Epidermal Growth Factor Receptor Antibody Research Articles

In vivo fluorescence imaging for cancer diagnosis using receptor-targeted epidermal growth factor-based nanoprobe

Receptor-targeted imaging is emerging as a promising strategy for diagnosis of human cancer. Herein, we developed an epidermal growth factor-based nanoprobe (EGF-NP) for in vivo optical imaging of epidermal growth factor receptor (EGFR), an important target for cancer imaging. The self-quenched EGF-NP is fabricated by sequentially conjugating a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3) to EGF, a low-molecular weight polypeptide (6.2 kDa), compared to EGFR antibody (150 kDa). The self-quenched EGF-NP presented great specificity to EGFR, and rapidly internalized into the cells, as monitored by time-lapse imaging. Importantly, the self-quenched EGF-NP boosted strong fluorescence signals upon EGFR-targeted uptake into EGFR-expressing cells, followed by lysosomal degradation, as confirmed by lysosomal marker cell imaging. Consistent with cellular results, intravenous injection of EGF-NP into tumor-bearing mice induced strong NIR fluorescence intensity in the target tumor tissue with high specificity against EGFR-expressing cancer cells. Signal accumulation of EGF-NP in tumor was much faster than that of EGFR monoclonal antibody (Cetuximab)-Cy5.5 conjugates due to the rapid clearance from the body and tissue permeability of low-molecular weight EGF. This self-quenched, EGF-based imaging probe can be applied for diagnosis of various cancers.

Epidermal growth factor receptor, p16, cyclin D1, and p53 staining patterns for inverted papilloma

The aim of this study was better characterize the staining patterns of inverted papilloma (IP) with and without carcinoma by performing immunohistochemistry for p16, epidermal growth factor receptor (EGFR), p53, and cyclin D1 antibodies in a large patient cohort. A total of 162 IP specimens were collected from 147 patients treated at the University of Michigan between 1996 and 2011. Twenty-two specimens contained carcinoma. Tumor was extracted for construction of 2 tissue microarrays and stained for p16, EGFR, p53, and cyclin D1. Tumor staining intensity and percentage staining were scored. Benign disease was positive for p16 in 64%, EGFR in 50%, p53 in 30%, and cyclin D1 in 76%. IP with carcinomatous degeneration was positive for p16 in 14%, EGFR in 71%, p53 in 62%, and cyclin D1 in 76%. The differences in staining positivity between benign and malignant disease reached significance for p16 and p53 only. Mean percentage staining by tumor surface area for IP and IP with carcinoma was 12% vs 7% for p16 (no statistical significance [NS]), 20% vs 34% for EGFR (NS), 4% vs 24% for p53 (p < 0.001), and 17% vs 21% for cyclin D1 (NS). Important characteristic staining pattern for IP with and without carcinoma are highlighted in this study. Unlike recent trends in human papilloma virus (HPV)-related head and neck malignancies, low expression of p16 is a marker for malignancy in this series. Positive staining for p53 correlates with the development of carcinoma in IP.

Preclinical modeling of EGFR-specific antibody resistance: oncogenic and immune-associated escape mechanisms

To define the molecular basis of secondary resistance to epidermal growth factor receptor (EGFR)-specific antibodies is crucial to increase clinical benefit in patients. The limited access to posttreatment tumor samples constitutes the major barrier to conduct these studies, representing preclinical experimentation as a useful alternative. Anti-EGFR antibody-based therapy has been reported to mediate tumor regression by interrupting oncogenic signals and, more recently, by inducing antitumor immunological responses. However, resistance models have been focused only on tumor escape associated with EGFR blockade, whereas studies describing immune-associated escape mechanisms have not been reported thus far. To address this idea, we modeled resistance induction in D122 metastasis-bearing C57BL/6 mice treated with 7A7 (an anti-murine EGFR antibody). Similarly to patients receiving EGFR-specific antibodies, 7A7 resistance promotion represents an important drawback to successful therapy. Characterization of primary cultures derived from metastasis in 7A7-treated mice revealed a high frequency of tumor variants resistant to in vivo and in vitro antibody treatment. We showed, for the first time, the convergence of alterations in oncogenic and immunological pathways in 7A7-resistant variants. To identify key molecules behind resistance, seven 7A7-resistant variants were screened. HER3 overexpression and PTEN deficiency leading to hyperactivation of protumoral downstream signaling were found in these variants as a consequence of 7A7-mediated EGFR inhibition. Concomitantly, we found a high percentage of resistant variants carrying abnormalities in the constitutive and/or interferon gamma (IFN-γ)-inducible major histocompatibility complex I (MHC-I) expression. A significant decrease in mRNA levels for MHC-I heavy chains, β2-microglogulin and antigen processing machinery genes as well as transcriptional alterations in IFN-γ pathway components were identified as the main mechanisms underlying MHC-I expression defects in 7A7-resistant variants. Notably, these defects have not been previously associated with EGFR-specific antibody resistance, providing novel immunological escape mechanisms. This study has strong implications for the development of new combination strategies to overcome anti-EGFR antibodies refractoriness.

Treatment of Metastatic Colorectal Cancer with Cetuximab: Influence on the Quality of Life.

Epidermal Growth Factor Receptor (EGFR) antibodies are innovative anti-cancer drugs prolonging survival in metastatic colocrectal cancer. However, due to adverse drug reactions, patients develop acneform skin toxicities. We hypothesized that the skin reaction leads to a decline in general (QOL) and dermatological health related quality of life (HQOL). Furthermore, we aimed at evaluating predictors for QOL and HQOL to improve individual adjustment of therapy. 40 outpatients with metastatic colocrectal cancer were involved in this study. According to their KRAS status, patients were allocated to 2 groups: The CTCX group (n = 20; KRAS wild-type) was treated with the EGFR-antibody Cetuximab plus chemotherapy, the CT group (n = 20; KRAS mutation) was receiving chemotherapy only. Psychological assessment consisted of questionaires to evaluate QOL and HQOL, depression, coping-styles, health beliefs and the patient´s personality. Between the two groups, no significanct difference in QOL was found, QOL remained stable over the course of treatment. Yet, the severity of the skin reactions had a significant influence on HQOL. Internal health beliefs and high compliance were found to be protective factors, while passive coping strategies, depression and the personality trait neuroticism were identified as risk factors. Interdisciplinary cooperation between medical professionals and psycho-oncologists is strongly recommended to encourage patients to embark on and to retain EGFR-antibody therapy. If risk factors are present, psycho-oncological therapy should focus on the minimization of depression and on the development of active coping strategies.

Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity

Despite advances in surgery and radiotherapy of uveal melanoma (UM), many patients develop distant metastases that poorly respond to therapy. Improved therapies for the metastatic disease are therefore urgently needed. Expression of the epidermal growth factor receptor (EGFR), a target of kinase inhibitors and humanised antibodies in use for several cancers, had been reported.Forty-eight human UMs were analysed by expression profiling. Signalling was tested in three EGFR expressing UM cell lines by Western blotting using phosphorylation specific antibodies for EGFR and the downstream mediators AKT (v-akt murine thymoma viral oncogene homolog) and extracellular signal-regulated kinase (ERK). Evidence for signalling in tumours was obtained through the application of a UM-specific EGF-signature. The EGFR specific kinase inhibitor, Gefitinib and the humanised monoclonal antibody, Cetuximab, were tested for their effect on EGFR signalling. Natural killer cell mediated antibody-dependent cellular cytotoxicity (ADCC) and tumour necrosis factor α (TNF-α) release was analysed for Cetuximab.Fourteen of 48 UMs and three of 14 cell lines (over-)express EGFR, at least in part due to trisomy of the EGFR locus on chromosome 7p12. EGFR and the downstream mediator, AKT, are phosphorylated upon stimulation with EGF in EGFR expressing cell lines. EGFR over-expressing tumours but not EGFR negative tumours show an activated EGF-signature. Gefitinib inhibits EGFR and AKT phosphorylation and Cetuximab induces EGFR phosphorylation but inhibits signalling to AKT induced with EGF. Cetuximab triggers natural killer (NK) cells to lyse EGFR+ cell lines and to release TNF-α.EGFR appears suited as a novel molecular drug target for therapy of uveal melanoma.

Cancer Therapy: Diamond‐Lipid Hybrids Enhance Chemotherapeutic Tolerance and Mediate Tumor Regression (Adv. Mater. 26/2013)

To facilitate targeted drug delivery using nanodiamond (ND) vehicles, epirubicin-functionalized ND/lipid hybrids are coupled to epidermal growth factor receptor (EGFR) antibodies. The resulting particles mediate tumor regression, and markedly improve drug tolerance in a triplenegative breast cancer model in vivo. Furthermore, comprehensive biocompatibility analysis following intravenous ND administration indicate that the particles are well tolerated by mice. Further information can be found in the article by Dean Ho and co-workers on page 3532.

Genomic alterations in advanced esophageal cancer may lead to subtype-specific therapies.

The development of targeted agents for metastatic esophageal or gastroesophageal junction (GEJ) tumors has been limited when compared with that for other common tumors. To date, the anti-human epidermal growth factor receptor-2 (HER-2) antibody, trastuzumab, in combination with chemotherapy, is the only approved novel agent for these cancers, and its use is limited to the small population of patients whose tumors overexpress HER-2. Despite recent progress in the field, median overall survival remains only 8-12 months for patients with stage IV esophageal or GEJ cancer. In this article, we examine the molecular aberrations thought to drive the development and spread of esophageal cancer and identify promising targets for specific tumor inhibition. Data from clinical studies of targeted agents are reviewed, including epidermal growth factor receptor antibodies, tyrosine kinase inhibitors, HER-2, and vascular endothelial growth factor-directed therapy. Current and future targets include MET, fibroblast growth factor receptor, and immune-based therapies. Evidence from trials to date suggests that molecularly unselected patient cohorts derive minimal benefit from most target-specific agents, suggesting that future collaborative investigation should focus on preselected molecular subgroups of patients with this challenging heterogeneous disease.

Electrochemical impedance immunosensor based on gold nanoparticles–protein G for the detection of cancer marker epidermal growth factor receptor in human plasma and brain tissue

A sensitive label-free impedimetric immunosensor for the detection of cancer biomarker epidermal growth factor receptor (EGFR) was developed with a limit of detection as low as 0.34pgmL−1 in PBS and 0.88pgmL−1 in human plasma. The gold nanoparticles were electrodeposited to modify the gold surface and to increase the electrochemical active area by a factor of approximately 3, i.e. by 68%. Protein G was used as scaffold for well oriented EGFR antibodies immobilization. Under optimal experimental parameters, the impedance changes were used for the detection of EGFR with a wide dynamic range of 1pgmL−1–1μgmL−1. The immunosensor showed an excellent reproducibility and selectivity against biomarkers, murine double minute 2 and platelet derived growth factor receptor. The excellent analytical performance of the EGFR immunosensor in terms of selectivity, sensitivity and low detection limit might be attributed to the synergetic effect between the Au nanoparticles and the protein G scaffold. The matrix effect from mouse brain tissue homogenate was also studied and the immunosensor showed excellent recoveries ranging from 98.3% to 115% and RSD of 1.55–6.17. Finally, our developed strategy could open new avenues for clinical screening and prognosis of tumors.

New Developments in the Second-Line Treatment of Metastatic Colorectal Cancer: Potential Place in Therapy

In this review article we discuss the evolution of second-line treatment options for patients with metastatic colorectal cancer (mCRC). The benefits of second-line chemotherapy have been established for some time, but in the last decade a number of trials have evaluated combinations of irinotecan- and oxaliplatin-based chemotherapy with molecular-targeted agents; e.g., vascular endothelial growth factor (VEGF)-targeting agents (bevacizumab, aflibercept), epidermal growth factor receptor antibodies (cetuximab, panitumumab), and tyrosine kinase inhibitors (vatalanib). Recent developments include the availability of the new VEGF-targeted agent aflibercept and the new concept of continuing bevacizumab after failure of first-line bevacizumab, which is likely to become a new treatment option in the second-line setting. Choosing the most appropriate second-line treatment regimen for mCRC patients remains a complex issue. All of the currently available molecular-targeted agents seem to be active even after patients have received a bevacizumab-based first-line regimen. Overall, the selection of second-line treatment for mCRC depends on several variables and should be determined taking into account the patient's performance and disease status.

인테그린 α2와 상피성장인자수용체 차단항체의 저해작용을 통한 구강편평상피암 세포의 선택적 제거

Purpose: This study was to find efficacy of integrin alpha2 (<TEX>${\alpha}_2$</TEX>) and epidermal growth factor receptor (EGFR) as tumor marker of oral squamous cell carcinoma (SCC) and clarify the selective cell death effect of anti-integrin <TEX>${\alpha}_2$</TEX> and anti-EGFR on SCC cells, additionally testify conjugated gold nanoparticles (GNP) with air plasma for selective cell death of oral SCC. Methods: Expression of integrin <TEX>${\alpha}_2$</TEX>, EGFR on human SCC cells (SCC25) were examined by western blot. SCC25 cells were treated with anti-integrin <TEX>${\alpha}_2$</TEX>, anti-EGFR and analysed by Hemacolor staining, immunoflorescence staining, FACS flow cytometry. Conjugated GNP with integrin <TEX>${\alpha}_2$</TEX>, EGFR antibody were treated by air plasma on SCC cells. Results: Integrin <TEX>${\alpha}_2$</TEX> and EGFR were over-expressed on SCC25 cells than normal lung WI-38 cells. The cell viability rate of SCC25 cells treated with anti-integrin <TEX>${\alpha}_2$</TEX>, anti-EGFR was lower than WI-38 cells. The concentration changes of nucleus, releasing cytochrome c and apoptosis inducing factor (AIF) from mitochondria to cytosol were observed. The changes of proteins related with apoptosis were observed. Increase of bax, bcl-xL, activation of caspase-3, -7, -9, and fragmentation of PARP, DFF45 and decrease of lamin A/C in SCC25 cells were observed. In FACS, increase of sub-<TEX>$G_1$</TEX> and S phase was observed. Cell cycle related proteins, Such as cyclin D1, cyclin dependent kinase (CDK) 4, cyclin A, cyclin E, CDK 2, p27 were decreased. After SCC25 cells treated with conjugatged GNP-Integrin <TEX>${\alpha}_2$</TEX>, GNP-EGFR, additionally air plasma, the cell death rate was significantly increased. Conclusion: Integrin <TEX>${\alpha}_2$</TEX>, EGFR were over-expressed in oral SCC cells. Anti-integrin <TEX>${\alpha}_2$</TEX>, anti-EGFR in SCC25 cells induced apoptosis selectively. When GNP-anti integrin <TEX>${\alpha}_2$</TEX>, GNP-anti EGFR were treated with air plasma on SCC25 cells, cancer cells were died more selectively. GNP-anti integrin <TEX>${\alpha}_2$</TEX>, GNP-anti EGFR with air plasma could be treatment choice of oral SCC.

A randomized clinical trial of chemotherapy compared to chemotherapy in combination with cetuximab in k-RAS wild-type patients with operable metastases from colorectal cancer: The new EPOC study.

3504 Background: Resection of liver metastases from colorectal cancer with or without neoadjuvant chemotherapy is the standard of care. The EPOC study (Nordlinger et al, Lancet 2008) randomised patients between surgery and surgery with chemotherapy and demonstrated an improvement in 3 year progression free survival (PFS) of 7·3% (from 28·1% to 35·4%). As a rational extension to the EPOC study data, the New EPOC study evaluates the benefit of cetuximab, an EGF receptor antibody, in addition to standard chemotherapy in patients with operable liver metastases. Methods: 272 patients were randomised between February 2007 and November 2012 into the New EPOC study. Eligible patients were required to be k-RAS wild type, have operable liver metastases and to be sufficiently fit for chemotherapy and surgery. Patients with the primary tumour in situ, and those who required short course rectal radiation were eligible. Patients were randomised to receive a fluoropyrimidine and oxaliplatin plus or minus cetuximab for 12 weeks before, then 12 weeks following surgery. Patients who had been treated with adjuvant oxaliplatin could receive irinotecan and 5 – fluorouracil. Results: Following a recommendation from the Independent Data Monitoring Committee on 19/11/2012, the New EPOC study was stopped when the study met a protocol pre-defined futility analysis. With 45.3% (96/212) of the expected events observed, progression free survival was significantly worse in the cetuximab arm (14.8 vs 24.2 months, HR (95%CI) 1.50037 (1.000707 to 2.249517) p&lt; 0.048). The result of a pre-planned analysis excluding the 23 patients treated with irinotecan based chemotherapy was similar (15.2 vs 24.2 months, HR 1.565546 (1.014967-2.414793) P&lt;0.043). Conclusions: Although the data are immature, the accumulation of more events is unlikely to change this result. In patients with resectable liver metastases and K-RAS wt tumours the addition of cetuximab to chemotherapy is not beneficial. Clinical trial information: ISRCTN22944367.

Molecular alterations of EGFR in small intestinal adenocarcinoma

Molecular testing for epidermal growth factor receptor (EGFR) mutations has recently become a standard practice for the management of patients with non-squamous none small cell lung cancer. Primary small intestine adenocarcinoma (SIA) is an uncommon malignancy, and EGFR mutation in the cancer has not been well characterized due to its rarity. A micro-tissue array with 53 SIAs and 24 surgically resected primary non-ampullary SIAs were studied. EGFR mutations were analyzed by DNA sequencing in 24 cases with formalin-fixed paraffin-embedded blocks. All 77 cases were examined by immunohistochemistry (IHC) using antibodies specific for the EGFR E746-A750 deletion in exon 19 (DEL), L858R point mutation in exon 21 (L858R), and total EGFR. EGFR amplifications were detected by fluorescence in situ hybridization. A positive reaction of DEL-specific, L858R-specific, and total EGFR antibodies was detected in seven (9.1%), 5 (6.5%) and 35 (45.5%) of 77 SIAs by IHC, respectively. Positive reaction of the three antibodies was not significantly correlated with patient's age, gender, differentiation, and stage. EGFR gene amplification was assayed in 77 SIAs in micro-tissue array. Of 24 SIA samples that had DNA sequencing, two (8.3%) harbored exon 19 deletion and one (4.2%) harbored L858R point mutation. Only one case with EGFR amplification and two cases with polysomy were shown. Our findings suggested that mutations and amplification in EGFR genes are minor events, and most of SIAs may be unsuitable to EGFR-TKIs treatment.

Fehler und Gefahren: Medikamentöse und strahlentherapeutische Behandlung von ­Kopf-Hals-Malignomen

Adverse effects and hazards which have their origin from radiation using conventional techniques like 3-D conformal radiotherapy and total radiation doses are well known. However little is known about the sprectum of especially late toxicity after radiation using new technologies like intensity modulated radiotherapy (IMRT) combined with novel target volume and dose concepts. Since IMRT allows for selective protection of the large salivary glands this technique improves the intermediate term quality of life and is the standard of care despite many details need further prospective evaluation. Combining cytotoxic drugs and radiotherapy yield improved survival in well-defined high risk patients. However morbidity and mortality of these protocols are high and deserve special expertise and supportive therapy. EGF-receptor antibodies have gained well defined indications, albeit specific toxicities in combination with irradiation deserve prospective studies and special attention.

Abstract 870: A clinical study of ABT-806, a humanized recombinant anti-EGFR monoclonal antibody, in patients with recurrent glioblastoma multiforme.

Abstract Background: Glioblastoma (GBM), the most common primary CNS malignancy, has a dismal prognosis with a median survival of under 15 months. Epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in GBM and has been implicated in tumor growth and resistance to therapy. ABT-806 is a humanized recombinant IgG1k monoclonal antibody that binds to a unique EGFR epitope exposed only in the untethered, (activated) state of EGFR or when the de2-7 (EGFRvIII) deletion mutation is present. ABT-806 has shown significant antitumor activity over other EGFR antibodies in both in vitro and in vivo models of GBM. We evaluated the safety, pharmacokinetics (PK), and antitumor activity of ABT-806 in a cohort of recurrent GBM patients treated at the recommended phase 2 dose determined from the dose-escalation portion of this phase 1 study. Methods: Patients with recurrent, EGFR-amplified GBM and measurable disease by RECIST (v1.1) were included. ABT-806 was administered at 24 mg/kg intravenously every other week until disease progression or study withdrawal. Tumor response by MRI or CT was assessed every 8 weeks. Adverse events (AEs) were graded by NCI CTCAE v4.0. Serum samples for determination of ABT-806 concentration and antidrug antibody were collected at multiple time points throughout the study. Archival tumor analysis for EGFR amplification/mutation was also performed. Results: Among 11 patients; median age was 60 years (range 39 to 69) and 9 were male. Median number of prior therapies was 2 (range 1 to 7). Six patients had prior bevacizumab exposure. No patients required dose modification. Hypophosphatemia was the only treatment-related emergent AE that occurred in 2 or more patients. Best responses were n=3 stable disease, n=7 progressive disease. One patient withdrew consent prior to the first post-baseline tumor assessment. Median time to disease progression and median progression free survival [95% CI] were 43 [22,57] and 29 [25,57] days, respectively. PK and EGFRde2-7 data were not available at the time of abstract submission. Conclusions: While ABT-806 related AEs were infrequent, there was little evidence of a clinical benefit in this refractory GBM population. Whether the lack of significant anti-tumor effect was due to poor binding to intracranial tumors, lack of response to the mechanism of action, or the refractory population is to be determined. A study is ongoing to evaluate the biodistribution of 111Indium-labeled ABT-806 in pts with advanced solid tumor types, including GBM. Further preclinical efforts to increase the potency of ABT-806 are also ongoing. Citation Format: David A. Reardon, James M. Cleary, Hao Xiong, Wijith P. Munasinghe, Ya-Hui Hsu, Edward B. Reilly, Jiang Qian, Michelle S. Pedersen, Rod A. Humerickhouse, Kyle D. Holen. A clinical study of ABT-806, a humanized recombinant anti-EGFR monoclonal antibody, in patients with recurrent glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 870. doi:10.1158/1538-7445.AM2013-870

Abstract 5586: Interpatient differences in colon tumors reflected in kinase activity and inhibition profiles related to KRAS and BRAF mutation status.

Abstract Background. Due to the biological heterogeneity, response to systemic therapy is variable in colon cancer. Chemotherapy improves median survival from 6-12 months to 18-24 months. Epidermal growth factor receptor (EGFR) antibodies have shown to improve survival, although modest, even further. KRAS mutations were identified as negative predictive markers for EGFR directed therapy. The response rate to EGFR targeted therapy remains low. Therefore there is an urgent need to find additional therapeutic options and accompanying biomarkers. The analysis of kinase activity in colon tumors may yield tumor-specific information on aberrant cell signaling pathways and may be usefull in a better patient stratification. Aim of this study is to generate overall kinase activity profiles from colon cancer tissues to explore if subtypes of this cancer show distinct kinase activity profiles. This can help to find new therapeutic options or predictive markers for response to therapy. Methods. Normal and tumorous colon tissue from patients who had given informed consent was fresh frozen after surgical resection. All tumor specimens were analyzed for BRAF and KRAS mutation status. Tissue cryosections were lysed in buffer with phosphatase and protease inhibitors. Tyrosine kinase (PTK) and serine/threonine kinase (STK) activity profiles of normal and tumor tissue were generated on PamChip® peptide microarrays comprising peptide sequences derived from known human phosphorylation sites. The ex vivo effect of kinase inhibitors on these kinase activity profiles was also determined. Peptide phosphorylation was monitored using fluorescently labeled antibodies. Data were analysed with Bionavigator software. Results. Tumor tissue has a higher PTK activity than normal tissue, whereas STK activity was equal to or lower than in normal tissue. STK activity profiles of 23 colon tumors could not be grouped according to mutation status, percentage of tumor tissue, differentiation grade or TNM classification. The STK activity profiles showed that within the group of wild type KRAS tumors, clearly different clusters were found. In the group with KRAS mutations (G12V, G12D, G12C, G12R) the STK activity profiles differed between the tumors. A subset of tumors, (two KRAS mutants, a BRAF mutant and a WT tumor) were tested ex vivo with a selection of 11 kinase inhibitors and showed different sensitivity to these inhibitors. Conclusions. Kinase activity and the response to inhibitors can be measured ex vivo in colon tumor tissue to capture the diversity in kinase activity and response to inhibitors between patients. The differences in kinase activity profiles of colon cancer samples could not be explained by subtypes according to mutation (KRAS, BRAF) status. Interesting subclusters could be identified using the kinase activity profiling providing a higher granularity than KRAS mutation scoring allows. Citation Format: Rosanne van den Oord, Hans Pruijt, Adriaan van den Brule, Peet Nooijen, Rik de Wijn, Monique Mommersteeg, Riet Hilhorst, Rob Ruijtenbeek. Interpatient differences in colon tumors reflected in kinase activity and inhibition profiles related to KRAS and BRAF mutation status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5586. doi:10.1158/1538-7445.AM2013-5586

Abstract 4209: Genotypic agreement between histological specimen and preoperative cytology in small lung adenocarcinoma.

Abstract Background. Histological subtype and genotype is critical for making treatment decisions and improving patient outcomes in non-small cell lung cancer (NSCLC). Approximately 70% of lung cancers are diagnosed in advanced stages where small biopsies and cytological specimens are the only source of material for both diagnosis and genotyping. Patients with specific gene alterations in epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) likely receive more benefit from EGFR TKI or ALK TKI therapy, respectively. Although, genotyping agreement from cytological and histological samples is critical, more investigational studies need to be explored to assess the concordance of genotype. Purpose. We performed a comparing investigation on concordance rate for pathological diagnosis and EGFR gene mutation status between surgical specimen and preoperative cytological specimen in small lung cancer. Additionally we reviewed immunohistochemical (IHC) staining with two different ALK specific antibodies. Materials and Methods. 55 patients who had surgically resected small (≦3cm) lung adenocarcinoma with known preoperative bronchial brushing cytology were examined retrospectively. IHC staining was conducted by mutation-specific antibodies using E746-A750 deletion and L858R for EGFR or 5A4 and D5F3 for ALK. DNA analysis was performed by PNA-RNA PCR clamp assay. Results. 1) Diagnostic concordance rate between cytological and histological specimen was 90.9%. Disagreement cases were included one case of squamous cell carcinoma, two cases of small cell carcinoma, one case of melanoma and one case of mucoepidermoid carcinoma by cytology. 2) In IHC positive for mutation-specific EGFR antibodies, the sensitivity was 83% in E746-A750 and 100% in L858R, respectively. In EGFR IHC positive cases, well to moderate differentiation were dominant. 3) In ALK IHC, one case was observed stronger cytoplasmic staining in D5F3 than 5A4. Furthermore, the cribriform pattern was observed in H&amp;E staining. Distinctive cytological feature was not observed. Conclusion. Our results showed that high genotype concordance rates of histology and cytology agreement in small lung adenocarcinoma. Citation Format: Noriko Hirai, Sasaki Takaaki, Yoshinori Minami, Masahiro Kitada, Naoyuki Miyokawa, Yoshinobu Ohsaki. Genotypic agreement between histological specimen and preoperative cytology in small lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4209. doi:10.1158/1538-7445.AM2013-4209

Abstract 933: Modeling acquired resistance to EGFR-directed therapies in mouse models of lung cancer.

Abstract Seventy percent of patients with Epidermal Growth Factor Receptor (EGFR) mutant lung cancer respond to treatment with the tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib. Despite this high response rate, patients almost inevitably develop resistance to these drugs on average within a year of starting drug treatment. Acquired resistance to EGFR TKIs is most commonly due to the emergence of a secondary mutation (T790M) in EGFR (in 50% of cases), amplification of the genes encoding the ERBB2 and MET receptor tyrosine kinases in 12 and 5% of cases, respectively, and phenotypic transformation of the adenocarcinomas to small cell lung cancer (5% of cases). Previously, in an effort to develop strategies to overcome T790M-mediated resistance, we generated tetracycline-inducible transgenic mice that express the EGFRL858R+T790M in the lung epithelium. Upon administration of doxycycline these mice develop lung adenocarcinomas that are resistant to TKIs. However, the combination of the irreversible TKI afatinib and the EGFR antibody cetuximab showed dramatic responses in these transgenic mice. These preclinical studies led to a clinical trial of these agents, which has showing a promising 30% response rate in patients with EGFR mutant tumors resistant to TKIs. However, tumors also acquire resistance to this drug combination and the mechanisms of resistance to afatinib+cetuximab are currently unknown. We set out to identify these mechanisms using xenograft and transgenic mouse models of EGFR mutant lung cancer. Transgenic mice with EGFRL858R+T790M-induced tumors were treated with afatinib+cetuximab using an intermittent dosing strategy that we had previously used to generate erlotinib-resistant tumors in mice with EGFRL858R and EGFRDEL-induced tumors. 75% of mice develop afatinib+cetuximab resistant tumors after three month-long rounds of treatment. The same treatment strategy applied to xenografts harboring subcutaneous tumors induced by EGFRDEL+T790M gave rise to resistant tumors in 20% of cases. Analysis of the afatinib+cetuximab resistant tumors performed to date has not revealed additional mutations in the EGFR transgene, the ERBB2 kinase domain or KRAS. Additional sequencing studies and examination of signaling pathway alterations in the resistant tumors are ongoing. Uncovering mechanisms of resistance to this drug combination will allow the development of strategies to treat tumors that acquire resistance to EGFR-directed therapies. Citation Format: Valentina Pirazzoli, Elisa de Stanchina, Jungfeng Xia, Zhongming Zhao, William Pao, Katerina A. Politi. Modeling acquired resistance to EGFR-directed therapies in mouse models of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 933. doi:10.1158/1538-7445.AM2013-933

Promotion of neuronal differentiation of neural progenitor cells by using EGFR antibody functionalized collagen scaffolds for spinal cord injury repair

The main challenge for neural progenitor cell (NPC)-mediated repair of spinal cord injury (SCI) is lack of favorable environment to direct its differentiation towards neurons rather than glial cells. The myelin associated inhibitors have been demonstrated to promote NPC differentiation into glial lineage. Herein, to inhibit the downstream signaling activated by myelin associated inhibitors, cetuximab, an epidermal growth factor receptor (EGFR) neutralizing antibody, functionalized collagen scaffold has been developed as a vehicle for NPC implantation. It was found that collagen-cetuximab 1 μg scaffolds enhanced neuronal differentiation and inhibited astrocytic differentiation of NPCs exposed to myelin proteins significantly in vitro. To test the therapeutic effect in vivo, NPCs expressing green fluorescent protein (GFP)-embedded scaffolds have been implanted into the 4 mm-long hemisection lesion of rats. We found that the collagen-cetuximab 5 μg scaffolds induced neuronal differentiation and decreased astrocytic differentiation of NPCs, enhanced axon regeneration, and promoted functional recovery markedly. A well-functionalized scaffold was constructed to improve the recovery of SCI, which could promote the neuronal differentiation of neural progenitor cells in vivo.

Oxaliplatin for colorectal cancer: recent evidence from clinical trials

SUMMARY Oxaliplatin, a second-generation platinum analog, has evolved as one of the most important therapeutic agents in the treatment of both metastatic colorectal cancer and stage II/III colon cancer. Moreover, oxaliplatin is currently being investigated in the perioperative treatment of locally advanced rectal cancer. Oxaliplatin can be safely combined with fluoropyrimidines, irinotecan, bevacizumab and EGF receptor antibodies, resulting in increased response rates and delayed progression. In combination with EGF receptor antibodies, fluoropyrimidine schedules need to be cautiously considered. Treatment strategies to limit oxaliplatin-induced neurotoxicity by discontinuous administration schedules (e.g., induction followed by maintenance, followed by reinduction or intermittent treatment) are available.

A new nanoconstruct for epidermal growth factor receptor-targeted photo-immunotherapy of ovarian cancer

Targeted photodynamic therapy (TPDT) involves the administration of a photosensitizer (PS) conjugated with a targeting moiety followed by light activation. The systemic toxicity associated with conventional therapy may thus be significantly reduced in TPDT due to the dual selectivity provided by the spatial localization of the illumination as well as the target-localizing ability of the conjugate. Herein, a photo-immuno-conjugate-associating-liposome (PICAL) for TPDT has been developed in which the FDA approved benzoporphyrin derivative monoacid A (BPD) and the Cetuximab antibody for epidermal growth factor receptor (EGFR) were associated into a stable Preformed Plain Liposome (PPL) by passive physical adsorption. Results have shown that the BPD molecules adsorbed into PICAL have stable optical behavior and a higher fluorescence quantum yield than free-BPD. The Cetuximab adsorbed into PPL selectively binds to cells that overexpress EGFR. The inhibition of EGFR signaling by PICAL has enhanced PDT-mediated ovarian cancer cell death. From the Clinical EditorIn this basic science study, a photo-immuno-conjugate-associating-liposome for targeted photodynamic therapy is investigated. The FDA-approved benzoporphyrin derivative monoacid A and an epidermal growth factor receptor antibody were assembed into a stable Preformed Plain Liposome (PPL) by passive physical adsorption. The authors demonstrate therapeutic efficacy of the above construct in an ovarian tumor system.