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Originally published as JCO Early Release 10.1200/JCO.2005.12.157 on January 31 2005 © 2005 American Society of Clinical Oncology. Study of the Biologic Effects of Lapatinib, a Reversible Inhibitor of ErbB1 and ErbB2 Tyrosine Kinases, on Tumor Growth and Survival Pathways in Patients With Advanced Malignancies
From the Department of Discovery Medicine and Clinical Pharmacology, GlaxoSmithKline, Research Triangle Park; Division of Hematology-Oncology, Duke University Medical Center, Durham; Lineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, NC; Tennessee Oncology Associates, Sarah Cannon Cancer Center, Nashville, TN; Division of Hematology-Oncology, Case Western Reserve Medical School, Cleveland, OH; Prediction Sciences, San Diego, CA; Quantitative Diagnostics Laboratory, Westmont, IL Address reprint requests to Neil L. Spector, MD, Discovery Medicine and Clinical Pharmacology, GlaxoSmithKline, Five Moore Dr, Research Triangle Park, NC 27709-3398; e-mail: Neil.L.Spector{at}gsk.com.
PURPOSE: This was a pilot study to assess the biologic effects of lapatinib on various tumor growth/survival pathways in patients with advanced ErbB1 and/or ErbB2-overexpressing solid malignancies. PATIENTS AND METHODS: Heavily pretreated patients with metastatic cancers overexpressing ErbB2 and/or expressing ErbB1 were randomly assigned to one of five dose cohorts of lapatinib (GW572016) administered orally once daily continuously. The biologic effects of lapatinib on tumor growth and survival pathways were assessed in tumor biopsies obtained before and after 21 days of therapy. Clinical response was determined at 8 weeks. RESULTS: Sequential tumor biopsies from 33 patients were examined. Partial responses occurred in four patients with breast cancer, and disease stabilization occurred in 11 others with various malignancies. Responders exhibited variable levels of inhibition of p-ErbB1, p-ErbB2, p-Erk1/2, p-Akt, cyclin D1, and transforming growth factor alpha. Even some nonresponders demonstrated varying degrees of biomarker inhibition. Increased tumor cell apoptosis (TUNEL) occurred in patients with evidence of tumor regression but not in nonresponders (progressive disease). Clinical response was associated with a pretreatment TUNEL score > 0 and increased pretreatment expression of ErbB2, p-ErbB2, Erk1/2, p-Erk1/2, insulin-like growth factor receptor-1, p70 S6 kinase, and transforming growth factor alpha compared with nonresponders. CONCLUSION: Lapatinib exhibited preliminary evidence of biologic and clinical activity in ErbB1 and/or ErbB2-overexpressing tumors. However, the limited sample size of this study and the variability of the biologic endpoints suggest that further work is needed to prioritize biomarkers for disease-directed studies, and underscores the need for improved trial design strategies in early clinical studies of targeted agents.
Historically, cytotoxic cancer therapies have largely been developed without identifying a molecular tumor profile predicting for their response. Consequently, patients are often subjected to highly toxic therapies with only limited therapeutic benefit. Recent advances in our understanding of the regulation of tumor growth and survival have led to the development of rationally designed tumor-targeted therapies. ErbB1 (epidermal growth factor receptor [EGFR]) and ErbB2 promote tumor growth and survival in a variety of epithelial tumors, where their expression or overexpession in some tumors correlates with a poor clinical outcome, making them attractive therapeutic targets.1,2 Although ErbB1 and ErbB2 contain multiple tyrosine phosphorylation sites, autophosphorylation of specific tyrosine residues within the highly conserved catalytic kinase domains of ErbB1 and ErbB2 establishes binding sites for Src-homology 2 and phosphotyrosine binding-domain containing proteins linking ErbB receptors to downstream cell proliferation (mitogen-activated protein kinase [MAPK]; also known as Erk1/2) and survival (phosphatidylinositol-3-kinase [PI3K]) pathways.3-8 Therapeutic monoclonal antibodies and small molecule tyrosine kinase inhibitors targeting ErbB1 or ErbB2 have been developed. Trastuzumab, a humanized anti-ErbB2 monoclonal antibody, is approved for treating patients whose breast cancers overexpress ErbB2 protein or demonstrate ErbB2 gene amplification,9 and gefitinib is approved for third-line treatment of non–small-cell lung cancer.10 The inability of the mono-ErbB1 inhibitor gefitinib to demonstrate a survival advantage when added to first-line chemotherapy in metastatic non–small-cell lung cancer raises questions regarding the optimal use of these agents in the clinic and underscores the need to identify biomarkers to guide their clinical development. Lapatinib (GW572016) is an orally active small molecule that reversibly inhibits ErbB1 and ErbB2 tyrosine kinases, which in turn blocks phosphorylation and activation of Erk1/2 (p-Erk1/2) and Akt (p-Akt) in ErbB1- and/or ErbB2-expressing tumor cell lines and xenografts.11-14 Lapatinib elicits cytostatic or cytotoxic antitumor effects depending on the cell type.11,12 Because ErbB2-containing heterodimers exert potent mitogenic signals, simultaneously interrupting both ErbB1 and ErbB2 signaling is an appealing therapeutic approach. To date, the effects of anti-ErbB1 therapies on signaling pathways have been primarily assessed in skin, an accessible ErbB1-expressing tissue, although data concerning the correlative extrapolation from effects on skin to effects in tumor or clinical response have not been demonstrated.15,16 The objective of this study was to explore a range of putative biologic markers in tumor biopsies from patients before and after therapy with lapatinib. The results presented demonstrate the difficulties in establishing clear dose-response relationships in early clinical studies of targeted agents and support efforts to refine technical and clinical design methodologies to enhance the relevance of these early investigations.
Clinical Study Design The institutional review boards at the Sarah Cannon Cancer Center (Nashville, TN), University of North Carolina-Chapel Hill/Lineberger Comprehensive Cancer Center (Chapel Hill, NC), Duke University Medical Center (Durham, NC), and Case Western Reserve Medical Center (Cleveland, OH) approved this study. Patients providing informed consent were enrolled on this open-label, randomized trial if their tumors (1) overexpressed ErbB2 and/or ErbB1 (2 to 3+ immunohistochemistry [IHC] staining in >10% of tumor cells), or (2) demonstrated ErbB2 gene amplification by fluorescent in situ hybridization. Additional eligibility criteria included: age  18 years; measurable metastatic solid malignancies not amenable to established standard therapies; Karnofsky performance status 70; no prior chemo-, radio-, hormonal or immunotherapy within the previous 4 weeks; left ventricular ejection fraction 40%; hemoglobin 9 g/ml; absolute neutrophil count 1,500/µL; platelet 100,000/µL; total bilirubin < 2.0 mg/dL, and transaminases < 3x upper limit of normal unless due to tumor. Patients were randomly assigned to one of five dose cohorts of lapatinib (500, 650, 900, 1,200, or 1,600 mg) administered orally, once daily, on a continuous basis until evidence of disease progression or intolerable side effects. Tumor biopsies were obtained within 3 days before initiating lapatinib (day 0) and again on day 21 within 4 to 12 hours after administration of lapatinib. Patients were monitored by physical examination, clinical chemistry, and hematology blood tests, and formally restaged after 8 weeks of therapy using Response Evaluation Criteria in Solid Tumors criteria to assess clinical response in appropriate target lesions.17 Responding patients (complete response, partial response [PR], or stable disease) continued on therapy with restaging every 8 weeks thereafter.
Reagents
IHC
Statistical Analysis
Patients and Sequential Tumor Biopsy Specimens Demographics of the 33 patients are shown in Table 1. Expression of ErbB1, ErbB2, p-ErbB1, p-ErbB2, Erk1/2, p-Erk1/2, Akt, p-Akt, TGF-  , IGF-1R, and cyclin D1 was assessed by IHC with automated image analysis using established assays; OD values were assigned to each specimen. For reference, OD values > 15 are analogous to 3+ grading.2 TUNEL scores indicate the percent of tumor cells staining positive. Day 21 tumor biopsies were obtained within 4 to 12 hours following lapatinib dosing to maximize the probability of detecting a biologic response. Preliminary data describing clinical responses, safety profile, and pharmacokinetics for this study have been previously reported.20,21
Biologic Effects of Lapatinib on Growth and Survival Pathways in Clinical Tumor Biopsies Forty-two percent of the patients on study were women with metastatic breast cancer all previously treated with multiple chemotherapeutic regimens, most in combination with trastuzumab. Four PRs were observed, all in patients with breast cancer whose disease had progressed despite multiple prior chemotherapies with and without trastuzumab, and in some cases, hormonal therapy. Biologic responses in the four responders were characterized by variable levels of inhibition of (1) p-ErbB1, (2) p-ErbB2, (3) p-Erk1/2, (4) p-Akt, (5) cyclin D1, or (6) combinations of the above. Figure 1 shows representative IHC analyses from day 0 and day 21 tumor biopsies in a responding breast cancer patient. In contrast to p-ErbB2, expression of total ErbB2 protein was relatively unchanged by lapatinib (Fig 1). Similar results were observed in ErbB1-expressing tumors (data not shown). One of the objectives of the study was to identify biologic features (eg, tumor type, baseline tumor biomarker profile, or characterization of the biologic response to lapatinib at day 21) that might distinguish responders from nonresponders. In this regard, all of the PRs occurred in ErbB2-overexpressed breast cancers that also exhibited markedly elevated levels of activated, p-ErbB2 (Table 2). One biologic effect shared by all the responders was inhibition of cyclin D and the EGFR ligand TGF- (Fig 2; Table 3). Activated p-Akt was inhibited in three of the four responders with the fourth demonstrating no change (Fig 2; Table 3). Similarly, activated p-Erk1/2 was markedly inhibited in three of the four responders; the fourth responder (Fig 3) had no evidence of Erk1/2 activation at baseline (Fig 2; Table 3). p-ErbB2 and p-ErbB1 were variably modulated in responding patients.
Even some of the nonresponders demonstrated varying degrees of p-ErbB2, p-ErbB1, p-Erk1/2, p-Akt, cyclin D, or TGF- inhibition (Fig 2; Tables 2 and 3). In contrast to responders, nonresponding patients tended to have lower baseline levels of ErbB2 and p-ErbB2 expression (Table 2). No uniform pattern of biologic effect on ErbB-MAPK-Erk1/2 or ErbB-PI3K-Akt signaling pathways was seen in nonresponding patients.
Preliminary Assessment of Markers in Responding and Nonresponding Patients
Biomarkers in Responding Patients Compared With Nonresponders Biomarker expression using quantitative IHC (OD values) was determined in pretreatment tumor biopsies, and changes between pre- and post-treatment values were examined in both breast and non–breast cancer patients. Results of this analysis and their relationship to clinical outcome are summarized. Breast cancer patients achieving a PR tended to have higher pretreatment expression of p-ErbB2 compared with nonresponders (Table 2). In contrast to ErbB2, baseline ErbB1 expression did not discriminate between responders and nonresponders. Inhibition of p-ErbB1 at day 21 was seen in those patients who achieved a PR (Table 2). Inhibition of p-Akt and p-Erk1/2 at day 21 was associated with clinical responses (PR; Table 3). Additional markers that were increased at baseline (day 0) in responders compared to nonresponders included IGF-1R, p70 S6 kinase, and TGF- . In addition, pretreatment TUNEL scores > 0 were associated with clinical responses (Table 4).
Cancer treatment will be increasingly based on molecular profiling of tumors rather than histology alone. Elucidating the biologic effects of targeted therapies in tumor tissue and correlating these effects with clinical response will be important to identify the predominant growth and/or survival pathways operative in tumors, thereby establishing tumor profiles to select patients more likely to respond to lapatinib. The purpose of this study was to assess the biologic effects of lapatinib on growth and survival pathways in sequential clinical tumor biopsies. Data from this study serve to generate hypotheses regarding the use of biomarkers to guide the clinical development of lapatinib. Although this is an exploratory study, several observations are nonetheless intriguing and merit further investigation. First, responses (PR) were only seen in patients with ErbB2-overexpressing breast cancers. ErbB2 is the preferred heterodimeric partner for other ErbB receptors. ErbB2-ErbB3 heterodimers, which play a role in breast cancer tumorigenesis, are potent activators of the PI3K-Akt survival pathway.3,22-24 ErbB3 is kinase-dead, relying on ErbB2 for transactivation. It is therefore not surprising that responses were seen in patients with ErbB2-overexpressing breast cancers and are consistent with current criteria for selecting patients to receive trastuzumab, namely breast cancers that overexpress ErbB2 protein or demonstrate ErbB2 gene amplification. Despite this selection, the majority of patients do not respond to trastuzumab. In this regard, ErbB2 overexpression does not necessarily indicate the presence of an activated receptor,25,26 which was confirmed in the current study as < 50% of ErbB2 overexpressing tumors exhibited evidence of increased p-ErbB2, although the anti-phospho-ErbB2 antibody used is specific for tyrosine 1248, only one of several autophosphorylation sites. Whether additional tyrosine phosphorylation sites will serve as better biomarkers for lapatinib remains to be determined. Additional data are required in order to determine whether qualitative or quantitative analysis of ErbB2 receptor status will be better at identifying patients who are more likely to respond to lapatinib. The variable biologic effect of lapatinib is similar to that seen with EGFR inhibitors, where most of the pharmacodynamic have been demonstrated in normal EGFR-expressing tissue (skin). In these studies, inhibition of p-ErbB1, p-Erk1/2, Ki67, and induction of p27 in keratinocytes and hair follicles have been demonstrated.16,19,27,28 However, these biologic effects in skin did not necessarily correlate with clinical response. Similarly, in our study, some nonresponders still had evidence of biologic response to lapatinib, suggesting that redundant signaling pathways might be regulating the growth and/or survival of those particular tumors. There is less biologic data on the effects of ErbB-targeted therapies in clinical tumor biopsies. Recently, Rojo et al29 demonstrated that single-agent therapy with gefitinib resulted in the inhibition of p-ErbB1 and p-Erk in clinical tumor biopsies from patients with advanced-stage gastric carcinoma enrolled in a phase II clinical trial. In our study, lapatinib had variable effects on p-ErbB1 expression, although in contrast to the study reported by Rojo et al, we enrolled heterogeneous tumor types and studied multiple dose cohorts, thereby making it difficult to reach solid conclusions from the data. Regardless of the effects of lapatinib on p-ErbB1 or p-ErbB2, it is the repercussions of ErbB inhibitors on downstream signaling pathways, not on ErbB1 or ErbB2 phosphorylation state, that correlates with tumor growth inhibition.30 As demonstrated previously in preclinical studies11,12 and now confirmed here, treatment with lapatinib resulted in the inhibition of downstream mediators of tumor growth and/or survival in clinical tumor biopsies. Although pretreatment ErbB1 and p-ErbB1 did not appear to discriminate between responders and nonresponders, day 21 inhibition of p-ErbB1 did correlate with response to lapatinib in breast cancer patients. With the exception of glioblastoma multiforme31 and a small subset of non–small-cell lung cancers,32,33 where ErbB1 activating mutations exist, there is little evidence to support the role of ErbB1 as the predominant growth and survival regulator of most epithelial carcinomas. This does not necessarily exclude a functional role for ErbB1 through its dimerization with ErbB2 or other ErbB receptors. Although all responders in our study demonstrated elevated expression levels of ErbB2 and not necessarily ErbB1, inhibition of p-ErbB1 at day 21 still correlated with response underscoring the potential advantage of a potent inhibitor of ErbB2 and ErbB1 tyrosine kinases compared with inhibitors of either receptor alone. Several patients had disease progression despite inhibition of p-ErbB1, p-ErbB2, p-Erk1/2, and p-Akt. Only patients with evidence of tumor regression by clinical or radiographic parameters demonstrated increased tumor cell apoptosis at day 21. These results are reminiscent of those reported by Chang et al,34 in which increased tumor cell apoptosis was observed 8 days after treating breast cancer patients with trastuzumab in the neoadjuvant setting. In the present study, tumor regression was only observed in those patients whose tumors had a pretreatment TUNEL score > 0. A baseline TUNEL score of 0 suggests the presence of an inoperative apoptotic pathway (eg, inactivating mutations in effector caspases, selection of tumors with activated antiapoptotic mediators, Bcl-2).35 If the proapoptotic pathway in a tumor can not be activated, then tyrosine kinase inhibitors, which exert their antitumor effects by inducing apoptosis, will likely be ineffective. The high incidence of tumors with baseline TUNEL scores of 0 in the current study may be an indication of the heavily pretreated nature of the study population, where selective pressures for tumor survival favor cells with inoperative apoptotic machinery. Although we only had baseline levels of IGF-1R and p70 S6 kinase expression from 15 patients (three PR and 12 PD), elevated levels of these two markers were associated with clinical response (Table 4). These results are in contrast to that of other ErbB2 inhibitors, where elevated IGF-1R and p70 S6 kinase have been linked to resistance to trastuzumab and gefitinib in preclinical models and recently in patients with breast cancer receiving trastuzumab therapy.36-38 IGF-1R and ErbB2 form heterodimers that are characterized by a hierarchical signaling pattern—IGF-1R signaling propagated through ErbB2.39 It is tempting to speculate that increased coexpression of IGF-1R and ErbB2 favors formation of ErbB2/IGF-1R complexes whose signaling can then be blocked by a potent ErbB2 kinase inhibitor such as lapatinib. Conversely, in the absence of elevated ErbB2 expression, IGF-1R may activate the PI3K-Akt-mTOR-p70 S6 kinase pathway through ErbB independent mediators such as insulin responsive substrate 1 and 2, which would be insensitive to lapatinib. Elevated p70 S6 kinase in the presence of concomitant, high ErbB2 and IGF-1R may indicate a tumor survival pathway amenable to ErbB2 inhibition by lapatinib. These complex receptor interactions will require further investigation.
In addition to ErbB receptors, ErbB receptor ligands expressed in many epithelial tumors through autocrine or paracrine mechanisms stimulate tumor proliferation and survival.40 Increased expression of ligands in the tumor microenvironment activate their respective cognate receptors.41 One particular ErbB1 ligand, TGF- There are several aspects of this study that make it difficult to reach any solid conclusions. First, although expression of ErbB1 or overexpression of ErbB2 in tumors was required for entry into this study, these included patients with heterogeneous epithelial malignancies. Despite expressing or overexpressing ErbB1 or ErbB2, respectively, the dependence of different tumor types upon ErbB receptors for growth and survival most likely varies. Second, variability in biomarker data may, in some cases, relate to technical issues (eg, procuring tumor biopsies without activating phosphatases). Third, interpatient variability in pharmacokinetics might influence pharmacodynamic effects. And fourth, multiple dose cohorts were studied, adding an additional layer of variability in interpreting the data. With these caveats in mind, we submit that lapatinib elicits (1) biologic effects on growth and survival pathways in tumors, and (2) clinical activity in heavily pretreated patients at doses that appear to be well-tolerated grade I/II transient skin and gastrointestinal toxicity.21 Clinical responses occurred in patients whose breast cancers had progressed on prior trastuzumab therapy, implying that lapatinib is not cross-resistant with trastuzumab. Identification of the pathways regulating the survival of particular tumors and whether these pathways are operative may ultimately enable individual therapeutic decisions to be based on tumor biology rather than histology alone. Further testing of hypotheses generated in this study will hopefully enhance our ability to identify patients who are more likely to respond to lapatinib.
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Owns stock (not including shares held through a public mutual fund): Wenle Xia, GlaxoSmithKline; Deborah A. Smith, GlaxoSmithKline; Kevin M. Koch, GlaxoSmithKline; Andrew Stead, GlaxoSmithKline; Steven Mangum, GlaxoSmithKline; Jennifer Harris, GlaxoSmithKline; Neil L. Spector, GlaxoSmithKline. Acted as a consultant within the last 2 years: Howard Burris III, Sarah Cannon Cancer Center. Performed contract work within the last 2 years: Sarah Bacus, QDL; Albert K. Man, Prediction Sciences Inc; Troy M. Brenner, Prediction Sciences Inc.
We thank Drs Allen Oliff, Peter Goodfellow, Peter Ho, Tona Gilmer, Alan Baxter, and Lini Pandite for their critical review of the manuscript and for providing valuable discussion. In addition, we would like to thank Dot Chin for his technical assistance and Michael Stocum, Ed Kirk, Aiden Flynn, and Dr Christopher Herold for their valuable assistance in the biomarker analysis. We also would like to thank the dedicated medical, nursing, and ancillary healthcare staffs at the participating institutions for providing excellent patient care and without whose help this study would not have been feasible.
Results from this study were presented at the 40th Annual Meeting of the American Society of Clinical Oncology, New Orleans, June 5-8, 2004. Funding for this study was provided by GlaxoSmithKline. Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
10, 10 to 15, and 
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), progressive disease (x), and a patient with progressive disease despite marked inhibition of biomarkers (*) are highlighted. OD, optical density.
