Pharmacodynamic studies with the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839

Pharmacodynamic Receptor

Studies With the Epidermal Growth Tyrosine Kinase Inhibitor ZD I839 Joan

ZD I839

is an orally

active,

selective

Albanell,

Federico

epidermal

growth

Rojo,

and Jose

Factor

Baselga

From the Medical Oncology Seruice, Vail d’Hebron Hospital, Barcelona, Spain. Supported in part by a grant from the Spanish Health Ministry “Fonda de Investigucidn Sanitaria” grant no. 99/0020-01. Address reprint requests to Jose’ Baselga, MD, Medical Oncolog)l Service, Hospital General Universitari Vail d’Hebron, Paseo Vail d’Hebron 119-I 29, Barcelona, Spain 08035. Copyright 0 2001 by W.B. Saunders Company 0093-7754/01/2805-1609$35.00/O doi:10.1053/sonc.2001.28551

ROWTH FACTORS and growth factor receptors play important roles in the pathogenesis of several human malignancies.1 Among the most-studied growth factor receptors is the epiderma1 growth factor receptor (EGFR), a 170-kd plasma membrane glycoprotein that belongs to the ErbB receptor tyrosine kinase family.2 The EGFR is composed of an extracellular ligand-binding domain, a transmembrane lipophilic segment, and an intracellular protein kinase domain with a regulatory carboxyl terminal segment. The EGFR can be activated by a variety of ligands including epiderma1 growth factor, transforming growth factor-a (TGF-(u), amphiregulin, and betacellulin. After binding of ligand, EGFR dimerization occurs, which results in high-affinity ligand binding, activation of the intrinsic protein tyrosine kinase activity, and tyrosine autophosphorylation.3 Activation of the EGFR tyrosine kinase has been identified as a key event that initiates the cascade of intracellular signaling events leading to proliferation, cell survival, angiogenesis, and metastasis (Fig 1). Several lines of evidence support EGFR as a target for cancer therapy: coexpression of high levels of EGFR and its ligands leads to a transformed cellular phenotype+$ EGFR is expressed in most normal epithelial cells but is elevated in many epithelial tumors and tumor-derived cell lines7; this overexpression correlates with a poor clinical outcome in a number of malignancies7-9; and monoclonal antibodies directed at the EGFR and inhibitors of the tyrosine kinase activity of the receptor can suppress the growth of EGFR-expressing cancer cells.rO-14 For these reasons, inhibition of EGFR function is clearly an attractive target for novel cancer therapies. A particularly promising approach is the development of small molecules to inhibit the tyrosine kinase activity of EGFR. However, tyrosine kinases also are important in many signaling pathways involved in normal cellular function. Therefore, EGFR tyrosine kinase inhibitors that are developed as anticancer agents should ideally show high selectivity for the EGFR tyrosine kinase, with little or no activity against other kinases.rsJ6

56

Seminars in Oncology, Vol 28, No 5, Suppl I6 (October),

factor

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PHARMACODYNAMIC

STUDIES WITH

ZD I839

57

EGF, TGF-a, HB-EGF, AR, Epi, VGF

Overexpression Transactivation (ErbB2, ErbB3, ErbB) 4

\

Fig I. tyrosine

Signaling molecules

The EGFR is composed of an extracellular binding domain, a transmembrane kinase domain with a regulatory carboxyl terminal segment. Monomeric

lipophilic receptors

segment, and an intracellular are inactive. Dimerization

protein triggers

receptor activation. In addition to ligand binding, mechanisms that promote the formation of dimers include receptor overexpression and heterodimerization. Following receptor dim&z&ion, activation of the intrinsic protein tyrosine kinase activity and tyrosine autophosphorylation occurs. These events lead to activation of a cascade of biochemical and physiological responses that are involved in the transduction of mitogenic signal. R, EGF receptor; K, intracellular protein tyrosine kinase domain; HB-EGF, heparin bindingEGF; AR, amphiregulin; Epi, epiregulin; VGF, vaccinia growth factor; pY, phosphotyrosine.

ZD1839

ZD1839 is a novel, low-molecular-weight synthetic anilinoquinazoline [(4-3-chloro-4-fluoroaniline)-7-methoxy-6-(3-morpholinopropoxy) quinazoline)] (Fig 2). I n vitro, ZD1839 potently inhibited tyrosine kinase activity of EGFR isolated from human vulva1 squamous carcinoma cells, with an ICsa of 0.023 to 0.079 pmol/L.15 In contrast, ZD1839 showed minimal activity against other tyrosine kinases such as erbB2, KDR, and c-flt, or serinelthreonine kinases including protein kinase C, MEK-1, and ERK-2, with IC5, values at least 100.fold higher than for EGFR.15 Therefore, ZD1839 is a potent and selective inhibitor of EGFR tyrosine kinase at concentrations that do not affect other kinases tested. ZD1839 prevented autophosphorylation of EGFR in a number of tumor cell lines in culture (KB oral squamous, A549 lung, DU145 prostate, and HT29 colorectal; IC&, 0.16 to 0.8 pmol/ L).16xi7 This results in the inhibition of both the

activation of downstream signaling molecules and tumor cell growth. In preclinical models, oral dosing can cause growth inhibition of multiple xenografts and regression of A431 xenografts that greatly overexpress EGFR. In animals treated with ZD1839, toxicities were exclusively seen in tissues that express EGFR, such as skin, cornea, or kidney. Based on its promising antitumor activity and faf vorable toxicity profile, ZD1839 has recently entered clinical trials. Preliminary results from phase I trials in patients with advanced disease and a wide variety of tumor types suggest that ZD1839 has an acceptable tolerability profile and promis, ing clinical efficacy, particularly in non-small cell lung cancer.isJssi9 ZD1839 is currently in phase III clinical development for the treatment of advanced non-small cell lung cancer. In addition, further trials are ongoing or planned in a number of other tumor types such as gastric, colorectal, prostate, head and neck, and breast. As we move forward with the clinical development of this ex-

58

ALBANELL,

l

Chemical

l

Orally

l

Selective

Class:

BASELGA

Quinazoline

available inhibitor

l

Competitive

l

Inhibits

inhibitor

EGFR tyrosine I&,=

0.023 pmol/L

erbB2

lC,=

1.2-3.7

established human in nude mice

pmol/L

of ATP

ligand-induced complete

kinase

EGFR

cell growth I&,=

* Induces

ROJO, AND

80 nmol/L

regression

of well-

carcinoma

xenografts

Signal Transduction Fig 2.

Properties

of ZDI 839. R, EGF receptor;

citing compound we face challenges not shared with conventional cytotoxic agents. As a result, original clinical trials have been designed. A critical goal in these trials is to explore the biological (pharmacodynamic) activity of ZD1839 against the receptor and receptor-dependent processes in tumor and normal tissues from patients treated with this novel drug. Some of the challenges of ZD1839 that need to be addressed in such pharmacodynamic studies are: (1) Definition of the optimal dose; (2) Could pharmacodynamic studies from tumors and skin biopsies be useful?; (3) Could biological profiling of the tumor and early pharmacodynamic changes predict response?; and (4) Is there a need for EGFR overexpression to benefit from ZD1839? We will review the preclinical studies that have been conducted to identify good surrogate markers of EGFR inhibition, studies aimed at characterizing the in vivo patterns of expression of EGFR signal transduction molecules, and the current status of the pharmacodynamic studies with ZD1839 in clinical trials. PRECLINICAL IDENTIFICATION OF PHARMACODYNAMIC MARKERS A key issue for pharmacodynamic studies are preclinical studies aimed at the identification of potential surrogate markers (Fig 3). In the case of

K, kinase;

ATP,

adenosine

triphosphate.

ZD1839, such preclinical studies focused on possible correlations between inhibition of EGFR phosphorylation, inhibition of receptor transduction pathways such as the Ras-Raf-mitogen-activated protein kinase (MAPK) or PI3K/Akt pathways, and effects on receptor-dependent processes such as tumor cell proliferation or apoptosis. A major signaling route of EGFR is the Ras-RafMAPK pathway. 2 Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs.~O The MAPKs ERKl and ERK2 are activated by dual phosphorylation on a tyrosine and a threonine residue by dualspecificity kinases, and subsequently regulate cell transcription. Mitogen-actived protein kinases have been linked to cell proliferation, survival, and transformation in laboratory studies.20 Based on these observations, we explored whether the activation state of EGFR and MAPK would correlate with the antiproliferative effects of ZD1839 to support the use of the activated forms of these molecules as surrogate markers of EGFR antitumor activity. The activation of EGFR and MAPK was assayed using antibodies that specifically detect the activated (phosphorylated) forms of these molecules.21 Using two human tumor cell lines that have been extensively characterized to be EGFRdependent, A43 1 (squamous carcinoma) and DiFi

PHARMACODYNAMIC

Proliferation

STUDIES

WITH

I Maturation

59

ZD I839

Survival

I Apoptosis

Angiogenesis

Metastasis

useful markers of EGFR activity may be tested in clinical specimens to document the biological effects Fig 3. A series of potentially of ZDl839. These markers can be categorized into four groups: (I) phosphorylation and levels of EGFR; (2) phosphorylation of downstream receptor-signaling molecules, such as MAPK, Akt, or Stat3; (3) nuclear proteins regulated by EGFR, such as p27 ot- cyclin D; and (4) phenotypic changes in cell proliferation, apoptosis, or maturation. R, EGF receptor: K, kinase; pY, phosphotyrosine.

(adenocarcinoma), we observed that the concentrations that inhibited tumor cell growth were the same concentrations that inhibited activated EGFR and MAPK (Fig 4).22x23 This was unrelated to downregulation of EGFR or MAPK because the levels of total (activation-independent) protein were not reduced in tumor cells treated with ZD1839. We also analyzed the effects of ZD1839 on the cellular distribution of total and activated MAPK in a series of immunocytochemistry assays. In both A431 and DiFi cells, treatment with ZD1839 markedly decreased activated (phosphorylated) MAPK staining and shifted total (phosphorylation-independent) MAPK staining to a mainly cytoplasmic localization. These results suggested that treatment with ZD1839 prevents phosphorylation of MAPK and results in the presence of nonphosphorylated MAPK localized mainly in the cytoplasm. Similar results were seen with the anti-EGFR monoclonal antibody C225. In concordance with our findings, in TGF-a/HER2 bigenic

4% 100 E E 2 = 8 L ‘P 3 5%

1550 25O-

Act-EGFR

-b

Fig 4. Correlation between inhibition of the activated EGFR and inhibition of growth by ZD 1839. The EGFR-wet-expressing A43 I cells with the indicated concentrations of ZD I839 and a dose-dependent inhibition of cell proliferation in monolayer cultures was observed. In parallel cultures, we examined the effects of ZD I839 on EGFR phosphorylation by Western blot using an antibody that specifically reacts with the activatedphosphorylated EGFR.

60

mice treated with the EGFR tyrosine kinase inhibitor AG1478, regression of mammary tumors was associated with the abolishment of constitutive MAPK activity.24 Overall, the close correlation between inhibition of growth and inhibition of activated EGFR and MAPK raised the prospect to assess these two markers in biopsies from patients treated in phase I ZD1839 clinical trials. The studies of biopsies are discussed later. Another signal-transduction pathway activated by EGFR is the PI3K/Akt pathway, which plays an important role in cell survival.*s-*7 PUK is a lipid kinase activated by EGFR that generates phosphorylated phosphoinositides. These phosphorylated lipids then bind and recruit the proto-oncogene Akt via its pleckstrin homology domain to the cell membrane. These events result in activation of Akt kinase activity on phosphorylation at Thr 308 and Ser 473. The Akt antiapoptotic role appears to be mediated by phosphorylation of multiple targets such as BAD and caspase-9.26 Recent evidence indicates that its role in promoting cell survival is critical for tumor development in vivo.27 Akt also may have a role in the control of cell proliferation. For example, Akt phosphory lates and inhibits glycogen synthase kinase 3, resulting in an increase in cyclin Dl and entry into the cell cycle. 26 Recently it has been shown that specific EGFR tyrosine kinase inhibitors prevent Akt phosphorylation, and this appears to result in marked upregulation of ~27, retinoblastoma hypophosphorylation, and reversible cell-cycle arrest.28 We also have observed inhibition of activated Akt following ZD1839 therapy in EGFR model cell lines (unpublished results), suggesting that Akt could be an additional useful marker of complete EGFR inhibition for pharmacodynamic studies. In another study, the effect of ZD1839 on the nuclear transcription factor, c-fos mRNA, which is a downstream biomarker for EGFR-tyrosine kinase and MAPK activation, was investigated.15 Timecourse studies in A43 1 xenograft samples obtained from ZD1839-treated mice showed that inhibition and recovery of c-fos transcription following oral doses of ZD1839 were time- and dose-dependent. Following a single oral dose, the nadir in c-fos mRNA was reached 6 hours after dosing, while normal levels were restored after approximately 36 hours. ZD1839 inhibited transcription of c-fos mRNA to 6% and 0.4% of control after four oral doses of 50 mg/kg and 200 mg/kg, respectively.

ALBANELL,

ROJO,

AND

BASELGA

In addition, ZD1839 has been shown to delay cell-cycle progression by disrupting regulation of cyclin-dependent kinase 2, which is a downstream component of the signaling cascade initiated by EGFR-tyrosine kinase activation.29 Growth inhibition by ZD1839 was associated with a dose- and time-dependent upregulation of the cyclin-dependent kinase inhibitor ~27~‘~‘. Expression of ~27~‘~’ reached a peak after 24 hours, coinciding with a dose-dependent downregulation of cyclindependent kinase 2 activity. This suggests that ~27~‘~~ plays a key role in ZD1839-induced cellcycle perturbation by decreasing cyclin-dependent kinase 2 activity and leading to G, growth arrest. A recent study that used another EGFR tyrosine kinase inhibitor (AG1478) further suggested that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. 28 From these observations, it is tempting to speculate that ZD1839 should increase p27 levels in responding tumors and that tumor cells lacking p27 should be less sensitive to ZD1839 than tumor cells with at least normal p27 levels. In addition to studying specific molecules that are regulated following EGFR tyrosine kinase activation, the effects on inhibition of EGFR by ZD1839 should also be detected at a phenotypic level (eg, reduced proliferation, reduced angiogenesis,i+ increased maturation, and increased apoptosis). Along this line, a recent study showed that ZD1839 inhibited proliferation of xenografts of surgically removed ductal carcinoma in situ breast tissue implanted into nude mice.30 The tyrosine kinases EGFR and c-erbB2 are expressed, and may be overexpressed, in ductal carcinoma in situ of the breast. Decreased proliferation and increased apoptosis in ductal carcinoma in situ xenografts was observed following ZD1839 treatment, whereas treatment with trastuzumab (Herceptin; Genentech, Inc, South San Francisco, CA) a monoclonal antibody directed against ceerbB2, did not have an effect. These data suggest that inhibition of EGFR tyrosine kinase by a small molecule may be more effective against ductal carcinoma in situ than a monoclonal antibody directed toward c-erbB2. This study also lends support to the measurement of proliferation rates and apoptosis in tumors from patients treated with ZD1839 as a measure of biological activity of the compound.

PHARMACODYNAMIC

STUDIES

WITH

ZDl839

CHARACTERIZATION OF EPIDERMAL GROWTH FACTOR RECEPTORDEPENDENT PATHWAYS IN HUMAN TUMORS As ZD1839 moved into the clinic, a better understanding of the EGFR-dependent pathways in vivo and their pattern of expression/activation was considered of interest for at least two reasons. First, it might be of assistance in predicting the subset of EGFR-positive tumors that will benefit from therapy. Second, downstream signal transduction molecules may prove to be useful surrogate markers of complete receptor blockade. This latest point is particularly relevant with this novel type of agent because an optimal biological dose (ie, a dose resulting in complete receptor inhibition) would be preferred to the maximally tolerated dose that is being used with conventional nontargeted chemotherapeutic agents.31)32 Based on our data in EGFR model cell lines showing that activated MAPK might be a good marker of ZD1839 antitumor effects, we decided to analyze the expression of activated MAPK and its relationship with EGFR/TGF-a expression and proliferation in human tumors in a clinical setting.33 Elevated levels of MAPK activation in tumor tissues compared with their corresponding non-neoplastic tissues have recently been reported in several studies of human tumors.34-41 However, its association with EGFR or TGF-CY expression was poorly characterized. To characterize this potential association in vivo, we studied a large series of head and neck cancers because they are well known for their common EGFR overexpression and role of the receptor and of the TGF-cr ligand in their clinical behavior.7,9 Epidermal growth factor receptor and TGF-a were assayed by immunohistochemistry. The activated MAPK was assessed by immunostaining with the same antibody that we used for the tumor cell line experiments described earlier (ie, specific for the dually phosphorylated and activated MAPKs ERKl and ERK2 [phospho-p44/42 MAPK]). The use of this antibody for immunostaining allows us to assess the activation state of MAPK in situ and provides subcellular resolution of their nuclear translocation. Proliferation rates were assayed by using the Ki-67 antigen as a marker because it is expressed only in proliferating cells.42,43 To score a tumor cell as positive for these markers, complete mem-

61

brane staining was required for EGFR, cytoplasmic or membrane staining for TGF-(u, and nuclear staining for activated MAPK or Ki-67. The percentage of stained tumor cells was scored, and grading of positivity ranged from a score of 1% to 100%. This scoring was used for statistical analysis. We found that activated MAPK was present at different levels in the majority of head and neck tumors. Mitogen-activated protein kinase activation levels were higher in tumors with advanced regional lymph node metastasis and in relapsed tumors compared with their corresponding paired primary tumors.33 The expression levels of activated MAPK were highly correlated with EGFR/ In a multivariate analysis, TGF-ol expression. TGF-or (but not EGFR) independently associated with MAPK activation, suggesting that autocrine/ paracrine stimulation of EGFR by ligand may be required for MAPK activation. In head and neck tumors the presence of TGF-CC may be required in addition to the almost universal expression of EGFR to activate the receptor tyrosine kinase activity and drive critical downstream events. This would be predicted from laboratory studies showing that genetically induced overexpression of EGFR ligands leads to cell transformation in the presence of receptor.6 The levels of activated MAPK were correlated in turn with the tumor proliferative index. Further supporting the association between activation of MAPK and tumor cell proliferation, we observed that the pattern of staining for both activated MAPK and Ki-67 was predominantly located in the periphery of the tumors, a finding that may be related to the highest level of tumor proliferation at the edges. This pattern of staining suggested that EGFR may be predominantly activated in these areas. Preliminary experiments in a limited number of specimens with an antibody to the activated/phosphorylated form of EGFR suggests this might be the case (Fig 5). 0 verall, these findings additionally supported the potential role for activated MAPK as a surrogate marker of EGFR activity that may be useful to characterize in vivo the biological effects and optimal biological dose of anti-EGFR treatments such as ZD1839. Given that MAPK may be activated by several pathways, the significant correlations observed between EGFR and TGF-a! with activated MAPK points to an important role of EGFR in activating MAPK in many head and neck tumors. However,

62

ALBANELL,

Fig 5.

Colocalization

of activated

EGFR,

activated

MAPK,

it is possible that other receptors or Ras mutations could lead to activation of MAPK, as some specimens had high levels of activated MAPK without high levels of any of the EGFR family members analyzed. Conversely, in some tumors with high levels of one or more of the EGFR family members analyzed, the level of activated MAPK was low or even undetectable. In such tumors it is possible that other EGFR signal transduction pathways, such as JAK/STAT or PI3K, are preferred instead.z7,+4 This complexity will have to be taken into account when we attempt to identify those patients who are more likely to benefit from ZD1839 therapy. We recently undertook the same approach to characterize EGFR in gastric carcinomas. In a series of 74 gastric carcinomas (50 intestinal-type and 24 diffuse-type adenocarcinomas), we studied the expression of EGFR and TGF-a by immunohistochemistry (unpublished data). In this series, 49 of 74 gastric tumors (66.2%) showed tumor cell staining for EGFR and 60 tumors (81%) were positive for TGF-a. There was a significant corre-

and proliferation

in the

edges

of head

ROJO,

and neck

AND

BASELGA

tumors.

lation between percentage of tumor cells staining for EGFR and for its natural ligand TGF-a. Subgroup analysis of intestinal-type versus diffuse-type gastric cancers showed no differences in the expression of EGFR and TGF-a. The expression of activated MAPK was also characterized and correlated with EGFR and TGF-cr results. Activated MAPK was assessed by immunohistochemistry using the same antibody used for the head and neck studies and was detected to varying degrees in 68% of the gastric tumors. In a fashion similar to what was observed in head and neck cancers, we found a significant relationship between increasing percentages of tumor cells with EGFR staining and activated MAPK. A higher proportion of intestinaltype tumors (38/50) expressed activated MAPK compared with diffuse-type tumors (12/24). Collectively, these data point to a potential autocrine EGFR and TGF-or loop in many gastric carcinomas that is associated with the presence of activated MAPK. We are currently extending this study to a larger set of gastric tumors; we are also assaying the activated-phosphorylated EGFR,

PHARMACODYNAMIC

STUDIES

WiTH

ZD I839

63

phosphorylated Akt, tumor proliferation rates (using Ki-67 as a marker), and p27 levels in the whole series. The goal of this study is to characterize the levels of expression of two critical EGFR pathways, Ras-Raf-MAX and PI3K/Akt, in human tumors. Furthermore, their relationship with EGFR activation, proliferation, and p27 levels will be explored. In a recent study in human breast cancer, activated MAPK was associated with poor prognosis and decreased sensitivity to endocrine therapy and with the expression of phosphorylated Jun, a transcription factor activated by MAPK.41 In this series of breast adenocarcinomas, activated MAPK was correlated with the expression of EGFR, which adds support, in a different tumor type, to our findings of a link between activation of MAPK and EGFR expression in clinical tumors of different sites and histologies. Taken together, these studies show that activated MAPK is present and is correlated with the expression of EGFR/TGF-a in a variety of carci, nomas. These data also point to the potential value of activated MAPK as a surrogate marker of EGFR activation or inhibition that should be further explored in pharmacodynamic studies. We are currently addressing whether a similar relationship occurs between EGFR and Akt. As a result of these studies, assessment of MAPK activation levels in pre- and post-therapy tissue biopsies from patients treated with the anti-EGFR tyrosine kinase inhibitor ZD1839 was incorporated to provide evidence for successful inhibition of EGFR function in vivo, and to determine whether there is a correlation between levels of activated MAPK and response to therapy. Newer ZD1839 studies also plan to assesschanges in activated Akt during therapy in serial biopsies. PHARMACODYNAMIC CLINICAL

STUDIES TRIALS

IN ZD I839

The early phase I clinical trials with ZD1839 included a pharmacodynamic endpoint to measure the biological effects of this compound. In particular, skin pharmacodynamic studies were conducted in two phase I trials of ZD1839 performed at six centers in the United States (study 0011) and 10 centers in Europe/Australia (study 0012).r3J9 In these studies, escalating doses of oral ZD1839 were administered at dose levels ranging from 150 mg to 1,000 mg per day. In patients consenting to the procedure, a normal skin biopsy

Skin biopsy

Dose

R&at

levels

(mg

daily)

150

biopsy

Dose-escalation

schedule

14 patients per level/trial until:

225 300

400

- maximum - IOOO-mg

800

dose

tolerated dose level

dose

800 1000 Fig 6. Design of the skin pharmacodynamic studies of ZD I839 (phase I trials 00 I I and 00 12). PD, progressive disease.

sample was collected within 2 weeks before the first dose of ZD1839 and again as close to day 28 as possible (Fig 6). The skin was chosen for pharmacodynamic studies because it is easily accessible and has high levels of EGFR expression. In fact, EGFR is a key regulator of epidermal development and physiology.45,4sAdditionally, in keratinocyte cultures, specific EGFR tyrosine kinase inhibitors or neutralizing monoclonal antibodies of EGFR potently inhibit proliferation of keratinocytes, block keratinocyte migration, and induce keratinocyte terminal differentiation and apoptosis.+9 There was also a clinical basis to study the skin because skin rashes affect some patients treated with different anti-EGFR tyrosine kinase inhibitors and with antibodies targeting EGFR, such as the chimeric monoclonal antibody C225.‘2~13,32 This observation clearly pointed to an EGFR-related mechanism of toxicity in the skin. Preliminary analysis of skin biopsies has shown that ZD1839 results in substantial changes in EGFR-dependent molecules, such as phosphorylated MAPK, ~27, phosphoSTAT3, and others (Fig 7).19J3~5OJrThe presence of EGFR and its ligand.TGF-a! was confirmed in all pre- and posttherapy skin biopsies. The expression in keratinocytes of both activated MAPK and the proliferative marker Ki-67 decreased during therapy. Concomitantly, there was an increase in the cyclin-dependent kinase inhibitor ~27~‘~r, in the keratinocyte maturation-related marker phosphory lated STAT3,5z and in cytokeratin K6. We are currently assessing the activated phosphorylated EGFR pre- and post-therapy to determine the effects of ZD1839 at the direct target. At the his-

ALBANELL,

64

Fig 7. ZD I839 challenges: Could pharmacodynamic were immunostained with an antibody specific for expressing

phospho-MAPK.

(Right)

Hair

follicle

the

showing

studies from skin dually activated decreased

topathologic level, increased apoptosis, lymphocytic infiltrates, and dermal edema were common during therapy. These biological changes were seen at all dose levels of ZD1839 (less than 150 mg/day), a finding that supports the hypothesis that biologically relevant concentrations are achieved.19J3xj0 This finding of biological activity at all dose levels is in agreement with the previously reported plasma concentrations of ZD1839, which are above the threshold concentration that results in growth inhibition in preclinical studies.13 This also is in agreement with the observations that clinical benefit and responses were not restricted to the highest dose levels.i9Js~50 We are extending these studies to the full set of skin biopsies harvested at other institutions within ZD1839 trials 11 and 12 to correlate the degree of the pharmacodynamic effects with clinically meaningful endpoints. These pharmacodynamic endpoints will be assessed for potential correlations with clinical benefit to establish if we can select those patients who are more likely to benefit from ZD1839 and whether early pharmacodynamic changes predict response. It would be important to determine whether a dose effect can be observed and whether the minimal dose resulting in maximal receptor inhibition could be identified. Additionally, these data suggest that inhibition of EGFR by ZD1839 affects skin biology and points to a potential interest in testing this compound in the treatment or prevention of EGFR-dependent skin diseases, such as psoriasis or epithelial tumors.27,53 The challenge now is to study these pharmaco-

biopsies be useful? and phosphorylated

expression

Skin

of phospho-MAPK

ROJO, AND

specimens containing ERKllZ MAPKs. (Left) following

ZD I839

BASELGA

hair follicles Hair follicle

therapy.

dynamic endpoints in serial tumor biopsies from patients treated in ZD1839 trials. Studies assessing activated EGFR, activated MAPK, and other selected tumor markers in phase II trials from patients treated with ZD1839 are currently planned based on the expression of activated MAPK in tumors and its relationship with EGFR. Additionally, the encouraging data obtained in the preliminary study with nontumor skin biopsies postZD1839 therapy further raised prospects for serial biological studies in tumor biopsies. A possible correlation between ZD1839 biological effects in skin versus tumor from the same patients will also be analyzed to explore the possibility of predicting benefit early in the course of therapy using skin biopsies. THE NEED FOR EPIDERMAL GROWTH FACTOR RECEPTOR OVEREXPRESSION TO BENEFIT FROM ZDl839 To date, there is no evidence for a direct relationship between EGFR expression levels and responsiveness to ZD1839, although this possibility is under investigation. In vivo ZD1839 preclinical studies showed substantial activity against small cell lung (LX-l), non-small cell lung (A549, SKLC-16), prostate (PC-3, TSU-PRl), and vulva1 (A43 1) xenografts .47 Antitumor activity was greatest in the A431 tumor, which had the highest EGFR expression, and lowest in the LX-1 tumor, which had no detectable EGFR expression. However, response was similar among A549, SKLC-16, PC3, and TSU-PRl tumors, which had modest levels of EGFR expression. Although

PHARMACODYNAMIC

STUDIES

WITH

ZD I839

overexpression of EGFR is generally considered to be the main mechanism of increased EGFR-mediated signaling, the pathway also can be activated by increased concentration of ligands.6 Although it might be tempting to establish a parallel between trastuzumab (and the need for HER2 overexpression) and ZD1839, the biology of EGFR is different from that of the HER2 receptor. The EGFR has a series of well-known ligands, and ligand binding to the receptor triggers homodimer and heterodimer formation. On the other hand, the HER2 is a ligand-less receptor, and receptor overexpression may be required to activate downstream signaling. The initial studies with ZD1839 were not aimed at determining clinical activity, and it was not required that EGFR expression be determined in the tumor.” Therefore, it will be critical in the new series of clinical trials to analyze the level of EGFR expression in the tumor and, perhaps, the level of expression of selected ligands, such as TGFor, which are believed to be required for the maintenance of an active EGFR autocrine loop. REFERENCES 1. Aaronson SA: Growth factors and cancer. Science 254: 1146-1153, 1991 2. Klapper LN, Kirschbaum MH, Sela M, et al: Biochemical and clinical implications of ErbB/HER signaling network of growth factor receptors. Adv Cancer Res 77:25-79, 2000 3. Lemmon MA, Schlessinger J: Regulation of signal transduction and signal diversity by receptor oligomerization. Trends Biochem Sci 19:459-463, 1994 4. DiFiore PP, Pierce JH, Fleming TP, et al: Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell 51:1063-1070, 1987 5. Rosenthal A, Lindquist PB, Bringman TS, et al: Expression in rat fibroblasts of a human transforming growth factoralpha cDNA results in transformation. Cell 46:301-309, 1986 6. Di Marco E, Pierce JH, Fleming TP, et al: Autocrine interaction between TGF alpha and the EGFzreceptor: Quantitative requirements for induction of the malignant phenotype. Oncogene 4:83 l-838, 1989 7. Salomon D, Brandt R, Ciardiello F, et al: Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 19:183-232, 1995 8. Sainsbury JR, Malcolm AJ, Appleton DR, et al: Presence of epidermal growth factor receptor as an indicator of poor prognosis in patients with breast cancer. J Clin Path01 38:12251228, 1985 9. Grandis JR, Melhem MF, Gooding WE, et al: Levels of TGF-a and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Nat1 Cancer Inst 90:824-832, 1998 10. Mendelsohn J: Epidermal growth factor receptor inhibl-

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