Epidermal Growth Factor Receptor Positive Tumors Research Articles

Challenges for the application of EGFR-targeting peptide GE11 in tumor diagnosis and treatment

Abnormal regulation of cell signaling pathways on cell survival, proliferation and migration contributes to the development of malignant tumors. Among them, epidermal growth factor receptor (EGFR) is one of the most important biomarkers in many types of malignant solid tumors. Its over-expression and mutation status can be served as a biomarker to identify patients who can be benifit from EGFR tyrosine kinase inhibitors and anti-EGFR monocloncal antibody (mAb) therapy. For decades, researches on EGFR targeted ligands were actively carried out to identify potent candidates for cancer therapy. An ideal EGFR ligand can competitively inhibit the binding of endogenous growth factor, such as epidermal growth factor (EGF) and transforming growth factor-α(TGF-α) to EGFR, thus block EGFR signaling pathway and downregulate EGFR expression. Alternatively, conjugation of EGFR ligands on drug delivery systems (DDS) can facilitate targeting delivery of therapeutics or diagnostic agents to EGFR over-expression tumors via EGFR-mediated endocytosis. GE11 peptide is one of the potent EGFR ligand screened from a phage display peptide library. It is a dodecapeptide that can specifically binds to EGFR with high affinity and selectivity. GE11 has been widely used in the diagnosis and targeted delivery of drugs for radiotherapy, genetherapy and chemotherpy against EGFR positive tumors. In this review, the critical factors affecting the in vivo and in vitro targeting performance of GE11 peptide, including ligand-receptor intermolecular force, linker bond properties and physiochemical properties of carrier materials, are detailedly interpreted. This review provides a valuable vision for the rational design and optimization of GE11-based active targeting strategies for cancer treatment, and it will promote the translation studies of GE11 from lab research to clinical application.

Toward the Development of GE11-Based Radioligands for Imaging of Epidermal Growth Factor Receptor-Positive Tumors.

The epidermal growth factor receptor (EGFR) is closely associated with tumor development and progression and thus an important target structure for imaging and therapy of various tumors. As a result of its important role in malignancies of various origins and the fact that antibody-based compounds targeting the EGFR have significant drawbacks in terms of in vivo pharmacokinetics, several attempts have been made within the last five years to develop peptide-based EGFR-specific radioligands based on the GE11 scaffold. However, none of these approaches have shown convincing results so far, which has been proposed to be attributed to different potential challenges associated with the GE11 lead structure: first, an aggregation of radiolabeled peptides, which might prevent their interaction with their target receptor, or second, a relatively low affinity of monomeric GE11, necessitating its conversion into a multimeric or polymeric form to achieve adequate EGFR-targeting properties. In the present work, we investigated if these aforementioned points are indeed critical and if the EGFR-targeting ability of GE11 can be improved by choosing an appropriate hydrophilic molecular design or a peptide multimer system to obtain a promising radiopeptide for the visualization of EGFR-overexpressing malignancies by positron emission tomography (PET). For this purpose, we developed several monovalent 68Ga-labeled GE11-based agents, a peptide homodimer and a homotetramer to overcome the challenges associated with GE11. The developed ligands were successfully labeled with 68Ga3+ in high radiochemical yields of ≥97% and molar activities of 41–104 GBq/μmol. The resulting radiotracers presented logD(7.4) values between −2.17 ± 0.21 and −3.79 ± 0.04 as well as a good stability in human serum with serum half-lives of 112 to 217 min for the monovalent radiopeptides and 84 and 62 min for the GE11 homodimer and homotetramer, respectively. In the following in vitro studies, none of the 68Ga-labeled radiopeptides demonstrated a considerable EGF receptor-specific uptake in EGFR-positive A431 cells. Moreover, none of the agents was able to displace [125I]I-EGF from the EGFR in competitive displacement assays in the same cell line in concentrations of up to 1 mM, whereas the endogenous receptor ligand hEGF demonstrated a high affinity of 15.2 ± 3.3 nM. These results indicate that it is not the aggregation of the GE11 sequence that seems to be the factor limiting the usefulness of the peptide as basis for radiotracer design but the limited affinity of monovalent and small homomultivalent GE11-based radiotracers to the EGFR. This highlights that the development of small-molecule GE11-based radioligands is not promising.

Synthesis, characterization, and targeted chemotherapy of SCT200-linker-monomethyl auristatin E conjugates.

Antibody-drug conjugates (ADCs) are currently among the most successful and important strategies for treating patients with solid tumors. ADCs are composed of a monoclonal antibody and warhead, which are conjugated via a linker. Currently, monomethyl auristatin E (MMAE) is the most widely applied warhead in the development of ADCs. However, MMAE-based ADCs are generally constructed using the MC-VC-PABC linker, and this design has limited structural diversity and some disadvantages. Accordingly, in this study, we generated three types of novel linker-MMAE (with alterations in the spacer, catabolizing area, and self-immolative compared with MC-VC-PABC-MMAE) in ADCs, termed SCT200-linker-MMAE conjugates, and then evaluated the linker-drug plasma stability and the rate of drug release by cathepsin B. The binding ability, internalization rates, and efficacy of all SCT200-linker-MMAE ADCs were systematically studied, and the expression of apoptosis-associated proteins and the therapeutic efficacies of SCT200-M-2, -C-2, and -C-4 were evaluated. The results showed that the activities of some of these ADCs were increased for epidermal growth factor receptor-positive tumors. Moreover, the novel linkers designed in this study can be linked with other antibodies to treat other types of cancer. Overall, these findings provide important insights into the application of SCT200-based linkers in ADCs.

Enzyme mediated incorporation of zirconium-89 or copper-64 into a fragment antibody for same day imaging of epidermal growth factor receptor.

Identification of tumors which over-express Epidermal Growth Factor Receptor (EGFR) is important in selecting patients for anti-EGFR therapies. Enzymatic bioconjugation was used to introduce positron-emitting radionuclides (89Zr, 64Cu) into an anti-EGFR antibody fragment for Positron Emission Tomography (PET) imaging the same day as injection. A monovalent antibody fragment with high affinity for EGFR was engineered to include a sequence that is recognized by the transpeptidase sortase A. Two different metal chelators, one for 89ZrIV and one for 64CuII, were modified with a N-terminal glycine to enable them to act as substrates in sortase A mediated bioconjugation to the antibody fragment. Both fragments provided high-quality PET images of EGFR positive tumors in a mouse model at 3 hours post-injection, a significant advantage when compared to radiolabeled full antibodies that require several days between injection of the tracer and imaging. The use of enzymatic bioconjugation gives reproducible homogeneous products with the metal complexes selectively installed on the C-terminus of the antibody potentially simplifying regulatory approval.

Necitumumab plus gemcitabine and cisplatin versus gemcitabine and cisplatin alone as first-line treatment for stage IV squamous non-small cell lung cancer: A phase 1b and randomized, open-label, multicenter, phase 2 trial in Japan

ObjectivesThis open-label, multicenter, phase 1b/2 study assessed necitumumab plus gemcitabine and cisplatin (GC + N) in patients with previously untreated squamous non-small cell lung cancer in Japan. Materials and methodsThe phase 1b part determined the gemcitabine dose for the phase 2 part, in which patients were randomized 1:1 to GC + N or gemcitabine and cisplatin (GC) (gemcitabine 1250 mg/m2 on days 1 and 8; cisplatin 75 mg/m2 on day 1 of maximum four 3-week cycles; nectimumab 800 mg on days 1 and 8 of a 3-week cycle continued until progressive disease or unacceptable toxicity). The primary endpoint of the phase 2 part was overall survival. ResultsIn the phase 2 part, 181 patients received GC + N (N = 90) or GC (N = 91). Overall survival was significantly improved with GC + N versus GC (median, 14.9 months vs 10.8 months; hazard ratio [HR] = 0.66, 95% CI: 0.47 – 0.93, p = 0.0161). Improvements were also observed in progression-free survival (median, 4.2 months vs 4.0 months; HR = 0.56; p = 0.0004) and objective response rate (51% vs 21%; p < 0.0001). Survival was also significantly prolonged with GC + N versus GC for patients with epidermal growth factor receptor-positive tumors. Grade ≥3 treatment-emergent adverse events at ≥5% higher incidence with GC + N than GC were neutrophil count decreased (42% vs 35%), febrile neutropenia (12% vs 3%), decreased appetite (11% vs 4%), and dermatitis acneiform (6% vs 0%). ConclusionGC + N is well tolerated and has significant and clinically meaningful treatment benefit in the first-line treatment of patients with squamous non-small cell lung cancer in Japan. Clinicaltrials.gov identifier: NCT01763788.

Aberrant status and clinicopathologic characteristic associations of 11 target genes in 1,321 Chinese patients with lung adenocarcinoma.

The aberrant status of target genes and their associations with clinicopathologic characteristics are still unclear in primary lung adenocarcinoma. The common mutations and translocations of nine target genes were evaluated in 1,247 specimens of surgically-resected primary lung adenocarcinoma. Immunohistochemistry was used to analyze the expressions of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) in 731 specimens. The frequency of the aberrations and their associations with clinicopathologic characteristics were analyzed. Overall, 952 (76.3%) of 1,247 patients harbored at least one target mutation or translocation: epidermal growth factor receptor (EGFR) (729, 58.5%), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) (83, 6.7%), human epidermal growth factor receptor 2 (HER2) (82, 6.6%), anaplastic lymphoma kinase (ALK) (23, 1.8%), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) (20, 1.6%), Ret proto-oncogene RET (15, 1.2%), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1) (12, 1.0%), B-raf proto-oncogene (BRAF) (9, 0.7%), neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) (3, 0.2%). Fourteen (1.9%) of 731 patients were PD-1 positive and 95 (13.0%) were PD-L1 positive in tumor cells. In men and smokers, there were more frequent KRAS mutations (both P<0.001) and PD-L1 positive tumors (P<0.001, P=0.005, respectively), and less frequent EGFR mutations (P=0.049, <0.001, respectively). In ground-glass opacity (GGO) or ground-glass nodules (GGN), there were more HER2 (P=0.033) but less EGFR (P=0.025) and PIK3CA mutations (P=0.012), and ALK translocations (P=0.014). EGFR (P<0.001), KRAS mutations (P=0.004) and PD-L1 positive tumors (P=0.046) were more frequent in older patients, while HER2 (P<0.001), ALK (P=0.005) and ROS1 aberrations (P=0.044) were less frequent. Invasive mucinous adenocarcinoma was significantly associated with KRAS and ALK aberrations (both P<0.001), while solid predominant adenocarcinoma was associated with ROS1 translocations (P=0.036) and PD-L1 expression (P<0.001). KRAS, HER2, and ALK aberrations were scarce in patients with EGFR mutations (all P<0.001), while PD-L1 positive tumors positively correlated with ALK translocations (P=0.031) and negatively correlated with HER2 mutations (P=0.019). Most patients with primary lung adenocarcinoma harbored target gene aberrations. The frequency of each alteration differed in patients depending on clinicopathologic characteristics.

Abstract 1875A: Preclinical proof of concept for the first Nanocyclix TKI-PET radiotracer targeting activated EGFR positive lung tumors

Abstract Background: IMAkinib® program is an innovative approach, based on Nanocyclix® chemistry technology, which aims to develop new Tyrosine Kinase Inhibitors (TKIs) radiotracers used for Positron Emission Tomography (PET). The epidermal growth factor receptor (EGFR) is an established target for the treatment of advanced non-small cell lung cancer (NSCLC). Gefitinib (Iressa®), erlotinib (Tarceva®) and afatinib (Giotrif®), TKIs targeting EGFR have already been approved for treatment of advanced non-small cell lung cancer (NSCLC). Unfortunately, the majority of patients develop a resistance to the TKI, which is for most of them (&amp;gt; 50%) related to an acquired T790M mutation of EGFR. TKI PET-imaging can provide a tool to determine and predict the activity of EGFR and the responsiveness to EGFR TKI. Methods: New compound ODS2004436 was characterized in vitro for its activity and metabolism. After radiolabeling with [18F], the compound was evaluated in vivo in three clinically relevant lung cancer cell lines (NCI-H441 – EGFR_wild-type (WT); NCI-H3255 - EGFR_L858R; NCI-H1975 – EGFR_L858R/T790M) xenografted in nude rats. Results: The biochemical and cellular profiles were comparable to gefitinib on EGFR_WT and activated EGFR_L858R mutant. The cellular activity was in the nanomolar range (5 ± 2 nM) on NCI-H3255 and weak on NCI-H441 (42 ± 27 µM) suggesting that our compound might inhibit to a lesser extent non activated WT-EGFR. We observed a better biochemical inhibition (254 nM vs 1350 nM) and cellular activity on NCI-H1975, the EGFR double mutant (4.8 ± 0.7 µM vs 18 ± 8.7 µM) of ODS2004436 compared to gefitinib. These data suggest that our compound is suitable to bind in vivo activated EGFR. In vitro binding experiments showed that [18F]-ODS2004436 specifically bound to patient tumor samples expressing EGFR. The in vivo experiments in rats showed that [18F]-ODS2004436 was rapidly cleared from the blood. Nevertheless the tumor uptake is stable overtime (up to 180 min) with a mean tumor/muscle ratio &amp;gt; 4 in NCI-H3255 and &amp;gt; 2 in NCI-H1975 at 90 min instead no specific binding was observed in the WT tumor, NCI-H441. Moreover, we observed a good correlation between the radiotracer uptake in the tumors and pEGFR immunostaining, suggesting that [18F]-ODS2004436 is a good biomarker of activated EGFR, regardless of the mutation. Conclusions: Based on this study, we proved in vivo that [18F]-ODS2004436 binds selectively to activated EGFR, and is a good candidate to evaluate the EGFR activity in NLCSC using PET imaging. Clinical evaluation of this novel radiotracer is ongoing (first in-man phase 0/I clinical trial NCT02847377). Citation Format: Philippe Genne, Cyril Berthet, Olivier Raguin, Sylvie Chalon, Xavier Tizon, Sophie Serriere, Peggy Provent, Marc Hillairet de Boisferon, Johnny Vercouillie, Céline Mothes, Fabienne Gourand, Louisa BARRE, Denis Guilloteau, Pétra Blom, Jan Hoflack. Preclinical proof of concept for the first Nanocyclix TKI-PET radiotracer targeting activated EGFR positive lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1875A. doi:10.1158/1538-7445.AM2017-1875A

Abstract 2586: ASP8273 selectively inhibits mutant EGFR signal pathway and induces tumor shrinkage in EGFR mutated tumor models

Abstract Activating mutations of epidermal growth factor receptor (EGFR) are associated with the high sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) like erlotinib. However, acquired resistance limits the clinical efficacy of EGFR-TKIs, which is the most commonly caused by T790M mutation in EGFR. Second generation EGFR-TKIs such as afatinib are able to inhibit T790M mutation but the clinical efficacy in T790M positive patients is limited due to severe side effects associated with wild type (WT) EGFR inhibition. ASP8273 is a mutant-selective irreversible EGFR inhibitor currently in clinical trials (ClinicalTrials.gov Identifier: NCT02113813, NCT02192697). We have previously reported that ASP8273 covalently binds to mutant EGFR via C797 and inhibits kinase activity of mutant EGFR, which results in anti-tumor activity in xenograft models. To further explore the selectivity and the activity of ASP8273 on mutant EGFR, we evaluated effects of ASP8273 and other EGFR-TKIs on EGFR signal pathway, cell growth and anti-tumor activity. Phosphorylation of EGFR, ERK and Akt was determined by Western blot after treatment with EGFR-TKIs at 10, 100 and 1000nM. Apoptosis induction was analyzed by detecting caspase activity after 24h treatment with EGFR-TKIs. Anti-tumor activity of ASP8273 was evaluated in mice xenografted with HCC827 (deletion in exon 19 [del ex19]), NCI-H1975 (T790M/L858R), A431 (WT), and a patient derived LU1868 (T790M/L858R). ASP8273 selectively inhibited phosphorylation of EGFR and its down-stream signal pathway, ERK and Akt from 10nM in HCC827 and NCI-H1975 while inhibitory effects were only detected at 1000nM in A431.In NCI-H1650 (del ex19), ASP8273 inhibited cell growth with an IC50 value of 70nM while other EGFR-TKIs were only partially effective. ERK and Akt phosphorylation were diminished after ASP8273 treatment at 1000nM, however, other EGFR-TKIs only partially reduced the phosphorylation levels of these proteins. ASP8273 potently enhanced the caspase activity in NCI-H1650 after 24h treatment, which is concordant with signal and cell growth inhibitory effect. In HCC827 and NCI-H1975 xenograft models, ASP8273 induced tumor regression at 10, 30 and 100mg/kg without affecting body weight. ASP8273 also produced tumor growth inhibition from 10mg/kg in the NSCLC patient derived tumor xenograft, LU1868 which express T790M/L858R. On the other hand, ASP8273 did not produce significant tumor growth inhibition at 10 and 30mg/kg in A431 xenograft model. ASP8273 selectively inhibited mutant EGFR compared to WT EGFR in preclinical models, showing activity in mutant EGFR cell line which is resistant to other EGFR-TKIs including AZD9291 and CO-1686. These results indicate the potential of ASP8273 to induce tumor shrinkage in patients with mutant EGFR positive tumors including those that do not respond to other EGFR-TKIs despite EGFR mutation. Citation Format: Satoshi Konagai, Hideki Sakagami, Hiroko Yamamoto, Hiroaki Tanaka, Takahiro Matsuya, Shinya Mimasu, Yusuke Tomimoto, Masamichi Mori, Hiroyuki Koshio, Masaaki Hirano, Sadao Kuromitsu, Masahiro Takeuchi. ASP8273 selectively inhibits mutant EGFR signal pathway and induces tumor shrinkage in EGFR mutated tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2586. doi:10.1158/1538-7445.AM2015-2586

Effects of EGFR, CK19, CK20 and Survinin gene expression on radiotherapy results in patients with locally advanced head and neck cancer.

To investigate the effects of epidermal growth factor receptor (EGFR), cytokeratin 19 (CK19), cytokeratin 20 (CK20) and survinin gene expression on local control (LC) and overall survival (OS) in patients with locally advanced head and neck cancer (LAHNC) who were administered radiotherapy (RT). Twenty-six patients who were admitted to Uludag University Medical Faculty Department of Radiation Oncology with a diagnosis of LAHNC (GIII-GIV) were included in this study. Gene expression was evaluated in tumor tissues and peripheral blood. RNA isolation was performed on paraffinized tumor tissues and peripheral blood samples obtained before RT (BR). The densities of the obtained RNAs were analyzed at 260/280 nm. cDNA samples obtained from total RNA,EGFR, CK19, CK20 and survinin gene expression levels were assessed via the Sybr Green method and data were analyzed with the ΔΔCt method. The same process was repeated for peripheral blood samples taken after RT (AR). The female/male ratio was 3:23 and the mean age was 56.5 years (38-75 years). After radiotherapy, CK19 and CK20 levels in the peripheral blood were found to be correlated according to Pearson correlation analysis(p=0.049). This result indicates a possibility of remaining positive for CK19 and CK20 in the peripheral blood even after RT in patients with CK19, CK20, and EGFR positive tumors before RT. There was a statistically significant correlation between survinin levels measured BR and AR (p=0.028). In this study, we found that patients with any EGFR, CK19, CK20 or survinin positivity in their peripheral blood obtain less benefit from radiotherapy. A wider patient population and advanced protein analyses are necessary in order to increase the reliability of our findings.

Over-Expression of Egfr is Closely Correlated to Poor Prognosis in Tunisian Patients with Non-Small Cell Lung Adenocarcinoma

We studied epidermal growth factor receptor (EGFR) expression profile with the aim of an individualized therapy for patients with non-small cell lung cancer (NSCLC) from whom tumor materials are not sufficient for molecular investigations. Using immunohistochemistry, we found a markedly increased EGFR expression with significant difference in term of intensity and distribution from normal mucosa to primary tumors (p < 0.05). Furthermore, patients with EGFR positive tumors had significantly shorter survival than those with EGFR negative tumors (p = 0.0001). Thus, EGFR over-expression is a valuable prognostic marker to predict poor outcome in Tunisian patients with NSCLC.

Abstract P4-15-04: Synthesis and characterization of EGFR antibody-mediated tumor targeted “gold nanobombs” for treatment of locally advanced breast cancer

Abstract Purpose: Epidermal growth factor receptor (EGFR) represents an important target for treatment of locally advanced breast cancer (LABC), since about 30% of patients with LABC have triple negative breast tumor, and these tumors are often EGFR-positive. Our aim was to synthesize and characterize a novel form of “gold nanobomb” (GNB) for localized targeted radioimmunotherapy of LABC. GNB uses gold nanoparticles (AuNPs) as a carrier to conjugate both EGFR targeting monoclonal antibody (panitumumab) and the β-particle emitting radionuclide 177Lu. Ultimately, these GNB would be administered locally in LABC for tumor treatment while minimizing normal tissue exposure. Methods: The basic idea for construction of this new-targeted agent is to use different length of poly(ethylene glycol) (PEG) spacers to conjugate antibody and DOTA metal chelators for 177Lu. First, OPSS-PEG-NH2 was used to synthesize OPSS-PEG-DOTA for complexing 177Lu and OPSS-PEG-SVA was used to link antibody. Then these PEG spacers were linked to 30 nm AuNPs to form “GNB”. OPSS-PEG-antibody was analyzed by SDS-PAGE and trinitrobenzenesulfonic acid (TNBSA) assay to determine the degree of amine substitution after PEGylation. A radiometric assay was used to determine the number of PEGylated antibody and DOTA-PEG-OPSS per AuNPs. Specificity of GNB for EGFR and internalization into MBA-MD-468 (1×106 EGFR/cell), MBA-MD-231 (1×105 EGFR/cell) and MCF-7 (1×104 EGFR/cell) was confirmed by confocal and darkfield fluorescence microscopy. Binding assay and cell fractionation study were used to determine the binding affinity to EGFR and the total amount of GNB internalized. Results: Reacting panitumumab with increasing ratios of PEG resulted in an increase in molecular weight from 147 to 250 kDa. This result was in accordance with TNBSA assay where increasing ratio of PEG was associated with increasing lysine substitution (1.4 to 50.9 PEGylated lysine per antibody). PEGylation of 1.4 ± 1.7 lysine per antibody resulted in retention of immunoreactivity and when the immunoconjugates were linked to AuNPs, 8.09 ± 0.38 antibodies were attached to one AuNP. The total number of OPSS-PEG-DOTA per AuNP was estimated to be 812 ± 53. The total size of GNB was 67.3 ± 0.6 nm and there was no particle precipitation or aggregation after incubation in PBS. Competitive binding assay revealed that GNB was targeted strongly to EGFR (Kd antibody: 2.7 ± 0.3 nM; Kd GNB: 5.0 ± 4.9 nM) and darkfield microscopy showed that low PEG to antibody ratio increased the targeting efficiency when compared to high PEG to antibody ratio. Confocal microscopy revealed specific GNB uptake in MDA-MB-468 cells and cell fractionation study demonstrated receptor-mediated internalization of GNB, where the total amount of GNB internalized was about 3.1 and 22.2 fold higher in MDA-MB-468 cells than in MDA-MB-231 or MCF-7 cells. Conclusion: This GNB is very effective in targeting and in internalizing into EGFR, thus the GNB labeled with 177Lu emitting β-particles may be a promising new localized treatment for LABC. We plan to now examine the cytotoxicity of GNB both in vitro and in vivo will be explored for treatment of EGFR positive tumors. Supported by a grant from the Canadian Breast Cancer Foundation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-04.

Antibody Conjugated PLGA Nanoparticles for Targeted Delivery of Paclitaxel Palmitate: Efficacy and Biofate in a Lung Cancer Mouse Model

Aberrant signaling of the epidermal growth factor receptor (EGFR) is common to a variety of human cancers and is also found to be over-expressed in most cases of non-small cell lung cancer. For the development of a molecularly targeted therapy, cetuximab-conjugated nanoparticles (immunonanoparticles, INPs) are designed and loaded with the lipophilic paclitaxel palmitate (pcpl) prodrug. Oleyl cysteineamide (OCA) is synthesized whereby its amphiphilic nature enables interfacial anchoring and thiol surface functionalization of PLGA NPs, facilitating bioconjugation to cetuximab by thioether bonds. It is demonstrated that the in vitro targeting efficiency and improved cellular internalization and cytotoxicity of this targeted delivery system in lung cancer cells over-expressing EGFR. A quantitative measure of the high binding affinity of INPs to EGFR is demonstrated using surface plasmon resonance. In vivo tolerability and enhanced efficacy of cetuximab pcpl INPs in a metastatic lung cancer model are reported. Its therapeutic efficacy in A549-luc-C8 lung tumors is shown using non-invasive bioluminescent imaging. Intravenous administration of cetuximab pcpl INPs to mice results in significantly higher inhibition of tumor growth and increased survival rates as compared to the non-targeted drug solution, drug-loaded nanoparticles or blank INPs. Pharmacokinetics and organ biodistribution of the prodrug and parent drug are evaluated by LC-MS/MS in lung tumor bearing mice. No enhanced total accumulation of nanoparticles or INPs is found at the tumor tissue. However, persistent pcpl levels with sustained conversion and release of paclitaxel are observed for the encapsulated prodrug possibly suggesting the formation of a drug reservoir. The overall results indicate the potential of this promising targeted platform for the improved treatment of lung cancer and other EGFR positive tumors.

The Role of p53 in Combination Radioimmunotherapy with 64Cu-DOTA-Cetuximab and Cisplatin in a Mouse Model of Colorectal Cancer

Radioimmunotherapy has been successfully used in the treatment of lymphoma but thus far has not demonstrated significant efficacy in humans beyond disease stabilization in solid tumors. Radioimmunotherapy with (64)Cu was highly effective in a hamster model of colorectal cancer, but targeted radiotherapies with this radionuclide have since not shown as much success. It is widely known that mutations in key proteins play a role in the success or failure of cancer therapies. For example, the KRAS mutation is predictive of poor response to anti-epidermal growth factor receptor therapies in colorectal cancer, whereas p53 is frequently mutated in tumors, causing resistance to multiple therapeutic regimens. We previously showed that nuclear localization of (64)Cu-labeled DOTA-cetuximab was enhanced in p53 wild-type tumor cells. Here, we examine the role of p53 in the response to radioimmunotherapy with (64)Cu-DOTA-cetuximab in KRAS-mutated HCT116 tumor-bearing mice, with and without cisplatin, which upregulates wild-type p53. Experiments with HCT116 cells that are p53 +/+ (p53 wild-type) and -/- (p53 null) grown in cell culture demonstrated that preincubation with cisplatin increased expression of p53 and subsequently enhanced localization of (64)Cu from (64)Cu-acetate and (64)Cu-DOTA-cetuximab to the tumor cell nuclei. Radioimmunotherapy studies in p53-positive HCT116 tumor-bearing mice, receiving either radioimmunotherapy alone or in combination with cisplatin, showed significantly longer survival in mice receiving unlabeled cetuximab or cisplatin alone or in combination (all, P < 0.01). In contrast, the p53-negative tumor-bearing mice treated with radioimmunotherapy alone or combined with cisplatin showed no survival advantage, compared with control groups (all, P > 0.05). Together, these data suggest that (64)Cu specifically delivered to epidermal growth factor receptor-positive tumors by cetuximab can suppress tumor growth despite the KRAS status and present opportunities for personalized clinical treatment strategies in colorectal cancer.

In vivo and in vitro evaluation of Cy5.5 conjugated epidermal growth factor receptor binding peptide

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and plays an important role in carcinogenesis. In this study, the epidermal growth factor receptor binding peptide (EGBP) was identified using a phage display method and evaluated in U87MG cells in order to investigate the possibility to target the EGFR using an optical imaging system. Cyanine dye 5.5 (Cy5.5) was conjugated with EGBP-GGG-SC, EGBP-AOC-SC, and EGBP-AM2BA-SC. Cellular binding study of EGBP-Linker-Cy5.5 conjugates or (125)I-EGBP-Linker compounds was performed in U87MG cells. Optical imaging studies were performed in U87MG bearing mice. Three of seven clones from the 12-mer peptide library showed a specific binding affinity to rhEGFR, and they encoded the same 12 amino acid peptide sequence, FPMFNHWEQWPP. Confocal images show that the fluorescent signal of EGBP-Linker-Cy5.5 conjugates was decreased in the order: EGBP-AOC-Cy5.5≫EGBP-AM2BA-Cy5.5>EGBP-GGG-Cy5.5. EGBP-AOC-Cy5.5 appeared in cell cytoplasm and surface, and it was inhibited by free EGBP apparently. The cellular binding of EGBP-AOC-Cy5.5 exhibited a higher average radiance value than EGBP-GGG-Cy5.5 and EGBP-AM2BA-Cy5.5. Among various (125)I-EGBP-Linker compounds, EGBP-GGG showed a higher binding than other compounds. However, uptake of (125)I-EGBP-AOC was clearly inhibited by free EGBP in inhibition study. In an in vivo study, the fluorescent signal of EGBP-AOC-Cy5.5 treated mouse was mainly detected in the tumor and kidney. Among the three derivatives, EGBP-AOC-Cy5.5 was the optimized optical imaging agent for U87MG EGFR positive tumors in the animal model. This study demonstrated the EGBP-Linker-Cy5.5 conjugates may be useful as a potential EGFR target optical probe.

Phosphorylation of EGFR measured with in situ proximity ligation assay: Relationship to EGFR protein level and gene dosage in cervical cancer

PurposeWe have applied the sensitive and specific in situ proximity ligation assay (PLA) to characterize Tyr1068 phosphorylation of the epidermal growth factor receptor (EGFR) in cervical cancer in relation to the protein level and gene dosage. Materials and methodsPretreatment tumor biopsies from 178 patients were analyzed. EGFR protein level was determined by immunohistochemistry, and Tyr1068 phosphorylation was detected with PLA in 97 EGFR positive tumors. EGFR gene dosage was derived from array comparative genomic hybridization of 86 cases. ResultsEGFR was expressed in most tumors, whereas phosphorylation was seen in about half of the EGFR positive ones. A correlation was found between the expression of EGFR and phosphorylated EGFR (p=0.016, membrane; p=0.012, cytoplasm). However, tumor regions with high protein level without phosphorylation were occasionally seen and the percentage of EGFR positive cells was higher than the phosphorylated percentage (p<0.001). Moreover, an increase in the phosphorylation in both the membrane (p=0.014) and cytoplasm (p=0.002) was seen in 11 tumors with gain of EGFR. The protein level was not correlated with gene dosage. ConclusionIn contrast to gain of the EGFR chromosomal region, high EGFR protein level may not necessarily indicate Tyr1068 phosphorylation and thereby receptor activation in cervical cancer.

Prognostic value of epidermal growth factor receptor expression in operable non-small cell lung carcinoma

&#x0D; &#x0D; &#x0D; Background and aim: Increased expression of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC), supporting the tumor growth by a possible endocrine mechanism, affects patient survival negatively. We designed a study to test EGFR expression by immunohisto- chemistry (IHC) in resected stage I–II NSCLC and to correlate its overexpression with survival.&#x0D; Methods: EGFR expression was evaluated in 98 consecutive NSCLC patients after complete resection (53 squamous cell carcinomas, 40 adenocarcinomas, 5 large cell carcinomas: stage I, 57 (58%) and stage II, 41 (42%). IHC was used to examine the expression of EGFR in resected lung tumor sam- ples obtained from these patients, who had no pre- or post- operative chemotherapy or radiotherapy. Univariate and multivariate analyses were performed for factors influencing patient survival.&#x0D; Results: EGFR was expressed in 51 (52%) of 98 NSCLC sam- ples. More squamous tumors (61%) were EGFR-positive than adenocarcinomas (38%) (p = 0.038). There was a statistically significant correlation between EGFR expression and stage (p = 0.04). No difference was found between EGFR positive and negative tumors in the 5-year overall survival (57% vs. 73%, p = 0.13).&#x0D; Conclusion: The level of EGFR expression in tumors was not a successful predictor of survival in resected NSCLC.&#x0D; &#x0D; &#x0D;

Prognostic value of epidermal growth factor receptor expression in operable non-small cell lung carcinoma

Background and aimIncreased expression of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC), supporting the tumor growth by a possible endocrine mechanism, affects patient survival negatively. We designed a study to test EGFR expression by immunohistochemistry (IHC) in resected stage I-II NSCLC and to correlate its overexpression with survival.MethodsEGFR expression was evaluated in 98 consecutive NSCLC patients after complete resection (53 squamous cell carcinomas, 40 adenocarcinomas, 5 large cell carcinomas: stage I, 57 (58%) and stage II, 41 (42%). IHC was used to examine the expression of EGFR in resected lung tumor samples obtained from these patients, who had no pre- or post-operative chemotherapy or radiotherapy. Univariate and multivariate analyses were performed for factors influencing patient survival.ResultsEGFR was expressed in 51 (52%) of 98 NSCLC samples. More squamous tumors (61%) were EGFR-positive than adenocarcinomas (38%) (p = 0.038). There was a statistically significant correlation between EGFR expression and stage (p = 0.04). No difference was found between EGFR positive and negative tumors in the 5-year overall survival (57% vs. 73%, p = 0.13).ConclusionThe level of EGFR expression in tumors was not a successful predictor of survival in resected NSCLC.

Immunohistochemical expression of EGFR in colorectal carcinoma correlates with high but not low level gene amplification, as demonstrated by CISH

To assess and compare immunohistochemical expression of epidermal growth factor receptor (EGFR) with gene amplification as demonstrated by chromogenic in situ hybridisation (CISH), in colorectal adenocarcinoma. Sections from 100 consecutive colorectal cancer resection specimens were stained for EGFR using immunohistochemistry and CISH. Immunohistochemical assessment was independently performed at two laboratories, using the same antibody and protocols. With immunohistochemistry, strong circumferential membrane staining (3+ staining) was demonstrated in only 5% of cases, and this was only focal in three of five cases. At one laboratory, weak or incomplete staining (1+ or 2+) was observed in five further cases (5%), which had been negative at the other laboratory. CISH demonstrated high level gene amplification (>10 copies/nucleus) in the same five cases which had demonstrated 3+ staining with immunohistochemistry, and in those cases where the staining was focal, the amplification was demonstrated in the same foci of the tumour. Five further cases (5%) had low level amplification (5-10 copies per nucleus); these cases did not exhibit significant positive staining with immunohistochemistry. All the cases which demonstrated gene amplification (high or low level) arose in the distal colon. There was no correlation between gene amplification status and a variety of other variables, including stage at diagnosis, mucinous differentiation, neuroendocrine differentiation, or loss of expression of mismatch repair proteins. Immunohistochemical expression of EGFR is variable between laboratories, even using standardised protocols. 3+ staining is predictive of high level gene amplification, but correlates very poorly with low level amplification, which may still be clinically significant. In some cases gene amplification was only focal, offering a potential explanation for poor response to targeted therapy in patients with EGFR positive tumours.

Phase II study of weekly administration of docetaxel (D) in combination with the epidermal growth factor receptor (EGFR) inhibitor erlotinib (E) in metastatic breast cancer (MBC).

Abstract Abstract #3124 Background: Single agent weekly docetaxel (D) is an active agent in the treatment of metastatic breast cancer (MBC) with response rates of 29% - 53%. Erlotinib (OSI-774, Tarceva®) is a tyrosine kinase inhibitor directed against EGFR, which is overexpressed in 30-40% of breast cancers, making EGFR an attractive treatment target. This study was designed to assess the combination of D and E in previously untreated recurrent and/or MBC.&amp;#x2028; Methods: Adult patients with histologically confirmed MBC without prior chemotherapy for recurrence or metastases were eligible. Treatment plan was: D (initially 35 mg/m2 intravenous infusion weekly x 3 every 4 weeks) and E 150 mg orally daily uninterrupted. In patients with responding or stable disease, E was continued in 4 week cycles following a minimum of 6 cycles of D and E. Estimates of overall survival (OS) and progression free survival (PFS) were made by Kaplan-Meier method and the difference between groups by log-rank test. Tumor EGFR expression by immunohistochemisty and ER/PR was correlated with OS and PFS.&amp;#x2028; Results: 39 female pts were enrolled between 12/02 and 8/06. The median age was 51 yrs (range 28-78). The median number of cycles of D and E received was 4 (range 1-26) and of E following D and E was 11 (range 2-18). EGFR, ER/PR and Her-2/neu status was determined on 35/39 patients. EGFR: 23 positive, 12 negative. ER/PR: 25 positive, 10 negative. Her-2/neu: 2 positive, 33 negative. Ten pts. were not evaluable for survival or response due to toxicity occurring within the first cycle. Best responses (n=29) ; PR 11(39%), SD 4 (14%), PD 13 (45%), and clinical benefit (PR+SD) 15 (54%). Median PFS was 8 mos (95% CI: 4.4-12.2). PFS for EGFR negative tumors appeared better than EGFR positive tumors (12 mos PFS 33% vs. 23%) but was not significant (p = 0.53). There was no difference in OS between these groups (p=0.38). PFS and OS for ER/PR positive pts was significantly higher than ER/PR negative pts 6 mos PFS 67% vs. 25% (p= 0.009) and 2 yr OS 53.9% vs. 12.5% (p=0.015). , All patients were included for toxicity assessment (n-39). The first 26 pts received planned D dose of 35mg/m2. Because of non-hematologic toxicity, trial was subsequently modified to start D at 25 mg/m2. Grade 3 or 4 Leukopenia was seen in 15% pts. Principal non-hematologic grade 3-4 toxicities included anorexia, diarrhea, and fatigue (18% pts).&amp;#x2028; Conclusions: Combination therapy for advanced breast cancer with docetaxel and erlotinib shows promising activity with favorable response compared with other studies. There was no significant association with EGFR expression and PFS, however this combination is more favorable for ER positive patients. Randomized trials for ER positive disease is warranted to further investigate the efficacy of this combination compared to single agent docetaxel. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3124.

Expression of EGFR and c‐kit is associated with the basal‐like phenotype in breast carcinomas of African women

Epidermal growth factor receptor (EGFR) and c-kit are tyrosine kinase growth factor receptors which are frequently expressed in basal-like breast carcinomas, and tyrosine kinase inhibition is now a promising strategy in treatment of breast cancer. The aim of this study was to evaluate the expression of EGFR and c-kit in breast cancer with special focus on the basal-like phenotype (BLP) and other prognostic factors in an African population. We analyzed 65 archival tissues immunohistologically. EGFR and/or c-kit were expressed in 55% of basal-like tumors. Expression of EGFR and/or c-kit was strongly associated with high histologic grade (P=0.001), high nuclear grade (P=0.017), high mitotic counts (P=0.002), ER negativity (P=0.003), PR negativity (P=0.007), and HER2 negativity (P=0.014). EGFR and/or c-kit positive tumors were more likely to express the BLP (OR 9.1, CI 2.6-32.0, P<0.0005) than the negative tumors. In conclusion, there is a high expression of EGFR and/or c-kit in basal-like breast carcinoma in this series from Uganda and their expression is associated with features of poor prognosis. More studies are required to assess the clinical significance of EGFR and c-kit in breast cancer patients in Uganda.