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© 2002 American Society for Clinical Oncology Pharmacodynamic Studies of the Epidermal Growth Factor Receptor Inhibitor ZD1839 in Skin From Cancer Patients: Histopathologic and Molecular Consequences of Receptor InhibitionByFrom the Oncology Service, Vall d’Hebron University Hospital; Dermatology Service, Hospital Clinic; Transfusio i Banc de Teixits, Vall d’Hebron Vall d’Hebron University Hospital, Barcelona, Spain; AstraZeneca Pharmaceuticals, Wilmington, DE; Oncology Service, M.D. Anderson Cancer Center, Houston, TX; Oncology Service, Harper Hospital, Detroit, MI; AstraZeneca Pharmaceuticals, Alderley Park; Department of Pharmacology, Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom; and Division of Haematology and Medical Oncology, Peter MacCallum Cancer Institute, Melbourne, Australia. Address reprint requests to Jose Baselga, MD, Oncology Service, Vall d’Hebron University Hospital, Paseo Vall d’Hebron 119-129, Barcelona 08035, Spain; email: baselga{at}hg.vhebron.es
PURPOSE: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (Iressa; AstraZeneca Pharmaceuticals, Alderley Park, United Kingdom) is under development as an anticancer agent. We studied the pharmacodynamic effects of ZD1839 on EGFR in the skin, an EGFR-dependent tissue, in cancer patients participating in ZD1839 phase I clinical trials.
PATIENTS AND METHODS: We studied 104 pre– and/or on–ZD1839 therapy ( RESULTS: Histopathologically, the stratum corneum of the epidermis was thinner during therapy (P < .001). In hair follicles, prominent keratin plugs and microorganisms were found in dilated infundibula. ZD1839 suppressed EGFR phosphorylation in all EGFR-expressing cells (P < .001). In addition, ZD1839 inhibited MAPK activation (P < .001) and reduced keratinocyte proliferation index (P < .001). Concomitantly, ZD1839 increased the expression of p27KIP1 (P < .001) and maturation markers (P < .001) and increased apoptosis (P < .001). These effects were observed at all dose levels, before reaching dose-limiting toxicities. CONCLUSION: ZD1839 inhibits EGFR activation and affects downstream receptor-dependent processes in vivo. These effects were profound at doses well below the one producing unacceptable toxicity, a finding that strongly supports pharmacodynamic assessments to select optimal doses instead of a maximum-tolerated dose for definitive efficacy and safety trials.
THE EPIDERMAL GROWTH factor receptor (EGFR) plays an important role in epithelial biology and in many human malignancies.1-6 The EGFR is a 170-kd plasma membrane glycoprotein composed of an extracellular ligand-binding domain, a transmembrane lipophilic segment, and an intracellular protein kinase domain with a regulatory carboxyl terminal segment.7,8 On binding of ligand, EGFR dimerization occurs, which results in high-affinity ligand binding, activation of the intrinsic protein tyrosine kinase (TK) activity, and tyrosine autophosphorylation.7,8 The EGFR can also be activated by ligand-independent mechanisms.8 Activation of the EGFR TK has been identified as a key initiating event that initiates the cascade of intracellular signalling events that regulate cell proliferation, differentiation, survival, angiogenesis, and metastasis.9 ZD1839 (Iressa; AstraZeneca Pharmaceuticals, Alderley Park, United Kingdom) is an oral nonpeptide anilinoquinazolone compound developed to inhibit selectively the TK activity of the EGFR.10-12 ZD1839 inhibits EGFR TK in vitro at concentrations at least 100-fold lower than that for many other kinases tested. In a number of cultured tumor cell lines, ZD1839 prevented autophosphorylation of EGFR, resulting in the inhibition of the activation of downstream signaling molecules. In preclinical models, oral dosing caused growth inhibition of tumor xenografts and complete regression of well-established A431 xenografts that express high EGFR levels.11,12 ZD1839 also markedly enhances the antitumor activity of conventional chemotherapeutic agents.13,14 Based on its promising preclinical antitumor activity, ZD1839 recently entered clinical trials in cancer patients with the goal to define the safety profile and pharmacokinetics and to select the optimal dose for future clinical studies.15,16 The selection of dose with conventional nontargeted chemotherapeutic agents has been usually based on the maximally tolerated dose. This same principle does not apply for targeted therapies, where an optimal biologic dose would be preferred instead. The definition of optimal dose may be established based on pharmacokinetic end points or, preferably, by demonstrating the desired effect on the target molecule.17-20 Because ZD1839 is a selective EGFR inhibitor, we incorporated pharmacodynamic studies to study inhibition of EGFR activation and EGFR-dependent processes in sequentially performed skin biopsies in patients participating in two phase I studies with escalating doses of ZD1839. Skin, in addition to its ease of access, was the selected tissue on which to perform the current studies because of the important role that EGFR plays in skin biology. In normal adult human skin, the EGFR is strongly expressed in keratinocytes and in cells of eccrine and sebaceous glands. In keratinocytes, the expression is highest in the basal layer of epidermis and in the outer root sheath of hair follicles.21,22 This high level of EGFR expression colocalizes with the population of proliferating, undifferentiated keratinocytes.4,23-27 As keratinocytes migrate to the suprabasal layers, they exit the cell cycle and enter a terminal maturation program that results in the formation of the stratum corneum, the outermost layer where the barrier function of the epidermis resides.24-27 In keratinocytes cultures, specific EGFR TK inhibitors or blocking monoclonal antibodies (Mabs) to the EGFR inhibit proliferation and block migration and induce terminal differentiation and apoptosis.22,28-30 Animal models have established that EGFR signaling plays a key role in the development of hair follicles and skin.4-6,31-34 Further support for a role of EGFR in skin biology is provided by the observation that some patients treated with ZD183915,16,35 and other EGFR TK inhibitors36,37 or with blocking anti-EGFR MAbs20 developed skin reactions, suggesting that EGFR inhibition results in alteration of normal skin homeostasis. In the current study, we report ZD1839-induced changes in EGFR activation, mitogen-activated protein kinase (MAPK) phosphorylation, p27KIP1 levels, signal transducer and activator of transcription (STAT)-3 phosphorylation, proliferation indexes, and skin maturation markers. In addition, we have characterized the histopathologic consequences of EGFR inhibition in the skin. All these effects on the EGFR and receptor-dependent processes were seen at doses below the one resulting in unacceptable toxicity. Our findings indicate effective EGFR inhibition by ZD1839 in vivo and support the use of doses below the maximum-tolerated dose for future clinical studies with these compounds.
Clinical Studies Two identical phase I trials of ZD1839 were performed at six centers in the United States (study 0011) and 10 centers in Europe/Australia (study 0012). Inclusion criteria were age older than 18 years, life expectancy more than 12 weeks, advanced or metastatic tumors expected to express EGFR, and performance status of 0 or 1.15,16,35 Patients with a history of or concurrent skin diseases were not eligible. Escalating doses of ZD1839 were administered continuously as a single daily oral dose. Dose levels ranged from 150 mg/d to 1,000 mg/d. Treatment was continued until disease progression or dose-limiting toxicity. All patients gave written informed consent to participate in the trial. Full details on the clinical and pharmacokinetic data of these studies will be reported separately.15,16,35 A second written informed consent was obtained from patients participating in the skin pharmacodynamic study. In consenting patients, skin specimens were obtained from an area of normal skin by an 8 mm (depth) x 4 mm (width) punch biopsy to the level of subcutaneous tissue or by an incisional biopsy (a minimum of 0.5 x 0.5 cm tissue was required). The skin biopsies were taken in the upper thorax/supraclavicular area in the majority of patients. In some patients, the biopsies were from upper extremities or upper back. In patients that had paired pre– and on–ZD1839 therapy samples, both biopsies were obtained from analogous sites.
Light-Microscope Analysis
Antibodies
Immunohistochemistry To score a keratinocyte as positive, complete membrane staining was required for total EGFR, cytoplasmic or membrane staining for activated EGFR, cytoplasmic staining for K1, and nuclear staining for activated MAPK, Ki67, p27KIP1, or phospho-STAT3. Qualitative changes in the expression of markers were assessed in a blind fashion by a panel of investigators (F.R., J.A., A.F., J.G., R.I.N., J.B., and Graham Bettom; AstraZeneca, Alderely Park, United Kingdom). For quantitative analysis, the percentage of stained keratinocytes with each antibody in interfollicular epidermis was scored from representative sections in 10 high-power fields (x400), and the average percentage of cells staining was calculated in every sample. Scoring was performed in a blind fashion (F.R.) with regards to clinical data and was used for statistical analysis. Hair follicles, dermal microvessels, and eccrine and sebaceous glands were qualitatively analyzed when present, but no statistical analysis was conducted because many samples lacked these elements.
Immunocytochemistry
Analysis of ZD1839 Plasma Concentrations
Statistical Methods
Patients and Skin Biopsies A total of 104 skin specimens from 65 cancer patients treated with escalating doses of ZD1839 were analyzed (Table 1).15,35 Biopsies from an area of clinically normal skin were collected within 2 weeks before the first dose of ZD1839 and/or as close to day 28 of ZD1839 therapy as possible. Sites of biopsies healed normally without delays or complications in all 45 patients who underwent skin biopsy during treatment period.
Histopathologic Effects of ZD1839 The epidermis is a keratinizing stratified squamous epithelium composed of four layers. These layers are the basal cell layer, the squamous layer, the granular layer, and the stratum corneum, the latter being the outermost layer formed by anucleated dead cells. The epidermis is contiguous with the outer root sheath of the hair follicle, which is a complex structure specialized in making a hair shaft.25-27 In the present study, at hematoxylin and eosine examination, epidermal thickness did not vary in pretherapy (Fig 1A) compared with on-therapy (Fig 1B) skin biopsies (n = 38; average thickness [mean ± SD] 37.5 ± 7.4 µm v 34.6 ± 8.1 µm; P = .67). The granular cell layer seemed to be thinner and interrupted in on-therapy biopsies, but because it was very faint in most samples, this variable could not be properly quantified. An evident observation when comparing pretherapy (Fig 1A) and on-therapy (Fig 1B) biopsies was that on-therapy, the stratum corneum was thinner, had lost its normal basket-weave configuration, and appeared more compact and eosinophilic, with the occasional presence of foci of parakeratosis. In 35 of the 38 paired cases, the stratum corneum was thinner during ZD1839 treatment (n = 38; average thickness [mean ± SD], pre-ZD1839 27.8 ± 18 µm v on-ZD1839 15.0 ± 9 µm; P < .001; Fig 1C). In one pair, stratum corneum thickness was not assessable.
In some specimens, focal mononuclear infiltrates with vacuolar degeneration of the basal cell layer and apoptotic keratinocytes (a lichenoid tissue reaction) were found in hair follicles and interfollicular epidermis. The apoptotic index (apoptotic cells by 10,000 assessed morphologically) in epidermis increased during therapy in 27 of the 39 paired samples (Fig 1A and 1B; P < .001, Fig 1D). Of note, some biopsies from skin rashes that developed during ZD1839 therapy also showed prominent lichenoid changes (not shown). Follicular changes could not be documented properly because most biopsy specimens lacked hair follicles. In a few specimens, prominent keratin plugs and microorganisms were found in dilated infundibula (Fig 1E), a finding suggestive of comedo. In other specimens, an acute neutrophilic folliculitis was found (Fig 1F). Biopsies from skin rashes that developed during ZD1839 therapy also showed acute folliculitis (not shown). Regarding other skin elements, no evident changes in the dermal capillaries, eccrine glands, or sebaceous glands were seen.
Effects of ZD1839 on EGFR Activation
To detect the activated, phosphorylated form of the EGFR we used a MAb that recognizes the ligand-activated and phosphorylated form of the EGFR and does not recognize other tyrosine-phosphorylated proteins.38,41 We confirmed in the present study that immunostaining generated using this test antibody was highly specific, both by the use of a control antibody on matched skin sections (data not shown) and by application of the test antibody to a human tumor cell line that we have demonstrated to activate EGFR signaling after EGF treatment and that is sensitive to EGFR TK inhibitors.39,40 Using this antibody, staining in nonstimulated DU145 tumor cells was almost absent (Fig 3A). However, we detected marked increases in granular membrane and, especially, cytoplasmic staining on stimulation with the receptor ligand EGF (Fig 3B). This staining was consistent with the described internalization of phosphorylated EGFR and its localization in intracellular vesicles after ligand binding.42,43 After in vitro treatment with ZD1839, both membrane and cytoplasmic staining induced by EGF were blocked (Fig 3C).
In skin cells, subcellular staining for activated EGFR using the same antibody38 and an enhanced signaling amplification system44 was similar to the one seen in ligand-stimulated human EGFR tumor cells (ie, granular membrane and mainly cytoplasmic). The activated EGFR was consistently detected in all of the cell types expressing total EGFR, ie, epidermal keratinocytes (Fig 4A), follicular keratinocytes (Fig 4B), cells of the eccrine glands (Fig 4C), cells of the sebaceous glands, and in occasional endothelial cells (not shown). As expected, activated EGFR was not detected in the cell types lacking total EGFR.
For scoring, we only assessed epidermal keratinocytes because the other skin elements were absent in many biopsies. In 49 pretherapy samples, the mean ± SD percentage of basal keratinocytes showing cytoplasmic and/or membrane staining for activated EGFR was 44 ± 3.4% (range, 0% to 90%) and was undetected in only one specimen. Membrane staining was always associated with granular cytoplasmic staining and was observed predominantly in basal keratinocytes (mean ± SD percentage of basal keratinocytes with membrane staining, 10.5 ± 6.8% SD; range, 0% to 22.8%). In 41 on-therapy samples, the mean percentage of basal keratinocytes showing cytoplasmic and/or membrane staining for activated EGFR was 2.1% (SD, ± 0.8%; range, 0% to 27%) and was completely absent in 27 biopsy samples. Inhibition of EGFR activation during treatment was achieved in all of the EGFR-positive cell types; ie, epidermal and hair follicle keratinocytes (Fig 4A and 4B), epithelial cells of the eccrine (Fig 4C) and sebaceous glands, and endothelial cells (not shown). There was no significant difference in ZD1839 dose (Mann-Whitney U, P = .43) or plasma levels (Mann-Whitney U, P = .76) when comparing the group of on-therapy samples with undetected EGFR activa- tion to the group with detected (although at low level) EGFR activation. In all paired cases evaluated for activated EGFR (n = 32), receptor activation was abolished or markedly reduced after ZD1839 treatment. Overall, the decrease in the expression of activated EGFR in epidermal keratinocytes during ZD1839 treatment compared with their corresponding paired pretherapy samples was significant (P < .001, Fig 4D). Inhibition of activated EGFR in paired samples was also documented when we scored only keratinocytes with membrane staining (P < .001, not shown).
Effects of ZD1839 on EGFR Signaling and Proliferation
Cell proliferation rates were assayed using Ki-67, a nuclear proliferation-associated antigen present only in proliferating cells.49-51 Ki-67 staining colocalized with activated MAPK; ie, mainly in basal layers (Fig 6A) and in the outer root sheath (Fig 6B). The Ki67 proliferative index in the basal layer decreased significantly during therapy in paired samples (n = 36, P < .001, Fig 6C). A decrease in Ki-67 staining was also seen in hair follicles during therapy (Fig 6B).
Because induction of the cyclin-dependent kinase inhibitor p27KIP1 is a hallmark of EGFR pathway inhibition and growth arrest in preclinical models,52-54 we assayed p27KIP1 expression. Pretherapy, staining for p27KIP1 was predominantly seen in the nuclei of suprabasal keratinocytes (Fig 6D), whereas staining in the basal layers was uncommon. In hair follicles, p27KIP1 staining was more common in cells of inner layers than in the outer root sheath (Fig 6E). In 23 of 23 paired samples evaluated for p27KIP1, there was an increase in p27KIP1 staining in the basal layers of epidermis (Fig 6D and 6F, P < .001) as well as in hair follicles (Fig 6E).
Effects on ZD1839 on Maturation
Pharmacodynamic Effects by ZD1839 Dose Levels and Plasma Levels Sequential cohorts of patients were treated with escalating doses of oral ZD1839 (150, 225, 300, 400, 600, 800, and 1,000 mg/d) (Table 1). No significant correlations were seen between dose levels of ZD1839 and the on-therapy scores of activated EGFR, activated MAPK, p27KIP1, K1, phospho-STAT3, or apoptosis. The only significant relationship was between increasing dose levels and reduced Ki67 indexes (Pearson’s r = -0.34; P = .021). The steady-state plasma ZD1839 concentrations obtained approximately on day 28 of therapy (the same day or close to the day of skin sampling) correlated with the dose of ZD1839 received (Pearson’s r = 0.63; P < .001), but there was no significant correlation with the on-therapy scores of activated EGFR, Ki67, p27KIP1, K1, phospo-STAT3, or apoptosis. The only significant relationship was between increasing plasma concentrations and increasing levels of activated MAPK (Pearson’s r = 0.38; P = .021). The correlations between Ki67 and dose or activated MAPK and plasma levels were not accompanied by other correlations and suggest that more patients would be needed to draw any conclusion in this regard. Although biologic effect of EGFR inhibition was observed at all studied dose levels and irrespective of steady-state plasma concentrations, dose-limiting toxicity was not reached until the dose of 1,000 mg/d, which consisted of gastrointestinal toxicities.15,35
ZD1839 Pharmacodynamic Effects and Skin Adverse Events
In this study, inhibition of EGFR activation was achieved in a variety of human skin cell types in vivo after ZD1839 treatment. In association with EGFR inhibition, MAPK activation and keratinocyte proliferative rates decreased, and, concomitantly, there was an increase in the expression of the CDK inhibitor p27KIP1. A change in the maturation of epidermal keratinocytes and an increase in the apoptotic index also occurred during therapy. The most striking histopathologic changes on-treatment were noted in the stratum corneum, which was markedly thinner and more compact, with a loss of its normal basket-weave pattern. In hair follicles, prominent keratin plugs and microorganisms were found in dilated infundibula. These changes may be the consequence of an altered terminal keratinocyte maturation in suprabasal keratinocytes as a result of EGFR blockade.4-6,56 Furthermore, they may be responsible for the acneiform rashes (ie, hair follicle changes) and desquamation (ie, interfollicular epidermal changes) that are seen in some patients treated with ZD1839. These findings in epidermis are very similar to those found in knockout mice lacking EGFR.4-6 Compared with wild-type litter mates, these animals have a thinner epidermis and granular layer, and the stratum corneum is almost absent. Although we did not find abnormalities in epidermal thickness, it is possible that biopsies at later time points would be needed to demonstrate potential changes in epidermal thickness because epidermal turnover time (ie, the time taken for a keratinocyte to pass from basal layer to the surface skin) is 52 to 75 days.25 Before ZD1839 treatment, total and activated EGFR, activated MAPK, and Ki67 were preferentially expressed in the proliferating basal layers of the epidermis and in the outer root sheath of the hair follicles, whereas p27KIP1, K1, and phospho-STAT3 were preferentially expressed in the differentiated suprabasal layers of epidermis and inner layers of hair follicles. Activated EGFR was seen in all the EGFR-positive cell types present in the skin. The pattern of staining for the activated EGFR was granular and mainly cytoplasmic, indicating that the activated receptor is internalized and located in intracellular vesicles in vivo in human skin cells. This pattern of staining is similar to the one in cell lines (Fig 3)42 and in a breast ductal carcinoma-in-situ xenograft model (Chan et al, manuscript submitted for publication). During ZD1839 treatment, EGFR activation was abolished in the majority of skin samples, indicating that the orally administered ZD1839 reached the EGFR and inhibited its activation in skin cells. The EGFR was inhibited in all the skin cell types expressing the EGFR. A few patients had detectable EGFR activation in a minority of keratinocytes during treatment. This was unrelated to ZD1839 dose or plasma levels, suggesting lack of complete receptor inhibition for reasons that remain unknown. The expression of activated MAPK, a signaling molecule activated in keratinocytes by EGFR ligands,51 was reduced during ZD1839 therapy, although it was still detectable. This suggests that, in addition to the EGFR, other pathways may activate MAPK in epidermal keratinocytes. However, the significant decrease in activated MAPK points to a key role of the EGFR in MAPK signaling in keratinocytes in vivo. This is in agreement with our previous finding that ZD1839 inhibits MAPK activation at concentrations that inhibit EGFR activation and cell growth in human EGFR model tumor cells (Albanell et al, manuscript submitted for publication) and with the in vivo inhibition of activated MAPK in mammary tumors in transforming growth factor alpha/HER2 bigenic mice treated with an EGFR TK inhibitor.48 Inhibition of MAPK activation by ZD1839 has also been shown in a xenograft model of human ductal carcinoma-in-situ (Chan et al, manuscript submitted for publication). Inhibition of activated MAPK is likely mediated by inhibition of EGFR TK because ZD1839 is a potent specific inhibitor of the TK activity of EGFR isolated from human vulval squamous carcinoma cells (IC50 of 0.023 to 0.079 µmol/L), whereas it is minimally active against other TKs, such as HER2, KDR, c-flt, or serine/threonine kinases including protein kinase C, MEK-1, and the MAPK ERK-2.11,15,16 Keratinocyte proliferation rates were reduced during ZD1839 treatment. This was accompanied by an induction of the CDK inhibitor p27KIP1. p27KIP1 is suggested to play a key role in ZD1839-induced cell cycle perturbation by decreasing CDK2 activity and leading to G1 growth arrest.53 Association of p27KIP1 with CDK2 has also been correlated with maturation and withdrawal from the cell cycle of primary keratinocytes. Indeed, we observed that in patients treated with ZD1839, basal keratinocytes increased the expression of K1, a specific marker of keratinocyte maturation, and this was accompanied by an increase in phosphorylated, activated STAT3, which is activated during keratinocyte differentiation.22,55 A recent study suggested that the EGFR has an antidifferentiation role on basal keratinocytes, whereas EGFR activation promotes rather than inhibits the terminal differentiation of suprabasal epidermal keratinocytes. This view of a differential role of the EGFR in basal versus suprabasal keratinocyte maturation is consistent with our finding of an induction of K1 and phospho-STAT3 in basal layers and by a thinner stratum corneum during therapy. The increased apoptotic index during ZD1839 treatment is consistent with a role of the EGFR in promoting keratinocyte survival28,51 and with studies in human keratinocytes showing that they undergo apoptosis when incubated with blocking EGFR antibodies or EGFR TK inhibitors.22,28 The observed effects of ZD1839 treatment on skin biology in humans indicate that the EGFR plays an important role in normal adult skin biology and suggest that this drug may be useful in the treatment or prevention of skin disorders where the EGFR has been implicated, such as psoriasis,57,58 keratinization disorders, or epithelial skin tumors.23,51 The lack of a dose-response effect in our pharmacodynamic studies may be a reflection that our starting dose of 150 mg/d already resulted in potential optimal receptor inhibition. An initial study with ZD1839 indicated that doses above 100 mg/d resulted in steady-sate plasma concentrations that would have resulted in more than 90% cell growth inhibition in cell culture.59 The possibility that potentially biologically active concentrations were achieved at all dose levels in our study is further strengthened by the fact that clinical benefit and antitumor responses were seen at all dose levels.15,35,59 It is noteworthy to mention that the dose-limiting toxicity was not reached until a dose level of 1,000 mg/d, with the occurrence of unacceptable gastrointestinal toxicity. Because all the effects of receptor inhibition were profound at doses well below the one associated with unacceptable toxicity, the present study strongly supports the use of pharmacodynamic assessments to select optimal doses instead of a maximum-tolerated dose for definitive efficacy and safety trials. In the next generation of ZD1839 trials, the two dose levels used are 250 mg/d and 500 mg/d. These dose levels are high enough to result in pharmacodynamic effects on the EGFR pathway and in clinical antitumor activity,15,35 without unacceptable toxicity. The next challenge is to study pharmacodynamic markers in serial tumor biopsies from patients treated in new ZD1839 trials. In support of these studies, we have observed a significant relationship between expression of EGFR and downstream molecules (ie, activated MAPK) in various tumor types, such as head and neck squamous carcinoma47 and breast60,61 and gastric62 adenocarcinomas. Based on these studies47,60-62 and on the current data obtained in nontumor skin biopsies during ZD1839 therapy, studies assessing pre- and on-therapy tumor biopsies, activated EGFR, and downstream markers, such as MAPK, p27KIP1, Ki67, or apoptosis, are currently planned or ongoing. A possible correlation between ZD1839 biologic effects in skin versus tumor will be analyzed to explore whether the effects on keratinocyte parallel the effects on tumors, which could be useful to predict potential benefits early in the course of therapy from skin biopsies.
APPENDIX
We thank the patients that generously participated in the pharmacodynamic study.
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ABSTRACT
INTRODUCTION
at day 28 of therapy) skin biopsies from 65 patients receiving escalating doses of daily oral ZD1839. We measured ZD1839 effects on EGFR activation by immunohistochemistry using an antibody specific for the activated (phosphorylated) EGFR. Effects on receptor signaling (activated mitogen-activated protein kinase [MAPK]), proliferation, p27KIP1, and maturation were also assessed. 
bigenic mice. Proc Natl Acad Sci USA 97: 9609-9614, 2000
