Epidermal growth factor receptor: a re-emerging target in glioblastoma.

PURPOSE OF REVIEW: Amplification and overexpression of the epidermal growth factor receptor (EGFR) gene are a hallmark of primary glioblastoma (45%), making it a prime target for therapy. In addition, these amplifications are frequently associated with oncogenic mutations in the extracellular domain. However, efforts at targeting the EGFR tyrosine kinase using small molecule inhibitors or antibodies have shown disappointing efficacy in clinical trials for newly diagnosed or recurrent glioblastoma. Here, we review recent insights into molecular mechanisms relevant for effective targeting of the EGFR pathway. RECENT FINDINGS: Molecular workup of glioblastoma tissue of patients under treatment with small molecule inhibitors has established drug concentrations in the tumor tissue, and has shed light on the effectiveness of target inhibition and respective effects on pathway signaling. Further, functional analyses of interaction of small molecule inhibitors with distinct properties to bind to the active or inactive form of EGFR have provided new insights that will impact the choice of drugs. Finally, vaccination approaches targeting the EGFRvIII mutant featuring a tumor-specific antigen have shown promising results that warrant larger controlled clinical trials. SUMMARY: A combination of preclinical and clinical studies at the molecular level has provided new insights that will allow refining strategies for targeting the EGFR pathway in glioblastoma.

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.

Oral epidermal growth factor receptor tyrosine kinase inhibitors for the treatment of non-small cell lung cancer: comparative pharmacokinetics and drug-drug interactions.

The development of orally active small molecule inhibitors of the epidermal growth factor receptor (EGFR) has led to new treatment options for non-small cell lung cancer (NSCLC). Patients with activating mutations of the EGFR gene show sensitivity to, and clinical benefit from, treatment with EGFR tyrosine kinase inhibitors (EGFR-TKls). First generation reversible ATP-competitive EGFR-TKls, gefitinib and erlotinib, are effective as first, second-line or maintenance therapy. Despite initial benefit, most patients develop resistance within a year, 50-60% of cases being related to the appearance of a T790M gatekeeper mutation. Newer, irreversible EGFR-TKls - afatinib and dacomitinib - covalently bind to and inhibit multiple receptors in the ErbB family (EGFR, HER2 and HER4). These agents have been mainly evaluated for first-line treatment but also in the setting of acquired resistance to first-generation EGFR-TKls. Afatinib is the first ErbB family blocker approved for patients with NSCLC with activating EGFR mutations; dacomitinib is in late stage clinical development. Mutant-selective EGFR inhibitors (AZD9291, CO-1686, HM61713) that specifically target the T790M resistance mutation are in early development. The EGFR-TKIs differ in their spectrum of target kinases, reversibility of binding to EGFR receptor, pharmacokinetics and potential for drug-drug interactions, as discussed in this review. For the clinician, these differences are relevant in the setting of polymedicated patients with NSCLC, as well as from the perspective of innovative anticancer drug combination strategies.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses.

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

In vivo therapeutic synergism of anti-epidermal growth factor receptor and anti-HER2 monoclonal antibodies against pancreatic carcinomas.

PURPOSE: Pancreatic carcinoma is highly resistant to therapy. Epidermal growth factor receptor (EGFR) and HER2 have been reported to be both dysregulated in this cancer. To evaluate the in vivo effect of binding both EGFR and HER2 with two therapeutic humanized monoclonal antibodies (mAb), we treated human pancreatic carcinoma xenografts, expressing high EGFR and low HER2 levels. EXPERIMENTAL DESIGN: Nude mice, bearing xenografts of BxPC-3 or MiaPaCa-2 human pancreatic carcinoma cell lines, were injected twice weekly for 4 weeks with different doses of anti-EGFR (matuzumab) and anti-HER2 (trastuzumab) mAbs either alone or in combination. The effect of the two mAbs, on HER receptor phosphorylation, was also studied in vitro by Western blot analysis. RESULTS: The combined mAb treatment significantly inhibited tumor progression of the BxPC-3 xenografts compared with single mAb injection (P = 0.006) or no treatment (P = 0.0004) and specifically induced some complete remissions. The two mAbs had more antitumor effect than 4-fold greater doses of each mAb. The significant synergistic effect of the two mAbs was confirmed on the MiaPaCa-2 xenograft and on another type of carcinoma, SK-OV-3 ovarian carcinoma xenografts. In vitro, the cooperative effect of the two mAbs was associated with a decrease in EGFR and HER2 receptor phosphorylation. CONCLUSIONS: Anti-HER2 mAb has a synergistic therapeutic effect when combined with an anti-EGFR mAb on pancreatic carcinomas with low HER2 expression. These observations may open the way to the use of these two mAbs in a large panel of carcinomas expressing different levels of the two HER receptors.

Coactivated platelet-derived growth factor receptor {alpha} and epidermal growth factor receptor are potential therapeutic targets in intimal sarcoma.

Intimal sarcoma (IS) is a rare, malignant, and aggressive tumor that shows a relentless course with a concomitant low survival rate and for which no effective treatment is available. In this study, 21 cases of large arterial blood vessel IS were analyzed by immunohistochemistry and fluorescence in situ hybridization and selectively by karyotyping, array comparative genomic hybridization, sequencing, phospho-kinase antibody arrays, and Western immunoblotting in search for novel diagnostic markers and potential molecular therapeutic targets. Ex vivo immunoassays were applied to test the sensitivity of IS primary tumor cells to the receptor tyrosine kinase (RTK) inhibitors imatinib and dasatinib. We showed that amplification of platelet-derived growth factor receptor α (PDGFRA) is a common finding in IS, which should be considered as a molecular hallmark of this entity. This amplification is consistently associated with PDGFRA activation. Furthermore, the tumors reveal persistent activation of the epidermal growth factor receptor (EGFR), concurrent to PDGFRA activation. Activated PDGFRA and EGFR frequently coexist with amplification and overexpression of the MDM2 oncogene. Ex vivo immunoassays on primary IS cells from one case showed the potency of dasatinib to inhibit PDGFRA and downstream signaling pathways. Our findings provide a rationale for investigating therapies that target PDGFRA, EGFR, or MDM2 in IS. Given the clonal heterogeneity of this tumor type and the potential cross-talk between the PDGFRA and EGFR signaling pathways, targeting multiple RTKs and aberrant downstream effectors might be required to improve the therapeutic outcome for patients with this disease.

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.

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.

An European Organisation for Research and Treatment of Cancer phase I study of lapatinib and docetaxel as neoadjuvant treatment for Human Epidermal Growth Factor Receptor 2 (HER2) positive locally-advanced/inflammatory or large operable breast cancer.

BACKGROUND: Lapatinib is an effective anti-HER2 therapy in advanced breast cancer and docetaxel is one of the most active agents in breast cancer. Combining these agents in pre-treated patients with metastatic disease had previously proved challenging, so the primary objective of this study aimed to determine the maximum tolerated dose (MTD) in treatment-naive patients, by identifying acute dose-limiting toxicities (DLT) during cycle 1 in the first part of a phases 1-2 neoadjuvant European Organisation for Research and Treatment of Cancer (EORTC) trial. PATIENTS AND METHODS: Patients with large operable or locally-advanced HER2 positive breast cancer were treated with continuous lapatinib, and docetaxel every 21days for 4 cycles. Dose levels (DLs) were: 1000/75, 1250/75, 1000/85, 1250/85, 1000/100 and 1250/100 (mg/day)/(mg/m(2)). RESULTS: Twenty-one patients were included. Two DLTs occurred at dose level 5 (1000/100); one grade 4 neutropenia ⩾7days and one febrile neutropenia. A further 3 patients were therefore treated at the same dose with prophylactic granulocyte-colony stimulating factor (G-CSF), and 3 patients at dose level 6. No further DLTs were observed. CONCLUSIONS: Our recommended dose for phase II is lapatinib 1000mg/day and docetaxel 100mg/m(2) with G-CSF in HER2 positive non-metastatic breast cancer. The dose of lapatinib should have been 1250mg/day but we were mindful of the high rate of treatment discontinuation in GeparQuinto with lapatinib 1250mg/day combined with docetaxel. No grade 3-4 diarrhoea was observed. Pharmacodynamics analysis suggests that concomitant medications altering P-glycoprotein activity (in addition to lapatinib) can modify toxicity, including non-haematological toxicities. This needs verification in larger trials, where it may contribute to understanding the sources of variability in clinical toxicity and treatment discontinuation.

TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95).

A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Comparative functional analysis of two fibroblast growth factor receptor 1 (FGFR1) mutations affecting the same residue (R254W and R254Q) in isolated hypogonadotropic hypogonadism (IHH).

FGFR1 mutations have been identified in both Kallmann syndrome and normosmic HH (nIHH). To date, few mutations in the FGFR1 gene have been structurally or functionally characterized in vitro to identify molecular mechanisms that contribute to the disease pathogenesis. We attempted to define the in vitro functionality of two FGFR1 mutants (R254W and R254Q), resulting from two different amino acid substitutions of the same residue, and to correlate the in vitro findings to the patient phenotypes. Two unrelated GnRH deficient probands were found to harbor mutations in FGFR1 (R254W and R254Q). Mutant signaling activity and expression levels were evaluated in vitro and compared to a wild type (WT) receptor. Signaling activity was determined by a FGF2/FGFR1 dependent transcription reporter assay. Receptor total expression levels were assessed by Western blot and cell surface expression was measured by a radiolabeled antibody binding assay. The R254W maximal receptor signaling capacity was reduced by 45% (p<0.01) while R254Q activity was not different from WT. However, both mutants displayed diminished total protein expression levels (40 and 30% reduction relative to WT, respectively), while protein maturation was unaffected. Accordingly, cell surface expression levels of the mutant receptors were also significantly reduced (35% p<0.01 and 15% p<0.05, respectively). The p.R254W and p.R254Q are both loss-of-function mutations as demonstrated by their reduced overall and cell surface expression levels suggesting a deleterious effect on receptor folding and stability. It appears that a tryptophan substitution at R254 is more disruptive to receptor structure than the more conserved glutamine substitution. No clear correlation between the severity of in vitro loss-of-function and phenotypic presentation could be assigned.

Correlation of CXCL10, tumor necrosis factor receptor type II, and galectin 9 with disease activity in juvenile dermatomyositis.

OBJECTIVE: Juvenile dermatomyositis (DM) is a systemic autoimmune disorder of unknown immunopathogenesis in which the immune system targets the microvasculature of skeletal muscles, skin, and other organs. The current mainstay of therapy is a steroid regimen in combination with other immunosuppressive treatments. To date, no validated markers for monitoring disease activity have been identified, which hampers personalized treatment. This study was undertaken to identify a panel of proteins specifically related to active disease in juvenile DM. METHODS: We performed a multiplex immunoassay for plasma levels of 45 proteins related to inflammation in 25 patients with juvenile DM in 4 clinically well-defined groups, as determined by clinical activity and treatment. We compared them to 14 age-matched healthy children and 8 age-matched children with nonautoimmune muscle disease. RESULTS: Cluster analysis of circulating proteins showed distinct profiles for juvenile DM patients and controls based on a group of 10 proteins. In addition to CXCL10, tumor necrosis factor receptor type II (TNFRII) and galectin 9 were significantly increased in active juvenile DM. The levels of these 3 proteins were tightly linked to active disease and correlated with clinical scores (as measured by the Childhood Myositis Assessment Scale and physician's global assessment of disease activity on a visual analog scale). CONCLUSION: Our findings indicate that CXCL10, TNFRII, and galectin 9 correspond to disease status in juvenile DM and thus could be helpful in monitoring disease activity and guiding treatment. Furthermore, they might provide new knowledge about the pathogenesis of this autoimmune disease.