Gene Signatures in Breast Cancer: Current and Future Uses

Gene signatures have been developed for estrogen receptor-positive breast cancer to complement pathological factors in providing prognostic information. The 70-gene and the 21-gene signatures identify patients who may not require adjuvant chemotherapy. Gene signatures in triple-negative disease and HER2-positive disease have not been fully developed yet, although studies demonstrate heterogeneity within these subgroups. Further research is needed before genotyping will help in making clinical decisions in triple-negative and HER2-positive disease. Molecular subtyping of breast cancer led to define luminal, basal, and HER2-enriched subtypes, which have specific clinical behavior. This approach may lead to identify new subgroups requiring specific therapies. Standardization of techniques will be required to translate investigations to the clinic.     

Détermination de HER2 dans les adénocarcinomes pour établir le pronostic et prédire la réponse aux traitements

The significant advances registered from targets definition to the use of targeted therapies are a model of innovative development in Oncology. HER2 overexpression has first been reported as progno stic factor before its consideration as a main parameter to predict treatment efficacy. In breast cancer HER2 determination is considered as a revolution for therapeutic strategy of about 15 to 20% of the patients. On the other hand, while HER2 is considered as a prognostic factor for many other cancers, its ability to predict efficacy of trastuzumab is much lower than in breast cancer.     

Bayesian Two-Stage Biomarker-Based Adaptive Design for Targeted Therapy Development

We propose a Bayesian two-stage biomarker-based adaptive randomization (AR) design for the development of targeted agents. The design has three main goals: (1) to test the treatment efficacy, (2) to identify prognostic and predictive markers for the targeted agents, and (3) to provide better treatment for patients enrolled in the trial. To treat patients better, both stages are guided by the Bayesian AR based on the individual patient’s biomarker profiles. The AR in the first stage is based on a known marker. A Go/No-Go decision can be made in the first stage by testing the overall treatment effects. If a Go decision is made at the end of the first stage, a two-step Bayesian lasso strategy will be implemented to select additional prognostic or predictive biomarkers to refine the AR in the second stage. We use simulations to demonstrate the good operating characteristics of the design, including the control of per-comparison type I and type II errors, high probability in selecting important markers, and treating more patients with more effective treatments. Bayesian adaptive designs allow for continuous learning. The designs are particularly suitable for the development of multiple targeted agents in the quest of personalized medicine. By estimating treatment effects and identifying relevant biomarkers, the information acquired from the interim data can be used to guide the choice of treatment for each individual patient enrolled in the trial in real time to achieve a better outcome. The design is being implemented in the BATTLE-2 trial in lung cancer at the MD Anderson Cancer Center.     

Role of HER2 gene overexpression in breast carcinoma

The HER2 proto-oncogene encodes a transmembrane glycoprotein of 185 kDa (p185(HER2)) with intrinsic tyrosine kinase activity. Amplification of the HER2 gene and overexpression of its product induce cell transformation. Numerous studies have demonstrated the prognostic relevance of p185(HER2), which is overexpressed in 10% to 40% of human breast tumors. Recent data suggest that p185(HER2) is a ligand orphan receptor that amplifies the signal provided by other receptors of the HER family by heterodimerizing with them. Ligand-dependent activation of HER1, HER3, and HER4 by EGF or heregulin results in heterodimerization and, thereby, HER2 activation. HER2 overexpression is associated with breast cancer patient responsiveness to doxorubicin, to cyclophosphamide, methotrexate, and fluorouracil (CMF), and to paclitaxel, whereas tamoxifen was found to be ineffective and even detrimental in patients with HER2-positive tumors. In vitro analyses have shown that the role of HER2 overexpression in determining the sensitivity of cancer cells to drugs is complex, and molecules involved in its signaling pathway are probably the actual protagonists of the sensitivity to drugs. The association of HER2 overexpression with human tumors, its extracellular accessibility, as well as its involvement in tumor aggressiveness are all factors that make this receptor an appropriate target for tumor-specific therapies. A number of approaches are being investigated as possible therapeutic strategies that target HER2: (1) growth inhibitory antibodies, which can be used alone or in combination with standard chemotherapeutics; (2) tyrosine kinase inhibitors (TKI), which have been developed in an effort to block receptor activity because phosphorylation is the key event leading to activation and initiation of the signaling pathway; and (3) active immunotherapy, because the HER2 oncoprotein is immunogenic in some breast carcinoma patients.     

Simultaneous Inhibition of Estrogen Receptor and the HER2 Pathway in Breast Cancer: Effects of HER2 Abundance

The estrogen receptor (ER) pathway and the epidermal growth factor receptor (EGFR) pathway play pivotal roles in breast cancer progression. Targeted therapies able to intercept ER or signaling downstream to EGFR and its kin, HER2, are routinely used to treat distinct groups of breast cancer patients. However, patient responses are limited by resistance to endocrine therapy, which may be due to compensatory HER2/EGFR signaling. This raises the possibility that simultaneous interception of HER2 and ER may enhance therapeutic efficacy. To address the question, we treated breast cancer cells with both fulvestrant (ICI 182780), an ER antagonist with no agonist effects, and lapatinib, an orally available tyrosine kinase inhibitor specific to EGFR and HER2. Our results indicate that the combination of drugs is especially effective when applied to HER2-overexpressing, ER-positive cancer cells. Interestingly, fulvestrant activated the mitogen-activated protein kinase (MAPK) pathway of these cells, but complete inhibition of MAPK signaling was observed on cotreatment with lapatinib. Taken together, our observations reinforce the possibility that the effectiveness of combining anti-ER and anti-HER2/EGFR drugs may be especially effective on a relatively small subtype of HER2-overexpressing, ER-positive tumors of the breast.     

曲妥珠单抗治疗HER2阳性乳腺癌的耐药机制及新疗法探索

研究表明大约有20％的乳腺癌患者存在HER2过表达现象。HER2的异常表达及异常信号通路与乳腺癌的侵袭转移、治疗抵抗及不良预后密切相关。在临床上,对于HER2阳性的初期乳腺癌患者常联合曲妥珠单抗及化学药物治疗,但部分患者对曲妥珠单抗产生耐药。因此,研究其耐药机制对于HER2阳性乳腺癌患者的治疗、预后及新疗法的探索具有重要的临床意义。目前引起曲妥珠单抗抵抗的主要机制有：p95-HER2累积、P13K／AKT／mTOR信号异常激活、HER家族受体和IGF-1R信号增加、非受体酪氨酸激酶c-SRC活性增加等。将对上述机制及治疗HER2阳性乳腺癌的新疗法进行综述。     

Insight into p95HER2 in breast cancer: molecular mechanisms and targeted therapies

Breast cancer afflicts more than 1.3 million people worldwide and is the main cause of cancer-related deaths among women. Many efforts are underway to develop new therapeutic and biomarker strategies for the management of this disease. Hormone receptors and human epidermal growth factor receptor 2 (HER2) are currently the most important molecular tools in this regard. Moreover, targeted therapies including trastuzumab in particular are the primary treatment in both the adjuvant and recurrent settings. However, many studies reported that selected patients may present with resistance to trastuzumab due to the presence of p95HER2 fragments. To address this challenge, drugs such as lapatinib and others described in recent patents promise alternative therapeutic options. We discuss the most recent patents related to HER2 and p95HER2 fragments for breast cancer treatment.     

PRP4K is a HER2-regulated modifier of taxane sensitivity

The taxanes are used alone or in combination with anthracyclines or platinum drugs to treat breast and ovarian cancer, respectively. Taxanes target microtubules in cancer cells and modifiers of taxane sensitivity have been identified in vitro, including drug efflux and mitotic checkpoint proteins. Human epidermal growth factor receptor 2 (HER2/ERBB2) gene amplification is associated with benefit from taxane therapy in breast cancer yet high HER2 expression also correlates with poor survival in both breast and ovarian cancer. The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), which we identified as a component of the U5 snRNP also plays a role in regulating the spindle assembly checkpoint (SAC) in response to microtubule-targeting drugs. In this study, we found a positive correlation between PRP4K expression and HER2 status in breast and ovarian cancer patient tumors, which we determined was a direct result of PRP4K regulation by HER2 signaling. Knock-down of PRP4K expression reduced the sensitivity of breast and ovarian cancer cell lines to taxanes, and low PRP4K levels correlated with in vitro-derived and patient acquired taxane resistance in breast and ovarian cancer. Patients with high-grade serous ovarian cancer and high HER2 levels had poor overall survival; however, better survival in the low HER2 patient subgroup treated with platinum/taxane-based therapy correlated positively with PRP4K expression (HR = 0.37 [95% CI 0.15-0.88]; p = 0.03). Thus, PRP4K functions as a HER2-regulated modifier of taxane sensitivity that may have prognostic value as a marker of better overall survival in taxane-treated ovarian cancer patients.     

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼15–20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2−) breast cancer tissues, whereas both HER2+ and HER2− tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser⁶⁷⁴ by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.     

Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by immunotherapeutical approaches based on HER2-directed monoclonal antibodies and cancer vaccines. We describe the adoptive transfer of autologous HER2-specific T-lymphocyte clones to a patient with metastatic HER2-overexpressing breast cancer. The HLA/multimer-based monitoring of the transferred T lymphocytes revealed that the T cells rapidly disappeared from the peripheral blood. The imaging studies indicated that the T cells accumulated in the bone marrow (BM) and migrated to the liver, but were unable to penetrate into the solid metastases. The disseminated tumor cells in the BM disappeared after the completion of adoptive T-cell therapy. This study suggests the therapeutic potential for HER2-specific T cells for eliminating disseminated HER2-positive tumor cells and proposes the combination of T cell-based therapies with strategies targeting the tumor stroma to improve T-cell infiltration into solid tumors.     

Coupling proteomics and transcriptomics in the quest of subtype‐specific proteins in breast cancer

Breast‐cancer subtypes present with distinct clinical characteristics. Therefore, characterization of subtype‐specific proteins may augment the development of targeted therapies and prognostic biomarkers. To address this issue, MS‐based secretome analysis of eight breast cancer cell lines, corresponding to the three main breast cancer subtypes was performed. More than 5200 non‐redundant proteins were identified with 23, four, and four proteins identified uniquely in basal, HER2‐neu‐amplified, and luminal breast cancer cells, respectively. An in silico mRNA analysis using publicly available breast cancer tissue microarray data was carried out as a preliminary verification step. In particular, the expression profiles of 15 out of 28 proteins included in the microarray (from a total of 31 in our subtype‐specific signature) showed significant correlation with estrogen receptor (ER) expression. A MS‐based analysis of breast cancer tissues was undertaken to verify the results at the proteome level. Eighteen out of 31 proteins were quantified in the proteomes of ER‐positive and ER‐negative breast cancer tissues. Survival analysis using microarray data was performed to examine the prognostic potential of these selected candidates. Three proteins correlated with ER status at both mRNA and protein levels: ABAT, PDZK1, and PTX3, with the former showing significant prognostic potential.     

Relationships of lipocalin 2 with breast tumorigenesis and metastasis

Breast cancer is one of the most common cancers in women worldwide and accounts for one‐sixth of cancer deaths in the United States. Breast cancer consists of a heterogeneous group of tumours classified into five types, in which the HER2/neu positive and the basal type (most are ER and HER2 negative) have the worst clinical prognosis. In recent years, prognostic/predictive markers such as ER/PR or HER2/neu have been widely used in the selection of the optimal breast cancer treatments for individual patients, which have been proven to be very effective in disease control. These results suggest that further examination of the molecular mechanisms underlying the breast tumorigenesis and identification of the potential biomarkers in different types of breast cancers will greatly benefit clinical diagnosis and facilitate the design of more effective personalized therapies to increase patient survival. This review aims to summarize recent research findings on lipocalin 2 (LCN2), a newly identified biomarker and a potential therapeutic target for breast cancer, and the possible mechanisms underlying its role in tumorigenesis and metastasis. J. Cell. Physiol. 226: 309–314, 2011. © 2010 Wiley‐Liss, Inc.     

Identification of a risk prediction model for clinical prognosis in HER2 positive breast cancer patients

BackgroundNew biomarkers are needed to identify different clinical outcomes for HER2+ breast cancer (BC).MethodsDifferential genes of HER2+ BC were screened based on TCGA database. We used WGCNA to identify the genes related to the survival. Genetic Algorithm was used to structure risk prediction model. The prognostic model was validated in GSE data.ResultsWe constructed a risk prediction model of 6 genes to identify prognosis of HER2+ BC, including CLEC9A, PLD4, PIM1, PTK2B, AKNAD1 and C15orf27. Kaplan-Meier curve showed that the model effectively distinguished the survival of HER2+ BC patients. The multivariate Cox regression suggested that the risk model was an independent predictor for HER2+ BC. Analysis related to immune showed that significant differences in immune infiltration between high- and low-risk groups classified by the prognostic model.ConclusionsOur study identified a risk prediction model of 6 genes that could distinguish the prognosis of HER2+ BC.     

Folate Receptor-α (FOLR1) Expression and Function in Triple Negative Tumors

Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense overexpression of FOLR1 in breast tumors and poor prognosis, yet there is limited examination of the distribution of FOLR1 across clinically relevant breast cancer subtypes. To explore this further, we used RNA-seq data from multiple patient cohorts to analyze the distribution of FOLR1 mRNA across breast cancer subtypes comprised of estrogen receptor positive (ER+), human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) tumors. FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors. However, subsets of high level FOLR1 expressing tumors were observed in all clinical subtypes. These observations were supported by immunohistochemical analysis of tissue microarrays, with the largest number of 3+ positive tumors and highest H-scores of any subtype represented by triple negatives, and lowest by ER+ tumors. FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status. To delineate the importance of FOLR1 overexpression in triple negative cancers, RNA-interference was used to deplete FOLR1 in overexpressing triple negative cell breast lines. Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions. Taken together, our data suggests patients with triple negative cancers expressing high FOLR1 expression represent an important population of patients that may benefit from targeted anti-FOLR1 therapy. This may prove particularly helpful for a large number of patients who would typically be classified as triple negative and who to this point have been left without any targeted treatment options.     

Making efficient use of patients in designing phase III trials investigating simultaneously a set of targeted therapies with different targets

Targeted therapies are a recent development in cancer treatment research. As these therapies can only be administered to patients with certain individual characteristics, it is a straightforward idea to investigate several of such therapies simultaneously in a given patient population in order to compare each targeted therapy with the current standard therapy. This raises the question how patients satisfying several characteristics should be handled. We consider in this paper several designs to allocate treatments in a random manner to these patients, such that the evaluation of each single targeted therapy can be based on a simple comparison of patients receiving the targeted therapies versus those receiving the standard therapy within a well defined subgroup of patients satisfying the corresponding characteristic. We show how one can ensure that patients with several characteristics can contribute simultaneously to the evaluation of several targeted therapies and that this is the key point for an efficient use of the patients available. We further discuss some ethical and practical issues in applying the new designs and outline strategies to evaluate the overall effect of all targeted therapies together.     

Comparison of Pathologic Response Evaluation Systems after Anthracycline with/without Taxane-Based Neoadjuvant Chemotherapy among Different Subtypes of Breast Cancers

Purpose

Several methods are used to assess the pathologic response of breast cancer after neoadjuvant chemotherapy (NAC) to predict clinical outcome. However, the clinical utility of these systems for each molecular subtype of breast cancer is unclear. Therefore, we applied six pathologic response assessment systems to specific subtypes of breast cancer and compared the results.

Patients and Methods

Five hundred and eighty eight breast cancer patients treated with anthracycline with/without taxane-based NAC were retrospectively analyzed, and the ypTNM stage, residual cancer burden (RCB), residual disease in breast and nodes (RDBN), tumor response ratio, Sataloff’s classification, and Miller—Payne grading system were evaluated. The results obtained for each assessment system were analyzed in terms of patient survival.

Results

In triple-negative tumors, all systems were significantly associated with disease-free survival and Kaplan-Meier survival curves for disease-free survival were clearly separated by all assessment methods. For HR+/HER2- tumors, systems assessing the residual tumor (ypTNM stage, RCB, and RDBN) had prognostic significance. However, for HER2+ tumors, the association between patient survival and the pathologic response assessment results varied according to the system used, and none resulted in distinct Kaplan—Meier curves.

Conclusion

Most of the currently available pathologic assessment systems used after anthracycline with/without taxane-based NAC effectively classified triple-negative breast cancers into groups showing different prognoses. The pathologic assessment systems evaluating residual tumors only also had prognostic significance in HR+/HER2- tumors. However, new assessment methods are required to effectively evaluate the pathologic response of HR+/HER2+ and HR-/HER2+ tumors to anthracycline with/without taxane-based NAC.     

Profiling the HER3/PI3K pathway in breast tumors using proximity-directed assays identifies correlations between protein complexes and phosphoproteins

Background

The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples.

Methodology and Principal Findings

Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation.

Significance

This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models.     

The impact of HER2-directed targeted therapy on HER2-positive DCIS of the breast

BackgroundIn invasive breast cancer, HER2 is a well-established negative prognostic factor. However, its significance on the prognosis of ductal carcinoma in situ (DCIS) of the breast is unclear. As a result, the impact of HER2-directed therapy on HER2-positive DCIS is unknown and is currently the subject of ongoing clinical trials. In this study, we aim to determine the possible impact of HER 2-directed targeted therapy on survival outcomes for HER2-positive DCIS patients.Materials and methodsThe National Cancer Data Base (NCDB) was used to retrieve patients with biopsy-proven DCIS diagnosed from 2004–2015. Patients were divided into two groups based on the adjuvant therapy they received: systemic HER2-directed targeted therapy or no systemic therapy. Statistics included multivariable logistic regression to determine factors predictive of receiving systemic therapy, Kaplan-Meier analysis to evaluate overall survival (OS), and Cox proportional hazards modeling to determine variables associated with OS.ResultsAltogether, 1927 patients met inclusion criteria; 430 (22.3%) received HER2-directed targeted therapy; 1497 (77.7%) did not. Patients who received HER2-directed targeted therapy had a higher 5-year OS compared to patients that did not (97.7% vs. 95.8%, p = 0.043). This survival benefit remained on multivariable analysis. Factors associated with worse OS on multivariable analysis included Charlson-Deyo Comorbidity Score ≥ 2 and no receipt of hormonal therapy.ConclusionIn this large study evaluating HER2-positive DCIS patients, the receipt of HER2-directed targeted therapy was associated with an improvement in OS. The results of currently ongoing clinical trials are needed to confirm this finding.     

Efficacy and Safety of HER2-Targeted Agents for Breast Cancer with HER2-Overexpression: A Network Meta-Analysis

BackgroundClinical trials of human epidermal growth factor receptor 2 (HER2)-targeted agents added to standard treatment have been efficacious for HER2-positive (HER2+) advanced breast cancer. To our knowledge, no meta-analysis has evaluated HER2-targeted therapy including trastuzumab emtansine (T-DM1) and pertuzumab for HER2-positive breast caner and ranked the targeted treatments. We performed a network meta-analysis of both direct and indirect comparisons to evaluate the effect of adding HER2-targeted agents to standard treatment and examined side effects.MethodsWe performed a Bayesian-framework network meta-analysis of randomized controlled trials to compare 6 HER2-targeted treatment regimens and 1 naïve standard treatment (NST, without any-targeted drugs) in targeted treatment of HER2+ breast cancer in adults. These treatment regimens were T-DM1, LC (lapatinib), HC (trastuzumab), PEC (pertuzumab), LHC (lapatinib and trastuzumab), and PEHC (pertuzumab and trastuzumab). The main outcomes were overall survival and response rates. We also examined side effects of rash, LVEF (left ventricular ejection fraction), fatigue, and gastrointestinal disorders, and performed subgroup analysis for the different treatment regimens in metastatic or advanced breast cancer.ResultsWe identified 25 articles of 21 trials, with data for 11,276 participants. T-DM1 and PEHC were more efficient drug regimens with regard to overall survival as compared with LHC, LC, HC and PEC. The incidence of treatment-related rash occurs more frequently in the patients who received LC treatment regimen than PEHC and T-DM1 and HC. In subgroup analysis, T-DM1 was associated with increased overall survival as compared with LC and HC. PEHC was associated with increased overall response as compared with LC, HC, and NST.ConclusionsOverall, the regimen of T-DM1 as well as pertuzumab in combination with trastuzumab and docetaxel is efficacious with fewer side effects as compared with other regimens, especially for advanced HER2+ breast cancer.ImpactThis study suggests that both T-DM1 and PEHC therapy are potentially and equally useful treatments for HER2+ breast cancer.