This is a US government work and, as such, is in the public domain in the United States of America.
The primary objective of this study was to evaluate the biochemical effects of gefitinib on its target signal-transduction pathways in patients with recurrent epithelial ovarian cancer (EOC). The secondary objectives included assessing clinical activity and toxicity and determining the association between biochemical and clinical outcomes.
Twenty-four heavily pretreated patients with EOC who had good end-organ function and performance status and who had measurable disease received gefitinib 500 mg daily. Prospectively planned core-needle tumor biopsies were obtained before treatment and after 4 weeks. Protein expression of total and phosphorylated (p) epidermal growth factor receptor (EGFR), protein kinase B (AKT), and extracellular regulated kinase (ERK) was quantified in microdissected tumor cells using tissue lysate array proteomics.
All tumor samples had detectable levels of EGFR and p-EGFR. A decrease in the quantity of both EGFR and p-EGFR was observed with gefitinib therapy in >50% of patients. This was not associated with clinical benefit, nor were responses observed. However, trends for increased gastrointestinal and skin toxicity were observed with greater phosphorylation or quantities of EGFR, ERK, and AKT in tumor samples (P ≤ .05). Gefitinib had limited clinical activity as monotherapy despite documented target inhibition.
The results from this study demonstrated that gefitinib inhibited the phosphorylation of EGFR in EOC tumor cells, providing proof of target in a clinical setting. Combinatorial therapy with molecular therapeutics against complementary targets may prove successful. Cancer 2007. Published 2007 by the American Cancer Society.
Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy among American women.1 Debulking surgery followed by adjuvant platinum and paclitaxel chemotherapy is highly effective in remitting disease.2 However, most women who are diagnosed with advanced-stage disease eventually develop recurrences. Various cytotoxic agents are used that produce response rates in the range of 10% to 30%. In all, approximately 70% of all women who are diagnosed with EOC will die of their disease. Thus, there is a critical need for the development and assessment of new agents.
One potential target for anticancer therapy is the epidermal growth factor receptor (EGFR), which is a regulator of cell growth and differentiation in normal tissues and plays a role in promoting cell proliferation, apoptosis inhibition, and angiogenesis in malignancy.3–5 EGFR is overexpressed in from 35% to 70% of epithelial ovarian cancers, depending on the assessment technique used.6, 7 Increased EGFR signaling has been associated with the development of an invasive phenotype in ovarian cancer cell lines8, 9 and is detected more often in metastases than in primary tumor samples.10 The relation between EGFR overexpression and clinical prognosis is less clear, with some reports suggesting a prognostic importance5, 7 and others refuting that association.11 EGFR homodimerizes or heterodimerizes with other members of the erbB family upon ligand binding.12, 13 This leads to activation of the intrinsic tyrosine kinase domain, autophosphorylation and transphosphorylation, and initiation of downstream signaling cascades.5, 14
EGFR is a promising target for molecular therapeutics because of its well-studied prosurvival role. Both monoclonal antibodies and small-molecule tyrosine kinase inhibitors of EGFR have been tested in the clinic.4, 15, 16 Gefitinib (Iressa) is a small-molecule inhibitor of the tyrosine kinase of EGFR. It attaches to the adenosine triphosphate (ATP)-binding domain of the receptor, preventing phosphorylation and activation of both EGFR and important downstream signaling molecules.17 Gefitinib inhibits EGF-stimulated growth of ovarian cancer cells in vitro; this effect is cytostatic, with an increase in apoptosis observed at higher doses and when, used in combination with conventional chemotherapy, producing supraadditive growth arrest.18, 19 Gefitinib also has been tested in xenograft models of ovarian cancer, resulting in inhibitory effects on cell growth and increased survival of mice in the treatment groups.16, 20
Phase I trials of gefitinib in solid tumors, including EOC, have demonstrated tolerable toxicity and promising clinical results.15, 21–24 We hypothesized that gefitinib would inhibit the activation of EGFR in patients with ovarian cancer, effect downstream signal-transduction events, and lead to regression or stabilization of disease. The primary objective of this study was to evaluate the biochemical effects of gefitinib on signal-transduction pathways in tumor. The secondary objectives included assessing the clinical activity and toxicity of gefitinib in EOC and determining the association between biochemical and clinical outcomes.
MATERIALS AND METHODS
This study was approved by the Institutional Review Board of the National Cancer Institute (Bethesda, MD). Written informed consent was obtained before enrollment. Eligible patients had histopathologically confirmed EOC. All patients had progressing disease and were ≥4 weeks from their most recent therapeutic intervention. Other criteria included an Eastern Cooperative Oncology Group performance status of 0 to 2, a leukocyte count ≥3000/mm3, platelets ≥100,000/mm3, serum creatinine level ≤1.5 mg/dL, transaminase levels (alanine and aspartate aminotransferase) ≤2.5 × the upper limit of normal, bilirubin ≤1.5 mg/dL, measurable disease on a computed tomography (CT) scan, and a sentinel lesion amenable to percutaneous biopsy. Toxicity from prior therapies must have recovered to grade ≥1. Patients with evidence of central nervous system involvement, a history of myocardial infarction or angina within the previous 6 months, a history of another invasive malignancy within 5 years, ongoing or active infection, previous treatment with any EGFR inhibitor, and concurrent treatment with alternative or complementary medications were excluded from study.
Patients received oral gefitinib 500 mg daily on 28-day cycles until progressive disease, unacceptable toxicity, or withdrawal. They were seen every 4 weeks for history, physical, and pelvic examinations and laboratory tests, including CA125. Response was assessed every 8 weeks by imaging studies and was scored according to the Response Evaluation Criteria in Solid Tumors.25 All patients were required to keep a diary to document compliance and adverse events. Toxicity was assessed by using the National Cancer Institute Common Toxicity Criteria (version 2.0). Symptomatic management was provided to patients with gastrointestinal and dermatologic toxicities. Grade 3 or hematologic grade 4 toxicities required treatment interruption until they resolved to grade 1. Patients were not eligible to resume therapy if the time to resolution was >2 weeks or 4 weeks if they had evidence of clinical benefit. Patients had their gefitinib dose reduced to 250 mg daily in the setting of grade 3 or 4 toxicity or of recurrent grade 2 dermatologic toxicity. Further dose reductions were not allowed.
The study prospectively planned the collection of percutaneous 18-g core-needle tumor biopsies to evaluate target modulation. Biopsies were obtained under imaging guidance prior to initiation and after 4 weeks of gefitinib treatment and were cryopreserved immediately in OCT compound (Sakura Finetek, Torrance, CA). Biopsy sections that measured 6 μm were cut for pathology review and laser-capture microdissection (LCM) (Molecular Devices, Sunnyvale, CA). Samples with predominant necrosis or lymphocytic infiltration were not assayed. Tumor and stromal cells were collected by using LCM as reported previously26, 27 and were analyzed independently.
Tissue Lysate Array Preparation and Analysis
Captured cells were lysed, and the proteins were extracted as described previously28 in a 1:1 preparation of Tris-glycine/sodium dodecyl sulfate sample buffer (Tissue Protein Extraction Reagent; Pierce, Rockford, IL) plus 2.5% β-mercaptoethanol for 30 minutes at 75°C. An estimated 30,000 cells were obtained yielding 30 μL of lysate to print 30 replicate arrays. EGF-treated A431 cell lysate (BD Biosciences, San Jose, CA) was used as a positive control. Lysates were loaded into 384-well plates in a 5-point 1:1 dilution curve and printed in triplicate onto nitrocellulose-coated glass slides (Schleicher and Schuell Bioscience, Keene, NH) using a GMS 417 pin and ring arrayer (Affymetrix, Santa Clara, CA)29 and were stored desiccated at −20°C. Arrays were incubated with Reblot antibody stripping solution (Chemicon, Temecula, CA), rinsed in phosphate-buffered saline, and blocked in I-Block (Applied Biosystems, Foster City, CA). Each slide was probed with primary antibody (Table 1) using an automated slide stainer (Dako, Carpinteria, CA) and detected with the Dako Catalyzed Signal Amplification system.30 Primary antibody was omitted on 1 slide to serve as the nonspecific background control. One slide from each set was stained with Sypro ruby stain to quantitate total protein load (Molecular Probes, Eugene, OR) and was visualized on a FluorChem imaging system (Alpha Innotech, San Leandro, CA). Protein intensity was quantified from stained arrays using ImageQuant (version 5.2; Molecular Dynamics; Sunnyvale, CA). Expression signals were normalized to total protein content then standardized to a control lysate of A431 cells printed onto each slide.
The primary endpoint was defined prospectively as the modulation of EGFR, protein kinase B (AKT), and extracellular regulatory kinase (ERK) after 1 month of gefitinib therapy. Secondary endpoints included clinical response and toxicity and the association of clinical and biochemical effects. We estimated that a minimum of 15 paired tumor biopsies would be required to address the primary endpoint. This number would be adequate to detect a difference of 1 standard deviation of change with 80% power, assuming that a 1-tailed t test would be used at the .008 significance level based on an assumption of undertaking 6 paired comparisons to allow at least implicitly for a Bonferroni correction that accounted for multiple comparisons.
The data used were standardized values:
Triplicate values were averaged to yield the single NIV for a given tissue-lysate array (TLA) parameter at a given time (pretreatment or posttreatment) for either tumor or stroma. The relative difference used for analysis after determining that the actual difference was more dependent on pretreatment than the relative difference and, hence, was less suitable as an endpoint. It was defined as follows: (posttreatment value − pretreatment value)/pretreatment value. A 2-tailed Wilcoxon signed rank-test was used to determine whether the relative changes between posttreatment and pretreatment differed from zero. An exact Jonckheere-Terpstra trend test was used to test for an association between grade of gastrointestinal toxicity or worst toxicity and each TLA parameter. Parameters in patients with or without diarrhea or skin toxicity were compared using an exact Wilcoxon rank-sum test. In view of the large number of parameters that ultimately were evaluated in this exploratory study, although no formal correction for multiple comparisons was performed, a P value <.005 was considered necessary to interpret a result as statistically significant, whereas .005 < P < .05 would suggest a trend.
Twenty-four patients with recurrent EOC were enrolled between November 2002 and January 2005 to yield 15 patients with matched biopsies. Patient demographics are summarized in Table 2. No patient had a kinase-activating EGFR mutation (data not shown).
Table 2. Patient Characteristics, N = 24
No. of patients
Median age (range), y
Sites of disease
No. of prior treatments
Sixteen patients completed ≥2 cycles of therapy and, thus, were evaluable for clinical assessment. There were no complete or partial responses. The median time on treatment was 2 months (range, from 3 days to 5 months). Nine patients (37%) had stable disease for >2 months. CA125 values were consistent with radiographic changes: Fifteen patients had a steady rise in CA125 value during treatment, and 8 patients had a decrease at some point during treatment, including 4 patients who were on study for ≥4 months. One patient presented with a solid cervical lymph node mass that became soft and fluctuant 3 months into therapy. A CT scan showed the development of cystic regions in the mass and histologically necrotic tumor tissue with a few viable tumor cells in the floor of the cavity. The patient had progressive disease after 4 months of therapy despite this promising beginning.
Safety and Toxicity
Adverse events observed in this trial generally were mild. The most common adverse events attributed to gefitinib treatment are summarized in Table 3. Diarrhea was managed successfully with loperamide therapy in most patients. An acneiform rash was observed in 42% of patients and improved with topical clindamycin treatment. Five patients had asymptomatic elevations in hepatic transaminases and/or alkaline phosphatase. Grade 3 and 4 toxicities related to gefitinib administration were rare. Grade 4 hyponatremia was reported in a patient who had multiple baseline electrolyte abnormalities resulting from short bowel syndrome and underlying adrenal insufficiency: She experienced drug-related, grade 3 liver enzyme elevations. A patient who was receiving enoxaparin for a synthetic aortic graft developed a grade 4 hemorrhage because of splenic rupture. Its attribution to gefitinib treatment was unclear. Thirty percent of patients required dose modification to 250 mg per day; 1 patient was reduced during Cycle 1, 3 patients were reduced in Cycle 2, and 3 patients were reduced at or after Cycle 3. Seven patients required short treatment interruptions for recovery of diarrhea or fatigue (median, 3.5 days; range, 1–10 days). Both patients with grade 4 toxicity had treatment discontinued.
Table 3. Common Adverse Events Related to Gefitinib Administration Observed in ≥2 Patients
No. of patients (%)
ALT indicates alanine aminotransferase.
Pretreatment and posttreatment tumor specimens were obtained from 23 patients and 18 patients, respectively; the disease sites are listed in Table 4. No second biopsy was obtained if there was no solid tumor in the first biopsy or if treatment was discontinued early. No complications occurred during sample acquisition. Several biopsies were considered unsuitable for microdissection because of the absence of tumor, excessive lymphocyte infiltration, or overwhelming necrosis. Matched data on protein expression in stroma were available for a limited number of patients, because many biopsies had minimal dissectable stroma. Fifteen paired tumor biopsies were used for proteomic evaluation.
Table 4. Sites of Acquisition of Core Tumor Biopsies for Proteomic Analysis
No. of patients
Liver parenchymal mass
Psoas muscle mass
Demonstration of target modulation
Target quantity and phosphorylation could be measured in varied numbers of patients (Table 5). Figure 1 shows changes in the primary parameters of total and phosphorylated EGFR (p-EGFR) (pY-1148), AKT, and ERK with 1 month of gefitinib therapy. The ratio of NIV values are presented for expression of the protein in tumor obtained during gefitinib therapy as a function of baseline values. Reductions in total EGFR and p-EGFR were observed in several patients. However, no absolute or relative differences were considered suggestive of a significant change.
Table 5. Sample Statistics for Parameters With a Trend Toward an Association With Increasing Toxicity: Tumor Site
No. of patients
SEM indicates standard error of the mean; EGFR, epidermal growth factor receptor; AKT, protein kinase B; p-ERK, phosphorylated extracellular regulated kinase; GI, gastrointestinal; p-AKT, phosphorylated AKT; pY1148-EGFR, pY1173-EGFR, and pY992-EGFR, phosphorylated forms of epidermal growth factor receptor.
The extent of adverse events observed was associated with changes in signaling parameters in several instances. Tables 5 and 6 show sample statistics for signaling parameters and clinical toxicity by grade and type of toxicity for those parameters for which there was at least a trend toward an association between relative change in the parameter and the type of toxicity noted. Increasing EGFR, AKT, p-ERK, and p-EGFR moieties in tumor on treatment demonstrated at least a trend toward an association with increasing overall toxicity (P ≤ .05), gastrointestinal toxicity (P < .05), and skin toxicity (P = .03). No pretreatment biochemical parameters were predictive of toxicity. Several strong trends were observed in this study and will need to be confirmed in a larger cohort because of the small number of patients studied.
Demonstration of biochemical proof of action is important in the continued development of small-molecule, signal-transduction inhibitors like gefitinib. Newer semiquantitative and quantitative techniques are being developed to facilitate this important endpoint.31 The current results indicated that EGFR is present in EOC and is activated in most patients despite the lack of an activating mutation. We were able to demonstrate that gefitinib inhibits the phosphorylation of EGFR in ovarian cancer cells, providing proof of target in a clinical setting. It also has been shown that gefitinib inhibits the phosphorylation and activation of EGFR with the corresponding inhibition of downstream signaling cascades and a decrease in cell proliferation and tumor cell growth in laboratory and animal models.16 Prior preclinical and clinical studies have shown decreased EGFR and diminished activation in unproven surrogates.19, 32 To our knowledge, this is one of the first studies to provide tumor-specific evidence of tyrosine kinase inhibition by gefitinib in a clinical setting. We also demonstrated that inhibition of p-EGFR results in the inhibition of downstream signaling molecules known to transmit the EGFR growth response. A decrease in phosphorylation of both AKT and ERK was observed with gefitinib therapy. These results in ovarian cancer are similar to recent data in breast cancer that indicated a down-regulation of p-EGFR with gefitinib therapy, as measured by immunohistochemistry staining of sequential tumor biopsies.33 Less downstream inhibition was observed in the breast cancer cohort with a decrease in p-ERK but not p-AKT in tumor cells. A 500-mg dose of gefitinib daily was effective biochemically in tumor cells from patients with ovarian cancer.
Gefitinib had limited activity as monotherapy in this population of heavily pretreated patients with recurrent or refractory ovarian cancer despite some target inhibition in 82% of patients. No patient attained stabilization of disease of at least 6 months, which was the clinical target parameter. The gefitinib Phase II trial for ovarian cancer patients coordinated by the Gynecologic Oncology Group (GOG 170C) had a primary endpoint of progression-free survival,34 and the patients were limited to fewer prior treatment regimens. Similar to our trial, the median number of cycles received by patients in the GOG trial was 2 cycles. Four patients achieved stable disease for >6 months, and 1 partial response was observed in a patient who had an EGFR mutation. It has been demonstrated that activating mutations of EGFR predict the response to gefitinib in some patients. The majority of patients with nonsmall cell lung cancer (NSCLC) who responded to gefitinib (77% in recently compiled data from multiple trials) had mutations of EGFR in or near the ATP-binding region of the kinase domain; these mutations were observed rarely in patients with NSCLC who did not respond.35–38 The somewhat more promising results from the GOG trial may have been caused by differences in the patient populations. Our more heavily pretreated patient population was a group known to have limited response to new agents.39
The adverse events observed in the current trial generally were mild and were comparable to those observed in other Phase II trials of gefitinib33, 40–42: diarrhea and rash, which are common adverse events with gefitinib. Greater quantities or phosphorylation of EGFR or its downstream signaling partners were associated with increased overall, gastrointestinal, and skin toxicities in our study. These toxicities and their associations imply effective levels of EGFR inhibition in the skin and gastrointestinal tract. The lack of clinical activity of single-agent gefitinib, despite some target effects in tumor tissue, indicates that the presence and extent of these toxicities cannot be used as surrogates for intratumoral effects.
The question presented by this trial is the lack of an association between the biochemical and clinical effects of gefitinib. It appears that blocking the activity of the EGFR kinase is not sufficient to produce a clinical effect in ovarian cancer. There are 3 possible explanations: 1) the target is not important in the tumor biology; 2) the target is important, but its modulation is not sufficient; or 3) the target is important and is inhibited sufficiently, but this effect can be overcome by paracrine or parallel signaling to the same downstream targets. The second explanation implies a possible incomplete inhibition of EGFR phosphorylation. The necessary threshold for EGFR inhibition and the specific key phosphorylation sites are unknown. Although a decrease >50% was observed in the level of p-EGFR expression, detectable levels of p-EGFR still were present in all tumor biopsies, and the proportion of EGFR activation remained unchanged by treatment. This may be adequate to stimulate tumor growth and proliferation. There are other promalignant pathways that promote tumor development and survival in molecularly heterogeneous cancers like ovarian cancer.43 These progrowth molecular signals may compensate for or overcome inhibition of the EGFR pathway. The finding that EGFR phosphorylation has a limited clinical effect but has biochemical activity provides additional rationale for the exploration of gefitinib in combination with signal-transduction inhibitors that effect downstream targets. The inhibition of 1 pathway at multiple levels or of several pathways in parallel may have an additive or even supraadditive effect.
This study also demonstrated that tissue lysate arrays may be a sensitive technique for detecting protein expression and activation in ovarian cancer. They provide a method for quantifying protein expression, something that cannot be achieved easily with immunohistochemistry.28, 29 Tissue lysate arrays also allow the simultaneous study of a large number of protein endpoints. Our proteomic evaluation, as a pilot study, was relatively limited; potentially, up to 30 different proteins could be probed in the analysis of 1 small tumor biopsy. Although the use of tissue lysate arrays in the clinical setting still is exploratory, the current study has demonstrated the feasibility and applicability of the technique to monitoring biochemical responses to molecularly targeted therapies.
Table 6. Sample Statistics for Parameters With a Trend Toward an Association With Increasing Toxicity: Tumor Site
No. of patients
SEM indicates standard error of the mean; p-AKT, phosphorylated protein kinase C; pY1148-EGFR and pY992-EGFR, phosphorylated forms of epidermal growth factor receptor.