologic explanations were evident and multiplicity of testing may have introduced bias.Higher levels of HER2 protein, cytoplasmic PTEN, sHER2, c-Myc) of cases. While no new single marker could be confirmed as predictive of treatment benefit, these analyses revealed several important observations that will impact future trial designs.NeoSphere demonstrated that mandatory biomarker sampling is feasible in a clinical trial setting. Samples were available from nearly all patients and the planned panel of biomarkers could be assayed reliably in 99.8% (HER2) to 65.9%  using both the modified H-score and digital image analyses. The digital readout resulted in a wider dynamic range compared with the manual scoring, showing a more pronounced effect in predicting pCR. While digital image analyses allowed us to better discern the pCR and non-pCR subgroups, HER2 levels remained highly overlapping between patients experiencing pCR versus patients not experiencing pCR and did not allow us to define clearly distinct populations that were either clinically or biologically meaningful.PIK3CA mutational status appears to have a relevant role in defining the likelihood of pCR with all treatment regimens tested in NeoSphere. However, while a decreased benefit was observed, the magnitude of the treatment effect with the addition of pertuzumab was maintained . This was similar to associations reported in the CLEOPATRA trial [PIK3CA mutant tumors, regardless of treatment regimen [PIK3CA mutations in exon 9 were linked to a lack of sensitivity to HER2-directed monoclonal antibodies. Although independent confirmation is needed, due to the lower frequency and lack of statistical power for exon 9 mutations, this finding might be explained by the different mechanisms and downstream effects that are proposed based on preclinical observations for the gain of function mutations in helical (exon 9) versus kinase (exon 20) domain [The RA trial . CLEOPAT regimen . Interes) domain .PIK3CA mutations at exons 9 and 20, although the benefit from pertuzumab was maintained regardless of mutation status [PIK3CA mutant tumors regardless of treatment regimen [PIK3CA mutations had worse outcomes compared with those with wild-type tumors when treated with lapatinib plus capecitabine [PIK3CA mutation on outcome, either when given as monotherapy or in combination with other therapeutics such as PI3K inhibitors, with the goal of improving the poor prognosis of patients carrying PIK3CA mutations. The data from NeoSphere suggest that such an approach could be useful only in tumors carrying the activating exon 9 mutation.In CLEOPATRA, there was an association between poorer prognosis and pooled n status . Similar regimen . In addicitabine . This waOur results also provide clinical support for the blockade of HER2 cleavage by trastuzumab . TrastuzAnother relevant aspect that emerged from our analyses is the distinct difference between ER-positive and ER-negative tumors . HER2-poA limitation of the biomarker analysis was that there was a narrow range of HER2 protein levels with little numerical difference between cutoffs, hindering an appropriate exploration of HER2 protein-related impact on treatment outcome. In addition, there was no significant difference in the pCR rates of patients in the subgroups with higher and lower HER2 levels, who received pertuzumab plus trastuzumab without docetaxel (group C). As the chemotherapy backbone is not known to impact HER2-related effects, the association between HER2 protein levels and pCR rates should have been detectable in both of the groups receiving trastuzumab plus pertuzumab.PIK3CA mutation at exon 9 is limited by the small sample size and warrants further exploration in larger datasets.Given that we were limited by small numbers of patients in some subpopulations, our analyses did not provide any additional predictive markers supporting a refinement of the HER2-positive target population treated with trastuzumab, pertuzumab, and docetaxel. It should be acknowledged that the use of the median cutoff for biomarker assessment could have overlooked a predictive value of some of these biomarkers at different, biologically meaningful, cutoffs that have not been explored in this study. However, our analysis did highlight the different biology of ER-positive and ER-negative HER2-positive tumors. The observation of a lower likelihood of pCR with trastuzumab, pertuzumab, and docetaxel in tumors harboring In conclusion, our data show that conventional assessment of HER2 by IHC or FISH should continue at this time, and that HER2 remains the only biomarker suitable for patient and/or regimen selection in this population. The observations in NeoSphere, however, may guide future neoadjuvant trial designs.


HER2 is overexpressed in 20% of invasive breast cancers (BCs) and correlates with a more aggressive disease. Until the advent of targeted agents, HER2 was associated with worse outcomes. Rationally designed HER2-targeted agents have been developed and introduced into clinical practice for women with HER2-amplified BC, improving disease-free and overall survival for primary and metastatic tumors. Trastuzumab, a recombinant humanized anti-HER2 monoclonal antibody, combined with chemotherapy, remains the standard of care for patients with HER2-positive BCs. However, many patients do not respond to this agent, whereas newer drugs have proven to be efficacious in clinical trials. The identification of biomarkers that select sensitive tumors and patients who will benefit from these new agents would help the incorporation of these therapies, limiting the risk of side effects and overtreatment and improving the outcomes of all patients with early-stage HER2-positive BC. We review the mechanisms of action of HER2-targeting agents, focusing on the involvement of the immune system and related predictive biomarkers. The tyrosine kinase HER2, with the other members of the HER (human EGF receptor) family of receptor tyrosine kinases , controls many signaling pathways in various cellular functions, including proliferation, migration, survival, DNA repair, and angiogenesis  of HER2 and, with or without chemotherapy, is the basis for systemic treatment of metastatic and early HER2-positive BC . AlthougDespite the therapeutic options that are available, the clinical benefit of trastuzumab and its combination with other HER2-targeted therapies that have complementary mechanisms of action—the dual blockade approach—remain modest, with many patients who do not improve survival with these agents. Many studies have been performed on the mechanisms of trastuzumab action and the efficacy and resistance of second-generation HER2-targeting compounds to identify patients in whom the therapeutic effects of these tailored therapies can be optimized.We review mechanisms of action of these drugs, focusing on the involvement of the immune system and the related biomarkers that have potential value in selecting patients for the most appropriate treatment option in neoadjuvant-adjuvant settings.The treatment of HER2-positive BC with MAbs and TKIs aims to impede uncontrolled tumor growth and invasiveness by blocking the intracellular signals that are derived from HER2. In addition to inhibiting oncogenic stimuli, the efficacy of HER2-targeting agents in BC is also based on their ability to engage antitumor immunity .Trastuzumab  uses several mechanisms to block HER2 signaling  Figure , underlyMoreover, trastuzumab inhibits the proteolytic cleavage of HER2, preventing shedding of the extracellular domain (ECD) and the generation of phosphorylated truncated p95-HER2, which has been implicated in tumor growth and progression ; this acγR) . The efficacy of trastuzumab is lost in FcγR-deficient mice and on inhibition of FcγR engagement in preclinical models [γR reduces the efficacy of trastuzumab in BC patients [γRIIIA (CD16) and are the major effectors of ADCC. Consistently, it has been reported that patient response to trastuzumab monotherapy is associated with robust tumor infiltration of lymphoid cells [in vitro antibody-dependent cellular cytotoxicity [γ (IFNγ) [One of the most significant mechanisms of action of trastuzumab is the antibody-dependent cellular-mediated cytotoxicity (ADCC) . Trastuzl models . Similarpatients , 17. NK id cells —primarilid cells —and a grtoxicity , 21. We toxicity —NKG2D exγ (IFNγ) .γR on macrophages (Another recent study reported that trastuzumab induces antibody-dependent cellular phagocytosis- (ADCP-) mediated cell death through recognition of trastuzumab-opsonized cells by Fcrophages . Anti-HE−/− mice, which lack T and B lymphocytes [Other studies that have aimed to determine the function of the immune system in trastuzumab activity revealed a critical role of adaptive immune cells –27. Antiphocytes , and thephocytes , 26. CD8phocytes , 26. Accγ and IFNAR1 (type I IFN receptor) [γ-producing CD8+ T cells. In addition, trastuzumab, through its Fc region, increases HER2 uptake by dendritic cells, facilitating cross-presentation of HER2 peptides and activation of antigen-specific T cells [γ [Stagg et al. demonstrated that trastuzumab depends in part on the antitumor effects of IFNs using neutralizing antibodies to IFNeceptor) . They pr T cells . In supp T cells . These ccells [γ . The samcells [γ .γ, leading to their recognition and death [γ and tumor necrosis factor α (TNFα), contribute to the induction of a cytotoxic antitumor response that cooperates with trastuzumab to upregulate MHC-I on HER2-positive BC cells, facilitating their recognition and lysis by CD8+ lymphocytes [γ enzyme-linked immunosorbent spot analysis (ELISPOT) than non-pCR patients after treatment [Mortenson et al. demonstrated that also CD4+ T cell participates in anti-HER2 therapy: CD4 depleting antibodies reduce antitumor activity of anti-HER2 MAbs that exert their effect also through a CD4-dependent antitumor-specific response . Upon trnd death . CD4+ T-phocytes . Accordireatment . Moreovereatment . Signalireatment . Per thereatment .Although trastuzumab significantly increases anti-HER2 humoral responses (against the extracellular and intracellular domains) primarily in metastatic patients with objective responses , 36, prein vitro [The monoclonal antibody pertuzumab  binds to the extracellular domain of HER2 and prevents the ligand-mediated dimerization of HER2 with HER1 and HER3 by steric hindrance  Figure . In partin vitro .Based on its synergistic effects when combined with trastuzumab versus alone as a monotherapy , 41, mosADCC is one of the most important mechanisms of action of trastuzumab and pertuzumab, as evidenced by the rise in NK cells that infiltrate and penetrate deeper into the tumor burden of mice that harbor trastuzumab-resistant JIMT-1 cells and have been treated with trastuzumab and pertuzumab compared with mice that have been given each agent individually . Based oMany tyrosine kinase inhibitors (TKIs) have activity in trastuzumab-resistant BC and can be used as alternatives to block HER2 signaling. Among such emerging HER TKIs , lapatinin vitro and in vivo by inhibiting HSF1 (heat shock transcription factor 1) and its target, HSP90 [γH2AX foci [Lapatinib downregulates phospho-HER2 (p-HER2), p-EGFR, and p-ERK and promotes mutant p53 degradation t, HSP90 . In addi2AX foci . The ant2AX foci . In conj2AX foci .γ-secreting CD4+ and CD8+ T cells in a Stat1-dependent manner [γ-secreting CD8+ cells.By blocking the tyrosine kinase domain of HER2, lapatinib elicits the accumulation of HER2 on the cell membrane  in the Bt manner . In contin vitro and in cells with innate and acquired trastuzumab resistance [Neratinib  is an oral pan-HER inhibitor that bonds covalently with a conserved cysteine residue (Cys-773) in the kinase domain of HER, leading to its irreversible inhibition and thus blocking the pathways that lie downstream of EGFR, HER2, and HER4 . Its spesistance , 57. Liksistance , 57. Nersistance .Trastuzumab was approved for metastatic HER2-positive BCs in 1998 by the FDA after a phase III trial demonstrated that its addition to standard chemotherapy extended the time to progression from 4.6 to 7.4 months and reduced the relative risk of death by 20% .p value < 0.001) with no additional toxicity [p value < 0.001) in HER2-positive patients who received trastuzumab versus those who did not [Neoadjuvant treatment with trastuzumab and chemotherapy was introduced into clinical practice, based on results of 3 phase III trials that recorded higher pathologic complete response (pCR) rates in the trastuzumab arm , comparetoxicity . Moreove did not . Also, i did not .n = 13493 women) showed that survival in trastuzumab-treated patients was superior in terms of DFS (risk ratio (RR): 0.62; 95% CI: 0.56–0.68) and mortality  [In the adjuvant setting, based on the results of 4 large trials  .In analyses of “real-world” treatment, patients who were given trastuzumab for early-stage HER2-positive BC had 5-year DFS and OS rates that were comparable with those in randomized trials. Of 476 patients in the Netherlands, those who were treated with trastuzumab had a superior DFS  than subjects who underwent chemotherapy alone . A retrop < 0.0001) [Pertuzumab was approved by the FDA in 2012 for use in combination with trastuzumab and docetaxel for patients with HER2-positive MBC who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease, based on a multicenter, randomized, double-blind, placebo-controlled trial (CLEOPATRA) in which its addition to trastuzumab and docetaxel improved progression-free survival  . In 2013 0.0001) , whereas 0.0001)  (Table 1p = 0.045), despite the 3-year DFS rising modestly from 93.2% to 94.1% [Positive results were recently published for the phase III APHINITY trial, comparing the activity of adjuvant pertuzumab, trastuzumab, and chemotherapy versus trastuzumab and chemotherapy. The study met its primary endpoint and showed that the dual blockade approach effected a statistically significant reduction in the risk of recurrence of invasive disease or death compared with trastuzumab and chemotherapy alone , based on a phase III study that compared lapatinib/capecitabine with capecitabine alone in patients with MBC who progressed after chemotherapy/trastuzumab, in which TTP improved from 4.4 to 8.4 months . Lapatinp < 0.001) (779 patients) [p = 0.003) [Dual blockade of HER2 with trastuzumab and lapatinib has been examined in the neoadjuvant treatment setting in 4 randomized studies , compariatients) . Althoug= 0.003) .p = 0.0480) with concomitant dual blockade compared with trastuzumab alone was not statistically significant  [Dual blockade in the adjuvant setting with trastuzumab and lapatinib in combination with taxanes was tested in 8381 women in the phase III ALTTO trial, but the 16% improvement in DFS  .Neratinib has been investigated in all settings (reviewed in ). In thep = 0.0091) [p = 0.0013) than HR-negative patients .In the adjuvant setting, neratinib has recently been approved by the FDA for extended treatment of early-stage HER2-positive BC , based o 0.0091) . NotablyAll current data (reviewed in ) suggestGiven the availability of effective agents, biomarkers that differentiate patients who actually need new adjuvant therapies must be identified. Several efforts have been made in the last decade to discover biomarkers that predict who might benefit from trastuzumab, but most have failed to be consistently validated in tumor samples from randomized clinical trials (reviewed in ).New high-throughput genomic technologies have increased the rate of discovery of potential markers with prognostic or predictive value. These technologies demonstrated the intrinsic molecular heterogeneity in clinically HER2-positive BCs. The PAM50 classifier identified all of the intrinsic subtypes in HER2-amplified BCs, 50% of which are classified HER2-enriched (HER2-E) . These tERBB2 and ESR1 mRNA levels influence the degree of benefit that is received from adjuvant trastuzumab [ERBB2 and ESR1, is predictive of early relapse in adjuvant setting [ESR1 and ERBB2 levels, as determined by mRNAseq, when considered as continuous variables, were individually linked to pCR, and their incorporation into an exploratory multivariate model removed intrinsic subtype, HER2 amplicon signature, and clinical assays for ER or HER2 from the model in the CALGB 40601 trial [ERBB2 and ESR1 were confirmed in the NeoALTTO trial across arms [ESR1 and ERBB2 RNA can be implemented in the clinical setting, but their predictive ability in HER2-positive BC supports the superiority of evaluating their mRNA levels over standard IHC tests and suggests that they better mirror activity of HER2 and tumor-addiction to its downstream signals, as PAM50 did.A retrospective analysis of the NSABP B-31 study indicated that stuzumab . Similar setting . In the 01 trial . The levoss arms  as the moss arms  and in toss arms   , single-Although much work remains in the effort to refine and optimize biomarkers predictive of trastuzumab/HER2 double-blockade benefits and their application in patients, the reported robustness of immune infiltration/TIL evaluation  and the The implementation of immune markers in everyday clinical practice requires robust assessment of their clinical utility and of the test analytical and clinical validity, other than the understanding of their added predictive value, if any, on tumor intrinsic characteristics  and the identification of the best performing immune-related marker. Indeed, TIL evaluation on H&E slides is an easy method to provide raw information on the complexity of the tumor immune microenvironment, but it does not give information on the composition and functional status of the immune infiltrate that can be obtained using immune gene signatures, as evaluated by mRNA profiling. Several efforts have been made to standardize TIL assessment, and guidelines have been published and continuously improved , 113. ThIn an attempt to understand the clinical utility of immune markers in predicting response to trastuzumab in CHERLOB study, TILs failed to provide an independent prediction of pCR beyond PAM50 and were outperformed by immune-related gene signatures . These dSeveral key questions remain regarding the immune-biology of HER2-positive tumors that could help in developing new strategies to modulate immune response and improve anti-HER2 therapy efficacy; what drives immune infiltration in tumors is an ongoing area of research. It has been hypothesized that immune infiltrate is dictated by high mutational burden corresponding to a greater amount of neoantigens. Although cancer neoantigens are required for mounting an anticancer immune response, recent evidence showed that mutational burden does not correlate with the presence or absence of CD8+ T cells in the tumor microenvironment of melanoma and with T cell signature in any cancer type . AccordiIndependently from TIL recruitment mechanisms, another important issue in the prospect of augmenting trastuzumab activity by strategies of immune modulation regards the activation status of such infiltrating immune cells. It is possible that the presence of TILs in the tumor burden mirrors an exhausted immune response, the presence of intratumoral immune suppression or a near-equilibrium immune state with immune surveillance able to only partially control the tumor growth of immunogenic subclones slowing the genomic diversification of the cancer . In suppEfficacy of drugs in those patients with immune-enriched tumors suggests that chemotherapy and HER2-targeting agents may relieve the preexisting immune suppression and/or tilt the balance in favor of immune surveillance . In thisTo activate antitumor immunity, recent preclinical data suggested that one strategy could be the blocking of PD-1/PD-L1 interaction, since it synergizes with anti-HER2 therapy . Accordi


Hormone receptor status has major implications for treatment and survival of breast cancer. Yet the impact of hormone receptor status on outcome after Trastuzumab has received little attention. The objective here was to explore any differential effects of Trastuzumab treatment (Trast +ve) on Luminal B HER2 or HER2+(ER−) breast cancer subtypes.A cohort of 469 HER2 receptor-positive breast cancers was categorised by molecular subtype and Trastuzumab treatment. Effects of Trastuzumab treatment on survival, locoregional recurrence and distant metastasis were investigated by subtype, using univariate and multivariate analysis.p < 0.001) and OS (p < 0.001), while Trast +ve HER2+(ER−) patients had significant improvements in 5-year DFS (p = 0.012) alone. Only Trast +ve Luminal B HER2 cancers displayed a significant reduction in LRR rates (p < 0.001). A significant reduction in distant metastasis rates was seen in Trast +ve Luminal B HER2 (p < 0.001) and HER2+(ER−) (p = 0.009) cancers. Interestingly, bone metastasis rates in Trast +ve Luminal B HER2 cancers demonstrated the greatest reduction (36.2–6.7%). Multivariate analysis of Trast +ve patients found no difference in distant metastasis rates (p = 0.96) between subtypes. Significantly, lower LRR rates were seen in Trast +ve Luminal B HER2 cancers, compared to Trast +ve HER2+(ER−) (p = 0.018).Trast +ve Luminal B HER2 patients had significant improvements in 5-year DFS  contains supplementary material, which is available to authorized users. Advances in molecular profiling have allowed breast cancer to be categorised into clinically relevant molecular subtypes. –3. In apTrastuzumab is a monoclonal antibody that binds to the HER2 receptor and interferes with the HER2-mediated signalling cascade, preventing proliferation and eventually leading to cell death . TrastuzWhile improvements in survival in HER2 receptor-positive cancers have been demonstrated in multiple studies, few studies have examined if Trastuzumab treatment has a varied response between Luminal B HER2 and HER2+(ER−) breast cancers. The aim of this study was to assess the impact of Trastuzumab therapy on the survival or outcome on the two HER2 receptor-positive breast cancer subtypes, before and after the introduction of Trastuzumab as part of the adjuvant treatment regime.This study group consists of all patients with HER2 receptor-positive breast cancers treated at a tertiary referral unit entered into a prospectively maintained database from 1991 to 2014. Only patients with a definitive HER2 receptor-positive subtype were included. All clinical pathological details and treatment regimes were analysed. Hormone receptor-positive patients received hormone therapy, as per standard clinical treatment protocols at the time of diagnosis. Clinically, testing for HER2 receptor status began at our centre in 1999. In order to find HER2 receptor-positive patients not treated with Trastuzumab, we used a cohort of patients identified using retrospective testing of pathology samples. A cohort of HER2 receptor-positive patients who received no Trastuzumab treatment was provided by retrospective testing of HER2 receptor status on patients included on a prospectively collected tissue microarray (samples from 1994 to 2001). Historically in our program, Trastuzumab therapy was introduced as adjuvant therapy in 2006, prior to this it was available only to patients recruited on clinical trials. Patients were categorised as received adjuvant/neo-adjuvant Trastuzumab (Trast +ve) or no Trastuzumab treatment (Trast −ve).Breast cancer subtypes were defined using ER, PR and HER2 receptor status. Luminal B HER2 was defined as  and HER2+(ER−) as  according to standard clinical pathological guidelines. The ER and PR receptor status were determined independently by clinical pathologists using immunohistochemistry as per ASCO guidelines (ALLRED score >2 or more than 1% stain positive). The HER2 receptor status was identified by Herceptest, as part of the routine clinical evaluation, with a score of 3+ considered positive. Any +2 inconclusive results were confirmed using a FISH testing as per ASCO guidelines, with a HER2/CEP17 ratio greater than two considered amplified.Overall survival (OS), disease free survival (DFS), and patterns of recurrence were determined. The 5-year DFS & OS were determined and only patients who had completed 5 years of follow-up were included in the analysis.Breast cancer recurrence was defined as a return of cancer after treatment and after a disease free period. Only stage I–III breast cancers were included for this section of the analysis. Recurrence was divided into LRR and distant metastasis. LRR is defined as recurrence at the same site of the primary cancer or the regional lymph nodes, while distant metastasis is recurrence at a distant site from the primary cancer.p value of less than 0.05 was considered statistically significant. The Kaplan–Meier method was used to determine survival distributions. The log rank was used to determine any statistically significant differences in survival between the indicated groups. Cox regression was used for multivariate analysis, with logistic regression used to analyse categorical data.Statistical analysis was performed using R statistical software version 3.2.3. A This study was conducted in accordance with the granted National University of Ireland Galway and University College Hospital Galway ethical approval. Informed consent was obtained from all patients. All patients had histologically confirmed breast cancer and all relevant clinicopathological and demographic data were obtained from a prospective breast cancer database.The study consisted of 468 HER2 receptor-positive patients eligible for this study, treated in our institute between 1991 and 2014. From these, 287 (61%) were found to be Luminal B HER2, with the remaining 181 (39%) patients HER2+(ER−). The median age of patients was 63 and the median follow-up was 49 months. The majority of the overall cohort was recruited after Trastuzumab received approval for adjuvant treatment in 2006 (Table p < 0.001). Furthermore, 263 (91.6%) of the Luminal B HER2 cancers received adjuvant hormone therapy. In the series recurrence occurred in 94 (20.1%) patients, of which 15 (3.2%) had LRR alone. 54 (11.5%) patients had distant metastasis alone and 25 (5.3%) patients had both LRR and distant metastasis. There was no significant difference in the distribution of age, stage or treatment of cancers between the two subtypes  patients were treated with Trastuzumab (Trast +ve). The clinical pathological details of the cohort are listed in Table p < 0.001) and OS (p < 0.001)  but not OS (p = 0.135)  subtypes Figure S1) and Trastuzumab treatment significantly improved overall survival in both subtypes . No difference was seen in survival between the two subtypes in either the Trast –ve or Trast +ve patients. Next to assess the impact of Trastuzumab introduction on each subtype, the 5-year DFS and OS were compared between the Trast –ve and Trast +ve groups in both subtypes. Analysing the DFS and OS by subtype, an increased survival rate is seen for Trast +ve patients in both Luminal B HER2 and HER2+(ER−) patients. However, a greater improvement was seen in Luminal B HER2 patients. Luminal B HER2 cancers had a statistically significant improvement in both 5-year DFS ( and Trasp = 0.004) and OS . However, no significant increase in hazard ratio was seen in Trast –ve HER2+(ER−) cancers compared to the Trast +ve group in 5-year DFS  or OS .No significant increased risk was seen in the HER2+(ER−) subtype when compared to Luminal B HER2 cancers for either 5-year DFS  or 5-year OS  (Table S1). Analysing risk factors for survival, higher grade was not associated with worse outcome but Trastuzumab given in the neo-adjuvant setting was associated with a significant improvement in 5-year DFS . A multivariate cox proportional hazard model analysis of survival was performed, where the model was controlled for age, stage, grade and chemotherapy treatment  and in HER2+(ER−)  (Table p < 0.001); however, there was no significant reduction in HER2+(ER−) breast cancers . Trastuzumab treatment induced a significant reduction in distant metastasis rates in both subtypes, with a greater reduction observed in Luminal B HER2  compared to HER2+(ER−) .Recurrences occurred in 52 (18.1%) of Luminal B HER2 breast cancers and 42 (23.2%) of HER2+(ER−) breast cancers overall. A significant reduction in recurrence rates in Trast +ve patients was observed in both Luminal B HER2 . HER2+(ER−) breast cancers did show decreases in all metastatic sites except for brain in Trast +ve patients; however, these reductions only approached significance in bone (p = 0.075), lung (p = 0.086) and liver (p = 0.075).Analysis of the effects of Trastuzumab treatment on distant metastasis by site of recurrence and subtype was performed Table . For Trap = 0.557). Importantly in Trast +ve patients, a significantly lower odds ratio for LRR was seen in Luminal B HER2 cancers compared to HER2+(ER−) . Distant metastasis rates to bone were the only significant difference between the two subtypes, in the no Trastuzumab group . Following Trastuzumab treatment, no difference was seen between subtypes in bone metastasis  but a significantly lower risk in brain metastasis is seen in the Luminal B HER2 cancers .Performing a multivariate analysis of recurrence risk by treatment, metastatic site and subtype Table , no diffThe impact of Trastuzumab treatment has been well established, greatly improving survival and significantly reducing recurrence in HER2 receptor-positive breast cancers , 21, 22.Few previous studies have compared survival or recurrence rates between the two HER2 receptor-positive breast cancers since the introduction of Trastuzumab. Romond et al. compared survival between the two HER2 receptor-positive breast cancers and found at 4-year follow-up that hormone receptor status minimally influenced the response to Trastuzumab, although hormone receptor status was reported as a significant predictor in DFS . Our finPrevious studies demonstrated that bone was the most common recurrence site in Luminal B HER2 breast cancers and lung was most common for HER2+(ER−) , 9. We sA potential reason for variation between subtypes could be that HER2 receptor over expression reduces the response to hormone therapy. Studies have observed increased hormone resistance rates in Luminal B HER2 cancers compared to Luminal A cancers , 28. CroAnother potential difference between subtypes could be the increased number of grade 3 cancers seen in the HER2+(ER−) group. It has been shown that both HER2+(ER−) and triple-negative breast cancers have worse outcomes and present with higher-grade cancer than Luminal cancers . AlthougIn patients receiving neo-adjuvant Trastuzumab chemotherapy, around 40% of patients will have a complete pathological response , 32. ThiThis study once again shows the benefit of Trastuzumab treatment in HER2 receptor-positive breast cancers, demonstrating the effects on both survival and recurrence rates. It also highlights how a targeted therapy has altered responses in related breast cancer subtypes, emphasising their molecular differences. Demonstrating a more positive impact of Trastuzumab treatment on Luminal B HER2 cancers supports the need to further characterise the mechanism of action of Trastuzumab in each subtype, suggesting that key differences remain to be defined. This work highlights the need to fully understand the subtype-specific effects and mechanisms of action of Trastuzumab therapy, which will allow truly individualised breast cancer management regimes to be implemented.Supplementary material 1 (DOCX 267 kb)Below is the link to the electronic supplementary material.