Experiment 23 Advance Study Assignment Determination



Breast cancer is the most common cancer in women in the world1,2 and despite improvements in treatment, the survival rate for patients with metastatic breast cancer remains poor, with a 5-year survival of only 23%.2

Although there is evidence that recent additions to the armamentarium of cytotoxic and hormonal agents may be producing an improvement in survival,3 there is increasing interest in the development of agents that act against the molecular targets that dictate malignant growth.4 In breast cancer, this approach is exemplified by the successful targeting of the ErbB family of growth factor receptors by trastuzumab (Herceptin; Genentech Inc, San Francisco, CA). Trastuzumab is a humanized monoclonal antibody directed at the ErbB2 receptor that is active and results in improved survival in patients with advanced breast cancer that has ErbB2 amplification.5-7

The epidermal growth factor receptor (EGFR), like ErbB2, is a member of the ErbB family of receptors (also known as type I receptor tyrosine kinases). This family of receptors plays a major role in promoting proliferation and the malignant growth of breast cancer cells. The expression of EGFR in breast cancer has been studied extensively and has been associated with poor prognosis.8-14 As a consequence, inhibiting EGFR function may be a fruitful approach in breast cancer therapy.

Gefitinib (Iressa; AstraZeneca, Macclesfield, United Kingdom) is an oral nonpeptide anilinoquinazoline compound that inhibits the tyrosine kinase activity of EGFR with an inhibitory concentration (IC50) of 0.03 μM.15 Preclinical studies demonstrated that gefitinib inhibited proliferation of breast cancer cells both in vitro and in vivo.16-20 Interestingly, the antitumor activity in breast cancer cells has been seen in cells expressing varying degrees of EGFR and also in cells expressing high levels of ErbB2. In the clinical setting, phase I trials of gefitinib monotherapy showed that this agent was generally well tolerated, with the majority of adverse events (AEs) being grade 1 to 2 gastrointestinal or skin events.21-24 Furthermore, antitumor activity was demonstrated in a range of tumor types, including the observation of prolonged stable disease in patients with breast cancer. Phase II trials of gefitinib monotherapy in previously treated patients with advanced non–small-cell lung cancer resulted in a response rate ranging from 9% to 19% and disease control in more than 40% of patients.25,26 In addition to non–small-cell lung cancer, responses have been reported in patients with advanced head and neck carcinomas.27

The primary aim of this study was to determine the response rate of gefitinib 500 mg/day in pretreated patients with locally advanced or metastatic breast cancer. Another important goal was to analyze the pharmacodynamic effects of gefitinib in the tumor, in order to further clarify the mechanism of action of this agent. We had initially proposed and reported that skin is a good surrogate tissue to study EGFR inhibition with anti-EGFR agents.21,22,28 Subsequent studies have confirmed a significant inhibition of EGFR phosphorylation and downstream signaling in the skin with other anti-EGFR inhibitors, including the tyrosine kinase inhibitorerlotinib (OSI-774, Tarceva; OSI Pharmaceuticals, Genentech, and Roche, Melville, NY)29 and preliminary confirmatory results with CI-103330 and the monoclonal antibody EMD72000.31 However, an important question that was not addressed in our initial studies was whether there was a correlation between inhibition of EGFR in the skin and in the tumor, as well as the downstream effects of EGFR inhibition in the tumors. Therefore, this study included evaluation of sequential tumor biopsies in order to answer this question.



Study Design

This was a multicenter, phase II, pharmacodynamic trial. Patients received gefitinib 500 mg/day, apart from day 1, when, in order to ensure that steady-state levels were rapidly reached, the patient received gefitinib 500 mg followed by a second 500 mg dose 12 hours later. Each treatment cycle was 28 days and patients received gefitinib until disease progression, unacceptable toxicity, or withdrawal of consent. The maximum period of treatment was 6 months after entry of the last patient (total maximum duration of 15 months). Administration of gefitinib could be prolonged at the discretion of the investigator, based on patient benefit and tolerability.

The primary objective was to evaluate the objective tumor response rate to gefitinib 500 mg/day in patients with locally advanced or metastatic breast cancer previously treated with chemotherapy. Determination of the pharmacodynamic profile of gefitinib in skin versus tumor tissues and evaluation of the safety of gefitinib were secondary objectives.


This trial involved women with histologically confirmed stage IIIb/IV advanced breast cancer that was resistant to one or two prior chemotherapy regimens. Although the study was not limited to EGFR-positive patients, it was intended that at least half the patients should be EGFR positive in order to investigate whether the activity of gefitinib is related to the presence of the receptor. It was mandatory that all patients have a baseline tumor sample. Before concluding the inclusion process, it was checked that at least 50% of the patients were EGFR positive.

The minimum anticipated number of patients was 21, based on a two-stage design for phase II studies, where the drug would be considered inactive in terms of response index if more than 5% of patients responded, giving a maximum response index of 20%. If a response of ≤ 1 was observed from the 21 patients, more patients would not be included. If the response was more than 1 of 21 patients, recruitment would continue until 41 patients had been included to permit a better estimate of the response index. The probability of accepting a drug as active with a response index less than 5% is 5% (α). The probability of rejecting a drug with a response index greater than 20% is 10% (β).

To be eligible for inclusion, patients were required to be 18 years of age or older with a life expectancy of ≥ 12 weeks and WHO performance status of 0 or 1. Patients provided written, informed consent. Any one of the following was regarded as a criterion for exclusion: more than two previous chemotherapy regimens; other antitumor therapy ≤ 4 weeks before day 1 of study treatment (≤ 6 weeks for nitrosoureas or mitomycin); unresolved AE higher than National Cancer Institute Common Toxicity Criteria (CTC) grade 2 from previous antitumor therapy (except hair loss); extensive radiotherapy ≤ 6 weeks before start of trial treatment; bone or cerebral metastases as only site of tumor; severe or uncontrolled disease; any other clinical disorder or laboratory finding that made it undesirable for the patient to participate; neutrophils < 1.5 × 109/L or platelets < 100 × 109/L; serum bilirubin > 1.25× upper limit of reference range; risk of transmitting HIV or hepatitis B; pregnant or lactating women.

The trial was conducted in accordance with the Declaration of Helsinki32 and the principles of Good Clinical Practice, and with the approval of appropriate ethics committees.


Medical history and a physical examination were carried out at the screening visit. Biochemical and hematologic assessments were carried out at baseline and at each clinic visit.

Response and survival

Response was assessed using Response Evaluation Criteria In Solid Tumors (RECIST).33 Disease control included those patients with objective response (complete or partial response) or stable disease, confirmed and maintained for at least a further 4 weeks. Tumor status was assessed at baseline, every 4 weeks after starting treatment, then every 8 weeks after the fourth treatment period.

Progression-free survival was assessed from the date of random assignment until the date at which disease progression was observed. If death occurred before the disease had progressed, this was considered to be progression. Patients who did not have documented objective progression on the date of the last analysis were excluded. Overall survival was evaluated from the date of random assignment until the date of death, or until the last available date for the living patient.


All AEs were reported (including evaluation of causality) and graded according to the CTC scale (version 2.0). Dose reduction from 500 to 250 mg/day was allowed in the case of toxicity (CTC grade 3 or 4 toxicity reversible within 14 days). Administration of gefitinib could be interrupted for a maximum of 14 days in the event of CTC grade 3 or above or unacceptable toxicity. The patient could resume taking the assigned dose, or a reduced dose, once the severity of the AE decreased to CTC grade 1 to 2.

Pharmacodynamic assessments

In those patients who gave their consent, skin and tumor biopsies were taken before treatment (at the time of giving consent [baseline]), after 28 days of treatment, and at the time of disease progression, if possible. Samples of skin and tumor tissue were analyzed at Vall d'Hebron University Hospital, Barcelona, Spain.

The following pharmacodynamic markers were assessed with appropriate antibodies: EGFR (mouse monoclonal antibody [MAb] clone 2-18C9, DAKO, Carpinteria, CA), activated/phosphorylated (p) EGFR (mouse MAb clone 74, Chemicon, Temecula, CA), the ligand transforming growth factor alpha (TGFα; mouse MAb clone Ab-2, Oncogene, San Diego, CA), the downstream signaling markers phosphorylated mitogen-activated protein kinase (p-MAPK; rabbit polyclonal phospho-p44/42 MAPK at Thr 202/Tyr 204 antibody, Cell Signaling Technology, Beverly, MA) and pAkt (PKB or Rac; rabbit polyclonal pAkt at Ser 473 antibody, Cell Signaling Technology), the proliferation marker Ki67 (mouse MAb clone MIB1, DAKO) and the cyclin-dependent kinase inhibitor p27kip1 (mouse MAb clone SX53G8, DAKO). All markers were evaluated by immunohistochemistry to determine both the percentage of target cells stained by each marker and the staining intensity. For each antibody, all immunohistochemical determinations were performed in a single-run assay including baseline and on-study samples, to compare results under the same conditions, within the same experiment. Immunostaining was performed using 4 μmol/L tissue sections on positively charged glass slides, as previously described.28 For a positive score, complete membrane staining was required for total EGFR, cytoplasmic or membrane staining for p-EGFR, cytoplasmic with a faint membranous staining for pAkt, cytoplasmic staining for TGFα, and nuclear staining for p-MAPK, Ki67, and p27. The individuals scoring the immunohistochemistry were blinded to the dates of the biopsies. Apoptotic levels were determined by terminal deoxyneucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay using fluorescein-labeled-16-dUTP-TdT (Roche Diagnostics GmbH, Mannheim, Germany) after proteinase K digestion of the tissue. An apoptotic index was calculated as a percentage of green fluorescent cells in 10 high-power fields (×400 optical magnification) of the tumor tissue, using a Fluorescence Eclipse E400 Nikon microscope. A Spearman's correlation test was used to analyze any relationship between the expression levels of different markers and a Wilcoxon signed rank test was used to compare marker expression levels in paired basal and day-28 samples.




The first patient was recruited in April 2001 and the last in November 2002. In total, 31 patients were assessable, and their demographic characteristics are shown in Table 1

Table 1. Demographic Characteristics

. Nineteen patients had previously received first-line chemotherapy and 10 patients had received second-line chemotherapy. The majority of patients had previously received hormone therapy or trastuzumab in addition to chemotherapy.

Table 1. Demographic Characteristics


Assessable patients31
Age, years
Performance status
Visceral metastases1961
Other metastases
    Skin, soft tissue, and local1652
    Lymph nodes826
Menopausal state
Adjuvant chemotherapy1755
Adjuvant hormone therapy1239
Prior chemotherapy2994
Other prior immunotherapy- or hormone therapy–based treatment


Clinical Activity

The median length of treatment period was 56 days (range, 27 to 350 days). Of the 31 patients, 12 (38.7%) had stable disease, of whom 10 (32.3%) were stable for ≥ 3 months, 6 (19.4%) were stable for ≥ 4 months, and 3 (9.7%) were stable for ≥ 6 months. Duration of stable disease was 84 to 349 days. No complete or partial responses were observed. Progressive disease occurred in 19 patients (61.3%), with a median time to progression of 55 days (95% CI, 42 to 88).

Median overall survival was 503 days (95% CI, not assessable; range, 56 to 617 [censored] days). The proportion of patients alive at 6 months was 80.6% (95% CI, 66.7 to 94.6) and the proportion alive and progression free at 6 months was 9.7% (95% CI, 0.0 to 20.1).


Drug-related AEs (ie, considered by the investigator to have a reasonable possibility of being related to the study drug) that occurred in > 10% of patients are shown in Table 2

Table 2. Drug-Related Adverse Events Occurring in > 10% of Patients (CTC Grades)*

Table 2. Drug-Related Adverse Events Occurring in > 10% of Patients (CTC Grades)*

Grade 1 to 2

Grade 3 to 4
Adverse event
Skin rash†1238.7516.1

Emil Slowinski, Macalester College

Emil J. Slowinski is an Emeritus DeWitt Wallace Professor of Chemistry at Macalester College. He earned a B.S. degree from Massachusetts State College in 1946 and a Ph.D. in physical chemistry from the Massachusetts Institute of Technology in 1949. He taught at Swarthmore College, 1949-1952; the University of Connecticut, 1952-1964; and Macalester College, 1964-1988. His sabbatical leaves were at Oxford University in 1960 and the University of Warsaw in 1968. He is a co-author, with Bill Masterton and/or Wayne Wolsey, of more than 25 books on various areas of general chemistry. He was actively involved in all editions of CHEMICAL PRINCIPLES IN THE LABORATORY up through the 9th edition, and though now retired from active writing still offers insights, advice, and support to his coauthors.

Wayne C. Wolsey, Macalester College

Wayne C. Wolsey, an inorganic chemist, received his B.S. from Michigan State University in 1958 and his Ph.D. from the University of Kansas in 1962. He joined the Macalester College faculty in 1965 and is now in "semi-retirement." His last three sabbaticals were spent at the Oak Ridge National Laboratory. In 2001-2002, he investigated various complexing agents for their effectiveness in dissolving calcium oxalate kidney stones, in collaboration with a former student, now a urologist. He has received various awards, including the Minnesota College Science Teacher of the Year in 1989; Macalester's Thomas Jefferson Award in 1993; designation as a MegaMole contributor to Minnesota Chemical Education in 1997; and an award from the Minnesota State AAUP Conference in 2001 for his support of academic freedom and shared governance. He remains professionally active in a number of scientific organizations.

Robert Rossi, Macalester College

Robert C. Rossi is the Laboratory Supervisor in the Chemistry Department at Macalester College. He obtained a B.S. degree in chemical engineering from the University of Wisconsin - Madison in 1993 and upon graduation joined the Peace Corps, serving in the Fiji Islands. He then taught and carried out applied photoelectrochemistry and semiconductor physics research at the California Institute of Technology, earning a Ph.D. in 2001. After several years teaching as a visiting professor at Carleton College, he moved to Macalester College, where he has been since 2003. In 2011 he became a co-author of Chemical Principles in the Laboratory, first writing for the 10th Edition.

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