Outcome of Patients with Platelet-Derived Growth Factor Receptor Alpha-Mutated Gastrointestinal Stromal Tumors in the Tyrosine Kinase Inhibitor Era.

PURPOSE: Platelet-derived growth factor receptor-alpha (PDGFRA) mutations are found in approximately 5% to 7% of advanced gastrointestinal stromal tumors (GIST). We sought to extensively assess the activity of imatinib in this subgroup. EXPERIMENTAL DESIGN: We conducted an international survey among GIST referral centers to collect clinical data on patients with advanced PDGFRA-mutant GISTs treated with imatinib for advanced disease. RESULTS: Fifty-eight patients were included, 34 were male (59%), and median age at treatment initiation was 61 (range, 19-83) years. The primary tumor was gastric in 40 cases (69%). Thirty-two patients (55%) had PDGFRA-D842V substitutions whereas 17 (29%) had mutations affecting other codons of exon 18, and nine patients (16%) had mutation in other exons. Fifty-seven patients were evaluable for response, two (4%) had a complete response, eight (14%) had a partial response, and 23 (40%) had stable disease. None of 31 evaluable patients with D842V substitution had a response, whereas 21 of 31 (68%) had progression as their best response. Median progression-free survival was 2.8 [95% confidence interval (CI), 2.6-3.2] months for patients with D842V substitution and 28.5 months (95% CI, 5.4-51.6) for patients with other PDGFRA mutations. With 46 months of follow-up, median overall survival was 14.7 months for patients with D842V substitutions and was not reached for patients with non-D842V mutations. CONCLUSIONS: This study is the largest reported to date on patients with advanced PDGFRA-mutant GISTs treated with imatinib. Our data confirm that imatinib has little efficacy in the subgroup of patients with D842V substitution in exon 18, whereas other mutations appear to be sensitive to imatinib. Clin Cancer Res; 18(16); 4458-64. ©2012 AACR.

Peptabody-EGF: a novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells.

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

Epidermal growth factor enhances the expression of an endogenous lectin in aggregating fetal brain cell cultures.

Aggregating cell cultures prepared from fetal rat telencephalon express the two subunits [cerebellar soluble lectins (CSL) 1 and 2] of a soluble, mannose-specific endogenous lectin (CSL) in a development-dependent manner. Increased CSL synthesis was found at an early postmitotic stage as well as during the period of maximal myelination. Repetitive treatment of early cultures with epidermal growth factor (EGF, 3nM) caused a great stimulation of CSL biosynthesis. Immunocytochemical studies revealed particularly intense CSL-specific staining in small, EGF-responsive cells, presumably glial cells. Large quantities of CSL-immunoreactive material were found also in the extracellular space and on the external side of the plasma membrane, indicating abundant release of CSL. The present findings suggest that EGF or EGF-related factors in the brain are able to regulate the expression of an endogenous lectin, affecting brain ontogeny.

Influence of epidermal growth factor on the maturation of fetal rat brain cells in aggregate culture. An immunocytochemical study.

Maturation of astrocytes, neurons, and oligodendrocytes was studied in serum-free aggregating cell cultures of fetal rat telencephalon by an immunocytochemical approach. Cell type-specific immunofluorescence staining was examined by using antibodies directed against glial fibrillary acidic protein (GFAP) and vimentin, two astroglial markers; neuron-specific enolase (NSE) and neurofilament (NF), two neuronal markers, and galactocerebroside (GC), an oligodendroglial marker. It was found that the cellular maturation in aggregates is characterized by distinct developmental increases in immunoreactivity for GFAP, vimentin, NSE, NF, and GC, and by a subsequent decrease of vimentin-positive structures in more differentiated cultures. These findings are in agreement with observations in vivo, and they corroborate previous biochemical studies of this histotypic culture system. Treatment of very immature cultures with a low dose of epidermal growth factor (EGF, 5 ng/ml) enhanced the developmental increase in GFAP, NSE, NF and GC immunoreactivity, suggesting an acceleration of neuronal and glial maturation. In addition, EGF was found to alter the cellular organization within the aggregates, presumably by influencing cell migration.

Pathway analysis of glioblastoma tissue after preoperative treatment with the EGFR tyrosine kinase inhibitor gefitinib--a phase II trial.

Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4.1 μg/g), 20 times higher than respective plasma. EGFR-pathway activity was evaluated with phosphorylation-specific assays. The EGFR was efficiently dephosphorylated in treated patients as compared to a control cohort of 12 patients. However, no significant effect on 12 pathway constituents was detected. In contrast, in vitro treatment of a glioblastoma cell line, BS-153, with endogenous EGFRwt amplification and EGFRvIII expression resulted not only in dephosphorylation of the EGFR, but also of key regulators in the pathway such as AKT. Treating established xenografts of the same cell line as an in vivo model showed dephosphorylation of the EGFR without affecting downstream signal transductors, similar to the human glioblastoma. Taken together, gefitinib reaches high concentrations in the tumor tissue and efficiently dephosphorylates its target. However, regulation of downstream signal transducers in the EGFR pathway seems to be dominated by regulatory circuits independent of EGFR phosphorylation.

INK4a/Arf is required for suppression of EGFR/DeltaEGFR(2-7)-dependent ERK activation in mouse astrocytes and glioma.

Amplification of the epidermal growth factor receptor (EGFR) or expression of its constitutively activated mutant, DeltaEGFR(2-7), in association with the inactivation of the INK4a/Arf gene locus is a frequent alteration in human glioblastoma. The notion of a cooperative effect between these two alterations has been demonstrated in respective mouse brain tumor models including our own. Here, we investigated underlying molecular mechanisms in early passage cortical astrocytes deficient for p16(INK4a)/p19(Arf) or p53, respectively, with or without ectopic expression of DeltaEGFR(2-7). Targeting these cells with the specific EGFR inhibitor tyrphostin AG1478 revealed that phosphorylation of ERK was only abrogated in the presence of an intact INK4a/Arf gene locus. The sensitivity to inhibit ERK phosphorylation was independent of ectopic expression of DeltaEGFR(2-7) and independent of the TP53 status. This resistance to downregulate the MAPK pathway in the absence of INK4a/Arf was confirmed in cell lines derived from our mouse glioma models with the respective initial genetic alterations. Thus, deletion of INK4a/Arf appears to keep ERK in its active, phosphorylated state insensitive to an upstream inhibitor specifically targeting EGFR/DeltaEGFR(2-7). This resistance may contribute to the cooperative tumorigenic effect selected for in human glioblastoma that may be of crucial clinical relevance for treatments specifically targeting EGFR/DeltaEGFR(2-7) in glioblastoma patients.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS Treatment: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND: Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS: TREATMENT: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS: Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION: First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

A dramatic lung cancer course in a patient with a rare EGFR germline mutation exon 21 V843I: Is EGFR TKI resistance predictable?

We report on the medical history of a Caucasian smoker woman diagnosed with a stage IV NSCLC adenocarcinoma, characterized by a rare epidermal growth factor receptor (EGFR) point mutation in exon 21 codon 843 (p.V843I/c.2527G>A/COSMIC ID 85894). This genetic alteration revealed to be germline, after its presence was demonstrated in chondroblasts from the bone biopsy. While it is the first description of germline V843I mutation without concomitant additional known EGFR activating mutation, we modeled the EGFR ATP catalytic domain in complex with ATP, gefitinib and erlotinib using computer-aided approaches to estimate possible changes in affinity upon the V843I mutation.

Epidermal growth factor (EGF) stimulation of cultured brain cells. I. Enhancement of the developmental increase in glial enzymatic activity.

Serum-free aggregating cell cultures of fetal rat telencephalon grown in the presence of 3 ng/ml (5 X 10(-10) M) epidermal growth factor (EGF) until day 12 showed 2- to 3-fold increased activities in the two glial enzymes, glutamine synthetase (GLU-S) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This effect was concentration-dependent, with maximal stimulation in cultures treated daily with 3 ng/ml EGF. Addition of EGF during the first 10 culture days was sufficient to produce a maximal stimulation of both GLU-S and CNPase on day 19, whereas treatments starting on day 12 were ineffective. The stimulation of GLU-S preceded that of CNPase. The EGF-induced increase in GLU-S activity was not directly dependent on the presence of insulin, triiodothyronine, or hydrocortisone in the medium, whereas insulin was required for the stimulation of CNPase. A single dose of 5 ng/ml EGF on day 2 caused a slight but significant decrease in DNA synthesis after day 6. The present results indicate that in serum-free aggregating cell cultures of fetal rat telencephalon EGF partially inhibits DNA synthesis, and stimulates an early step in glial differentiation.

Epidermal growth factor and bovine growth hormone stimulate differentiation and myelination of brain cell aggregates in culture.

Bovine growth hormone (bGH) and epidermal growth factor (EGF) increased the activity of ornithine decarboxylase (ODC) in brain cell aggregates cultured in a serum-free chemically defined medium. ODC is considered as a marker of cell growth and differentiation. The effect of bGH and EGF on myelination was investigated by measuring two myelin markers, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin basic protein (MBP). EGF treatment at days 2 and 5 caused a dose-dependent increase of both myelin markers at culture day 12. This increase could still be observed at culture day 19, indicating a prolonged action of EGF. The continual presence of bGH in the culture medium produced a large accumulation of MBP at day 19. This effect was dose-dependent and required the presence of triiodothyronine (T3). In contrast, the effect of bGH on CNP activity did not require the presence of T3. This is the first report showing a direct effect of bGH on CNS myelination in vitro and of EGF on both MBP accumulation and ODC activity.

Isolation from mouse fibroblasts of a cDNA encoding a new form of the fibroblast growth factor receptor (flg).

Structural definition of the receptors for neurotropic and angiogenic modulators such as fibroblast growth factors and related polypeptides will yield insight into the mechanisms that control early development, embryogenesis, organogenesis, wound repair and neovessel formation. We isolated 3 murine cDNAs encoding different binding domains of these receptors (flg). Comparison of these ectoplasmic portions showed that two of the forms corresponded to previously described murine molecules whereas the third one had a different ectoplasmic portion generated by specific changes in two regions. Interestingly, expression of this third form seems to be restricted in its tissue distribution. Such modifications could influence the ligand specificity of the different receptors and/or their binding affinity.

Epidermal growth factor (EGF) stimulation of cultured brain cells. II. Increased production of extracellular soluble proteins.

The production of extracellular soluble proteins was studied in serum-free aggregating cell cultures of fetal rat telencephalon labeled on culture day 7 with a mixture of radioactive amino acid precursors. Cultures treated continuously with epidermal growth factor (EGF; 20 ng/ml) showed a generally increased protein secretion and a particularly enhanced production of a few distinct extracellular proteins. The time lag of this response after an initial dose of EGF (25 ng/ml) on day 7 was 48 h. The total macromolecular radioactivity that accumulated within 96 h of labeling in the media of EGF-treated cultures was 175% of untreated controls, whereas no difference was found in the proportions of intracellular amino acid incorporation. Cultures which received a single dose of EGF (25 ng/ml) on day 1 showed still a greatly increased protein secretion on day 7. Prevention of extracellular protein accumulation by reducing the initial cell number and increasing the rate of media changes did not affect the EGF-induced stimulation of the two glial enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase. The results suggest that both the increased production of extracellular proteins and the enhanced development of glial enzymatic activities reflect the stimulated phenotypic expression of EGF-sensitive brain cells.