Epidermal Growth Factor Receptor Blockade Research Articles

Cooperative interaction between protein inhibitor of activated signal transducer and activator of transcription‐3 with epidermal growth factor receptor blockade in lung cancer

Epidermal Growth Factor Receptor (EGFR) targeting in nonsmall cell lung cancer (NSCLC) is an established treatment modality; however, it only benefits a minority of patients. STAT3 (signal transducer and activator of transcription-3) plays an important role in the oncogenic signal transduction pathway of NSCLC. Inhibition of STAT3 results in NSCLC growth inhibition and apoptosis. We have previously shown that combined inhibition of EGFR and STAT3 by small molecules resulted in improved therapeutic efficacy as compared with blocking EGFR alone. However, the STAT3 protein has a number of endogenous negative regulators including PIAS3 (Protein Inhibitor of Activated STAT3). In this study, we investigated for the first time the role of PIAS3 in modulating oncogenic EGFR-STAT3 signaling pathway in lung cancer and the anti-proliferative effect of using PIAS3 in conjunction with EGFR blockade in NSCLC. We demonstrate that PIAS3 is expressed in variable degrees in all NSCLC cells. EGF and IL-6 stimulation resulted in the association of PIAS3 with STAT3. The PIAS3/STAT3 complex then bound the STAT3 DNA binding sequence resulting in STAT3 regulated gene expression. Over-expression of PIAS3, using a PIAS3 expression construct, decreases STAT3 transcriptional activity. Furthermore, over-expression of PIAS3 consistently decreased proliferation. EGFR blockade and PIAS3 over-expression in combination had significantly greater anti-proliferative effects as compared with either EGFR blockade or PIAS3 over-expression alone. In conclusion, PIAS3 is expressed in NSCLC cell lines and its over-expression decreased STAT3 transcriptional activity, decreased proliferation of NSCLC cells and when used in conjunction with EGFR inhibitors, increased the anti-proliferative effects.

Assessment of K‐ras mutation

Some of the most significant therapeutic advances in the treatment of cancer have occurred in the management of colorectal metastases. The introduction of new cytotoxic chemotherapeutic and biologic agents has changed the approach to these patients from both an oncologic and a surgical perspective. In addition, an understanding of the molecular mechanisms by which these agents affect tumors is developing. This molecular information will be critical in the future in designing therapeutic regimens based on an individual tumor's genetic profile rather than treatment for a specific tumor type. The rapidly evolving treatment of colon cancer has provided several interesting genetic biomarkers/pathways/genes-/kinases that have been targeted or seem to play an important role. Of particular interest is the blockade of epidermal growth factor receptor (EGFR) with monoclonal antibodies. This treatment is efficacious when used alone or combined with chemotherapy. However, recent data revealed that patients with tumors positive for the K-ras mutation do not benefit from EGFR blockade. Compelling evidence has indicated that mutated K-ras is an important oncogene involved at the early stage of the development of colorectal cancer. Furthermore, mutations in the K-ras gene have been associated with aggressive tumor biology. K-ras mutational analysis is an important step in the overarching goal of developing personalized medicine. New treatment strategies are needed to more effectively treat patients with the K-ras mutation.

Simultaneous blockade of the epidermal growth factor receptor/mammalian target of rapamycin pathway by epidermal growth factor receptor inhibitors and rapamycin results in reduced cell growth and survival in biliary tract cancer cells

The prognosis of patients with biliary tract adenocarcinomas (BTA) is still poor due to lack of effective systemic treatment options. Knowledge of the molecular mechanisms involved in the pathogenesis of this disease is of importance for the development of new treatment strategies. We determined the expression of epidermal growth factor receptor (EGFR) and activated mammalian target of rapamycin (p-mTOR) in paraffin-embedded surgical specimens of BTA (n = 89) by immunohistochemistry. Overall survival was analyzed with Cox models adjusted for clinical and pathologic factors. Combined EGFR/p-mTOR expression was significantly associated with relapse-free survival [adjusted hazard ratio for relapse, 2.20; 95% confidence interval (95% CI), 1.45-3.33; P < 0.001] and overall survival (adjusted hazard ratio for death, 2.32; 95% CI, 1.50-3.58; P < 0.001) of the patients. The effect of the EGFR inhibitors erlotinib or cetuximab and the mTOR inhibitor rapamycin on growth and survival of five BTA cell lines was tested in short-term 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and long-term colony formation assays. Simultaneous blockade of EGFR and mTOR in biliary tract cancer cell lines results in a synergistic inhibition of both phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways, leading to reduced cell growth and survival. These results suggest that combined targeted therapy with EGFR and mTOR inhibitors may potentially benefit patients with BTAs and should be further evaluated in clinical trials.

Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non-small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification. We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation. Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects. These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients.

Proliferation of Human Glioblastoma Stem Cells Occurs Independently of Exogenous Mitogens

Primary glial tumors of the central nervous system, most commonly glioblastoma multiforme (GBM), are aggressive lesions with a dismal prognosis. Despite identification and isolation of human brain tumor stem cells (BTSCs), characteristics that distinguish BTSCs from neural stem cells remain to be elucidated. We cultured cells isolated from gliomas, using the neurosphere culture system, to understand their growth requirements. Both CD133(+) and CD133(-) adult GBM BTSCs proliferated in the absence of exogenous mitogenic stimulation and gave rise to multipotent GBM spheres that were capable of self-renewal. Epidermal growth factor (EGF) and fibroblast growth factor-2 enhanced GBM BTSC survival, proliferation, and subsequent sphere size. Blockade of EGF receptor (EGFR) signaling reduced exogenous mitogen-independent GBM sphere growth. Implantation of as few as 10 exogenous mitogen-independent GBM BTSCs led to the formation of highly invasive intracranial tumors, which closely resembled human GBMs, in immunocompromised mice. These results demonstrate that exogenous mitogen independence, mediated in part through EGFR signaling, is one characteristic that distinguishes CD133(+) and CD133(-) GBM BTSCs from neural stem cells. This novel experimental system will permit the elucidation of additional constitutively activated mechanisms that promote GBM BTSC survival, self-renewal, and proliferation.

A novel mechanism for anti‐EGFR antibody action involves chemokine‐mediated leukocyte infiltration

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of squamous cell carcinoma of the head and neck (SCCHN). Monoclonal antibodies (mAbs) against EGFR are currently used for therapy of recurrent or metastatic disease; however, their mode of action is not completely understood. To investigate the immunological effects of anti-EGFR mAb, we generated a three-dimensional spheroid model of EGFR-expressing SCCHN and used this model to study the effect of anti-EGFR mAb on leukocyte migration toward tumors. Pretreatment with the blocking anti-EGFR mAb EMD 72000, its F(ab')2 fragments or an EGFR tyrosine kinase inhibitor led to substantially increased leukocyte infiltration into EGFR overexpressing tumor spheroids, but not into those with low EGFR expression. Nonblocking anti-EGFR mAb or fibroblast-specific mAb did not affect leukocyte infiltration, suggesting that the observed increase in leukocyte infiltration depends on interference with EGFR activation. Using a human cytokine macroarray, we demonstrated that the blockade of EGFR by anti-EGFR mAb in EGFR-overexpressing SCCHN cells leads to differential expression of several cytokines and chemokines, including the chemokine MCP-1/CCL-2. The significant upregulation of MCP-1/CCL2 on exposure to anti-EGFR mAb was confirmed by quantitative PCR and enzyme-linked immunospot analyses. Moreover, blocking anti-MCP-1 antibody inhibited leukocyte migration toward tumor cells induced by anti-EGFR mAb, pointing to an important role of MCP-1/CCL2 in anti-EGFR mAb-induced leukocyte migration. Our findings demonstrate that anti-EGFR mAb induces leukocyte infiltration to tumor spheroids by upregulating chemokine expression. This novel mechanism for anti-EGFR mAb action may contribute to the antitumor effects of anti-EGFR mAb in vivo.

Panitumumab-Related Hypomagnesemia in Patients with Colorectal Cancer

Purpose Hypomagnesemia is an adverse reaction associated with epidermal growth factor receptor (EGFR)-targeting monoclonal antibodies. Of the 2 EGFR antibodies approved by the US Food and Drug Administration—cetuximab and panitumumab—cetuximab-induced hypomagnesemia has been extensively characterized but panitumumab-induced hypomagnesemia has not. Methods In this retrospective study, the clinical course of hypomagnesemia is described in three 64- to 68-year-old men who received panitumumab monotherapy or panitumumab-plus-irinotecan therapy for colorectal cancer for 8 to 21 weeks. Results The onset of hypomagnesemia was variable, ranging from 1 week to 10 weeks following the initiation of panitumumab. Magnesium levels did not normalize until 4 to 8 weeks after discontinuation of the agent. Of the patients in the study, 2 had new onset of grade 3 hypomagnesemia 1 to 3 weeks after panitumumab was discontinued. Management was magnesium sulfate 2 g infusion weekly and magnesium oxide 1,200 mg oral repletion daily. With severe hypomagnesemia (grade 3 and higher) or significant diarrhea (grade 3 and higher), a daily infusion of magnesium sulfate 2 or 4 g was administered. Conclusion When administering panitumumab therapy, magnesium levels should be monitored from the initiation of the agent to at least 8 weeks following cessation. Hypomagnesemia usually can be managed with magnesium sulfate 2 to 4 g infusion weekly and magnesium oxide 1,200 mg oral repletion daily. Future research is warranted to identify simple and efficient strategies for monitoring and treating EGFR blockade with monoclonal antibody-associated hypomagnesemia.

Establishment and Characterization of a Model of Acquired Resistance to Epidermal Growth Factor Receptor Targeting Agents in Human Cancer Cells

The epidermal growth factor receptor (EGFR) is recognized as a key mediator of proliferation and progression in many human tumors. A series of EGFR-specific inhibitors have recently gained Food and Drug Administration approval in oncology. These strategies of EGFR inhibition have shown major tumor regressions in approximately 10% to 20% of advanced cancer patients. Many tumors, however, eventually manifest resistance to treatment. Efforts to better understand the underlying mechanisms of acquired resistance to EGFR inhibitors, and potential strategies to overcome resistance, are greatly needed. To develop cell lines with acquired resistance to EGFR inhibitors we utilized the human head and neck squamous cell carcinoma tumor cell line SCC-1. Cells were treated with increasing concentrations of cetuximab, gefitinib, or erlotinib, and characterized for the molecular changes in the EGFR inhibitor-resistant lines relative to the EGFR inhibitor-sensitive lines. EGFR inhibitor-resistant lines were able to maintain their resistant phenotype in both drug-free medium and in athymic nude mouse xenografts. In addition, EGFR inhibitor-resistant lines showed a markedly increased proliferation rate. EGFR inhibitor-resistant lines had elevated levels of phosphorylated EGFR, mitogen-activated protein kinase, AKT, and signal transducer and activator of transcription 3, which were associated with reduced apoptotic capacity. Subsequent in vivo experiments indicated enhanced angiogenic potential in EGFR inhibitor-resistant lines. Finally, EGFR inhibitor-resistant lines showed cross-resistance to ionizing radiation. We have developed EGFR inhibitor-resistant human head and neck squamous cell carcinoma cell lines. This model provides a valuable preclinical tool to investigate molecular mechanisms of acquired resistance to EGFR blockade.

Update on Anti-EGFR Targeted Therapy

Targeted therapy has evolved recently as an important treatment modality for cancer, and the most extensively studied pathways for targeted therapy are those related to epidermal growth factor receptor (EGFR). EGFR is widely expressed in different tumor types and strong expression is associated with higher risk of recurrence/metastasis, poorer survival, and resistance to chemotherapy/radiotherapy. Both monoclonal antibody against EGFR and/or tyrosine kinase inhibitors are now in use clinically for treatment of advanced cancer. With the increasing usage of these agents and the availability of more new agents in the future, knowledge on anti-EGFR therapy is highly relevant to the current state of cancer therapy and to new research directions in the field of oncology. The focus of this special issue is on the clinical applications of approved or investigational anti-EGFR therapy in solid tumors with emphasis on efficacy, toxicity, response, assessment, multimodality treatment, prognostic factors and predictive markers. The review by A. Harandi et al. provides a comprehensive and updated overview of the frequently used EGFR inhibitors and summarizes clinical efficacy data of these agents and their associated toxicity and management. This review clearly shows the impact of anti-EGFR therapy on cancer treatment since improved treatment results can be seen with the use of anti-EGFR therapy in many common cancers. One of these cancers is colorectal cancer and the approval of anti-EGFR monoclonal antibodies in the treatment of metastatic colorectal cancer has expanded the armamentarium against this disease. H. Loong et al. in their review summarize the historical progress and recent clinical developments of anti-EGFR therapies in the treatment of metastatic CRC and discuss the novel strategies of targeting the EGFR pathway to improve efficacy as well as ongoing research in identifying specific molecular predictors of response. While the approved use of drugs like the dual kinase inhibitor Lapatinib represents significant advances in the clinical management of breast cancer, confirmatory studies must be considered to foster the use of anti-EGFR therapies including safety, pharmacokinetics, and clinical efficacy in this common cancer. The article by J. Flynn et al. reviews the mechanism of anti-EGFR therapy in breast cancer and summarizes recent advances including the development of improved high-throughput analyses for identifying novel anti-EGFR activity, as well as advances in DNA/RNA-microarray technology for classification purposes, which will contribute to the overall understanding and development of targeted therapy in treatment of breast cancer. Cancers of the esophagus and stomach present a major health burden worldwide. The impact of cytotoxic agents on the disease has been modest. EGFR pathway has also been implicated in pathophysiology of esophageal and stomach cancers. Recently EGFR inhibitors have been explored in patients with esophageal and gastric cancers, and the results are summarized by T. Dragovich and C. Campen in their review. It also appears that tumors of the distal esophagus and gastroesophageal junction are more sensitive to EGFR blockade than distal gastric adenocarcinoma. Similar to cytotoxic agenst, drug resistance remains an important issue and research field in targeted therapy. The article by J. Rolff et al. studied the impact of different resistance markers at protein and mRNA level in patient-derived nonsmall cell lung cancer xenografts in response to erlotinib and other cytotoxic agents. The results suggest that the expression levels of multidrug resistant proteins and mRNA do not play an important role in the drug resistance of nonsmall cell lung cancer. Increased transforming growth factor-β (TGF-β) expression and EGFR amplification accompany the emergence of highly aggressive human carcinomas, and cooperative signaling between these two growth factor/receptor systems promotes cell migration and synthesis of stromal remodeling factors that in turn regulate tumor invasion, neo-angiogenesis and inflammation. R. Samarakoon et al. studied the role of plasminogen activator inhibitor-1 in carcinogensis and explored its potential role as a novel therapeutic target in combination with EGFR inhibition. We hope that this special issue will stimulate interests and new researches in the development of anti-EGFR therapy in cancers, particularly the development of personalized medicine based on predictive biomarkers so that therapy can be tailored and optimized in every patient. Daniel Chua Peter Fasching Brigette Ma Sumitra Thongprasert Lori Wirth

Anti-EGFR-Targeted Therapy for Esophageal and Gastric Cancers: An Evolving Concept

Cancers of the esophagus and stomach present a major health burden worldwide. In the past 30 years we have witnessed some interesting shifts in terms of epidemiology of esophago gastric cancers. Regardless of a world region, the majority of patients diagnosed with esophageal or gastric cancers die from progression or recurrence of their disease. While there are many active cytotoxic agents for esophageal and stomach cancers, their impact on the disease course has been modest at best. Median survival for patients with advanced gastroesophageal cancer is still less than a year. Therefore, novel strategies, based on our understanding of biology and genetics, are desperately needed. Epidermal growth factor receptor (EGFR) pathway has been implicated in pathophysiology of many epithelial malignancies, including esophageal and stomach cancers. EGFR inhibitors, small molecule tyrosine kinase inhibitors and monoclonal antibodies, have been explored in patients with esophageal and gastric cancers. It appears that tumors of the distal esophagus and gastroesophageal junction (GEJ) may be more sensitive to EGFR blockade than distal gastric adenocarcinomas. Investigations looking into potential molecular predictors of sensitivity to EGFR inhibitors for patients with esophageal and GEJ cancers are ongoing. While we are still searching for those predictors, it is clear that they will be different from ones identified in lung and colorectal cancers. Further development of EGFR inhibitors for esophageal and GEJ cancers should be driven by better understanding of EGFR pathway disregulation that drives cancer progression in a sensitive patient population.

Molecular Imaging of Therapeutic Response to Epidermal Growth Factor Receptor Blockade in Colorectal Cancer

To evaluate noninvasive molecular imaging methods as correlative biomarkers of therapeutic efficacy of cetuximab in human colorectal cancer cell line xenografts grown in athymic nude mice. The correlation between molecular imaging and immunohistochemical analysis to quantify epidermal growth factor (EGF) binding, apoptosis, and proliferation was evaluated in treated and untreated tumor-bearing cohorts. Optical imaging probes targeting EGF receptor (EGFR) expression (NIR800-EGF) and apoptosis (NIR700-Annexin V) were synthesized and evaluated in vitro and in vivo. Proliferation was assessed by 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) positron emission tomography. Assessment of inhibition of EGFR signaling by cetuximab was accomplished by concomitant imaging of NIR800-EGF, NIR700-Annexin V, and [18F]FLT in cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell line xenografts. Imaging results were validated by measurement of tumor size and immunohistochemical analysis of total and phosphorylated EGFR, caspase-3, and Ki-67 immediately following in vivo imaging. NIR800-EGF accumulation in tumors reflected relative EGFR expression and EGFR occupancy by cetuximab. NIR700-Annexin V accumulation correlated with cetuximab-induced apoptosis as assessed by immunohistochemical staining of caspase-3. No significant difference in tumor proliferation was noted between treated and untreated animals by [18F]FLT positron emission tomography or Ki-67 immunohistochemistry. Molecular imaging can accurately assess EGF binding, proliferation, and apoptosis in human colorectal cancer xenografts. These imaging approaches may prove useful for serial, noninvasive monitoring of the biological effects of EGFR inhibition in preclinical studies. It is anticipated that these assays can be adapted for clinical use.

Bile Acid Alone, or in Combination with Acid, Induces CDX2 Expression Through Activation of the Epidermal Growth Factor Receptor (EGFR)

ObjectivesBile acids and acid are implicated in the development of Barrett’s esophagus. Evidence suggests that Barrett’s esophagus intestinal metaplasia may occur via induction of caudal homeobox gene 2 (CDX2). We hypothesized that induction of CDX2 by bile acids may be due to ligand-dependent transactivation of epidermal growth factor receptor (EGFR). MethodsHuman mucosal epithelial cells (SEG-1) were treated for 0 to 24 h with up to 300μm deoxycholic acid (DCA) at pH 7 or 5 with or without (w/wo) antibodies against EGFR ligand-binding site (Mab528, 3–5 μg/ml). Treatment with 100 ng/ml EGF served as control. CDX2 mRNA expression was determined by real-time polymerase chain reaction. EGFR activation was analyzed by Westerns of phosphorylated EGFR tyrosines. ResultsAcid (pH 5) increased the induction of CDX2 mRNA expression caused by DCA. CDX2 mRNA induction was markedly reduced by EGFR blockade with Mab528. Each treatment (pH 5, DCA or pH 5 plus DCA) activated the EGFR on all tyrosines tested but in different time courses. Phosphorylation by DCA was inhibited by Mab528. Activation of EGFR by DCA at pH 5 resulted in EGFR degradation, while that by DCA alone did not. ConclusionThus, CDX2 induction by DCA w/wo acid occurs through ligand-dependent transactivation of the EGFR. The variations in EGFR degradation pattern with DCA or DCA at pH 5 indicate differential transactivation pathways. The molecular pathogenesis of Barrett’s esophagus may occur via bile-stimulated cell signaling through the EGFR.

Epidermal growth factor receptor status and persistent activation of Akt and p44/42 MAPK pathways correlate with the effect of cetuximab in head and neck and colon cancer cell lines

The aim of this study was to investigate the effect of epidermal growth factor receptor (EGFR) blockade on cell survival and on downstream signalling pathways using the monoclonal antibody cetuximab. We used three colon cancer cell lines, of which one was EGFR-negative, and two head and neck squamous cell carcinoma (HNSCC) lines. EGFR expression and gene copy number were measured by immunohistochemistry and FISH analysis, respectively. The effect of cetuximab, irradiation or the combination of both on cell growth was estimated by SRB assay. Western blotting was used to determine the phosphorylation of intracellular signalling proteins and cell cycle phase distribution was measured by flow cytometry. The addition of cetuximab had only limited effects on cell growth, with a maximum inhibition of approximately 30%, but was correlated with the amount of protein expression and gene copy number of EGFR. When combined with irradiation, the effect of cetuximab was only additive and not dependent on the inherent radio-sensitivity of the cell lines. Persistent phosphorylation of Akt and/or p44/42 MAPK was detected by western blot in all of the cell lines, whereas there was no phosphorylation of Jak2 or STAT3. None of these factors alone could predict the sensitivity to cetuximab. Rather, the results suggest that it might be necessary to determine the activation status of several intracellular signalling proteins to better predict the sensitivity to cetuximab treatment.

Skin toxicity caused by EGFR antagonists—An autoinflammatory condition triggered by deregulated IL‐1 signaling?

Acneiform skin eruptions associated with sterile inflammation frequently accompany pharmacological inhibition of signaling through the epidermal growth factor receptor (EGFR) in cancer patients. Here we discuss possible pathogenic mechanisms for this phenomenon linked to control of inflammatory mediators by EGFR blockade in keratinocytes of the outer root sheath of the hair follicle. This discussion is focused on the putative role of EGFR activation in restraining interleukin (IL)-1-dependent inflammatory networks at the hair follicle.

Phase II trial of cetuximab in patients (pts) with metastatic and/or locally advanced soft tissue and bone sarcomas

10537 Background: Cytotoxic therapy has resulted in limited improvement in outcomes of pts with advanced sarcoma. The epidermal growth factor receptor (EGFR) pathway plays a role in tumorigenicity and is expressed in many sarcoma subtypes. Blockade of EGFR in certain sarcoma cell lines has decreased cell growth and increased tumor apoptosis. Therefore, we conducted a phase II trial of the anti-EGFR monoclonal antibody cetuximab in EGFR positive(+) and negative sarcoma and report here results of the EGFR+ group. Methods: Pts ≥ 16 years old with advanced sarcoma who progressed on ≥ 1 prior therapy were eligible. Cetuximab was administered at 400mg/m2 on day 1 and 250mg/m2 during subsequent weekly infusions. A cycle was defined as 4 weeks of therapy with radiographic assessment every 2 cycles. The primary objective was 4-month progression-free survival (PFS) rate in EGFR+ pts. Using a Simon two-stage design and a 10 and 30% rate as the null and alternative hypothesis, 22 pts were to be accrued in the first stage, with an additional 11 pts if ≥ 3 pts met primary endpoint. EGFR+ was defined by immunohistochemistry (IHC) as 1+, 2+, or 3+. Secondary objectives included assessment of response rate and evaluation of molecular alterations in EGFR pathway by IHC and mutational analysis using PCR/direct sequencing. Results: 22 evaluable pts with EGFR+ sarcoma were enrolled. Subtypes included synovial (32%), liposarcoma (27%), malignant fibrous histiocytoma (18%), leiomyosarcoma (14%), myxofibrosarcoma (4%), and malignant peripheral nerve sheath (4%). The median number of prior therapies was 2; the median number of cycles was 2; the median PFS was 52 days (95% CI: 48 - 54). One patient was free of progression for over 4 months. No objective responses were seen. Two patients withdrew from study due to ileus and acute renal failure. MAP-K, PTEN, and phospho-EGFR staining revealed variable expression without discernible pattern. No EGFR mutations were detected in exons tested in available specimens. Conclusions: Cetuximab has minimal activity in advanced EGFR+ sarcoma, and further investigation as a single agent is not indicated. Cetuximab in combination with cytotoxic therapy or following definition of specific molecular abnormalities in sarcoma requires further study. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Bristol-Myers Squibb

Epidermal Growth Factor Receptor Blockade in Combination with Conventional Chemotherapy Inhibits Soft Tissue Sarcoma Cell Growth In vitro and In vivo

The epidermal growth factor receptor (EGFR) is highly expressed in many human soft tissue sarcomas (STS). However, EGFR blockade has not apparently been used for human STS therapy; therefore, we examined the in vitro and in vivo effects and the underlying mechanisms before considering EGFR blockade as a therapy for STS patients. Human STS tissues and cell lines were used to study EGFR expression and activation. Western blot analysis was used to evaluate effects of EGFR activation on downstream signaling. Cell culture assays were used to assess the effect of EGF stimulation as well as EGFR blockade (using an EGFR tyrosine kinase inhibitor, Iressa; AstraZeneca) on STS cell growth, apoptosis, and chemosensitivity. An in vivo study (HT1080 human fibrosarcoma cell line in nude/nude mice: Iressa, doxorubicin, Iressa + doxorubicin, vehicle) was used to examine tumor growth; pEGFR, proliferating cell nuclear antigen, and terminal deoxyribonucleotide transferase-mediated nick-end labeling staining helped assess the effect of therapy in vivo on STS EGFR activation, proliferation, and apoptosis. EGFR was expressed and activated in STS cell lines and tumors, probably due to ligand binding rather than EGFR mutation. Stimulation caused activation of AKT and mitogen-activated protein kinase pathways. EGFR blockade inhibited these effects and also caused increased apoptosis, a p53-independent G(0)-G(1) cell cycle arrest, and decreased cyclin D1 expression. In vivo, Iressa + doxorubicin had markedly synergistic anti-STS effects. EGFR blockade combined with conventional chemotherapy results in anti-human STS activity in vitro and in vivo, suggesting the possibility that combining these synergistic treatments will improve anti-STS therapy.

Enhanced EGFR inhibition and distinct epitope recognition by EGFR antagonistic MABS C225 and 425

Monoclonal antibodies (mAbs) that inhibit activation of the epidermal growth factor receptor (EGFR) have shown therapeutic potential in select malignancies including breast cancer. Here, we describe that combined use of two such mAbs, C225 (Cetuximab) and 425 (EMD55900), reduced growth and survival of EGFR overexpressing MDA-MB-468 breast cancer cells more effectively than either antibody alone. Similarly, the C225/425 antibody combination more effectively inhibited AKT and MAPK phosphorylation in MDA-MB-468 cells. Surface plasmon resonance, size exclusion chromatography, and analytical ultracentrifugation demonstrated that mAbs C225 and 425 simultaneously bind to distinct antigenic epitopes on domain III of the soluble wild-type EGFR. Furthermore, neither mAb competed with the other for binding to cells expressing either wild-type EGFR or a mutant EGFR (EGFRvIII) associated with neoplasia. Mutagenesis experiments revealed that residues S460/G461 in EGFR domain III are essential components of the 425 epitope and clearly distinguish it from the EGF/ TGF-α binding site and the C225 interaction interface. Collectively, these results support the conclusion that therapeutic EGFR blockade in cancer patients by combined use of mAbs C225 and 425 could provide advantages over the use of the two antibodies as single agents.

Does Gefitinib Shorten Lung Cancer Survival? Chaos Redux

Does Gefitinib Shorten Lung Cancer Survival? Chaos Redux

Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members

The epidermal growth factor receptor (EGFR) is a central regulator of proliferation and progression in human cancers. Five EGFR inhibitors, two monoclonal antibodies and three TKIs, have recently gained FDA approval in oncology (cetuximab, panitumumab, erlotinib, gefitinib and lapatinib). These strategies of EGFR inhibition demonstrate major tumor regressions in approximately 10-20% of advanced cancer patients. However, many tumors eventually manifest acquired resistance to treatment. In this study we established and characterized a model to study molecular mechanisms of acquired resistance to the EGFR monoclonal antibody cetuximab. Using high-throughput screening we examined the activity of 42 receptor tyrosine kinases in resistant tumor cells following chronic exposure to cetuximab. Cells developing acquired resistance to cetuximab exhibited increased steady-state EGFR expression secondary to alterations in trafficking and degradation. In addition, cetuximab-resistant cells manifested strong activation of HER2, HER3 and cMET. EGFR upregulation promoted increased dimerization with HER2 and HER3 leading to their transactivation. Blockade of EGFR and HER2 led to loss of HER3 and PI(3)K/Akt activity. These data suggest that acquired resistance to cetuximab is accompanied by dysregulation of EGFR internalization/degradation and subsequent EGFR-dependent activation of HER3. Taken together these findings suggest a rationale for the clinical evaluation of combinatorial anti-HER targeting approaches in tumors manifesting acquired resistance to cetuximab.

Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis.