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Hindawi Publishing Corporation Journal of Oncology Volume 2010, Article ID 568938, 20 pages doi:10.1155/2010/568938 Review Article Targeting the Epidermal Growth Factor Receptor in Epithelial Ovarian Cancer: Current Knowledge and Future Challenges 1 1 2 1 Doris R. Siwak, Mark Carey, Bryan T. Hennessy, Catherine T. Nguyen, 1 1 1 Mollianne J. McGahren Murray, Laura Nolden, and Gordon B. Mills Department of Systems Biology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA Department of Gynecologic Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA Correspondence should be addressed to Doris R. Siwak, firstname.lastname@example.org Received 1 May 2009; Accepted 31 August 2009 Academic Editor: Maurie M. Markman Copyright © 2010 Doris R. Siwak et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The epidermal growth factor receptor is overexpressed in up to 60% of ovarian epithelial malignancies. EGFR regulates complex cellular events due to the large number of ligands, dimerization partners, and diverse signaling pathways engaged. In ovarian cancer, EGFR activation is associated with increased malignant tumor phenotype and poorer patient outcome. However, unlike some other EGFR-positive solid tumors, treatment of ovarian tumors with anti-EGFR agents has induced minimal response. While the amount of information regarding EGFR-mediated signaling is considerable, current data provides little insight for the lack of eﬃcacy of anti-EGFR agents in ovarian cancer. More comprehensive, systematic, and well-deﬁned approaches are needed to dissect the roles that EGFR plays in the complex signaling processes in ovarian cancer as well as to identify biomarkers that can accurately predict sensitivity toward EGFR-targeted therapeutic agents. This new knowledge could facilitate the development of rational combinatorial therapies to sensitize tumor cells toward EGFR-targeted therapies. 1. Introduction therapies in ovarian cancer, focusing on epithelial ovarian cancer whenever possible. Epithelial ovarian cancer, deﬁned as cancers arising either from the mesothelial lining of the ovaries (either from the 1.1. The Epidermal Growth Factor Receptor Family. The epithelial surface lining or cortical ovarian cysts formed by EGFR family (also known as the HER or ERBB family) invaginations of the surface epithelium) or from the fallopian consists of 4 members: EGFR, HER2, HER3, and HER4 tube epithelium , accounts for 90% of ovarian malig- (alternately known as ERBB1–4). Structurally, the EGFR nancies . Epithelial ovarian cancers are further divided family consists of an extracellular ligand binding domain, a into 5 histologic subtypes: serous, endometrioid, mucinous, single transmembrane-spanning region, and an intracellular clear cell, and undiﬀerentiated. Aberrant epidermal growth region containing the kinase domain (Figure 1;reviewed factor receptor (EGFR) expression is detected in up to 60% in [7–10]). In humans, more than 30 ligands have been of ovarian cancers and occurs in all histologic subtypes identiﬁed that bind to the EGFR family, including EGF and [3, 4]. Further, aberrant EGFR expression is associated with EGF-like ligands, transforming growth factor (TGF)-α,and poor outcome of ovarian cancer patients [5, 6]. In this heregulins (HRGs, also known as neuregulins) . article, we review the EGFR family, the role of EGFR in EGFR is activated upon ligand binding, which results in ovarian cancer, and the methods used to determine this a conformational change in the extracellular domain, leading role. We also summarize the results of anti-EGFR therapies to homo- or heterodimerization with another EGFR family in ovarian cancer clinical trials and discuss challenges and member. The EGFR binding partner appears to depend future work in eﬀective treatments utilizing anti-EGFR on several properties, including the proportion of EGFR 2 Journal of Oncology NH mitogen-activated protein kinases (MAPKs), and AKT (also known as protein kinase B), resulting in perturbation of mul- tiple cellular responses including proliferation, diﬀerentia- tion, cell motility, and survival (reviewed in [9, 19]). A sum- mary of selected EGFR family pathways is shown in Figure 2. The EGFR family members can also be activated by other signaling proteins independent of addition of exogenous EGFR ligands. These include other receptor tyrosine kinases (RTKs) such as insulin-like growth factor-1 receptor (IGF- 1R) (reviewed in [20, 21]) and tyrosine kinase receptor B (TRKB, ) as well as other types of receptors such as 622 G protein-coupled receptors (GPCRs) (reviewed in ), the leptin receptor , and adhesion proteins such as E- 644 cadherin (reviewed in ) and integrins (reviewed in ). - T 654 Desensitization While the details of EGFR transactivation upon crosstalk - T 669 Internalization - T 671 are not yet fully elucidated, transactivation has been shown to occur by a variety of mechanisms. For example, there - Y 845 Src is evidence that EGFR can be transactivated by IGF-1R by direct binding . Additionally, EGFR transactivation by GPCR has been shown to occur intracellularly, such as by activation of SRC upon GPCR stimulation (e.g., ), as well as extracellularly, such as by GPCR activation by gastrin releasing peptide . This induces the formation of a GPCR 1022 - Y 1045 Ubiquitination complex containing SRC, Phosphatidylinositol 3 -kinase - Y 1046 Attenuation of - Y 1047 autokinase activity (PI3K), PDK1, and TNF-α converting enzyme (TACE), 1136 resulting in activation and translocation of TACE to the membrane where it releases the EGFR ligand amphiregulin, COOH resulting in subsequent EGFR activation . Lysophos- Figure 1: Structure of EGFR. EGFR consists of extracellular, trans- phatidic acid (LPA)-GPCR-induced ectodomain shedding of membrane, and intracellular domains. The extracellular domain is pro Heparin Binding-EGF also activates EGFR . LPA- the least conserved domain among the EGFR family members and mediated signaling is of particular importance in ovarian consists of 4 subdomains—two ligand-binding domains and two cancer as abnormalities in LPA metabolism and function receptor dimerization domains, which are cysteine-rich (reviewed likely contribute to initiation and progression of ovarian in ). The transmembrane domain, which spans the cell cancer [31–33]. Additionally, TRKB may also play a role in membrane, is hydrophobic. The cytoplasmic tail of the EGFR ovarian cancer as its activation has been shown to enhance family is highly conserved and contains the tyrosine kinase domain. migration and proliferation and suppress anoikis in human Activation of EGFR family members leads to autophosphorylation of the tyrosine residues in the cytoplasmic tail. The phosphorylated ovarian cancer cells [22, 34]. tyrosine residues become docking sites for proteins with SRC homology 2 and phosphotyrosine binding domains, which trans- 1.2. EGFR in Ovarian Cancer. The EGFR gene, located duce the signals downstream. EGFR phosphorylation at selected on chromosome 7p12, is ampliﬁed in ovarian cancer in residues and their functional outcomes are indicted in the diagram. approximately 4%–22% of cases [3, 6, 35, 36], including T: threonine; Y: tyrosine. about 13% in epithelial ovarian cancers . Activating EGFR mutations, as determined by sequence analyses of potential activating mutation sites in the catalytic domain, family members in the membrane, type and proportion of is rare in ovarian cancer, with a frequency of 4% or less ligand (reviewed in [10, 13]), and cell lineage likely reﬂected [6, 35, 37]. The constitutively active mutant EGFRvIII, in the expression of additional members of the signaling while reported earlier to be detected in 73% (24/32) of complex (see below). Strikingly, HER2 is the preferred ovarian cancers , was not detected in subsequent and binding partner for all EGFR family members , while more extensive studies examining serous orvarious HER3 is an obligatory partner , being inactive on its types of ovarian cancers . Overexpression of the EGFR own or as a homodimer as it lacks intrinsic kinase activity protein has been detected in 9%–62% of human ovarian due to mutation of critical amino acids in the kinase domain cancers [6, 36, 40, 41]; the diﬀerences in frequencies from [16, 17]. This combination has lead to the suggestion by these studies likely reﬂect utilization of diﬀerent antibodies Yarden and colleagues that HER2 and HER3 are “deaf and and cutoﬀs for overexpression. EGFR gene ampliﬁcation or dumb” members of the EGFR family, functioning in normal protein overexpression occurs across all epithelial ovarian physiology as part of signaling complexes with other EGFR cancer histotypes [3, 4]. Increased EGFR expression has family members . been associated with high tumor grade [3, 5, 6], high cell Activation of the EGFR family members results in trans- proliferation index , aberrant P53 expression , and duction of EGFR signals, via intracellular cascades, such as poor patient outcome [5, 6]. Tyrosine Autophosphorylation kinase EGF binding EGF binding Journal of Oncology 3 Epi- Beta- Amphi- regulin cellulin TNF-α EGF HB-EGF regulin NRG1 NRG2 NRG3 NRG4 Ligands (1, 4) (1) (1) (1, 4) (3, 4) (4) (4) (1) (1) (4) Receptor 14 1 3 1 1 1 2 2 3 3 4 dimers Adaptors Plc SrcCbl γ PI3K Shp2 GAP Shc Nck VA V Grb7 Crk and enzymes Rac PKC Akt Ras PAK Abl Raf JNKK Bad S6K Cascades MEK JNK MAPK Jun Sp1 Fos Elk Egr1 Transcription Myc factors Stat Figure 2: Selected representation of canonical EGFR family signaling pathways. The EGFR family consists of 4 members: EGFR, HER2, HER3, and HER4 (indicated by numbers 1–4 in the diagram). EGFR family ligands include EGF-and EGF-like ligands, transforming growth factor (TGF)-α and heregulins (HRGs, also known as neuregulins, NRGs). As indicated by the numbers in parentheses beneath the ligands, each ligand binds preferentially to a particular EGFR family member. HER2, while lacking any known ligand, is the preferred binding partner of for all EGFR family members. HER3 lacks intrinsic kinase activity due to mutation of critical amino acids in the kinase domain; therefore, it is inactive on its own or as a homodimer. Transduction of EGFR signals occurs through intracellular adaptor proteins, which transmit signals through cascades such as the RAS/RAF/MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 -kinase (PI3K)/AKT cascades. The downstream proteins in these signaling cascades can shuttle from the cytoplasm to the nucleus, where they signal to transcription factors and their complexes such as MYC, ELK, and FOS/JUN. Signal transduction through the EGFR family to downstream pathways and cascades controls diverse cellular responses such as proliferation, diﬀerentiation, cell motility, and survival as well as tumorigenesis. Figure adapted from . Abbreviations: PLCγ: Phospholipase Cγ; SHP2: SRC homology phosphatase 2; GAP: GTPase activating protein; SHC: SRC homology 2 domain and collagen-containing protein; PKC: Protein kinase C; MEK: MAPK/ERK kinase; PAK: P21-activated kinase; JNKK: JNK kinase; JNK: JUN N-terminal kinase; EGR1: Early growth response protein 1; STAT: Signal transducer and activator of transcription. One of the ﬁrst studies implicating the EGFR pathway in invasion as well as gelatinolytic, caseinolytic, and plasmin ovarian cancer was the detection of TGF-α in human ovarian activity in a dose-dependent manner . cancer eﬀusions as determined by radioimmunoassay . While initial studies suggested that EGF, due to the TGF-α was also shown to increase proliferation as measured inability to detect transcripts in Northern blotting, might by [ H]thymidine incorporation aswellasincrease not play a signiﬁcant role in ovarian cancer , subsequent levels of the tumor markers cancer antigen-125 and tissue studies indicated that exogenous EGF can also induce eﬀects polypeptide antigen  in EGFR-positive primary human associated with transformation. Like TGF-α,treatment of serous ovarian cancer cells. In the human ovarian adeno- OMC-3 cells with EGF induced cell migration and invasion carcinoma cell line OMC-3, TGF-α induced migration and and degradation of extracellular matrix components . 4 Journal of Oncology Additionally, human ovarian cancer cell lines treated with inhibitor tyrphostin AG1478 as well as the ET -speciﬁc EGF showed signiﬁcant increases in expression of proteins antagonist BQ-123 . associated with invasion (urokinase plasminogen activator More recent studies have found additional signaling and its receptor, and plasminogen activator inhibitor-1 ). molecules or pathways that contribute to EGFR-mediated EGF can also aﬀect pathways associated with angiogenesis, as malignant phenotype in human ovarian cancer cell lines, EGF stimulation of the human ovarian adenocarcinoma cell including EGFR-interleukin-6 crosstalk through Janus kinase line OVCAR-3 leads to increased H O levels, which in turn 2/Signal transducer and activator of transcription 3 sig- 2 2 activates the AKT-P70S6K pathway and increases vascular naling to mediate epithelial-mesenchymal transition , endothelial growth factor transcription through hypoxia- coactivation of Src/EGFR and axin/glycogen synthase kinase inducible factor-1α expression . (GSK)-3β pathways and induction of invasion by β-arrestin While earlier studies focused on EGFR ligands in ovarian activation of the ET-A receptor , and Src/EGFR trans- cancer, emerging studies examined the mechanism of EGFR activation, cyclooxygenase-2 expression, and cell migration activation itself. For example, Campiglio et al. detailed the upon LPA2 stimulation in CAOV-3 cells . activation characteristics of the EGFR family members upon addition of EGF or HRG in human ovarian cancer cell lines containing diﬀerent levels of EGFR family proteins 2. Disease Models, Knockouts, and . In this report, they show that the pattern of EGFR Assays for EGFR in Ovarian Cancer family activation in human ovarian cancer cells appears to be distinct from that of human breast cancer cell lines; In addition to the studies alluded to above in determining while EGFR and HER2 were consistently activated upon EGF the eﬀects of molecular modulations of EGFR and its treatment, HER3 and HER4 activation depended upon the biochemical and biological eﬀects, several other approaches relative abundance of each receptor in ovarian cancer cells. for studying EGFR have been used; these are summarized in Additionally, HER3 activation could occur independently of Table 1. As EGFR is an extracellular signaling protein, the HER2 . This complex pattern of EGFR family activation assays most commonly used in examining EGFR in human could in part explain the poor rate of response to EGFR ovarian cancer cell lines or tissues involve methods that inhibition in ovarian cancer. directly or indirectly measure EGFR activity. Assays include Further elucidation of the eﬀects of EGFR signaling in methods for detecting increased levels of the EGFR gene (e.g., ovarian cancer comes from inhibition of EGFR in cultured ﬂuorescence in situ hybridization) or protein (e.g., immuno- human ovarian cancer cells. For example, treatment of the histochemistry, Western blotting) as well as expression of human ovarian serous epithelial cancer cell line OVCA420 activating EGFR mutations (e.g., polymerase chain reaction with the anti-EGFR murine monoclonal antibody (mAb) + sequencing) or measurement of EGFR protein activity C225 resulted in decreased levels of cell cycle progression- (e.g., Western blotting of EGFR phosphorylation sites, in associated proteins Cyclin-dependent kinase (CDK) 2, vitro kinase assays). CDK4, and CDK6 and increased expression of the cell cycle- To determine the eﬀects of EGFR activation or inhibition Kip1 inhibiting protein P27 , along with increased association in tumor formation, human ovarian tumor cells are most Kip1 of P27 with the CDKs . Additionally, modulation of frequently implanted heterotopically (subcutaneously) in other cell cycle proteins was observed, including decreased immunocompromised mice (Table 1). No reports of “true expression and phosphorylation of the CDK substrates orthotopic” implantation such as in the ovarian bursa of RB and P130 and decreased protein levels of cyclin A. mice have been found in EGFR studies in ovarian cancer, Modulation of these proteins upon C225 treatment was presumably due to the complex and labor-intensive nature associated with an increase in the proportion of cells in of these procedures, while a few reports of “semiorthotopic” the G1 phase of the cell cycle. The eﬀects observed upon implantations via intraperitoneal (IP) injection were identi- EGFR inhibition were enhanced upon combined treatment ﬁed. While IP tumor implantation oﬀers a model potentially of human ovarian cancer cells with the anti-HER2 murine more reﬂective of advanced ovarian cancer in the patient mAb 4D5 . than subcutaneous injection , the diﬃculty in measuring As transactivation pathways in various cell systems have tumor volume in intact mice has precluded its widespread been delineated, so have the pathways associated with EGFR use in anti-EGFR drug studies. family activation in ovarian cancer. For example, Vacca et al. In addition to implantation of human tissues or cells have provided evidence that the GPCR ligand, endothelin via xenografts, animal models utilizing other methods of (ET)-1, can activate EGFR in the human ovarian cancer tumor formation have been used to study ovarian cancer. cell line OVCA 433 . ET-1 has been observed to play (For comprehensive reviews on animal tumor models, see a role in mitogenic autocrine loops in various cultured [58–61].) Most of these animal models utilize mice, and cell types including human ovarian cancer [51, 52]and the methods used to induce tumor formation include is proposed to contribute to tumor growth in vivo . (1) exposure to radiation (e.g., ) or chemicals (car- ET-1 treatment increased phosphorylation of EGFR and its cinogens or hormones) introduced at or near the ovary downstream proteins SRC homology 2 domain and collagen- (e.g., ), (2) syngeneic models in which spontaneously containing protein (SHC) and ERK2 as well as increased transformed murine ovarian epithelial cells are transplanted SHC-GRB2 association . These eﬀects were reversed into immunocompetent mice (e.g., ), and (3) knockout upon pretreatment of OVCA 433 cells with the EGFR or transgenic models in which selected genes are removed Journal of Oncology 5 Table 1: Summary of assays used in detecting EGFR in vitro and in vivo. Aside from high-throughput methods (such as cDNA arrays, comparative genomic hybridization, and reverse phase protein arrays) and xenograft tumor assays, more broadly encompassing biological methods such as assays for invasion, migration, or gene knockouts have been excluded. cDNA: complementary DNA; PCR: polymerase chain reaction. Performed in ovarian Platform for ovarian References for ovarian EGFR assay method Assay output cancer? cancer cancer Detection of mRNA Patient tissue, Human cDNA Array Yes  levels of various genes cell lines Detection of copy Comparative Genomic Patient tissue, Human number changes in Yes  Hybridization cell lines chromosomes Detection of stable Chromatin protein-DNA No Immunoprecipitation associations Detection of stable Coimmunoprecipitation protein-protein No + Western blotting associations Determination of entire structure or portions of Crystallography No molecule; interacting molecules Determination of Enzyme-linked amount of protein in Yes Patient tissue  Immunosorbent Assay sample Fluorescence/ Determination of gene Chromogenic in situ Yes Patient tissue [3, 6, 35, 36] copy number Hybridization Flow Cytometry/ Determination of Patient tissue, Human Fluorescence-Activated protein levels at cell Yes [175–179] cell lines Cell Sorting surface Immuno- histochemistry/ Determination of Patient tissue, Patient [4, 5, 35– Immunocyto-chemistry/ presence, location, or Yes eﬀusions, Human cell 37, 40, 41, 43, 46, 97, Immunoﬂuorescence amount of protein in lines 117, 123, 178, 180–195] (includes Tissue tissue/cell Microarrays) Measurement of In vitro Kinase Assay No intrinsic kinase activity Detection of protein Mass Spectrometry after modiﬁcation sites (e.g., Protein Enrichment phosphorylation, /Puriﬁcation (e.g., glycosylation); changes Immunoprecipitation, No in protein levels or Chromatographic proteomic proﬁles, Separation, Baculovirus protein-protein Expression) complexes Determination of Microscopic Techniques presence, location, or No (e.g., Confocal) amount of proteinincell Mulitplex Antibody Detection of multiple Patient serum, Human Arrays (Solid Phase or molecules (usually Yes [196, 197] cell lines Bead Based) proteins) of interest Determination of Patient tissue, Human Northern Blotting Yes [43, 186, 193, 198, 199] steady-state RNA levels cell lines PCR + DNA analysis (e.g., Sequencing, Detection of known Restriction Fragment Patient tissue, Human [6, 35– mutations/ Yes Length Polymorphisms, cell lines 37, 117, 130, 187, 200] polymorphisms Denaturing Gradient Gel Electrophoresis) 6 Journal of Oncology Table 1: Continued. Performed in ovarian Platform for ovarian References for ovarian EGFR assay method Assay output cancer? cancer cancer Measurement of RNA Quantitative PCR Yes Human cell lines [39, 174, 201] levels of interest Estimation of number of receptors; determination Patient tissue, Patient Radioligand Binding/ of ligand or agonist/ Yes eﬀusions, Human cell [42–45, 199] Radioimmunoassay antagonist binding lines kinetics Determination of levels Reverse Phase Protein of several proteins and Patient tissue, Patient Yes [202, 203] Array protein modiﬁcations of eﬀusions interest Reverse Determination of Human cell lines, Rat Transcription-PCR + Yes [198, 204] mRNA levels cell lines Southern Blotting Detection of gene of Southern Blotting Yes Rat cell lines  interest Tryptic Digests + Peptide Resolution (e.g., Determination of Reverse Phase High phosphorylation sites of No Performance Liquid protein Chromatography) Determination of protein abundance, [38, 39, 46, 48– protein-associated Patient tissue, Human 50, 56, 147, 175, 177, Western Blotting Yes modiﬁcations (e.g., cell lines 178, 181, 186, 196, 200, phosphorylation, 201, 204–212] cleavage, ubiquitination) Determination of eﬀect Human and mouse cell [47, 49, 147, 178, 213– Xenograft Tumors of gene/cell perturbation Yes lines 219] on tumor growth EGFR was detected and reported, but samples were not necessarily preselected for alteration of EGFR sequence, expression, or activity. or activated within the mouse. While none of these methods epithelial ovarian cancers (serous, mucinous, clear cell, have directly examined the role of EGFR aberrations in ovar- transitional) await further development. ian cancer, some of these methods have been applied to other tumor models (e.g., glioma , lung adenocarcinoma ) in which EGFR perturbations (activating mutations) have 3. Targeting EGFR in Ovarian Cancer been studied, indicating that EGFR-mediated tumor devel- opment can be successfully developed in transgenic mice. While several strategies have been attempted to block In one study where signaling proteins downstream of EGFR activity, two types of inhibitors are currently used EGFR induced ovarian cancer, transgenic mice harboring in the clinic: (1) monoclonal antibodies (mAbs), and (2) exogenously controllable (“ﬂoxed”) expression of phos- small molecule tyrosine kinase inhibitors (see [68, 69]for phatase and tensin homolog (PTEN ) and mutated K-RAS reviews). A summary of these inhibitors and their uses in genes were induced to gain oncogenic K-RAS and lose clinical trials is shown in Table 2. While the various natural tumor suppressing PTEN expression in the ovaries via functions of antibodies may contribute to their utility as injection of an adenovirus-Cre recombinase vector into the anticancer agents, including their role as modulators or infundibulum . All animals developed endometrioid eﬀectors of the immune response, molecular carriers, and adenocarcinoma of the ovary and, unlike previous ovarian pharmacologic agents that directly interfere with activation tumor models, were well diﬀerentiated, reﬂecting similar of the receptor and its downstream pathways (reviewed in histomorphology to human epithelial ovarian cancers. Thus, ), the focus of this paper will be on mAbs as pharma- this model allows for detailed study of the endometrioid cologic agents. As indicated above by the in vitro studies subtype of epithelial ovarian cancer at various stages of in human ovarian cancer cells, EGFR and its downstream tumor development and with some manipulations could be eﬀectors may be activated directly or indirectly by numerous used to study the eﬀects of EGFR aberrations in ovarian other signaling molecules. Since determination of which tumor development. Mouse models for other subtypes of molecules are key to EGFR signaling in ovarian cancers is Journal of Oncology 7 not completely understood, the focus will be on inhibition Among other anti-EGFR antibodies, a single multi- of EGFR and its family members. institution open-label phase II trial was reported in patients with ovarian cancer using matuzumab (EMD 72000) . 3.1. Anti-EGFR Monoclonal Antibodies. Anti-EGFR mAbs While screening for this phase II trial included EGFR positiv- that are used in the clinic typically bind to the extracellular ity in the ovarian tumor as determined by IHC, no responses to therapy were observed. To date there are no approved domain of EGFR (e.g., [71, 72]). While there are potentially many diﬀerent mechanisms of inhibition, in many of the anti-EGFR antibodies for ovarian cancer, and while there known cases, the antibodies prevent ligand binding (in was one clinical trial involving panitumumab (Vectibix) in combination with AMG 706 and gemcitabine-cisplatin in the case of wild-type EGFR), promote antibody-receptor complex internalization [73–75], induce transient decrease patients with advanced cancers (including ovarian), this trial of EGFR expression , inhibit EGFR heterodimerization was terminated. Currently, there are no full reports of clinical [72, 77, 78], and increase ubiquitin-mediated degradation trials for ovarian cancer with other anti-EGFR antibodies . The downstream eﬀects of inhibition in EGFR- such as zalutumumab (HuMax-EGFr) and nimotuzumab EGFR dependent cancer cells include decreased TGF-α secretion, (BIOMAb ). Among patented mAbs directed towards angiogenesis, cell migration, invasion (reviewed in ), EGFR that are not yet in clinical use, one has been proposed and induction of apoptosis . Additionally, certain engi- for use in ovarian cancer (patent number WO2005010151); however, as it is directed against deletion mutants of EGFR neered IgG subclass antibodies in which the F region is maintained can induce antibody-dependent cell-mediated (particularly EGFRvIII), its use in ovarian cancer is likely to cytotoxicity or complement activation (see [82, 83]for be limited. Due to potential EGFR transactivation by other EGFR comprehensive reviews). To reduce the likelihood of patient immune response against the therapeutic antibody, mouse family members, mAbs targeting other EGFR family mem- mAbs have been humanized (reviewed in ); these are bers have also been tested or used clinically against various reﬂected by their antibody names. For example, human- cancer types such as breast and urothelial malignancies mouse chimeric antibodies of 30% mouse composition (reviewed in ). This includes clinical trials targeting HER2 such as a phase II multi-institutional trial in ovarian are designated as “-ximab” (e.g., cetuximab); humanized antibodies with 10% mouse composition are given the “- cancer in which trastuzumab (Herceptin) was used as a single zumab” designation (e.g., trastuzumab, matuzumab), while agent in patients determined HER2 positive by IHC . An overall response rate of 7.3% (1 CR, 2 PR) was reported. fully humanized antibodies are designated as “-mumab” (e.g., panitumumab). However, the relatively low frequency of HER2 ampliﬁcation Cetuximab (Erbitux) was the ﬁrst anti-EGFR mAb in unselected ovarian cancers (e.g., 10%–23%; [35, 102]) has tested in the clinic. Cetuximab inhibits growth of a variety precluded more extensive studies. Pertuzumab (Omnitarg), of cultured cancer cells including breast, prostate, lung, a HER2 dimerization inhibitor, was administered with colon, kidney, head and neck (reviewed in ), pancreas gemcitabine (Gemzar) in platinum-resistant ovarian cancer , and bladder  and can induce regression (either patients in a phase II safety study ; eﬃcacy awaits alone or as a combined therapy) of a number of human further reports. Among antibodies targeted toward other signaling tumor xenografts such as epidermoid carcinoma , renal cell carcinoma , pancreatic cancer [86, 90], non-small molecules known to activate EGFR are monoclonals for IGF- cell lung cancer (NSCLC) , thyroid carcinoma , 1R, including 19D12 and EM164. These antibodies have been demonstrated to inhibit proliferation of human ovarian and glioblastoma multiforme . Cetuximab demonstrates activity in patients with colorectal, head and neck, and lung cancer cells aswellastumor growth in mousexenograft cancers [94, 95]. studies . However, whether EGFR aberrations aﬀect Reports for cetuximab in ovarian cancers have appeared response to anti-IGF-1R treatment or whether inhibition can recently (Table 2), including its use as a single agent in a be enhanced by anti-EGFR treatment is unknown. phaseIItrial  and in two other phase II trials in combi- nation with carboplatin with or without paclitaxel (Taxol) 3.2. Small Molecule EGFR Inhibitors. Small molecule inhibi- [97, 98]. In all studies, EGFR positivity was determined tors, based on modeling by structure-based drug design by immunohistochemistry (IHC) and in two cases was  or by screening (e.g., erlotinib, ), appear to used among the criteria for inclusion [96, 98]. Cetuximab act intracellularly by competing with ATP binding in the therapy alone showed 4% (1/25 patients) partial response catalytic region of the kinase domain, thereby abrogat- (PR) , while the cetuximab + carboplatin trial showed ing enzymatic activity of the kinase and its subsequent 12% (3/26 patients) complete response (CR) and 23% (6/26 downstream signaling eﬀects (reviewed in ). Small patients) PR . While no response rate was reported in the molecule inhibitors directed against EGFR generally prevent cetuximab + carboplatin + paclitaxel trial, progression-free homo- and heterodimerization between it and other EGFR survival (PFS) at 18 months was 39%, which did not meet the family members; however, in some cases the inhibitor allows authors’ criteria for meaningful response  and did not heterodimerization but prevents activation of these dimers proceed to the next phase of accrual. There was no evidence . While most mAbs are designed to target full length of correlation between EGFR levels and patient response in EGFR, many small molecule inhibitors can target mutant any of the reports. The implications of these and subsequent RTKs such as EGFRvIII that lack a critical extracellular results will be discussed in the “Next frontiers” section. regulatory region targeted by some of the antibodies. Small 8 Journal of Oncology Table 2: Summary of clinical trials using EGFR inhibitors in ovarian cancers References are in parentheses next to the ﬁrst author of the study. CT: clinical trial; IHC: immunohistochemistry; RPPA: reverse phase protein array; CR: complete response; PR: partial response; SD: stable disease; pt: patient; PFS: progression free survival; GOG: Gynecologic Oncology Group; VEGFR: vascular endothelial growth factor receptor. (a) Monoclonal Antibodies Study and Year CT no. Phase # Pts Therapy Selection criteria Outcome Comments CR: 3 pts Response rate criteria not met for Secord et al. NCT Cetuximab + Recurrent, platinum-sensitive next stage of accrual. 26 pts were II 28 PR: 6 pts 2008  00086892 Carboplatin disease EGFR positive by IHC. SD: 8 pts Median PFS: Konner et al. Cetuximab + Combination was adequately NCT Grade III-IV debulked tumor, II 40 14.4 months 2008  Paclitaxel + tolerated. No increase in PFS when 00063401 EGFR positive by IHC PFS at 18 Carboplatin compared to historical data. months: 39% 12 serologic markers examined Persistent or recurrent ovarian before and during treatment. No Schilder et al. PR: 1 pt II 25 Cetuximab or primary peritoneal disease, correlation between PFS and 2009  SD: 9 pts EGFR positive tumors by IHC marker changes, but high baseline of markers associated with earlier disease progression. No objective Seiden et al. Primary objective was NCT Recurrent platinum-refractory II 37 Matuzumab response 2007  pharmacodynamic; signal 00073541 disease, EGFR positivity by IHC SD: transduction evaluation. 75 pts 16%–22% were screened for EGFR status. Persistent and/or refractory CR: 1 pt Serum HER2 levels not associated Bookman et al. GOG-160 II 41 Trastuzumab disease with 2-3+ HER2 by IHC with clinical outcome. 2003  PR: 2 pts (b) Small Molecule Inhibitors Study and Year CT no. Phase # Pts Therapy Selection criteria Outcome Comments No objective Protein correlates done with RPPA. Posadas et al. NCT II 24 Geﬁtinib Platinum-refractory disease response No signiﬁcant correlation between 2007  00049556 SD: 37% for EGFR phosphorylation and tumor >2months response Analyses suggest trend towards responsiveness in EGFR positive (by Schilder et al. NCT IHC) pts. Activating mutations II 27 Geﬁtinib Persistent or recurrent disease PR: 1 pt 2005  documented in the PR pt. No objective NCT Geﬁtinib + Disease refractory or resistant to EGFR positivity not a prerequisite; Wagner et al. II 56 response 00189358 Tamoxifen platinum-taxane-based therapy EGFR status not determined 2007  SD: 16 pts PR: 2 pts Primary goal was to estimate the Gordon et al. Relapsed or progressive disease, II 34 Erlotinib objective tumor response rate to SD: 15 pts 2005  EGFR positivity by IHC erlotinib as a single agent. CR: 5 pts Erlotinib + Phase Ib dose ﬁnding study. Vasey et al. Ib 45 Docetaxel + Chemona¨ ıve pts Addition of erlotinib to other agents PR: 7 pts 2008  Carboplatin did not increase response rate. (23 evaluable) Journal of Oncology 9 (b) Continued. Study and Year CT no. Phase # Pts Therapy Selection criteria Outcome Comments Recurrent or refractory disease, No indication of improvement over Nimeiri et al. NCT Erlotinib + ≤2 prior cytotoxic CR: 1 pt bevacizumab treatment only. No II 13 2008  00126542 Bevacizumab chemotherapies; no previous EGFR mutations detected; one PR: 1 pt anti-EGFR or VEGFR therapies EGFR 2+ IHC staining detected. Kimball et al. NCT Lapatinib + Recurrent, platinum-sensitive PR: 3 pts No screening or measurement of I11 2008  00317434 Carboplatin disease EGFR or HER2 performed. SD: 3 pts No objective Baseline HER1-2 levels determined Campos et al. II 105 CI-1033 Relapsed or refractory disease response by IHC. No association between 2005  SD: 26–34% HER levels and SD. molecule inhibitors can bind reversibly (e.g., geﬁtinib or phase Ib trial . EGFR aberration or positivity was not erlotinib) or irreversibly (e.g., CI-1033) to EGFR. The clinical an inclusion criterion in either study, and EGFR status was signiﬁcance of these diﬀerent mechanisms of inhibition is reported in only one study , which examined EGFR not yet known. positivity via IHC and activating mutations in exons 19 Geﬁtinib (Iressa or ZD1839), which inhibits a variety and 21 via PCR ampliﬁcation and sequencing. The objective of cancer cell lines and xenograft tumors (reviewed in response rates were 15% (2/13 patients) for the erlotinib + ), including ovarian , was tested as a single agent bevacizumab therapy  and 52% (12/23 patients) for in two trials [112, 113]. In both trials, EGFR aberrations erlotinib + docetaxel + carboplatin . No EGFR muta- were not included as selection criteria but were assayed tions were detected, and one patient demonstrated EGFR via IHC for EGFR protein expression orvia reverse positivity, but this patient was unresponsive to erlotinib + phase protein array (RPPA) for total and phospho-EGFR bevacizumab therapy . Due to lack of improvement levels aswellasfor EGFR mutations in exons 18– over bevacizumab therapy alone and two incidents of fatal 21 via polymerase chain reaction (PCR) ampliﬁcation and gastric perforations, the erlotinib + bevacizumab study nucleotide sequencing . In both studies, there was no was discontinued . Whether these are due to the CR; 0%–4% had PR, and 4%–37% had stable disease (SD) combinatorial eﬀects of the drugs or due to bevacizumab [112, 113]. While decreased EGFR phosphorylation and alone, which has been reported to induce gastric perforation expression, as determined by RPPA, was observed in >50% , remains undetermined. The response rate of the of geﬁtinib-treated patients, this was not associated with erlotinib + docetaxel + carboplatin therapy was slightly lower clinical beneﬁt or response . However, EGFR positivity than that of a docetaxel + carboplatin therapy previously via IHC was associated with longer PFS . Additionally, conducted by the same group (52% versus 59%, [118, 120]), a mutation in exon 19 was detected in the one partially but due to good patient tolerance of the 3-drug combination, responding patient , a location that was shown to be it was recommended for further studies, particularly as responsive to geﬁtinib treatment in NSCLC patients . maintenance therapy. Geﬁtinib was also used in combination with tamoxifen Lapatinib (Tykerb, Tyverb), a dual EGFR-HER2 inhibitor in a phase II study in Germany involving patients refrac- , was tested in a multicenter phase I trial in combination tory or resistant to platinum-taxane-based treatment but with carboplatin in patients with platinum-sensitive recur- not prescreened for estrogen receptor or EGFR expression rent ovarian cancer . Patients were not prescreened or . While this combination therapy was well toler- measured for EGFR in this study. Three of 11 patients (27%) ated, it was reported to be ineﬀective against platinum had PR, and 3 patients (27%) had SD . This treatment refractory/resistant ovarian cancer as there were no tumor regimen was not recommended, as it had a low response responses. rate and signiﬁcant treatment toxicities, including grade 3– Another small molecule inhibitor, erlotinib (Tarceva), 4 neutropenia and grade 4 thrombocytopenia. In addition, demonstrated limited activity for ovarian cancer patients in 2 other patients had treatment delays due to development a multicenter phase II trial, with only 2 chemorefractory of nondose limiting grade 3 neutropenia using the initial patients in 34 demonstrating a partial response to treatment combination therapy regimen . . While EGFR expression was determined by IHC, low The irreversible pan-EGFR family inhibitor CI-1033 expression was not used as a criterion for exclusion. Erlotinib (Canertinib) was administered in a multicenter open-label has also been tested in combination with other chemothera- phase II trial for ovarian cancer patients who had failed peutic agents, including the antivascular endothelial growth prior platinum-based therapy . While baseline EGFR factor (VEGF) antibody bevacizumab (Avastin) in a phase family levels were determined via IHC from archival patient II trial , and docetaxel (Taxotere) with carboplatin in a tumor specimens, it was not used as a selection criterion. 10 Journal of Oncology No objective response was observed, although SD was of combined cetuximab and geﬁtinib therapy in patients conﬁrmed in 26%–34% of the patients (depending on the with advanced or metastatic NSCLC previously treated with dosage). There was no association between EGFR family platinum therapy . These patients had no detectable levels by IHC and stable disease. EGFR ampliﬁcations or K-RAS mutations. The regimen, Due to the relatively unremarkable results of anti-EGFR with the exception of the development of hypomagnesemia, small molecules in earlier clinical trials, more recent trials was well tolerated. There was no objective response; however, have focused on small molecules that bind irreversibly or 4 of 13 had SD. Based on these results, the group has have a broader target range. For instance, BIBW2992 (Tovok) recommended an optimum tolerated dose to use in a phase binds irreversibly to EGFR and HER2 and can inhibit both II trial. wild type EGFR and activated mutants of EGFR and HER2 While later studies selected patients based on EGFR . BIBW2992 was shown to inhibit growth of human positivity or overexpression via IHC, many of these trials still NSCLC cells implanted in nude mice more eﬀectively than demonstrated low eﬃcacy, suggesting that other methods erlotinib . Several phase I and II trials are underway of EGFR detection might be better suited for pre-drug with BIBW2992 as a single agent or in combination with screening. Quantitative approaches to assess protein level, various agents such as paclitaxel, cisplatin, or temozolomide RNA levels, gene ampliﬁcation, and mutations might prove (Temodar, Temodal) in patient groups consisting of various less subjective and more robust than IHC and could be solid tumors including glioma, NSCLC, prostate, breast, included as one of the predictors of patient response. In lung and colorectal cancer (http://www.clinicaltrials.gov/). A few cancer, gene copy number assessed by ﬂuorescence in situ trials will screen patients for EGFR or HER2 status, whether hybridization (FISH) has been reported to indicate sensitiv- by detection of gene ampliﬁcation or by activating EGFR ity to EGFR inhibition (reviewed in ). Whether EGFR mutations. An example of a small molecule with an even ampliﬁcation as determined by FISH is a reliable indicator broader target range is AEE788, which inhibits EGFR, HER2, of EGFR inhibitor sensitivity for other types of cancers has and vascular endothelial growth factor receptor (VEGFR) not yet been conclusively assessed. Additionally, it is possible . While the current focus of AEE788 is on glioblastoma, that gene increase is associated with mutational activation there is also a study that assesses the safety and clinical of EGFR, serving as a surrogate marker for mutation, and activity of AEE788 in various solid tumors. There is currently would suggest that screening by FISH might be limited to no complete report indicating which tumor types were cancers in which EGFR is frequently mutated. At any rate, included, patient response, and follow up. Other small clinical trials in which better-deﬁned measurements of EGFR molecule EGFR family inhibitors undergoing clinical trials status are taken into consideration have been emerging, such against solid tumors of various types (speciﬁc types not yet as screening of EGFR mutations in NSCLC patients prior to reported) include HKI-272 and EKB-569. administration of erlotinib. In lung cancers, sensitivity to EGFR inhibition by small An understanding of the mechanisms leading to resis- molecules such as geﬁtinib and erlotinib is associated with tance of EGFR inhibitors could help enrich for patients EGFR mutation [126–129]. Therefore, Lacroix et al. analyzed likely to respond to therapy and more importantly identify EGFR sequences from exons 18–24 in 18 advanced epithelial rational combinatorial therapy. Resistance of tumors to ovarian carcinoma specimens from patients that displayed anti-EGFR therapies has been discussed in a number of objective response or disease stabilization to carboplatin- reviews (e.g., ). Furthermore, various mechanisms of paclitaxel-geﬁtinib treatment, along with NSCLC . chemoresistance in tumor treatment have been described While 2 of 20 NSCLC samples displayed an activating (e.g., see [134, 135]). Resistance can be apparent from deletion in exon 19 (consistent with previous reports), no the onset of treatment (“intrinsic”) or develop over time EGFR mutations were detected in the ovarian carcinomas. (“acquired”). While resistance at the physiologic level has However, the potential role of mutations, insertions, or been attributed to mechanisms such as suboptimal immune deletions elsewhere in EGFR or other EGFR family members system activity or rapid metabolism or poor absorption of was not explored. the drug, resistance at the molecular level has been attributed to expression or activation of molecules or signaling path- ways that can directly or indirectly override the eﬀects of 4. Next Frontiers in Anti-EGFR Drug Discovery the drug (reviewed in ). This activation may occur via 4.1. Improving Response to EGFR Inhibitors in Ovarian Can- intracellular or intercellular mechanisms, and the activating cer. As detailed by the list of clinical trials, the use of EGFR intercellular source could either be another tumor cell or be inhibitors as single agents or in early combination studies in the surrounding stroma (reviewed in ). ovarian cancer has met with limited success. The regimens Anti-EGFR therapy resistance mechanisms include pro- have included EGFR-selective or less selective inhibitors duction of EGFR-activating ligands, receptor mutations, and administration as single agents or in combination with constitutive activation of downstream pathways, and acti- other non-EGFR antineoplastic agents. One not yet widely vation of alternative signaling pathways (reviewed in [138, 139]). Another mechanism recently suggested is explored possibility is whether using a combination of an externally targeting EGFR drug (i.e., mAb) with an internally increased resistance to autophagic cell death upon increased targeting drug (i.e., small molecule kinase inhibitor) would EGFR expression via stabilization of the facilitated glucose transporter sodium/glucose cotransporter 1 (SGLT1) . produce better results. So far, there is one complete report of a phase I study that has determined optimal doses SGLT1 can transport glucose “upstream” of a glucose Journal of Oncology 11 gradient, enabling cells to accumulate higher glucose con- such as NSCLC, colon, and bronchioalveolar carcinoma centrations than their environment, as in the case of cancer are resistant to anti-EGFR receptor agents and may have a cells, and providing more “food” for the cell . Increased worsened outcome with therapy [151–153]. This is leading SGLT1 stability is dependent on EGFR expression and not its to widespread testing of RAS mutations in patients (such activity . Thus, agents that target EGFR activity but not as the recent study in ovarian cancer ) and, indeed, is its expression are likely ineﬀective. approved by the European Medicines Agency as an exclusion Another potential mechanism of EGFR inhibitor resis- criterion for anti-EGFR therapy in colorectal cancer in tance is inﬂammation, such as by release of the inﬂammatory Europe. Optimal eﬃcacy of anti-EGFR therapy is likely to cytokine prostaglandin E2, which in lung cancer cells require concurrent targeting of the PI3K/AKT or RAS/MAPK induced phosphorylation of MAPK, indicating a bypass of pathways in patients with mutational activation of these EGFR activation (reviewed in ). One other consider- downstream components. To this end, trials that target both ation regarding chemoresistance is the sequence or timing EGFR and the PI3K/AKT pathway have been performed of multidrug administration. Proliferation of an esophageal or are underway, including cancers for glial cells and head squamous epithelial cancer cell line possessing autocrine and neck. While new agents that target the PI3K/AKT EGFR activity was either inhibited or enhanced depending pathway, including XL765 or XL147, are being tested against on whether a cytotoxic drug (platinum derivative or taxane) various solid tumors in combination with erlotinib, no was administered before or after an EGFR inhibitor . known combination trials exist in ovarian cancer. Also, while While many of these mechanisms have been studied in trials utilizing the farnesyl transferase inhibitor lonafarnib other cancer types, the data for ovarian cancer is currently (Sarasar), which targets RAS , are underway, none are sparse. currently examining the combination of EGFR and RAS Experimental results have also indicated the need to inhibition in any tumor type. better understand the interaction of EGFR with other family In addition to signaling across EGFR family members members, signaling events, and the tumor environment in and proteins downstream, consideration of other trans- ovarian as well as in other cancers. As noted earlier, relative membrane signaling molecules must be taken into account. diﬀerences in levels of EGFR family members induced Considerable data in various cell types including hepatoma diﬀerent dimerization partners upon stimulation by a given , prostate , and breast  has shown that ligand in ovarian cancer cell lines . Further, there is EGFR inhibition can be overridden by IGFR stimulation. evidence that HER3, a family member also present in ovarian Moreover, there is in vitro evidence in human NSCLC cancers and associated with increased tumor aggressiveness and head and neck squamous cell cancer cells to support  and poor prognosis , plays a critical role in EGFR- therapies combining EGFR and GPCR inhibitors, such as and HER2-driven tumors (reviewed in ). Therefore, antagonists for bradykinin (CU201) or gastrin (PD176252) only targeting EGFR will likely be insuﬃcient due to (e.g., [158, 159]). Recently, ampliﬁcation of the RTK gene functional overlap by other EGFR family members. Also, in MET has been shown to bypass EGFR receptor inhibition mouse studies using SU11925, a small molecule that targeted in human lung cancer cells and was present in 4 of 18 both EGFR and HER2, a higher concentration of SU11925 lung cancer specimens that developed resistance to geﬁtinib was required to inhibit HER2 phosphorylation in xenograft or erlotinib, supporting the idea that MET should also be tumors than in cultured human or murine cells when relative targeted in EGFR-dependent cancers . On the other HER2 levels in the cell were higher than EGFR . These hand, treatment of solid tumors with the dual EGFR- results point to a potential shortcoming of small molecule VEGFR inhibitor vandatanib (ZD6474 or Zactima) was inhibitors in vivo. ineﬀective . Based on these reports and the emergence As evident here and in numerous other reports on of numerous potential EGFR-mediated signaling proteins EGFR inhibitors in various cancer cell types, other signaling of interest in ovarian cancers, determination of which molecules aﬀected by or eﬀecting EGFR family members proteins play crucial roles in ovarian tumors might prove will have to be concomitantly examined in solid tumors. to be a challenging process. High-throughput methods First, signaling of the EGFR family occurs primarily in such as gene expression arrays and RPPA should help in trans with HER2 being the preferred binding partner . determining which genes and proteins are modulated upon Also, in human breast cancer cells, there is evidence that single and combination treatment of ovarian cancer cell cells can escape geﬁtinib treatment due to increased HER3 lines and tissues. For example, Skvortsov et al. have used expression induced by AKT-mediated negative feedback 2-dimensional gel electrophoresis and mass spectrometry signaling . Additionally, examining signaling proteins to identify proteins associated with sensitivity or resistance further downstream indicates that constitutive activation of to C225 in two colon cancer cell lines . Additionally, these pathways must also be taken into consideration. For development of robust algorithms to predict eﬀective drug example, EGFR-overexpressing human cell lines treated with combinations (e.g., ) should aid in streamlining high- geﬁtinib were resistant when PTEN, the negative regulator throughput studies and increase the likelihood of ﬁnding of the PI3K/AKT pathway, was not functional [149, 150]. In successful combinations. NSCLC, 0 of 8 patients with both EGFR ampliﬁcation and Despite these challenges, reports utilizing adherent K-RAS mutation responded to erlotinib treatment compared human epithelial cancer cell lines and tumor types suggest to 4 of 5 responders with EGFR ampliﬁcation alone . that mechanisms of resistance and methods to overcome Further, tumors with RAS mutations in several cell lineages resistance could be determined and incorporated into 12 Journal of Oncology ovarian cancer therapies. For instance, MAPK phospho- correlated with patient response. High-throughput methods rylation was not inhibited in an EGFR-positive, geﬁtinib- could also be used to aid in developing predictive models resistant human bladder cancer cell line upon geﬁtinib of drug combination in patients, such as by testing well- treatment, while MAPK phosphorylation decreased in an deﬁned chemotherapeutic drugs in a large number of cancer EGFR-positive, geﬁtinib-sensitive cell line . Moreover, cell lines and performing cell “population studies,” to better in the geﬁtinib-sensitive cell line, increased GSK-3β activity correlate drug response with precisely deﬁned oncogene and decreased cyclin D1 levels were observed upon geﬁtinib status (e.g., speciﬁc mutations, gene ampliﬁcation), such as treatment and correlated with responsiveness. Additionally, with EGFR . Further studies of other proteins aﬀecting platelet-derived growth factor receptor-β (PDGFR-β)was or aﬀected by EGFR activity, some of which have been observed to short circuit the EGFR/MAPK pathway in the discussed above, should also be performed to clarify their geﬁtinib-resistant cells . These results suggest that, in roles in ovarian cancer, both independently and in context bladder cancer, MAPK kinase phosphorylation could be a with EGFR activation. Further, the role of EGFR in diﬀerent marker for resistance while GSK-3β activation or cyclin ovarian cancer histotypes should be examined. Additionally, D1 levels could be a marker for sensitivity of EGFR drug preclinical combination therapy reports such as by Morelli et treatment, and that inhibition of both EGFR and PDGFR- al.  suggest that more studies should be performed on β wouldbemoreeﬀective in treatment of EGFR-positive determining proper scheduling of multiple therapies as well bladder cancers than EGFR alone. as examination of previously untested drug combinations. Also of great beneﬁt is designing more streamlined and 4.2. Improving Understanding of EGFR Processes in Ovarian rational methods for performing drug combination studies, such as by development of search algorithms to determine Cancer. With the emergence of high-throughput technolo- gies and their accompanying development and reﬁnement of optimal doses of combined drugs . data analyses, reports contributing to further understanding of ovarian cancers have emerged. Among the ﬁrst reports 5. Conclusion utilizing gene arrays was that of Wang et al., who identiﬁed genetic diﬀerences between human ovarian tumor specimens EGFR and its family members play a variety of roles in (comprising 5 diﬀerent histopathologic types) and normal oncogenesis and tumor progression in diﬀerent cancer and ovarian tissue . Later studies expanded the number cell types. To date, clinical studies using EGFR antagonists in and reﬁned the analyses of histopathologic types of samples ovarian cancer have shown limited eﬃcacy. As we learn more (serous papillary, clear cell, endometrioid, undiﬀerentiated, about the complexities of speciﬁc signaling changes associ- and adenocarcinomas) included in the analyses (e.g., ), ated with EGFR mutation and overexpression, future studies as well as compared drug (primarily platinum) sensitive using EGFR antagonists in ovarian cancer should focus on and resistant samples . While the number of samples determining reliable predictors for patient responsiveness to analyzed in depth is increasing, this number is still relatively anti-EGFR therapy such as by obtaining good biomarker small; whether the proﬁle of EGFR-positive ovarian cancers proﬁles and utilizing assays most appropriate to determine is diﬀerent from that of other prominent molecular markers EGFR status as well as developing rational combination ther- is unknown. Moreover, the most comprehensive proﬁles apies with EGFR inhibitors. These determinations should be characterized thus far have focused on gene alterations, facilitated by the use of high-throughput methods, as well via comparative genomic hybridization or gene microarrays as development of robust algorithms to help design experi- (reviewed in [168, 169]), which provide an incomplete ments and analyze results. 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