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Predictive Factors for Outcome in a Phase II Study of Gefitinib in Second-Line Treatment of Advanced Esophageal Cancer Patients

Maarten L. Janmaat, Mariëlle I. Gallegos-Ruiz, José A. Rodriguez, Gerrit A. Meijer, Walter L. Vervenne, Dick J. Richel, Cees Van Groeningen, Giuseppe Giaccone

From the Departments of Medical Oncology and Pathology, Vreije Universiteit Medical Center; and Academic Medical Center, Amsterdam, the Netherlands

Address reprint requests to Giuseppe Giaccone, MD, PhD, Department of Medical Oncology, Vreije Universiteit Medical Center, De Boelelaan 1117, PO Box 7057, MB 1007 Amsterdam, the Netherlands; e-mail: g.giaccone{at}vumc.nl

	ABSTRACT

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

 
PURPOSE: The efficacy of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib was assessed in a phase II study in patients with advanced esophageal cancer. Several biologic features were investigated as potential markers of gefitinib activity.

PATIENTS AND METHODS: Patients with advanced esophageal cancer, who had failed one line of prior chemotherapy, were administered gefitinib 500 mg/d. Response was evaluated every 8 weeks. Tumor material obtained before gefitinib treatment was investigated for gene mutations in EGFR, k-ras, and PIK3CA; protein expression levels of EGFR, p-Akt, and p-Erk; and EGFR gene amplification.

RESULTS: Of the 36 enrolled patients, one (2.8%) achieved a partial response, 10 (27.8%) had stable disease, 17 (47.2%) experienced progression on treatment, and eight (22.2%) were not assessable for response. The progression-free survival time was 59 days, and the median overall survival time was 164 days. Although EGFR or PIK3CA mutations were absent, k-ras mutations were found in two patients with progressive disease. High EGFR gene copy number was identified in two patients experiencing partial response or progressive disease. A higher disease control rate (response plus stable disease) was observed in females (P = .038) and in patients with squamous cell carcinoma (SCC; P = .013) or high EGFR expression (P = .002).

CONCLUSION: Gefitinib has a modest activity in second-line treatment of advanced esophageal cancer. However, the patient outcome was significantly better in female patients and in patients demonstrating high EGFR expression or SCC histology. The selection of esophageal cancer patients for future studies with EGFR-TKIs based on the level of EGFR expression in their tumors or SCC histology should be considered.

	INTRODUCTION

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

 
Esophageal cancer is a disease with a high mortality and is the fastest growing malignancy in the United States.¹ Survival depends on the stage of the disease. Surgical resection is the treatment of choice for early lesions. In recent years, possibly as a result of the introduction of broader use of flexible endoscopes, tumors that are confined to mucosa and submucosa are more frequently diagnosed.² However, even when surgery can be performed, survival is still poor, with only 5% to 20% of patients alive at 5 years.³ The introduction of neoadjuvant chemotherapy and chemoradiotherapy has improved survival in several series; however, there is still no consensus on whether neoadjuvant therapy is indicated in all operable patients.⁴ When patients are not operable or they relapse after operation, chemotherapy can induce response rates in approximately 30% to 40% of patients; however, survival of patients with advanced esophageal cancer is poor, with a median survival time of 7 to 8 months. Although there is no standard chemotherapy for advanced esophageal cancer, platinum-based regimens have been used mostly in fit patients. The addition of new cytotoxic agents like paclitaxel and irinotecan seems to lead to higher response rates. A number of patients who progress after first-line chemotherapy administered for advanced disease may still be fit for second-line treatment. There is no drug presently available with substantial activity in this setting.⁴

Gefitinib (ZD1839, Iressa; AstraZeneca, Wilmington, DE) is a specific tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR)⁵ that has been approved in Japan and a number of other counties as single-agent therapy for patients with refractory non–small-cell lung cancer (NSCLC). The response rate in white patients is 10% to 20%, whereas another 20% to 30% of the treated patients show stable disease for at least 2 months.⁶ Somatic mutations within the EGFR kinase domain correlate with a dramatic clinical response to gefitinib in NSCLC patients.^7,8 Moreover, gefitinib-treated NSCLC patients with nuclear phospho-Akt tumor staining demonstrated an improved outcome.⁹ Conversely, k-ras mutations are associated with primary resistance of NSCLC patients to EGFR-TKI therapy.¹⁰ In contrast, EGFR, human epidermal growth factor receptor 2, and phospho-Erk stainings did not predict for response of NSCLC patients to gefitinib.^11,12

EGFR is a membrane-bound tyrosine kinase receptor that mediates growth and survival signals.¹³ EGFR is activated on binding of ligand to its extracellular domain, resulting in autophosphorylation and activation of downstream signaling molecules, such as Ras, Erk, PI3K, and Akt. EGFR plays a prominent role in tumorigenesis because it promotes growth of cells and is highly expressed and/or mutated in a variety of solid tumor types, including esophageal cancer, NSCLC, and glioma.¹⁴ EGFR overexpression was observed in 29% to 92% of esophageal tumors, which was correlated with poor patient prognosis and inferior response to therapy.¹⁵

In this study, we present the results of a phase II study of second-line gefitinib monotherapy for patients with advanced esophageal cancer, after treatment failure with chemotherapy. We investigated the tumor material of these patients for a number of potential biologic markers of activity.

	PATIENTS AND METHODS

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

 
This was a phase II study of gefitinib in patients who experienced relapse after chemotherapy for advanced esophageal cancer (1839IL/0059). The primary objective of this study was to assess tumor response according to Response Evaluation Criteria in Solid Tumors criteria¹⁶; secondary end points were to estimate the duration of responses and progression-free survival, the disease control rate, and the tolerability of the treatment.

Patients
Entry criteria included histologically confirmed carcinomas of the esophagus, WHO performance status of 2 or less, and measurable or assessable disease. Patients with cancers of the gastroesophageal junction were included if more than 50% of the tumor was localized in the esophagus. Patients had to have experienced relapse after one chemotherapy regimen, administered at least 4 weeks before, and have WBC count of more than 4,000/µL, platelet count of more than 100,000/µL, serum creatinine within 1.5x the upper normal limit, and transaminases up to 5x the upper normal limit in case of liver metastases. Patients had to be greater than 18 years old, and life expectancy was to be 12 weeks or longer. Excluded were patients who received prior treatment with an EGFR inhibitor; patients with brain metastases, with unresolved toxicities from prior treatment, or with other active malignancies in the last 5 years; female patients who were pregnant or breast feeding; and patients using concomitant liver enzyme–inducing medicines (eg, phenobarbital, phenytoin). Patients provided written informed consent, and the study was approved by the ethical committees of the two institutions that participated in this study.

Treatment
Before enrollment, patients underwent clinical examination, ECG, full blood counts, chemistries and urinalysis, and tumor assessment by computed tomography scans of the chest and abdomen. During treatment, clinical examination, full blood counts, and chemistries were repeated every 2 weeks for the first 2 months and thereafter every month. Tumor assessment was repeated every 8 weeks. Tumor biopsies were performed at start of treatment and every 4 weeks whenever feasible. Gefitinib was administered at 500 mg/d (two 250-mg tablets at each dosing) by oral route without interruption, unless severe toxicity ensued. In case of excessive toxicity, interruptions were allowed up to 14 days. If the adverse effect did not return to lower than grade 2, dose reduction to 250 mg/d was allowed. No more than one dose reduction was permitted. Toxicity was assessed according to the National Cancer Institute Common Toxicity Criteria grading system. Treatment continued until excessive toxicity, disease progression, or patient request.

DNA Isolation, Polymerase Chain Reaction, and Sequencing
Tumor specimens obtained at the time of primary diagnosis or study entry were collected to analyze the gene status of EGFR, k-ras, and PIK3CA. Paraffin-embedded tissue sections were macrodissected, and total genomic DNA was isolated using QIAamp DNA extraction kits (Qiagen, Venlo, the Netherlands). Nested polymerase chain reactions were carried out using the isolated DNA as template and external and internal primers as previously described.¹⁷ Sequencing of polymerase chain reaction products was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), M13 primers,¹⁷ and the ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Immunohistochemical Staining
Tumor slides were deparaffinized, autoclaved in 10 mmol/L of citrate buffer (pH 6.0), and incubated with EGFR antibody (VUMC, homemade antibody), p-Akt (Ser 473) antibody (#9277; Cell Signaling, Beverly, MA), or P-p44/42 Mapk (Thr202/Tyr204) antibody (#9106; Cell Signaling) overnight at 4°C. The sections were developed using the DAKO EnVision visualization system (DAKO, Copenhagen, Denmark). At present, there are no validated scoring systems for interpreting immunohistochemical staining for EGFR, p-Akt, or p-Erk. We used a system for interpreting EGFR staining that was based on scoring human epidermal growth factor receptor 2 staining,¹⁸ as follows: 0, none of the tumor cells stained; 1+, the staining of the tumor cell membranes was weak and incomplete; 2+, the staining of the tumor cell membranes was moderate and complete; and 3+, the staining of the tumor cell membranes was strong and complete. Interpreting p-Akt and p-Erk staining was based on staining incidence and intensity, as follows: 0, none of the tumor cells stained; 1+, less than 10% of the tumor cells stained weakly; 2+, more than 10% of the tumor cells stained moderately; and 3+, more than 25% of the tumor cells stained strongly. Interpretation of immunohistochemically stained slides was performed by a pathologist (G.A.M.) who was blinded to the clinical data.

Chromogenic In Situ Hybridization
Tumor sections were deparaffinized, boiled for 10 minutes in 1 mmol/L of EDTA/Tris (pH 9.0), digested with 0.01% pepsin/0.2 N HCl at 37°C, washed, and dried. Subsequently, the slides were incubated with the Zymed POT-light EGFR amplification probe (Zymed Laboratories Inc, San Francisco, CA) for 16 to 24 hours at 37°C after denaturation at 80°C. After blocking with goat antiserum and incubation with a monoclonal antidigoxin antibody (Clone DI-22; Sigma, Zwijndrecht, the Netherlands), the sections were developed using the DAKO EnVision visualization system (DAKO). Similar to EGFR chromogenic in situ hybridization (CISH) analysis by Bhargava et al,¹⁹ tumors were considered to be CISH positive when more than 50% of the tumor cells showed tight EGFR clusters or had more than four EGFR gene copies. Evaluation of the slides was performed independently by two authors who were blinded to the patients' clinical characteristics and all other molecular variables.

Study Design and Statistical Analysis
The study was performed as a phase II single-agent trial using a Fleming's single-stage design. The minimum required response rate of interest was set at 5%, and the response of clinical interest was set at 20%. The required number of patients to be recruited was 38, based on a 90% power. Response rates and controlled disease rates were summarized by percentages and their 95% CIs. Survival curves were constructed using the Kaplan-Meier method, and differences between the two groups were analyzed using the log-rank test. Patient groups were compared with the Spearman's Rho or ² tests when appropriate. All statistical analyses were performed using SPSS software (version 11.0.1; SPSS Inc, Chicago, IL). Correlation was significant at the .05 level (two tailed) or the .01 level (two tailed).

	RESULTS

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Patient Characteristics and Treatment Outcome
Thirty-seven patients were screened and enrolled from February 2002 to February 2004, and 36 patients were eligible for the study and received at least one dose of gefitinib. One patient was found to be ineligible because of poor performance status and deteriorating conditions and did not start treatment. The main patient characteristics are listed in Table 1. The majority of patients were male, had good performance status, and had adenocarcinoma, and half of the patients had prior surgery.

View larger version (7K): [in this window] [in a new window]   Fig 1. Kaplan-Meier curve for overall survival of pretreated esophageal cancer patients treated with gefitinib. Survival was calculated from the date of gefitinib therapy initiation to the date of death. Median survival time was 164 days (95% CI, 0 to 333 days).

Expression of EGFR
Among the 24 tumors evaluated for EGFR expression, 15 (62.5%) had low or moderate (0, 1+, or 2+) EGFR expression and nine (37.5%) were highly positive (3+) for EGFR (Table 4; Fig 2). High EGFR expression was significantly associated with SCC histology but not with other clinical parameters (Table 4). The disease control rate was 66.7% for patients with high EGFR-expressing tumors, which was significantly higher than the rate for patients with low to moderate EGFR-expressing tumors (Table 5). The time to progression was longer for patients with high EGFR-expressing tumors (median, 153 days; 95% CI, 36 to 270 days) than for patients with low or moderate EGFR-expressing tumors (median, 55 days; 95% CI, 42 to 68 days), although this was not statistically significant (Fig 3A). The overall survival time for patients with high EGFR-expressing tumors was longer (median, 233 days; 95% CI, 110 to 356 days) compared with the overall survival time of patients with low or moderate EGFR-expressing tumors (median, 83 days; 95% CI, 25 to 141 days), but this was not significantly different (Fig 3B).

View larger version (147K): [in this window] [in a new window]   Fig 2. EGFR, p-Akt, and p-Erk staining as determined by immunohistochemistry. Panels show representative examples of esophageal tumor specimens stained for (A, D, G, and J) total EGFR, (B, E, H, and K) p-Akt, and (C, F, I, and L) p-Erk. Panels illustrate specimens with (A-C) 0, (D-F) 1+, (G-I) 2+, and (J-L) 3+ expression levels. All esophageal tumors were positive for p-Akt, hence SW1573 non–small-cell lung cancer cells were shown as a control for (B) negative staining (0) of p-Akt.

View larger version (14K): [in this window] [in a new window]   Fig 3. Kaplan-Meier curves for (A, C, and E) time to disease progression and (B, D, and F) survival according to protein expression levels of (A and B) epidermal growth factor receptor (EGFR), (C and D) p-Akt, and (E and F) p-Erk. Time to disease progression was calculated from the date of initiation of gefitinib treatment to the date of detection of progressive disease. Survival was calculated from the date of therapy initiation to the date of death. Statistical significance of differences between groups was evaluated with the log-rank test.

Expression of p-Erk
Of the 22 tumors that were stained for p-Erk, 14 (63.6%) had negative (0 or 1+) p-Erk expression, and eight (36.4%) showed positive (2+ or 3+) p-Erk expression (Table 4). High Erk phosphorylation was observed more frequently in adenocarcinomas and in patients who had no prior radiotherapy (Table 4). Disease control rates, the median time to progression (56 days; 95% CI, 41 to 70 days for patients with negative p-Erk expression v 62 days; 95% CI, 27 to 97 days for patients with positive p-Erk expression), and the median survival time (115 days; 95% CI, 82 to 148 days for patients with negative p-Erk expression v 234 days; 95% CI, 0 to 535 days for patients with positive p-Erk expression) were similar among patients whose tumors differed on the basis of p-Erk status (Figs 3E and 3F).

EGFR Gene Copy Number
EGFR gene copy numbers of 16 tumors were evaluated by CISH. Two tumors (12.5%) were considered CISH positive (data not shown); these tumors were from a patient with a partial response and a patient with disease progression on gefitinib treatment. Similar to the recent findings of Hanawa et al,²⁰ the two CISH-positive patients showed high (3+) EGFR protein expression and SCC histology.

	DISCUSSION

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

 
Gefitinib administered at 500 mg/d in second-line treatment of advanced esophageal cancer patients who experienced treatment failure after chemotherapy induced one partial response. Although this response rate (2.8%) is disappointing, 30.6% of patients demonstrated controlled disease. These results suggest that gefitinib as monotherapy is less effective than historical controls, which, with cytotoxic chemotherapy regimens, have recorded response rates of 12% to 30% for patients who have received second-line therapy, with progression-free survival times of approximately 3.5 months^21,22; the progression-free survival time was 2 months in this study. The safety profile of gefitinib administered at 500 mg is similar to other studies with this agent and demonstrates that the drug is well tolerated. The overall adverse events profile was, as expected, predominated by GI and skin-related events. Although the clinical results were disappointing, we attempted to identify the patients who benefited from gefitinib treatment, in particular the patients who had a response or stable disease (Table 6).

In addition to clinical parameters, we investigated several potential biologic markers of gefitinib activity. Given the fact that only a part of the tumors were assessable for biomarker analysis, the results presented in this study are, unfortunately, not conclusive. The finding that EGFR kinase domain mutations were not detected in esophageal tumors may be a result of the low frequency of EGFR mutations detected in tumors other than NSCLC.²⁴ In line with our results, no EGFR mutations were reported in a recent study evaluating 40 esophageal SCCs.²⁰ Conversely, we did not observe such dramatic clinical responses on treatment with gefitinib in this study as those that have been described for EGFR-mutant NSCLC patients treated with EGFR-TKIs.^8,7 However, patients with high EGFR expression experienced significantly better controlled disease and demonstrated a trend towards improved time to progression and overall survival while on treatment. Our data suggest that patients with high EGFR expression may have a better prognosis when treated with EGFR-TKIs because high EGFR expression has been shown to be an adverse prognostic factor for esophageal cancer patients.^25,26 Because esophageal cancers overexpress EGFR in 29% to 92% of patients,¹⁵ it is conceivable that a higher response or disease control rate might be achieved if patients were selected on the basis of high EGFR expression.

Two of four patients with high EGFR expression were also CISH positive (data not shown). This suggests that EGFR overexpression is caused by increased EGFR gene copy number in just a fraction of esophageal tumors, like Hanawa et al²⁰ reported recently. Our data further suggest that analysis of EGFR expression by immunohistochemistry is more effective to predict gefitinib efficacy in esophageal cancer patients than EGFR gene copy number, in contrast to NSCLC.^11,27,12 However, direct comparisons are difficult between the immunohistochemical data of the different studies because of differences in antibodies used and interpretation of the results. The introduction of a standardized system for scoring EGFR immunohistochemical stainings is needed.

EGFR-independent activity of the downstream Ras/Erk and PI3K/Akt pathways confers resistance of tumor cells to gefitinib treatment.²⁸ Similar to NSCLCs,^17,10 the presence of k-ras mutations in esophageal tumors may be a factor of resistance to gefitinib because two patients with k-ras mutant tumors experienced disease progression on gefitinib therapy. However, phosphorylation of Erk, downstream of k-ras,²⁹ was not elevated in tumors with k-ras mutations, and p-Erk status was not predictive for outcome to gefitinib treatment. No mutations were detected in the PIK3CA gene in 24 esophageal tumors. PIK3CA mutations are relatively frequent in colorectal, breast, and ovarian tumors but less frequent in other tumor types.^30,31 Low p-Akt staining was associated with better disease control and longer progression-free survival in the small group of patients evaluated. However, p-Akt status, downstream of PI3K,³² will not be a good predictive factor for gefitinib therapy in esophageal cancer because of the difficult evaluation of phosphorylated Akt levels.

In conclusion, gefitinib treatment of unselected patients with advanced, pretreated esophageal cancer has modest activity. However, female sex, high EGFR expression levels, and SCC histology are associated with better patient outcome. The selection of esophageal cancer patients for future studies with EGFR-TKIs based on the level of EGFR expression in their tumors or SCC histology should be considered.

	Authors' Disclosures of Potential Conflicts of Interest

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

 

Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other Maarten L. Janmaat AstraZeneca (B) Giuseppe Giaccone AstraZeneca (B)

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

	Author Contributions

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

 

Conception and design: Maarten L. Janmaat, José A. Rodriguez, Dick J. Richel, Giuseppe Giaccone Administrative support: Mariëlle I. Gallegos-Ruiz Provision of study materials or patients: Gerrit A. Meijer, Walter L. Vervenne, Dick J. Richel, Cees Van Groeningen, Giuseppe Giaccone Collection and assembly of data: Maarten L. Janmaat, Mariëlle I. Gallegos-Ruiz, Gerrit A. Meijer, Walter L. Vervenne, Cees Van Groeningen, Giuseppe Giaccone Data analysis and interpretation: Maarten L. Janmaat, Mariëlle I. Gallegos-Ruiz, José A. Rodriguez, Gerrit A. Meijer, Giuseppe Giaccone Manuscript writing: Maarten L. Janmaat, José A. Rodriguez, Gerrit A. Meijer, Giuseppe Giaccone Final approval of manuscript: Maarten L. Janmaat, Mariëlle I. Gallegos-Ruiz, José A. Rodriguez, Gerrit A. Meijer, Walter L. Vervenne, Dick J. Richel, Cees Van Groeningen, Giuseppe Giaccone

 

	Acknowledgment

 
We thank Karijn Floor for performing the chromogenic in situ hybridization experiments, and Carlos Ferreira for drafting the clinical protocol.

	NOTES

 
Supported in part by AstraZeneca.

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

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Submitted August 1, 2005; accepted January 24, 2006.

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