Tyrosine Kinase Inhibitor Lapatinib Research Articles

Fulvestrant regulates epidermal growth factor (EGF) family ligands to activate EGF receptor (EGFR) signaling in breast cancer cells

Estrogen receptor-α (ER) targeted therapies are routinely used to treat breast cancer. However, patient responses are limited by resistance to endocrine therapy. Breast cancer cells resistant to the pure steroidal ER antagonist fulvestrant (fulv) demonstrate increased activation of epidermal growth factor receptor (EGFR) family members and downstream ERK signaling. In this study, we investigated the effects of fulv on EGFR signaling and ligand regulation in several breast cancer cell lines. EGFR/HER2/HER3 phosphorylation and ERK1,2 activation were seen after 24-48 h after fulvestrant treatment in ER-positive breast cancer cell lines. 4-Hydroxy-tamoxifen and estradiol did not cause EGFR activation. Fulvestrant did not affect EGFR expression. Cycloheximide abolished the ability of fulv to activate EGFR suggesting the autocrine production of EGFR ligands might be responsible for fulvestrant induced EGFR signaling. qRT-PCR results showed fulv differentially regulated EGFR ligands; HB-EGF mRNA was increased, while amphiregulin and epiregulin mRNAs were decreased. Fulvestrant induced EGFR activation and upregulation of EGFR ligands were ER dependent since fulv treatment in C4-12, an ER-negative cell line derivative of MCF-7 cells, did not result in EGFR activation or change in ligand mRNA levels. ER downregulation by siRNA induced similar EGFR activation and regulation of EGFR ligands as fulvestrant. Neutralizing HB-EGF antibody blocked fulv-induced EGFR activation. Combination of fulv and EGFR family tyrosine kinase inhibitors (erlotinib and lapatinib) significantly decreased EGFR signaling and cell survival. In conclusion, fulvestrant-activated EGFR family members accompanied by ER dependent upregulation of HB-EGF within 48 h. EGF receptor or ligand inhibition might enhance or prolong the therapeutic effects of targeting ER by fulvestrant in breast cancer.

Abstract PR11: Characterizing the mechanism and the clinical relevance of the synthetic lethal interaction between STAT3 inhibition and HER2 overexpression in breast cancers

Abstract HER2 is a receptor tyrosine kinase found overexpressed in 20-40% of breast tumors correlating with a malignant phenotype and worse prognosis. Different targeted therapies have been developed to specifically inhibit its activity such the monoclonal antibodies Trastuzumab and Pertuzumab or the small tyrosine kinase inhibitor Lapatinib. Unfortunately, a large number of these patient tumors eventually progress, acquiring resistance. Thus, there is the need to find alternative tumor targets to develop specific and more efficient treatments for these patients. In a tumor cell, multiple regulatory networks have been rearranged in order to adapt to specific genomic abnormalities. Due to this divergence, transformed cells may acquire vulnerabilities that create opportunities for therapeutic intervention. Our main goal is to identify which pathways have become essential for HER2 overexpressing cells but are not needed for normal cells to survive. To determine this, we performed a pooled genome-wide shRNA screen that provides us with a list of HER2 synthetic lethal genes. By combining the analysis from RNAi loss-of-function screens with system biology interactome models, we have recently found that the activation of the JAK/STAT pathway is essential for HER2 mediated transformation, as we demostrate by knocking-down STAT3 expression either in vitro or in orthotopic mouse models. To investigate the mechanism mediating STAT3 activation, we compared the expression profile of parental and HER2 activated cells and found a strong upregulation of IL6 and its canonical receptor that was associated with an increase of IL6 in the media. Overall, our data shows that overexpression of HER2 in breast cancer cell lines leads to an increase in the expression and secretion of IL6, generating an autocrine loop that keeps JAK/STAT3 pathway constitutively active. Signal transducers and activators of transcription (STATs) are latent transcription factors that reside in the cytoplasm until they are phosphorylated and activated by Janus kinases (JAKs). STATs then dimerize and enter the nucleus, where they can activate or repress transcription of their target genes. To further study the transcriptional program regulated by STAT3 in HER2+ breast cancer cell lines, we analyzed the RNA profile in different conditions by using expression microarrays. Interestingly, our results included STAT3 as well as SOCS3, previously described targets of STAT3, but also two S100 family members: S100A8 and S100A9. Our data shows that the overexpression of these two genes is also essential for the transformation of HER2+ cells in vitro and in orthotopic mouse models. In order to translate our results to the clinical setting, we searched for compounds that could inhibit STAT3 activity in vivo. Ruxolitinib is a small-molecule inhibitor of JAK1 and JAK2 approved by the FDA for the treatment of patients with myelofibrosis and other myeloproliferative disorders, so we decieded to assess if Ruxolitnib was effective at inhibiting STAT3 signaling in our model. In fact, treatment with Ruxolitinib impairs in vitro transformation of cells that overexpress HER2 as shown by soft agar assays and 3D cultures in matrigel. More interestingly, oral administration of Ruxolitinib to mice significantly reduces the growth of the tumors implanted in the mammary fat pad. In summary, JAK/STAT pathway activation is essential for HER2-associated transformation suggesting this pathway inhibition as a promising therapeutic approach in HER2 positive tumors that do not respond to the conventional treatment. This abstract is also presented as Poster B35. Citation Format: Ruth Rodriguez-Barrueco, Jiyang Yu, Mariano Alvarez, Veronica Castro, Patricia Villagrasa, Netonia Marshall, Ruoxi Sun, Laura Castro, Andrea Califano. Characterizing the mechanism and the clinical relevance of the synthetic lethal interaction between STAT3 inhibition and HER2 overexpression in breast cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR11.

Radiological response of brain metastases to novel tyrosine kinase inhibitor lapatinib

A 54-year-old woman with stage IV right breast cancer presented to our oncology clinic with 2-week history of headache, nausea and gait instability. Six months ago, she was diagnosed with T4N1M1 lobular carcinoma of right breast, which was estrogen receptor and progesterone receptor negative but over expressed human epidermal growth factor receptor …

Abstract 950: Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models.

Abstract Background. The dual EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib is approved in metastatic breast cancer patients after trastuzumab failure. Unfortunately, several patients do not respond to treatment due to constitutive resistance, and even in responders the occurrence of disease progression is often observed. Recently, the activation of Src TK has been associated with resistance to anti-HER2 drugs, both trastuzumab and lapatinib, in HER2 overexpressing breast cancer cells. However, the molecular mechanisms by which Src activation may affect response to HER2 antagonists are still partially unclear. Methods. We used a panel of HER2 expressing human breast cancer cell lines with different degree of response to lapatinib: SKBR3, BT474 and MDA-MB-361 (high sensitivity), KPL-4 (moderate sensitivity), JIMT-1 (low sensitivity). Moreover, we generated a cell line with acquired resistance to lapatinib, MDA-MB-361-LR (Lapatinib Resistant). On these models, we investigated the effect of Src silencing via small interference RNA (siRNA) or pharmacological inhibition by saracatinib on lapatinib response, both in vitro and in vivo. Results. The combination of Src inhibition with lapatinib synergistically inhibited survival of human breast cancer cells with both intrinsic or acquired resistance to lapatinib, by interfering with signal transducers involved in cell proliferation and survival. Moreover, both saracatinib and Src specific siRNAs were able to reduce migration and invasion capabilities of breast cancer cells, by affecting Focal Adhesion Kinase (FAK) and paxillin activation. In nude mice xenografted with JIMT-1 cells, the combined treatment with saracatinib and lapatinib produced a cooperative antitumor effect with inhibition of tumor growth, interference with signal transduction and prolongation of mice survival. The combination strongly reduced the incidence of lung metastasis, as revealed through experimental metastasis assays. Interestingly, a significant reduction of EGFR/HER2 co-immunoprecipitation was found in resistant cells, thus suggesting that growth signals could be transduced by other type of TKRs dimers in these cells. Moreover, in breast cancer cell lines with both intrinsic or acquired resistance to lapatinib, Src interacted with EGFR rather than with HER2, in opposition to what was observed in sensitive cells. Consistently, EGFR inhibition through cetuximab or EGFR specific siRNAs was able to partially restore lapatinib sensitivity in resistant cells, by interfering with the reciprocal activation of EGFR and Src. Conclusions. Src plays an important role in the onset of resistance to lapatinib in breast cancer cell lines, and its interaction with EGFR may be involved in this event. Therefore, the combined inhibition of Src and EGFR/HER2 axis may represent a potentially effective therapeutic strategy in order to prevent or overcome resistance to lapatinib. Citation Format: Roberta Rosa, Lucia Nappi, Luigi Formisano, Claudia D'Amato, Valentina D'Amato, Vincenzo Damiano, Roberta Marciano, Ana Paula De Maio, Lucia Raimondo, Umberto Malapelle, Giancarlo Troncone, Sabino De Placido, Roberto Bianco. Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2013-950 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Dual Human Epidermal Growth Factor Receptor 2 (HER2) Blockade and Hormonal Therapy for the Treatment of Primary HER2-Positive Breast Cancer: One More Step Toward Chemotherapy-Free Therapy

During the last decade, the introduction of agents that target the human epidermal growth factor receptor 2 (HER2), such as the monoclonal antibody trastuzumab (Herceptin; Roche, Basel, Switzerland) or the tyrosine kinase inhibitor lapatinib (Tykerb; GlaxoSmithKline, Research Triangle Park, NC), in combination with chemotherapy, has changed the course of HER2-positive breast cancer. In the metastatic setting, the addition of trastuzumab to taxanes increases progression-free survival (PFS) and overall survival (OS), whereas the addition of lapatinib to capecitabine increases PFS after treatment with regimens that include an anthracycline, a taxane, and trastuzumab. In the adjuvant setting, trastuzumab increases diseasefree survival and OS. In addition to these impressive advances, clinical research to improve the outcome in HER2-positive breast cancer is as vibrant as it has ever been. A number of important questions are being addressed at this time, including the delineation of the role of neoadjuvant (presurgical) therapy, theconceptofdualHER2receptorblockade, theidentificationof markers of sensitivity or resistance to therapy, and, finally, the study of new agents. As reported in the article that accompanies this editorial, the timely Neoadjuvant Translational Breast Cancer Research Consortium 006 (TBCRC 006) trial by Rimawi et al has addressed three of these questionsandhas identifiedonemorepiece in thepuzzle: the importance ofaddinghormonal therapyinthesubsetofpatientswithHER2-positive/ hormone receptor (HR) –positive tumors. To place this study in context, let us look at what we know today. In the neoadjuvant setting, the addition of trastuzumab to chemotherapy and maintenance for 1 year increases pathologic complete response (pCR) rates and 3-year event-free survival (EFS). And there is increasing evidence that there is a good correlation between pCR and EFS in HER2-positive disease, which has led to the acceptance of pCR as the primary end point in a number of neoadjuvant studies. If some of these studies, such as the Neo-Adjuvant Lapatinib and/or Trastuzumab Treatment Organisation (NeoALTTO) study, further confirm that pCR is a surrogate marker of EFS, improved pCR in HER2-positive breast cancer could lead to regulatory approval of novel agents in this disease. In terms of dual HER2 blockade, laboratory studies have shown that a more complete blockage of the HER2 and/or the HER signaling pathway by combining two or three inhibitors with nonoverlapping mechanisms of action improves cell death and tumor shrinkage in HER2-positive models. These preclinical findings have now been confirmed in the clinical setting. Trastuzumab and lapatinib (or trastuzumab and pertuzumab [Perjeta; Genentech, South San Francisco, CA], a humanized monoclonal antibody binding to the HER2 dimerization domain), in combination with chemotherapy, results in pCR rates of 45.8% to 51.3% compared with pCR rates of 24.0% to 29.5% with a single anti-HER2 agent combined with the same chemotherapy schedule. Moreover, the addition of pertuzumab to trastuzumab and docetaxel increases PFS and OS in first-line metastatic HER2-positive disease. These results have led to regulatory approval of pertuzumab and have placed the dual HER2 blockade with pertuzumab and trastuzumab in combination with chemotherapy as the standard of care in first-line metastatic disease. Furthermore, dual HER2 blockade strategies could also become standard of care in other settings. For example, the ongoing Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO) phase III trial is evaluating the adjuvant dual HER2 blockade with trastuzumab and lapatinib afterchemotherapy,andAStudyofPertuzumabinAdditiontoChemotherapy and Herceptin (Trastuzumab) As Adjuvant Therapy in Patients With HER2-Positive Primary Breast Cancer (APHINITY), a phase III trial, is also currently evaluating adjuvant trastuzumab and pertuzumab with chemotherapy. Given that the dual HER2 blockade is more efficacious that single-agent HER2 therapy, a question that arises is whether the dual blockade may eliminate the need for chemotherapy in a subset of patients. In support of this proposal, the dual HER2 blockade without chemotherapy has shown high activity in a group of patients with metastatic and primary HER2-positive breast cancer. In HER2-positive metastatic breast cancer that was previously treated with trastuzumab, the addition of pertuzumab or lapatinib to trastuzumab showed higher clinical benefit than either pertuzumab or lapatinib alone. In treatment-naive JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 14 MAY 1

MEK Inhibition Increases Lapatinib Sensitivity Via Modulation of FOXM1

The standard targeted therapy for HER2-overexpressing breast cancer is the HER2 monoclonal antibody, trastuzumab. Although effective, many patients eventually develop trastuzumab resistance. The dual EGFR/HER2 small molecule tyrosine kinase inhibitor lapatinib is approved for use in trastuzumab-refractory metastatic HER2-positive breast cancer. However, lapatinib resistance is a problem as most patients with trastuzumab-refractory disease do not benefit from lapatinib. Understanding the mechanisms underlying lapatinib resistance may ultimately facilitate development of new therapeutic strategies for HER2-overexpressing breast cancer. Our current results indicate that MEK inhibition increases lapatinib-mediated cytotoxicity in resistant HER2-overexpressing breast cancer cells. We genetically and pharmacologically blocked MEK/ERK signaling and evaluated lapatinib response by trypan blue exclusion, anchorage-independent growth assays, flow cytometric cell cycle and apoptosis analysis, and in tumor xenografts. Combined MEK inhibition and lapatinib treatment reduced phosphorylated ERK more than single agent treatment. In addition, Western blots, immunofluorescence, and immunohistochemistry demonstrated that the combination of MEK inhibitor plus lapatinib reduced nuclear expression of the MEK/ERK downstream proto-oncogene FOXM1. Genetic knockdown of MEK was tested for the ability to increase lapatinib-mediated cell cycle arrest or apoptosis in JIMT-1 and MDA361 cells. Finally, xenograft studies demonstrated that combined pharmacological inhibition of MEK plus lapatinib suppressed tumor growth and reduced expression of FOXM1 in HER2-overexpressing breast cancers that are resistant to trastuzumab and lapatinib. Our results suggest that FoxM1 contributes to lapatinib resistance downstream of MEK signaling, and supports further study of pharmacological MEK inhibition to improve response to lapatinib in HER2-overexpressing trastuzumab-resistant breast cancer.

Epidermal growth factor receptor gene copy number may predict lapatinib sensitivity in HER2-positive metastatic breast cancer

Objective: Lapatinib, a dual HER2/EGFR tyrosine kinase inhibitor (TKI), associated to capecitabine represents the treatment of choice in HER2-positive metastatic breast cancer (BC) patients in progression after trastuzumab-based therapy. Though lapatinib-based therapy prolongs the time to progression, its efficacy is often limited by the development of drug resistance. It is aimed to evaluate novel biomarkers predictive of lapatinib response, we analyzed EGFR protein and gene status in a series of 50 metastatic HER2-positive BC patients.Methods: Lapatinib was given at 1250 mg/day continuously and capecitabine at 2000 mg/m2/day every 3 weeks. EGFR protein expression and gene copy number (GCN) were assessed by immunohistochemistry and FISH, respectively. Receiver operating curve (ROC) analysis identified the value of > 3.36 EGFR copies/nucleus as the cut-off point able to discriminate responders versus non-responders.Results: A statistical significant correlation between EGFR GCN value > 3.36 and response to lapatinib (p = 0.01) was found. Cox regression analysis further supported these findings evidencing that HER2 score 3+ and EGFR GCN increase are positive predictor factors of lapatinib response.Conclusions: Though further investigations are needed to confirm these findings, EGFR GCN could be a suitable screening to identify the subset of BC patients particularly responsive to the dual TKI lapatinib.

Synthesis of a new conjugated polymer for cell membrane imaging by using an intracellular targeting strategy

A water soluble polythiophene with tyrosine kinase inhibitor lapatinib as side chain moieties (PTL) was designed and synthesized. Together with its fluorescent characteristic and low cytotoxicity, PTL can be used for cell membrane imaging of living cells by targeting the intracellular domain of transmembrane proteins.

Design, Synthesis, and Biological Evaluation ofN-Aryl-salicylamide Derivatives as Potential Antitumor Agents

According to the scaffold hopping, a series of N-aryl-salicylamide derivatives, which have fragments of tyrosine kinase inhibitors lapatinib and neratinib were designed and synthesized. Their structures were identified by IR, H NMR, C NMR and MS techniques. The target compounds were tested for cytotoxic activity against four cancer cell lines, including A549, MCF-7, SGC-7901, Bel-7402, by methyl thiazolyl tetrazolium (MTT) in vitro. All the compounds demonstrated certain antitumor abilities, and some of them were better than gefitinib.

Activity‐based kinase profiling of approved tyrosine kinase inhibitors

The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors.

EGFR through STAT3 modulates ΔN63α expression to sustain tumor‐initiating cell proliferation in squamous cell carcinomas

Many squamous cell carcinomas (SCCs) are characterized by high levels of EGFR and by overexpression of the ΔNp63α isoform. Here, we investigated the regulation of ΔNp63α expression upon EGFR activation and the role of the EGFR-ΔNp63α axis in proliferation of SCC tumor-initiating cells (TICs). SCC cell lines A-431, Cal-27, and SCC-25 treated with EGF showed a time-dependent increase in ΔNp63α expression at the protein and mRNA levels, which was blocked by the tyrosine kinase inhibitor (TKI) Lapatinib. RNA interference experiments suggested the role of STAT3 in regulating ΔNp63α expression downstream of EGFR. Inactivation of EGFR by the monoclonal antibody Cetuximab and RNA interference against STAT3 or ΔNp63α impaired the TICs ability to grow under non-differentiating conditions. Radiation treatment, which triggers EGFR activation, induced ΔNp63α accumulation without affecting TICs proliferation, whereas the combination Cetuximab plus radiation significantly reduced TICs growth under non-differentiating conditions. Together, our findings provide evidence that ΔNp63α expression is regulated by EGFR activation through STAT3 and that the EGFR-ΔNp63α axis is crucial for proliferation of TICs present in SCCs.

Abstract S5-8: Parallel upregulation of Bcl2 and estrogen receptor (ER) expression in HER2+ breast cancer patients treated with neoadjuvant lapatinib.

Abstract Background: We previously showed in HER2+ models of breast cancer (BC) that potent inhibition of the HER receptor layer can lead to re-expression of estrogen receptor (ER) or activation of the ER pathway. Consequently, the anti-apoptotic ER gene product Bcl2 is upregulated, resulting in enhanced tumor cell survival and treatment resistance. In this study, we investigated whether Bcl2 and ER expression levels are simultaneously increased by neoadjuvant treatment with the dual HER1/2 tyrosine kinase inhibitor lapatinib in HER2+ BC patients. Methods: In a neoadjuvant phase II clinical trial 49 HER2+ BC patients were treated with lapatinib as a single agent for 6 weeks, followed by trastuzumab/docetaxel for 12 weeks before surgery. Tumor specimens were prospectively collected at different time-points during lapatinib treatment (baseline, and weeks 2 and 4). Bcl2, ER, progesterone receptor (PR), total (t) and phosphorylated (p)-HER2, and Ki67 were assessed by immunohistochemistry. Spearman correlation was used to evaluate the association among the biomarkers at baseline, and the correlation of their changes over time. Fisher's Exact test and non-parametric Wilcoxon rank sum test were used respectively to determine if the frequency and the magnitude of Bcl2 expression changes were associated with baseline ER status. Results: 35/49 HER2+ tumor specimens (71%) were available for baseline evaluation of Bcl2 and ER. Of those, 12 (34%) were ER-positive (Allred score ≥ 3) and 23 (66%) ER-negative. Baseline Bcl2 expression correlated positively with ER (r = .75; p < .0001) and PR (r = .53; p = .0015), and inversely with t-HER2 (r = −.43; p = .0097). ER baseline expression correlated positively with PR (r = .57; p = .0004), and inversely with t and p-HER2 (r = −.55; p = .0005, and r = −.37; p = .0282, respectively) and Ki67 (r = −.39; p = .0271). Bcl2 changes at week 2 (w2) positively correlated with changes in both ER and PR levels (r = .70; p = .0002 and r = .57; p = .0076, respectively). Additionally, the increase in Bcl2 expression, observed in 9 of the 23 (39%) tumors with tissue available at w2, was significantly more frequent (p = .0147) and of greater magnitude (p = .0001) in ER-pos vs. ER-neg tumors — 8/9 ER-pos tumors at w2 (including 3 converted from ER-neg by lapatinib) had increased Bcl2, while only 1 of the 14 (7%) ER-neg tumors (at baseline and w2) had increased Bcl2. The expression of ER itself at w2 also increased in 3 out of the 6 (50%) tumors which were originally ER-pos and had tissue available at w2, and in all of them Bcl2 increased in parallel. Of note, the single baseline ER-pos tumor that showed a reduction in ER at w2 had a parallel decrease in Bcl2. Similar observations or trends were found at week 4. Conclusion: Our study suggests that Bcl2 is upregulated as a result of enhanced/restored ER activity upon anti-HER2 therapy with lapatinib in HER2+ tumors. This further supports the use of endocrine along with anti-HER-2 therapy to block this escape pathway which could otherwise cause treatment resistance. In addition, the ER re-expression with lapatinib treatment observed in this study emphasizes the need to re-biopsy HER2+/ER− patients receiving anti-HER2 therapy and to add endocrine therapy if the tumor becomes ER-positive. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S5-8.

Abstract PD02-06: Concordance between immunohistochemistry and FISH(fluorescence in sity hybridization) & SISH(silver in situ hybridization) for assessment of the HER2

Abstract Goals: Accurate assessment of the human epidermal growth factor receptor 2 (HER2) of invasive breast cancer with Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) & Silver in situ hybridization(SISH) is very important to decide treatment of targeted therapy with the humanized monoclonal antibody trastuzumab and the dual tyrosine kinase inhibitor lapatinib. The accuracy of these three testing methods can be adversely affected by various factors. We performed a retrospective analysis of the concordance of IHC and FISH & SISH analysis for HER2/neu at Donga-A Medical Center and reviewed possible causes of disparity between these two methods. Methods: We performed retrospective review of a total of 416 invasive ductal carcinomas evaluated for HER2/neu at the Department of Surgery in Dong-A Medical Center between 2005 and 2011. We reviewed this data retrospectively and evaluated the concordance between IHC and FISH & SISH. Results: Comparison of the routinely assessed immunohistochemical HER2 status and FISH & SISH revealed that ten invasive carcinomas diagnosed immunohistochemically as highly overexpressed (score 3+) did not show amplification of the HER2 gene by FISH n that these are false positives (SISH: false positive: 7 cases). Eight invasive carcinomas diagnosed immunohistochemically as Negatively expressed (score 0) showed amplification of the HER2 gene by SISH on that these are false negative. Fourteen tumor, which scored as 2+ by IHC, was proven to be amplified on initial testing with FISH (SISH: 6 cases). Concordance between IHC and FISH analysis was good (171/181= 94.5%) (false positive: 5.5%) (SISH: 228/235=97.0%, false positive 3.4%, false negative 2.9%) and within the published range. Conclusion: IHC as first-line testing for HER2 may result in false positive and false negative results. The novel ASCO/CAP criteria for the immunohistochemical evaluation of the HER2 status may prevent some false positive results. The discordant cases between IHC and FISH & SISH represent a well-recognized subset of genetic heterogeneity in HER2/neu, which is known to contribute to positive IHC and negative FISH & SISH tests. The most powerful method for studying HER2 status is ISH. However, in heterogeneous carcinomas even FISH may not succeed in a correct evaluation of HER2. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD02-06.

Abstract PD10-05: HLA-DQA1*02:01/DRB1*07:01 as a biomarker for lapatinib-induced hepatotoxicity: prospective confirmation in a large randomised clinical trial (TEACH, EGF105485)

Abstract Hepatotoxicity is associated with small molecule tyrosine kinase inhibitors (TKI) in use for the treatment of a variety of cancers. Retrospective studies have identified and confirmed that specific Class II Human Leukocyte Antigen (HLA) alleles are strongly associated with ALT elevation in women treated with the TKI lapatinib for breast cancer. This study aimed to further evaluate and validate the role of the specified HLA alleles as predictors of elevated ALT in a pre-defined analysis of a large, randomized, double-blind, placebo-controlled study of lapatinib monotherapy in early stage HER2 positive breast cancer, the TEACH study (Tykerb Evaluation After Chemotherapy, EGF105485). This prospectively defined pharmacogenetic study compared the frequency of hepatobiliary adverse events between pre-specified Major Histocompatibility Complex (MHC) genetic variants, including the HLA alleles DQA1*02:01 and DRB1*07:01. The primary focus was on elevated ALT, as well as rare cases of concurrent ALT (>3x ULN) and bilirubin (>2x ULN) elevation, which represent possible Hy's Law cases and a high risk of acute liver failure, among 1194 patients randomized to lapatinib treatment from whom pharmacogenetic data was available. This study prospectively validated prior reports of the association of the specified MHC variants with elevated ALT among women treated with lapatinib. The strongest effects were observed for carriers of the HLA alleles DQA1*02:01 and DRB1*07:01, with odds ratios of 20 (95% CI: 8–40) between cases (n = 34) and controls (n = 807–808). These two HLA alleles are highly correlated, inherited together in most individuals and are consistent with a single genetic association. The overall risk of patients having an ALT (>3xULN) elevation was 3.0% and 0.7% during treatment with lapatinib and placebo respectively. Carriers of either HLA allele had a 12% chance (positive predictive value) of having an elevated ALT (>3xULN), in contrast to a 0.9% risk (negative predictive value, 99.1%) for non-carriers of the specified HLA alleles. These associations were maintained for higher ALT elevation thresholds and for cases of concurrent ALT and TBL elevation, consistent with possible Hy's Law cases. These results strongly support the role of Class II HLA-modulated immune mechanisms in lapatinib-induced hepatotoxicity. Our results validate the large strength of association of the HLA alleles DRB1*07:01 and DQA1*02:01 with hepatotoxicity and provide the possibility of managing the risk of hepatotoxicity in women receiving lapatinib for early or late stage HER2 positive breast cancer. This association with immune mechanisms may have implications for toxicities with other TKIs in current use in cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD10-05.

Lapatinib and 17AAG Reduce 89Zr-Trastuzumab-F(ab′)2 Uptake in SKBR3 Tumor Xenografts

Human epidermal growth factor receptor-2 (HER2) directed therapy potentially can be improved by insight in drug effects on HER2 expression. This study evaluates the effects of the EGFR/HER2 tyrosine kinase inhibitor lapatinib, the heat shock protein-90 inhibitor 17AAG, and their combination, on HER2 expression with in vivo HER2-PET imaging. Lapatinib and 17AAG effects on EGFR and HER2 membrane expression were determined in vitro using flow cytometry of human SKBR3 tumor cells. Effect of lapatinib on HER2 internalization was studied in vitro by (89)Zr-trastuzumab-F(ab')(2) internalization. For in vivo evaluation, (89)Zr-trastuzumab-F(ab')(2) μPET imaging was performed two times with a 7 day interval. Lapatinib was administered for 6 days, starting 1 day after the baseline scan. 17AAG was given 1 day before the second (89)Zr-trastuzumab-F(ab')(2) injection. Imaging data were compared with ex vivo biodistribution analysis and HER2 immunohistochemical staining. 17AAG treatment lowered EGFR expression by 41% (P = 0.016) and HER2 by 76% (P = 0.022). EGFR/HER2 downregulation by 17AAG was inhibited by lapatinib pretreatment. Lapatinib reduced internalization of (89)Zr-trastuzumab-F(ab')(2) with 25% (P = 0.0022). (89)Zr-trastuzumab-F(ab')(2) tumor to blood ratio was lowered 32% by lapatinib (P = 0.00004), 34% by 17AAG (P = 0.0022) and even 53% by the combination (P = 0.011). Lapatinib inhibits HER2 internalization and 17AAG lowers HER2 membrane expression. Both drugs reduce (89)Zr-trastuzumab-F(ab')(2) tumor uptake. Based on our findings, supported by previous preclinical data indicating the antitumor potency of lapatinib in combination with HSP90 inhibition, combination of these drugs deserves further investigation.

Method development and validation for the quantification of dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib and sunitinib in human plasma by liquid chromatography coupled with tandem mass spectrometry

To support pharmacokinetic-guided dosing in individual patients, a fast and accurate method for simultaneous determination of anticancer tyrosine kinase inhibitors (TKIs) dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib and sunitinib in human plasma was developed using high-performance liquid chromatography and detection with tandem mass spectrometry (HPLC-MS/MS). Stable isotopically labeled compounds of the eight different TKIs were used as internal standards. Plasma proteins were precipitated and an aliquot of supernatant was directly injected onto a reversed phase chromatography system consisting of a Gemini C18 column (50 × 2.0 mm i.d., 5.0 µm particle size) and then compounds were eluted with a gradient. The outlet of the column was connected to a triple quadrupole mass spectrometer with electrospray interface. Ions were detected in the positive multiple reaction monitoring mode. This method was validated over a linear range from 20.0 to 10,000 ng/mL for erlotinib, gefitinib, imatinib, lapatinib, nilotinib and sorafenib, and from 5.00 to 2500 ng/mL for dasatinib and sunitinib. Results from the validation study demonstrated good intra- and inter-assay accuracy (<13.1%) and precision (10.0%) for all analytes. This method was successfully applied for routine therapeutic drug monitoring purposes in patients treated with the investigated TKIs.

Recent advances in novel targeted therapies for HER2-positive breast cancer

The monoclonal antibody trastuzumab has improved the outcomes of patients with breast cancer that overexpresses the human epidermal growth factor receptor 2 (HER2). However, despite this advancement, many tumors develop resistance and novel approaches are needed. Recently, a greater understanding of cellular biology has translated into the development of novel anti-HER2 agents with varying mechanisms of action. The small molecule tyrosine kinase inhibitor lapatinib has demonstrated activity in HER2-positive metastatic breast cancer (MBC) and in the preoperative setting. Pertuzumab is a monoclonal antibody with a distinct binding site from trastuzumab, which inhibits receptor dimerization. In recent studies, the addition of pertuzumab to combination therapy has led to improvements in progression-free survival in patients with HER2-positive MBC and higher response rates in the preoperative setting. An alternative approach is the use of novel antibody-drug conjugates such as trastuzumab-emtansine, which recently demonstrated activity in MBC. Neratinib, a pan-HER tyrosine kinase inhibitor, which irreversibly inhibits HER1 and HER2, also has proven activity in MBC. A range of compounds is being developed to attempt to overcome trastuzumab resistance by targeting heat shock protein 90, a molecular chaperone required for the stabilization of cellular proteins. Furthermore, agents are being developed to inhibit the mammalian target of rapamycin, a downstream component of the PTEN/PI3K pathway, which has been implicated in trastuzumab resistance. Finally, there are emerging data indicating that combinations of anti-HER2 agents may circumvent resistance mechanisms and improve patient outcomes. In this review, recent data on these emerging agents and novel combinations for HER2-positive breast cancer are discussed.

The effects of lapatinib and neratinib on HER2 protein levels in breast cancer cell lines.

637 Background: HER2, a member of the c-erbB receptor tyrosine kinase family, is over-expressed (HER2 gene amplification/IHC 3+, &gt;30% cells) in approx. 25% of breast cancers. Pre-clinical studies have shown that the HER2-targeted small molecule tyrosine kinase inhibitor (TKI) lapatinib (LAP) can increase HER2 levels in cell line models and can potentiate trastuzumab-mediated antibody dependent cell-mediated cytotoxicity. To assess the potential effects of the next generation TKI neratinib (NER) on expression of HER2 in breast cancer, this study compares the effects of LAP and NER on HER2 protein levels in the HER2-amplified SKBR3 and HER2-non-amplified T47D cell lines in vitro. Methods: SKBR3 and T47D were treated with LAP or NER (0.2, 1 and 2 µM) for 12, 24 and 48 hours.HER2 protein levels were determined by ELISA, immunoblotting and high content analysis (HCA). HER2 protein was examined using two antibodies targeting the extracellular domain (ECD) and the intracellular domain (ICD) of HER2. pHER2, EGFR/pEGFR, MAPK/pMAPK and AKT/pAKT levels were determined by immunoblotting. Proliferation studies utilized an acid phosphatase-based assay. Results: ELISA analysis confirmedsignificantly lower HER2 expression in T47D (44 +/- 17 pg/µg) compared to SKBR3 (748 +/- 296 pg/µg), p = 0.015. NER proved a more potent inhibitor of pHER2 and pEGFR as analyzed by immunoblotting and in proliferation studies (NER IC50 - SKBR3 0.03 +/- 0.01 nM, T47D 199 +/- 70 nM, LAP IC50 - SKBR3 20 +/- 1 nM, T47D 1.2 +/- 0.2 µM). LAP induced an increase in both ECD and ICD-containing HER2 protein levels in SKBR3 and T47D cells. No increase in HER-2 levels was observed with NER treatment, as determined by HCA and immunoblotting. EGFR protein levels increased in response to lapatinib in both cell lines. Conclusions: Our results suggest that LAP and NER have differing effects on HER2 protein levels in the models examined. NER provides greater inhibition of HER2 signaling activity but does not increase HER2 protein levels as was observed with LAP. Further pre-clinical assessment of combinations of LAP and NER with HER2 and EGFR monoclonal antibody therapies is warranted in HER2-amplified, HER2-non-amplified and EGFR-expressing breast cancer models.

Pharmacological interactions of TKIs with the P-gp drug transport protein.

2536 Background: Tyrosine Kinase Inhibitors (TKIs) can interact with drug transport proteins. P-gp is a transporter with two important roles in cancer drug therapy. If overexpressed in tumour cells it can cause drug resistance. However, P-gp, expressed in tissues as part of normal drug clearance mechanisms, is also involved in termination of drug action. Hence, TKI-mediated interactions with P-gp have significant therapeutic consequences. Methods: P-gp over-expressing cancer cell lines were used to determine the inhibitor or substrate status of tyrosine kinase inhibitors (erlotinib, gefitinib, lapatinib, dasatinb, neratinib, afatinib and pazopanib). Cell proliferation assays in combination with a potent P-gp inhibitor, or P-gp substrate were also employed. Findings were augmented using LC-MS-based quantitation of cellular levels of target drugs. Results: We summarise our findings of four distinct interactions with P-gp among various TKIs. Some agents have little interaction at conventional doses; others can act as P-gp inhibitors without being substrates; substrates without being inhibitors or substrates which also prevent the actions of the transporter.Eachof the investigated TKIs has a distinct relationship with P-gp. As examples, lapatinib is an inhibitor but not a substrate, dasatinib is a substrate but not an inhibitor, while pazopanib has little interaction with P-gp. Other agents also have an effect on or are affected by P-gp to varying amounts with some of these interactions likely to be suprapharmacological. Conclusions: P-gp protein has important roles both in resistance and drug toxicology, hence, a clear understanding of the interaction of emerging drugs with this transporter is vital. Agents which are inhibitors of P-gp may have applications in drug resistance circumvention but may also greatly exacerbate the toxicity of concurrently administered P-gp substrate cytotoxics; conversely the activity of P-gp substrate TKIs may be reduced by tumour overexpression of the transporter. Hence in vitro screening of TKI-transporter interactions may identify putative TKI resistance mechanisms, help guide the development of combination schedule trials and/or reducing unwanted treatment side effects.

ALTERNATIVE (EGF114299): A study of lapatinib, trastuzumab, and endocrine therapy in patients who received neo-/adjuvant trastuzumab (IV) and endocrine therapy.

TPS661 Background: Approximately 50% of human epidermal growth factor receptor 2 positive (HER2+) breast cancers are also hormone receptor-positive (HR+). For patients with HER2+/HR+ disease, combining the aromatase inhibitor (AI) letrozole with the dual tyrosine kinase inhibitor lapatinib (L) has been shown to improve outcomes vs letrozole alone. Similar results were found for the combination of trastuzumab (T; a humanized monoclonal antibody targeting HER2) and anastrozole vs anastrozole alone. Finally, the combination of L and T has been shown to improve outcomes vs L alone. Methods: This phase III, randomized, open-label, multicenter trial (ALTERNATIVE) will enroll postmenopausal female patients with HER2+/HR+ metastatic breast cancer (MBC) who have not received prior treatment for MBC, have received neo/adjuvant T and endocrine therapy, and are not candidates for chemotherapy. Patients will be randomized 1:1:1 to 1 of 3 treatment arms: L plus T plus an AI; T plus an AI; or L plus an AI. The AI can be letrozole, anastrozole, or exemestane, to be selected by the investigator. The primary objective of ALTERNATIVE is to examine the efficacy of L/T/AI compared with T/AI alone. The primary efficacy endpoint is overall survival (OS; time from randomization until death due to any cause) for L/T/AI vs T/AI. Secondary efficacy objectives include: comparisons of OS between T/AI and L/AI as well as between T/L/AI and L/AI, comparisons of progression-free survival, overall response rate, time to response, duration of response, safety, and tolerability for all 3 treatment groups. The study is powered to detect a 42% reduction in risk of death (hazard ratio=0.70) in patients who receive L/T/AI (median 28.5 months) vs T/AI (median 20 months) using a 1-sided test for superiority with α=0.025. The required number of total events to achieve a power of 80% is 249. Secondary comparisons are not powered and will be based on the ITT population. This study is currently recruiting, with a target of 525 patients. If its objectives are reached, it will provide a valuable, chemotherapy-free treatment option for HER2+/HR+ MBC patients. Clinical trial registry number: NCT01160211.