Epidermal growth factor receptor targeting in cancer: A review of trends and strategies

The epidermal growth factor receptor (EGFR) is a cell surface receptor belonging to erythroblastic leukemia viral oncogene homologue (ErbB) family of tyrosine kinase. It plays critical role in the regulation of cell proliferation, survival and differentiation. The EGFR receptor is crucial in a variety of tumor development due to unlikely triggered by receptor overexpression, chromosomal mutation and or ligand-dependent receptor dimerization. The EGFR inhibition established a major therapeutic target in cancer therapy. The signal transduction pathway of EGFR is directly involved in tumor pathogenesis and progression. The combinatorial approach with EGFR inhibitors bring novel therapeutic regime with proved clinical efficacy. This critique briefly addressed EGFR receptor characteristics, worldwide report on various cancers and EGFR based potential targeting modalities in skin, breast, ovary, brain, lungs, pancreas, gastric and colorectal tumors and molecular pathways involved in EGFR targeting.

Targeting Epidermal Growth Factor Receptor for Cancer Treatment: Abolishing Both Kinase-Dependent and Kinase-Independent Functions of the Receptor.

While therapeutic strategies have improved, ∼500,000 women worldwide die from breast cancer each year. Many proteins have been implicated in breast cancer, including the HER family of receptors. Overexpression of epidermal growth factor receptor (EGFR) and one of its family members, HER2, each occur in ∼30% of breast cancers and correlates with poor clinical prognosis. Herceptin, an inhibitor of HER2, has been approved for treatment of breast cancer patients; however small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have not shown efficacy in breast cancer. To understand why breast cancers do not respond to EGFR tyrosine kinase inhibition, a panel of twenty breast cancer cell lines was characterized for EGFR expression. Thirteen of the twenty cell lines expressed EGFR; seven of which were resistant to EGFR TKIs. Unlike the response of HER2 positive breast cancers to Herceptin, increased expression of EGFR correlated with resistance to EGFR TKIs. One possible mechanism for resistance to EGFR TKIs is that EGFR expression is not required for growth. However, when EGFR expression was knocked down, cell growth was significantly decreased in four of the seven cell lines suggesting that EGFR expression is important for cell growth in a portion of EGFR TKI resistant breast cancers. A second mechanism for resistance to EGFR may be elevated HER2 and HER3 expression as observed in lung cancer. Conversely, in these cell lines, HER2 and HER3 expression levels did not correlate with response to EGFR TKIs. In addition, an EGFR family kinase inhibitor also was unable to abrogate the growth of these cells suggesting that HER2 expression and kinase activity was not a mediator of cell growth in the presence of EGFR TKIs. EGFR is known to localize within many sub-cellular regions including the nucleus, mitochondria, endosomes, and plasma membrane. Localization of EGFR to membrane microdomains (lipid rafts) is known to modulate EGFR signaling. Therefore, a third potential mechanism for EGFR TKI resistance may be localization of EGFR within lipid rafts. Using biochemical raft isolation and immunofluorescence, EGFR was shown to localize to lipid rafts in all four EGFR-dependent, EGFR TKI resistant breast cancer cell lines. To determine if this localization mediates resistance to EGFR TKIs, cholesterol (a main structural component of lipid rafts) was depleted using cholesterol biosynthesis inhibitors. While such inhibitors had no effect on cell growth alone, combinatorial depletion of lipid rafts and inhibition of EGFR-kinase activity resulted in decreased cell growth. Further, the interaction between cholesterol biosynthesis inhibitors and EGFR inhibitors was synergistic. Thus, we have discovered that EGFR expression and localization to lipid rafts correlate with EGFR TKI resistance in breast cancer, and that depletion of lipid rafts can overcome such resistance in EGFR expression-dependent breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 269.

Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein that constitutes one of the four members of ErbB family of tyrosine kinase receptors. Activation of EGFR leads to autophosphorylation of receptor tyrosine kinase that initiates a cascade of downstream signaling pathways involved in regulating cellular proliferation, differentiation, and survival. EGFR is abnormally activated by various mechanisms like receptor overexpression, mutation, ligand-dependent receptor dimerization, ligand-independent activation and is associated with the development of variety of human cancers. EGFR inhibition is one of the key targets for cancer chemotherapy. Approval of tyrosine kinase inhibitors such as erlotinib, gefitinib, and lapatinib for the treatment of non-small cell lung cancer led to tremendous development of novel EGFR inhibitors in the last decade. Diverse class of chemical compounds from the synthetic origin has been extensively studied. This review highlights the various classes of synthetically derived molecules which have been reported in the last few years as potential EGFR and EGFR/ErbB-2 dual inhibitors. A brief synthetic methodology to access these compounds has been highlighted along with the SAR. We strongly believe that this review will provide a platform to the synthetic chemists and biologists to design and synthesize new and potent compounds that inhibit EGFR and ErbB-2.

Pancreatic cancer is a lethal disease with little understanding of the etiology and no effective therapies. While aberrant epidermal growth factor receptor (EGFR), Src and Stat3 are detected in pancreatic cancer and implicated in the disease, their exact roles in the support of the disease phenotype remain poorly defined. Moreover, the potential for a cross-talk between EGFR, Src and Stat3 poses a challenge to any therapy that targets only one of these entities. In this study, we sought to define the EGFR, Src and Stat3 signaling integration and to investigate the contributory roles of the three entities to the pancreatic cancer phenotype. We found in Panc-1 and Colo-357 lines that phospho-Y845EGFR, pY1068EGFR and pY1086EGFR levels are responsive to c-Src inhibition, in contrast to pY1173EGFR that is solely EGFR kinase-dependent. Treatment of Panc-1 and Colo-357 cells with the EGFR inhibitor, Iressa (ZD 1839) or the Src inhibitor, Dasatinib (Das) suppressed the constitutively-active Stat3 activity, suggesting that both EGFR and Src activities promote aberrant Stat3 activation in pancreatic cancer cells. However, the early suppression of aberrantly-active Stat3 by the EGFR and Src inhibition in Panc-1 cells is countered by a Janus kinase (Jaks)-dependent re-activation, suggesting that Jaks activity could represent a compensatory mechanism for Stat3 induction. The abrogation of c-Src activity by either Das treatment or the over-expression of a kinase-dead c-Src mutant suppressed the levels of phospho-FAK, phospho-p130Cas, phospho-cortactin, phospho-p120catenin, and phospho-paxillin, suggesting that Src activity induces the mediators of motility and migration in pancreatic cancer cells. By contrast, neither EGFR nor Src inhibition modulated the baseline enhanced phospho-ErkMAPK or phospho-Akt levels in Panc-1 and Colo-357 cells, consistent with the observation that the inhibition of EGFR or Src alone only weakly suppressed cell growth and survival of pancreatic cancer cells. By contrast, the concurrent inhibition of Stat3 and EGFR or Src induced greater viability loss and apoptosis, and diminished the migration/invasion of pancreatic cancer cells in vitro. Of therapeutic significance, the concurrent inhibition of Stat3 and EGFR or Src, as compared to mono-targeting modality, induced a stronger human pancreatic tumor growth inhibition in xenografts models. We infer that the large tumor growth inhibition in vivo is due to the simultaneous suppression of the abnormal functions of Stat3 and EGFR or Src. These studies strongly suggest that the concurrent targeting of Stat3 and EGFR or Src could be a beneficial therapeutic approach for pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2528.

Epidermal Growth Factor Receptor Targeting in Cancer

Epidermal Growth Factor Receptor Is a Critical Mediator of Ultraviolet B Irradiation-Induced Signal Transduction in Immortalized Human Keratinocyte HaCaT Cells

Background: Non-small cell lung cancer (NSCLS) comprises about 80% of all lung cancers. The regular treatments for NSCLC are surgery, chemotherapy, radiotherapy and EGFR-targeted therapy. EGFR is frequently mutated and amplified in lung adenocarcinom as sensitive to EGFR inhibitors. The EGFR-targeted therapy, Gefitinib and Erlotinib, significantly improves disease free survival. However, it was not possible to predict super-responder of gefitinib degree of EGFR expression as a single agent. There is an imposing need for new rationally designed regimens to overcome Gefitinib resistance. The other tyrosine kinase inhibitor, Lapatinib, is a small-molecule inhibitor targeting both the HER2 and the epidermal growth factor receptor (EGFR) in solid tumors. We hypothesized that this aberrant activation of EGFR may contribute to Gefitinib resistance and dual blocking EGFR activation with EGFR and ERBB2 inhibitor will re-sensitize HER family in lung cancers. Experimental Design: In the in vitro study, cancer cells with EGFR wild type or mutation type were treated with Tyrosine kinase inhibitor, Gefitinib, Lapatinib, or combined treatments. The responses of cells to Gefitinib or/and Lapatinib treatments were evaluated by MTS proliferation, apoptosis assay, soft agar colony formation assay and western blot. In the in vivo study, pulmonary xenograft tumors were established in ICR/SCID mouse and treated with vehicle control, Gefitinib, Lapatinib, or Gefitinib plus Lapatinib for 4 weeks. Tumor volume were monitored and further studied by immunohistochemistry on collected tumors. Results: We selected multiple pairs of EGFR wild type and mutation type. The EGFR mutation cells have more effect under single tyrosine kinase inhibitor (Gefitinib) treatment compared with EGFR wild type cells. Dual inhibition of EGFR activation sensitized EGFR expression pulmonary cancer cells to Gefitinib treatment. Compared with single treatment, combined treatment led to a significantly more effective inhibition of cell proliferation and induced apoptosis. In a mouse pulmonary xenograft model, combination therapy with Gefitinib and Lapatinib dramatically inhibited EGFR wild type tumor growth. Conclusions: Since the EGFR family inhibitor, Lapatinib is currently under Phase III clinical trial, combining Gefitinib with this Lapatinib, dual EGFR inhibitors is a clinically applicable strategy and a promising combinatory targeted anti-cancer therapy to overcome EGFR wild type mediated Gefitinib resistance. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 619.

BackgroundThe efficacy of epidermal growth factor (EGF) receptor (EGFR) inhibitors in patients with non‐small cell lung cancer (NSCLC), pancreatic cancer (PC), or colorectal cancer (CRC) has been demonstrated. However, dermatological reactions to these inhibitors can cause significant physical and psychosocial discomfort. The objective of the present study was to evaluate the efficacy of EGF ointment for EGFR inhibitor‐related skin adverse events (ERSEs).Materials and MethodsThis placebo‐controlled, double‐blind, multicenter, pilot phase III trial enrolled patients with NSCLC, PC, or CRC treated with EGFR inhibitors. Patients with grade ≥2 ERSEs were included. Patients were randomized to three treatment arms: arm 1, placebo; arm 2, 1 ppm of EGF ointment; and arm 3, 20 ppm of EGF ointment. Patients applied ointment to their skin lesions twice daily.ResultsEfficacy evaluation was available for 80 patients (9 for PC, 28 for NSCLC, and 43 for CRC). Responses were 44.4% in arm 1, 61.5% in arm 2, and 77.8% in arm 3. There was a linear correlation between EGF concentrations and responses (p = .012). Quality of life (QoL) was assessed for 74 patients. Maximum changes in composite scores by Skindex‐16 after treatment were significantly different among arms (mean ± SD: −5.2 ± 8.6 for arm 1, −11.7 ± 14.2 for arm 2, and − 18.6 ± 17.7 for arm 3; p = .008). EGF arms showed significant improvement in emotions (p = .005) and functioning (p = .044) scores over the placebo arm.ConclusionEGF ointment is effective for managing ERSEs. It can also improve patients’ QoL compared with placebo. Clinical trial identification number. NCT02284139Implications for PracticePatients with non‐small cell lung cancer, pancreatic cancer, or colorectal cancer who are treated with epidermal growth factor (EGF) receptor (EGFR) inhibitors may experience dermatologic reactions to their treatment. This study investigated the benefit of an EGF ointment in the treatment of these adverse events and observed the ointment to be effective in managing EGFR inhibitor‐related skin adverse events.

Background: Platinum agents are a standard of care for treatment many cancers. Clinical data has shown that patients (pts) with CRC with mutant (MT) KRAS do not respond to EGFR inhibitors (Amado et al 2009). Furthermore, results from phase 3 clinical trials with erlotinib, cetuximab, or panitumumab have shown that pts whose tumors express MT KRAS have shorter progression-free survival and overall survival when an EGFR inhibitor is added to a platinum-containing regimen vs chemotherapy alone (Eberhard et al 2005, Bokemeyer et al 2011, Douillard et al 2010). However, KRAS status is not predictive of outcomes in pts receiving Ox- or irinotecan-containing regimens without an EGFR inhibitor (Richman et al 2009). This negative interaction has not been observed in pts whose tumors express either wild type (WT) KRAS receiving an EGFR inhibitor and chemotherapy or whose tumors express MT KRAS receiving an EGFR inhibitor with irinotecan (Peeters et al 2010, Van Cutsem et al 2009). Our goal was to gain a molecular mechanistic understanding of the negative interaction between EGFR inhibitors and Ox in KRAS MT CRC cells. Methods: To investigate the negative interaction between Ox and EGFR inhibitors, isogenic MT and WT KRAS-expressing HCT116 CRC cells were treated with Ox, SN-38 (the active metabolite of irinotecan), panitumumab, or gefitinib as single agents or in combination for 72 hrs. Viability was measured using an ATPlite assay. To determine the cellular distribution of EGFR, cells were serum-starved, labeled with a fluorescent anti-EGFR mAb, and visualized by a confocal microscopy. To analyze the effects of single agent or combination treatment on downstream phosphorylation of the PI3K or MAPK pathways, cells were treated for 24 hrs and phospho-proteins were detected by Western blotting. To investigate whether negative interaction between Ox and EGFR inhibitors could be reversed, cells were treated with Ox and gefitinib in combination with inhibitors to MEK, PI3K or Src for 72 hrs. Results: Treatment with gefitinib, but not panitumumab, reversed the anti-proliferative effects of Ox in the MT KRAS-expressing CRC cells vs Ox alone (p<0.0005). Neither treatment reversed the anti-proliferative effects of Ox observed in the WT KRAS-expressing cells or SN-38 in either the MT or WT KRAS-expressing cells. In the MT KRAS-expressing cells, EGFR was predominantly expressed intracellularly; in contrast, distinct cell surface staining was observed in the W T-KRAS expressing cells. This difference correlated with the inability of panitumumab to reverse the anti-proliferative effects of Ox in the CRC cells. Combination treatment of MT KRAS CRC cells with Ox and gefitinib increased pAKT which was not observed with Ox alone. Treatment of the mutant KRAS-expressing cells with a MEK, PI3K or Src inhibitor reversed the negative interaction between Ox and gefitinib (p<0.0005). Conclusion: We developed a preclinical model to further understand the negative interaction between MT KRAS-expressing cancer cells, platinum agents, and EGFR inhibitors. Feedback though AKT may contribute to the increased resistance to Ox in KRAS MT cells. Inhibitors of MEK, PI3K, or Src reversed the negative interaction between the EGFR inhibitors and Ox in a mutant-KRAS genetic background. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A150.

Programmed death-ligand 1 (PD-L1) is an immune checkpoint expressed in a wide range of malignancies leading to immune tolerance and cancer cell immune evasion. Receptor tyrosine kinases (RTKs) are key regulators of cancer cell proliferation, survival, and invasion. The Hepatocyte Growth Factor (HGF) receptor, MET, and the Epidermal Growth Factor Receptor (EGFR) are RTKs known for their tumorigenic potential in a variety of human malignancies. This study aimed to evaluate the effect of the small-molecule tyrosine kinase inhibitors (TKIs) on the expression of PD-L1 in breast cancer cells and the effect of their combination with inflammatory cytokines. The study also assessed the association of the expression of the tumoral PD-L1 mRNA with each of MET and EGFR mRNA expression and the tumor features and treatment outcomes in breast cancer patients using the METABRIC dataset publicly available from cBioPortal for Cancer Genomics. The TKIs crizotinib [a MET inhibitor] and gefitinib [an EGFR inhibitor] were used as a single treatment and in combination with the cytokines; tumor necrosis factor-α (TNF-α) or interferon-γ (INF-γ) in MCF7 and MDA-MB-231 breast cancer cells in vitro. The cells were also treated with the mitogens HGF and EGF. The viability of cells after the different treatments was determined using the MTT viability assay. The effects of the combination treatments were analyzed using combination index (CI) analysis. The expression level of PD-L1 in cancer cells was assessed using Western blotting. The combination treatment of crizotinib with TNF-α and INF-γ produced a synergistic growth inhibition of MCF7 and MDA-MB-231 cells with CI values of 0.31 and 0.55, respectively. Alternatively, combined crizotinib and TNF-α produced an antagonist effect on the viability of MCF7 cells (CI=2.49). The combination of gefitinib with TNF-α produced a synergistic growth inhibition in both MCF7 and MDA-MB-231 cells with CI values of 0.39 and 0.78, respectively. Alternatively, gefitinib resulted in an additive effect when combined with INF-γ (CI=0.99) and an antagonistic effect when combined with TNF-α (CI=2.68) in MCF7 and MDA-MB-231 cells, respectively. Treatment with HGF (50 and 100 ng/ml) increased the protein levels of PD-L1 while EGF (50 and 100 ng/ml) reduced its levels in MDA-MB-231 cells. Treatment with the TKIs crizotinib (0.1-4 µM) and gefitinib (1-40 µM) significantly reduced PD-L1 levels in MDA-MB-231 cells compared to vehicle-treated cells. The analysis of the METABRIC dataset revealed that the mRNA expression of PD-L1 was positively correlated with the mRNA expression of the EGFR gene (r= 0.081, p< 0.001) but not the MET gene. A double-high PD-L1/MET expression was significantly associated with younger age at diagnosis, high-grade carcinoma, greater tumor size, hormone receptor-negative status, HER2-positivity, and non-luminal disease compared to patients with a double-low PD-L1/MET expression. Similar findings were reported for patients with a double-high PD-L1/EGFR expression compared to patients with a double-low PD-L1/EGFR expression. Nevertheless, the mRNA expression of the PD-L1, MET, and EGFR genes as well as the co-expression of PD-L1/MET or PD-L1/EGFR genes did not affect the overall survival of breast cancer patients. Collectively, MET and EGFR TKIs could modulate the effects of inflammatory cytokines differently based on the type of cytokine and the molecular subtype of breast cancer cells. The expression of PD-L1 in breast cancer cells was upregulated by HGF while TKIs reduced its expression. The co-expression of the PD-L1 gene with MET and EGFR genes in breast cancer was associated with advanced disease presentation and worse prognosticators in patients. Together, TKIs that target MET or EGFR could be an appealing therapeutic target in breast cancer, particularly in younger patients with the non-luminal disease. Citation Format: Nehad Ayoub, Muhsen Al-Diabat, Moath Al-Shorman, Laith Al-Eitan. Programmed Death-Ligand 1 and Receptor Tyrosine Kinases in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-03.

NG01: Blockade of EGFR and HER3 enhances PI3K/Akt antitumor activity in triple negative breast cancer

HNSCC is the sixth most common cancer worldwide. EGFR is overexpressed in up to 90% of HNSCC and associated with poor outcome. An EGFR monoclonal antibody, cetuximab, was the only approved molecular targeted therapy for HNSCC; however, resistance eventually occurs in all patients. Functional screens including a small-molecule kinase inhibitor were used to identify agents that synergized with EGFR inhibitors in reducing viability in HNSCC patient-derived tumor cells. Effective combination therapies were validated in scale-up experiments, and their true targets were evaluated using siRNAs to rule out off-target effects of the drugs. Cell number, cell apoptosis, and colony formation ability were determined in tumor cells treated with single agents and drug combination. ALK expression and phosphorylation in tumor cells before and after anti-EGFR treatment was tested by Western immunoblotting. Two ALK inhibitors on the drug screen panel showed synergistic effects with EGFR inhibitors in 4/8 HNSCC patients9 tumor cells, despite ineffectiveness of single drug. siRNA targeting ALK synergized with gefitinib in reducing cell viability, indicating specificity to ALK. Scale-up dose-response experiments confirmed patient cell sensitivity to 4 different ALK inhibitors in combination with the EGFR inhibitor gefitinib. Combination indices were below 1 in the sensitive patients9 cells, indicating synergy. Cotargeting EGFR and ALK decreased HNSCC patients9 tumor cell number, colony formation ability, and increased apoptosis. Because of low expression of ALK mRNA in original tumors and patient tumor-derived cells, we hypothesized that EGFR inhibition induced ALK expression. Indeed, inhibition of EGFR by gefitinib increased ALK protein expression and phosphorylation, suggesting induction of ALK expression and activation as a novel mechanism of EGFR inhibitor resistance in HNSCC amenable to combination therapy. In conclusion, we identified induction of ALK by EGFR inhibitor as a potential novel mechanism relevant to resistance to cetuximab in the clinic, and EGFR/ALK inhibition as a potential combination therapeutic strategy for treating EGFR inhibitor resistant HNSCC. Citation Format: Xiaoming Ouyang, Ashley Barling, Aletha Lesch, Jeffrey Tyner, Sophia Jeng, Christina Zheng, Sara A. Courtneidge, Shannon McWeeney, Molly Kulesz-Martin. Inhibiting anaplastic lymphoma kinase (ALK) overcomes epidermal growth factor receptor (EGFR) inhibitor resistance in head and neck squamous cell carcinoma (HNSCC) patient-derived models [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 78.

Abnormal expression of the epidermal growth factor receptor (EGFR) has been reported in a wide range of human epithelial malignancies and in some studies has been associated with poor prognosis and treatment resistance. Currently, three different types of EGFR inhibitors have been approved by the FDA for the treatment of patients with head and neck, metastatic colorectal, pancreatic or breast cancers: anti-EGFR monoclonal antibodies [(mAbs), cetuximab and panitumumab], small molecule EGFR tyrosine kinase inhibitors [(TKIs) gefitinib, erlotinib] or a dual EGFR and HER-2 tyrosine kinase inhibitor (lapatinib) However, there has been no clear association between the expression levels of EGFR in the tumours determined by the FDA approved EGFR PharmDx™ kit (DakoCytomation) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors. One reason could be that the antibody used in the diagnosis of EGFR is not specific to the wild type EGFR and can also bind to the type-III mutant form of EGFR (EGFRvIII). We have extensively profiled our unique panel of high affinity rat anti-EGFR mAbs for use in the diagnosis and therapy of human cancers. The aim of the present investigation was to evaluate the potential of several of these antibodies (ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild type EGFR and/or EGFRvIII in formalin fixed paraffin embedded tumour specimens. We prepared 10% (v/v) neutral buffered formalin paraffin embedded sections of HN5 and HC2 20d2/c pelleted cells, which overexpress wild type EGFR and EGFRvIII respectively. Tumour sections were incubated with primary anti-EGFR mAbs followed by HRP linked secondary antibody (ABD Serotec Ltd., UK). The sections were then treated with DAB+ substrate-chromogen solution (Dako), counterstained with haematoxylin and mounted. The EGFR PharmDx™ kit, containing positive and negative control cell lines was purchased from Dako (UK) and the immunostaining of sections was conducted according to the manufacturer's protocol. We found that the mouse anti-EGFR mAb in the EGFR pharmDx™ kit stained both wild type and EGFRvIII expressing cells in the formalin fixed paraffin embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR9 and ICR10, which are directed against two distinct epitopes on the external domain of the EGFR, were specific for wild-type EGFR in formalin-fixed paraffin embedded tumour specimens. We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild type EGFR (membranous, nuclear, and cytoplasmic) in tumour specimens using immunohistochemistry and to determine their prognostic significance as well as predictive value for response to therapy with EGFR antibodies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 694.

Background: We previously found Epidermal Growth Factor Receptor (EGFR) inhibitor can block liposarcoma cell EGFR (HER1) survival signalling. However, it did not interfere with HER2 regulated Janus Kinases (JAK), and Signal Transducer and Activator of Transcription (STAT) signal transduction. Our research suggested JAK/STAT may be one mechanism for EGFR inhibitor resistance. Targeting both EGFR and STAT3, which has never been tested in sarcoma may overcome this problem. The principal aim of this preliminary study was to investigate the effect and mechanism of both STAT3 inhibitor alone and in combination with EGFR inhibitor in the treatment of soft tissue sarcoma (STS) cell lines. Methods: STAT3 inhibitor (S3I-201) mono-therapy or in combination with EGFR inhibitor Gefitinib was investigated in 7 STS cell lines (449b, 778, SW872, SW684, SW982, GCT and HT1080). Crystal-violet colorimetric and clonogenic assays were used to measure drug effects. For assessing a potential role of treatment, we investigated expression and activity of STAT and EGFR by Western blot and immunohistochemistry staining before and after treatment. Data were analysed using Chou & Talalay method and CalcuSyn software. Results: All 7 STS cell lines expressed phosphorylated/total STAT3 and EGFR. Anti-proliferative and anti-clonogenic effects of S3I-201 monotherapy on STS were dose- and time-dependent. Sensitive cells to S3I-201 (IC50 Δ50μM) were HT1080, SW684, GCT and SW982 (90% HT1080 were inhibited at day 3 post-administration of 25μM drug), partially sensitive cells were 449B and 778, and non-sensitive cells (IC50 ≤ 200μM) were SW872 (only 29% SW872 were inhibited at day 5 with 200μM S3I-201). Western blot analysis of whole-cell lysates from HT1080 showed phosphorylated STAT3 (pSTAT3) levels were significantly diminished after S3I-201 treatment, whereas pSTAT3 were not down-regulated in resistant cells SW872. IC50 of Gefitinib monotherapy on STS were 20 - 40μM, and 778 had minor effect (33% cells were inhibited after 5 days treatment). Combination therapy with Gefitinib and S3I-201 achieved synergistic antiproliferative effect (mean CIs < 0.90) in 6/7 STS cell lines (CI: 449b (0.3-1), 778 (0.1-0.2), SW872 (0.6-0.7), SW684 (0.2-0.5), GCT (0.3-0.4) and HT1080 (0.5-0.6)), except SW982 (1-1.2). For the most synergistic 778 cells, the drug reduction index for Gefitinib and S3I-201 was 5.9 and 12.7. Although treatment with Gefitinib alone inhibited pSTAT3 expression on serum-starved 778, EGF stimulation reversed the inhibition function. However in combination therapy, STAT3 phosphorylation was not induced by EGF stimulation. Conclusion: Combination therapy targeting both EGFR and STAT3 is a worthwhile treatment to pursue in anti-sarcoma therapy. Further studies will focus on potential mechanisms as well as the anti-sarcoma effect in the animal models. These results will have a clinical implication in treatment of STS in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3763. doi:1538-7445.AM2012-3763