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Bexarotene and Erlotinib for Aerodigestive Tract Cancer

From the Hematology/Oncology Section, Department of Medicine, Norris Cotton Cancer Center; Departments of Pharmacology and Toxicology and Pathology, Dartmouth Medical School and Dartmouth College Hanover; and Dartmouth-Hitchcock Medical Center, Lebanon, NH

Address reprint requests to Ethan Dmitrovsky, MD, Department of Pharmacology and Toxicology, Remsen 7650, Dartmouth Medical School, Hanover, NH 03755; e-mail: ethan.dmitrovsky{at}dartmouth.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: The epidermal growth factor receptor (EGFR) and cyclin D1 are overexpressed in lung carcinogenesis. The rexinoid, bexarotene, represses cyclin D1 and EGFR expression in vitro. It was hypothesized that combining bexarotene with the EGFR inhibitor, erlotinib, would augment clinical activity.

PATIENTS AND METHODS: In vitro studies and a phase I clinical trial were performed. Twenty-four patients with advanced aerodigestive tract cancers were enrolled; 79% had non–small-cell lung cancer (NSCLC). The primary objective was to determine the maximum-tolerated dose. Clinical activity was a secondary objective.

RESULTS: Combining erlotinib with bexarotene enhanced growth suppression in vitro compared with each single-agent treatment. This cooperatively repressed cyclin D1 expression. Clinically, the most frequent toxicities were mild hypertriglyceridemia and skin rash. Two serious treatment-related adverse events occurred (creatine phosphokinase elevation attributed to antilipid therapy and a case of generalized pain). Five objective responses (four partial and one minor) were observed in NSCLC patients. Responses were observed in males and smokers. EGFR sequence analyses did not reveal activating mutations in tumors from assessable responding patients. Median time to progression was 2.0 months; overall survival time was 14.1 months; and 1-year survival rate was 73.8%.

CONCLUSION: The recommended phase II doses are erlotinib 150 mg/d and bexarotene 400 mg/m²/d orally. These agents can be administered in combination at the recommended single-agent doses without added toxicity. Overall survival and clinical features of responding patients differ from prior reports of single-agent erlotinib treatment. These findings are encouraging and warrant further investigation of this regimen.

	INTRODUCTION

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Lung cancer is the leading cause of cancer-related mortality in the United States.¹ More effective lung cancer therapeutic and chemopreventive strategies are needed.² The epidermal growth factor receptor (EGFR) and its ligands are important in normal and neoplastic epithelial cell growth.³ Targeting the EGFR with the EGFR-tyrosine kinase inhibitor (TKI) erlotinib caused clinical responses and significantly prolonged survival in chemotherapy-refractory advanced non–small-cell lung cancer (NSCLC) patients.^4,5 Activating mutations of the tyrosine kinase domain of the EGFR were reported to predict sensitivity of NSCLC for response to EGFR-TKI treatments.^6,7 However, only a small subset of patients achieve objective responses to targeting the EGFR with an EGFR-TKI. Novel strategies are needed to broaden clinical efficacy of these agents.

We recently reported results from a mechanistic proof-of-principle trial at Dartmouth where the EGFR-TKI erlotinib (Tarceva; Genentech, South San Francisco, CA; OSI Pharmaceuticals, Melville, NY) was administered as short-term therapy in patients with aerodigestive tract tumors.⁸ Cyclin D1 expression was found as a critical biomarker of response. The epidermal growth factor can induce cyclin D1 expression through several pathways.^9-15 Targeting the EGFR with erlotinib caused G₁ arrest and inhibited an epidermal growth factor–mediated induction of cyclin D1 transcription and expression in erlotinib-sensitive cancer cells.⁸ Notably, cyclin D1 expression was unaffected by erlotinib treatment of erlotinib-resistant NSCLC cell lines.⁸ Taken together, these findings directly implicate cyclin D1 as a key signal in the EGFR pathway and suggest that targeting cyclin D1 with a combination regimen that affects this species would augment clinical activity of this EGFR-TKI.

The retinoids, natural and synthetic derivatives of vitamin A, exert antiproliferative, differentiation-inducing, and proapoptotic effects, as reviewed.¹⁶ Retinoid X receptor agonists (rexinoids) and classical retinoic acid receptor (RAR) agonists activate distinct nuclear receptors but can engage related pathways, as reviewed.¹⁷ A rexinoid can bypass RARß repression, which is frequent in NSCLC and likely contributes to resistance to classical retinoids that activate RARs.² The rexinoid bexarotene (Targretin; Ligand Pharmaceuticals, San Diego, CA) has shown activity in NSCLC.^18-20 Potential cooperation between the retinoid and EGFR pathways has been reported.^21,22 In the BEAS-2B immortalized human bronchial epithelial (HBE) cell line, both retinoids and rexinoids²³ were found to repress EGFR expression, inhibit cell cycle progression, and induce cyclin D1 proteolysis through proteasome-dependent degradation.^21,23-25

It was hypothesized that combining an EGFR inhibitor with a rexinoid would coordinately repress cyclin D1 expression and thereby confer cooperative clinical antitumor effects.^26,27 To explore this, in vitro studies reported here revealed that combining the rexinoid, bexarotene, with the EGFR-TKI, erlotinib, yielded at least additive growth-inhibitory effects and cooperative repression of cyclin D1 immunoblot expression in examined HBE and NSCLC cells. On the basis of these findings, an Investigational New Drug application was filed for a phase I clinical trial that combined erlotinib and bexarotene in patients with advanced aerodigestive tract tumors. The primary objective was to evaluate the safety of administering bexarotene in combination with erlotinib, both administered orally on a daily basis, and to determine the recommended phase II doses. The secondary objectives were to evaluate response rates, survival, and biomarker changes in harvested pretreatment versus post-treatment buccal-swab specimens. Findings reported here indicate the clinical activity of this targeted combination regimen.

	PATIENTS AND METHODS

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Cell Culture
BEAS-2B cells were derived from normal HBE cells by immortalization with SV-40, as previously described.²⁸ All-trans-retinoic acid–resistant BEAS-2B-R1 cells were derived from parental BEAS-2B cells and exhibit repressed RARß expression.²⁴ These lines were passaged in LHC-9 media.^23,25,29 Erlotinib and bexarotene were each dissolved in the vehicle dimethyl sulfoxide. The A427 lung cancer cell line was passaged as recommended by American Type Culture Collection (Manassas, VA).

Proliferation Assays
Cellular proliferation was measured using the MTT growth assay, as described previously.⁸ Cells were plated for assays with four to six replicates per experiment. Cells were then treated with erlotinib, bexarotene, or the combination of the two agents at various clinically achievable doses or with the vehicle, dimethyl sulfoxide, for 72 hours. Growth was determined by measuring absorbance at 72 hours, and results were normalized to the absorbance measured in vehicle-treated cells that served as controls. The combination index was calculated to assess potential cooperative interactions between agents administered in combination.^30,31

Immunoblot Assays
BEAS-2B-R1 and A427 cells were independently treated with erlotinib alone, bexarotene alone, combinations of these two agents, or vehicle alone. Cells were harvested and subjected to immunoblot analyses, as previously described.^8,23,25,29 Antibodies were purchased that recognized cyclin D1 (M-20; Santa Cruz Biotechnology, Santa Cruz, CA) or actin (C-11; Santa Cruz Biotechnology).

EGFR Genomic DNA Sequence Analyses
Genomic DNA from clinical lung cancer specimens was extracted from paraffin-embedded tissues using a DNEasy Tissue Kit (Qiagen, Valencia, CA) and the procedures recommended by the manufacturer. Polymerase chain reaction (PCR) assays were performed using established techniques to amplify EGFR exons 18, 19, and 21.^6-8 PCR products were purified using the QIAquick kit (Qiagen) and sequenced using established techniques.^6-8 These PCR products were also subjected to clonal sequence analysis, as described.⁸

Patients
Eligible patients had a histologic or cytologic diagnosis of carcinoma of the aerodigestive tract, including lung, head and neck, and esophageal cancers; advanced-stage disease with no known curative treatment options; Karnofsky performance status of 60% or greater; and age greater than 18 years. Prior chemotherapy or radiotherapy was allowed. Fasting triglycerides had to be less than the upper limit of normal (ULN). Effective contraception or sexual abstinence was required for female patients of childbearing potential or male patients with female partners of childbearing potential.

Exclusion criteria included hepatic dysfunction, as evidenced by either bilirubin greater than the ULN or transaminase (AST or ALT) greater than 2.5x ULN or greater than 5x ULN if there were known liver metastases; renal dysfunction (creatinine clearance < 30 mL/min); and a serious uncontrolled medical disorder or active infection that would have impaired the patient’s ability to receive study treatment. Patients with dementia or altered mental status prohibiting understanding or rendering of informed consent and compliance with the protocol were excluded. Prior use of bexarotene, erlotinib, or other EGFR inhibitors or concurrent use of other anticancer approved or investigational agents was not allowed. Patients with known hypersensitivity to bexarotene, erlotinib, or other components of the capsules or with risk factors for pancreatitis were also excluded.

All inclusion and exclusion criteria were assessed within 14 days before initiation of therapy with the exception of radiographic studies, which were performed within 28 days of screening. This clinical study was conducted after approval by the Committee for the Protection of Human Subjects at Dartmouth College and the Institutional Review Board. Informed consent was obtained from each patient enrolled onto the study.

Study Drugs
An Investigational New Drug application for the combination of erlotinib and bexarotene was submitted to the US Food and Drug Administration and was granted to the Principal Investigator (K.H.D.). Dose-limiting toxicity (DLT) was defined as greater than grade 3 hematologic or nonhematologic toxicity (except hyperlipidemia, nausea or vomiting, or anaphylactoid reactions; National Cancer Institute Common Toxicity Criteria version 2.0). The recommended phase II dose was defined as the highest dose level of bexarotene in combination with erlotinib that induced DLT in fewer than 33% of patients (ie, one dose level below that which induced DLT in two of six patients). Cohorts of three to six eligible patients were treated with escalating doses of oral erlotinib with bexarotene oral capsules. The agents were taken at the same time daily without interruption.

Three dose levels of bexarotene in combination with erlotinib were studied (Table 1). Eligible patients were entered in cohorts of three at each dose level. Doses were not escalated over the course of treatment of an individual patient. If a single patient experienced grade 4 hematologic or nonhematologic toxicity (excluding hyperlipidemia), three additional patients were entered at the same dose. Dose escalation was performed after all patients at the previous dose level received treatment for 4 weeks. Additional patients were treated at the recommended phase II dose levels to characterize the toxicity profile and to perform pharmacodynamic studies. Treatment continued until progression of disease, unacceptable adverse effects, or withdrawal of informed consent. Atorvastatin was started if abnormally high fasting triglyceride or cholesterol levels were detected.

Buccal Mucosa Specimens
Buccal specimens were harvested on days 1 (before treatment) and 15 by swabbing the buccal mucosa first with a soft-bristled brush (discarded) and then with a firm-bristled brush. The firm-bristled brush was immediately placed in ice-cold phosphate-buffered saline and transferred to a –85°C freezer within 1 hour. Cells were lysed using phenol-chloroform extraction, and equal amounts of protein were subjected to immunoblot analyses and optical densitometry to determine changes in cyclin D1 biomarker expression after normalizing to actin expression as a control.

Statistical Analysis
Time to progression and overall survival were determined using the Kaplan-Meier method. Changes in growth-suppressive effects in vitro were compared using the two-sample t test, with significance defined as a two-sided P < .05.

	RESULTS

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Effects of Bexarotene and Erlotinib on Cell Growth and Cyclin D1 Expression
Both erlotinib and bexarotene suppressed the growth of the all-trans-retinoic acid–resistant BEAS-2B-R1 cell line and the A427 lung cancer cell line at pharmacologically achievable doses. As shown in Figure 1, combining bexarotene and erlotinib significantly increased growth suppression compared with each single agent (P < .05). When BEAS-2B-R1 cells were treated with varying concentrations of erlotinib and bexarotene at a fixed ratio, the combination index was 1.0 across a broad dose range, indicating at least additive antiproliferative effects, as shown in Figure 2A. Low doses of these individual agents each had minimal effects on cyclin D1 immunoblot expression. However, treatment with the combination regimen caused substantial repression of this biomarker, as shown in Figure 2B. Pharmacokinetic studies from independently completed clinical trials with erlotinib and bexarotene indicate that the concentrations used in these in vitro studies were within clinically achievable dose ranges^8,32 (data not shown).

View larger version (10K): [in this window] [in a new window]   Fig 1. Growth suppression induced by bexarotene and erlotinib in BEAS-2B-R1 and A427 cells. BEAS-2B-R1 human bronchial epithelial cells and A427 lung cancer cells were independently treated with subtherapeutic doses of bexarotene, erlotinib, or a combination of both drugs. (*) Significantly increased growth suppression compared with each single-agent treatment (P < .05).

View larger version (9K): [in this window] [in a new window]   Fig 2. The combination of bexarotene and erlotinib induces at least additive inhibitory effects on growth and cyclin D1 protein expression. (A) The combination index method was applied to growth assay results from BEAS-2B-R1 cells. (B) Cooperative repression of cyclin D1 immunoblot expression was observed in BEAS-2B-R1 (as shown in this figure) and A427 (data not shown) cells.

Clinical Activity
Results are based on an intent-to-treat analysis. Four patients (NSCLC) had objective partial radiographic responses. One NSCLC patient with bronchioloalveolar carcinoma at dose level 1 had a minor response (23% reduction of the sum of the longest dimensions of all measurable lesions). Nine patients had stable disease (including the patient with the minor response), which was defined as changes not sufficient for diagnosis of response or progression, first detected at 4 weeks and confirmed at 6 weeks. The characteristics of these responding patients are listed in Table 5. Median time to progression for all patients was 2.0 months. The median overall survival time was 14.1 months for all patients and for the NSCLC patients. The 1-year survival rates for all patients and NSCLC patients were 73.8% and 72%, respectively. The median overall follow-up time was 8.3 months.

Cyclin D1 Expression in Buccal Swabs
Cyclin D1, a candidate biomarker of erlotinib and bexarotene response,^8,23 was assessed by immunoblot analyses performed in pretreatment (day 1) and in paired post-treatment (day 15) harvested buccal mucosal swabs. These samples were obtained from patients treated at dose level 3, as described in Patients and Methods. Normalized cyclin D1 protein expression was repressed in post-treatment buccal swabs compared with pretreatment buccal swabs in five assessable patients, as displayed in Figure 3A. A representative immunoblot is shown in Figure 3B.

View larger version (10K): [in this window] [in a new window]   Fig 3. Cyclin D1 protein expression in pretreatment versus post-treatment buccal mucosal swabs. (A) The normalized cyclin D1 protein signals are displayed for pretreatment and post-treatment buccal mucosal swabs. (B) The cyclin D1 immunoblot expression profile is shown for a representative patient, with actin expression serving as a loading control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

We hypothesized, on the basis of our prior work,^21,27 that combining an EGFR antagonist and a rexinoid would confer at least additive clinical benefits by cooperatively repressing the EGFR signaling pathway and cyclin D1 expression.^26,27 This is an anticipated outcome based on our findings that erlotinib and bexarotene have cyclin D1, a previously highlighted biomarker of clinical erlotinib response, as a common downstream target.⁸ In vitro studies of BEAS-2B-R1 HBE cells and A427 lung cancer cells have shown that combining bexarotene with erlotinib significantly enhances growth suppression (P < .05) and repression of cyclin D1 protein.

This phase I clinical trial established the safety of the combination regimen of erlotinib and bexarotene in patients with advanced aerodigestive tract tumors. Mild toxicities were noted (mainly hypertriglyceridemia, skin rash, and subclinical hypothyroidism) that were not different from the toxicities expected for single-agent bexarotene or erlotinib treatment.^{4,5,18,32,33,36,37} Both drugs were administered together at their individual US Food and Drug Administration–approved doses for single-agent use, without reaching the maximum-tolerated dose. The recommended phase II dose for the combination is erlotinib 150 mg/d and bexarotene 400 mg/m²/d. The results from the described preclinical data, the present targeted combination trial, and our prior work provide a strong rationale for exploring cooperation between rexinoids and EGFR-TKIs in the treatment of advanced aerodigestive tract cancer and, in particular, lung cancer.

Four patients achieved partial radiographic response. Because all observed objective clinical responses occurred in NSCLC patients, these clinical data point to activity in this group of patients. Further evidence for activity of this regimen comes from the observation of a survival time of greater than 14 months, which is longer than the previously reported survival time with single-agent erlotinib^4,5,34 or bexarotene.²⁰ This observed prolongation in survival is a somewhat surprising outcome given that this result was observed in the setting of a phase I trial. As such, this trial was not designed to establish a survival advantage of this combination regimen, which was administered at varying dose levels. Notably, objective clinical responses were observed at all the examined dose levels of this regimen. The characteristics of the responding patients differed from what was expected based on single-agent EGFR-TKI trials in lung cancer.^4-7,40,41

Prior EGFR-TKI studies highlighted clinical responders as more likely to be women, nonsmokers, and patients with bronchioloalveolar carcinoma histology and with activating EGFR mutations, as previously reviewed.^40,41 The molecular pharmacologic relationship between these prognostic features and potential survival benefit needs to be determined. The patients who responded to the combination regimen reported here were predominately men, largely current or former smokers, and infrequently patients with bronchioloalveolar carcinoma histology. There were no EGFR activating mutations identified in the examined tumor specimens from responding patients. This could reflect the small number of tumors that were available for sequence analyses. Alternatively, this finding may indicate that the combination of erlotinib and bexarotene has a broader range of activity than single-agent erlotinib treatment and that clinical cooperation between these agents might overcome a requirement for activating EGFR mutations to achieve clinical responses to EGFR-TKIs in NSCLC.

Taken together, these findings support the hypothesis that combining bexarotene with erlotinib increases the clinical activity of single-agent erlotinib. This hypothesis should be examined further in a confirmatory phase II trial or perhaps in a randomized phase III clinical trial designed to compare survival of patients treated with the bexarotene and erlotinib combination with survival of patients treated with single-agent erlotinib therapy.

	Authors' Disclosures of Potential Conflicts of Interest

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The author or immediate family members 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. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other Konstantin H. Dragnev Genentech (B); Ligand (B) James R. Rigas Ligand (A); OSI (A) Ligand (A); Genentech/OSI (A); Ligand (B); Genentech/OSI (B) Ethan Dmitrovsky Ligand (A)

Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C) $100,000 (N/R) Not Required

	NOTES

 
Presented in part at the 40th Annual Meeting of the American Society of Clinical Oncology, New Orleans, LA, June 5-8, 2004; and the 16th European Organisation for Research and Treatment of Cancer–National Cancer Institute–American Association for Cancer Research Symposium on Molecular Targets and Cancer Therapeutics, Geneva, Switzerland, September 28-October 1, 2004.

Supported by Community Clinical Oncology Program Grant No. CA37447-21 (K.H.D.), the National Institutes of Health and the National Cancer Institute Grant Nos. RO1-CA087546 (E.D.), R01-CA111422 (E.D.), and RO1-CA62275 (E.D.); a Samuel Waxman Foundation Cancer Research Award (E.D.); and the Oracle Giving Fund (E.D.). W.J.P. received grant support from the CHEST Foundation of the American College of Chest Physicians and the LUNGevity Foundation, the National Research Service Award No. T32-CA009658 from the National Institutes of Health, and an American Society of Clinical Oncology (ASCO) Young Investigator Award. K.H.D. was also supported by an ASCO Young Investigator Award. Additional funding was provided by Ligand and Genentech (K.H.D.).

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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Submitted March 11, 2005; accepted September 9, 2005.

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