Cetuximab Treatment Research Articles

A Phase II Clinical Trial of the Anti-EGFR Antibody Cetuximab in Patients with Refractory or Relapsed Multiple Myeloma: Final Results,

Abstract 3965 INTRODUCTION:So far, monoclonal antibodies have not yielded convincing therapeutic success in multiple myeloma (MM). Therefore, the discovery of an effective targeted antibody against MM would open new perspectives for patients with relapsed or refractory MM and for the combination with established therapies. Cetuximab is an anti-epidermal growth factor receptor (EGFR) antibody. EGFR is also expressed on multiple myeloma (MM) plasma cells and bone marrow stromal cells. Recently, the inhibition of EGFR by small molecule inhibitors has been shown to induce apoptosis in primary myeloma cells revealing a synergistic effect with dexamethasone. Therefore, the anti-EGFR antibody cetuximab might be of clinical benefit in the treatment of MM, especially in combination with dexamethasone. Here we show the final results of the first clinical trial with an anti-EGFR antibody in MM. METHODS:Cetuximab was administered once weekly in standard dose to patients with refractory or relapsed MM who had previously received at least one line of prior treatment including autologous stem cell transplant (ASCT). Patients who still were candidates for ASCT were not included. Dexamethasone 20 mg on day 1–3 of each cycle was added starting week 5 in case of tumor progression or week 9 if no partial response (PR) or complete response (CR) was achieved with cetuximab alone. Planned treatment duration was 16 weeks (primary endpoint) with the possibility to prolong treatment in case of stable disease (SD) or response. RESULTS:In total, 15 patients have been enrolled. Seven patients were treated for a minimum of 16 weeks and 5 of those patients received cetuximab for at least 28 weeks. One patient received cetuximab treatment as single agent for more than one year. Thrombocytopenia, hyponatremia and acneiform rash were the most common CTC grade 3 or 4 side effects. Acneiform rash CTC grade 1 occurred in all patients and 3 patients suffered from acneiform rash CTC grade 3.After 16 weeks (primary endpoint) cetuximab in combination with dexamethasone induced 4 responses (3 minimal responses (MR) and 1 partial response (PR)) and achieved stable disease (SD) in 1 patient. Cetuximab as single agent induced SD in 2 patients. This results in an intention-to-treat overall response rate (ORR; CR+PR+MR) of 27% and a clinical benefit rate (at least SD) of 47%. Five of the 15 patients included did not receive the planned 16 weeks of treatment due to progressive disease (PD); three patients stopped treatment due to intolerable side effects. Six patients were treated more than 16 weeks: 1 patient received cetuximab as single agent and had SD more than a year and 5 patients continued treatment with cetuximab and dexamethasone in combination. CONCLUSIONS:Cetuximab is feasible and safe in MM patients. It demonstrated moderate efficacy in highly pre-treated patients, especially in combination with dexamethasone. Due to its unique mode of action and favorable side effect profile, cetuximab should be evaluated as combination partner for established substances such as bortezomib or lenalidomide to increase response rates both in therapy naive and in refractory patients. Disclosures:No relevant conflicts of interest to declare.

Abstract B36: Exploring potential clinical application of cetuximab in NSCLC: Efficacy study in a large panel patient derived xenograft (PDX) model.

Abstract NSCLC is a common cancer with one of the highest mortality numbers and few effective treatment options, except those with EGFR and ALK activating mutations. Cetuximab has been approved for CRC and H&N, but not yet for NSCLC. In this study, we set out to explore potential clinical utilities by testing cetuximab in a large cohort of patient derived xenograft (PDX) models (over established 60 models). Among the 12 NSCLC models tested so far, 5 out of 12 were found to be sensitive (42%), with all of which have wild-type KRAS and EGFR except for 1 model that carries an EGFR exon-20 a short in-frame insertion. These data suggest that cetuximab could potentially benefit a subgroup of patients, although it remains to be seen whether cetuximab can offer additional benefit besides standard chemotherapy regimens through combinational therapies. Among the 7 resistant models, two of them have L858R mutation in EGFR, a known driver mutation for non-smoking Asian female patients who are sensitive to gefitinib treatment. This data suggests that although both cetuximab and gefitinib can block EGFR signaling, the mechanism of action may also play an important role in tumor response to drugs. Analysis of genomic analysis did not reveal obvious relationship between cetuximab sensitivity and EGFR expression level, nor EGFR copy number. This could be attributed the relative small cohort size so far. Although the current study suggests that EGFR L858R mutation might be negatively correlated with response to cetuximab, additional studies with more PDX models are needed to further substantiate this observation, and for uncovering other biomarkers which could potentially predict patient populations who can benefit from cetuximab treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B36.

Abstract A144: Therapeutically targeting high-affinity ligand activation of EGFR with MM-151, an oligoclonal therapeutic.

Abstract The Epidermal Growth Factor Receptor (EGFR/ErbB1) is a receptor tyrosine kinase whose activation has been shown to play a key role in tumor growth and development. The EGFR ligand family is comprised of seven transmembrane precursor proteins whose expression and processing is highly regulated. These ligands can be classified based upon their affinity to EGFR; epidermal growth factor (EGF), betacellulin (BTC), heparin-binding epidermal growth factor (HB-EGF), and transforming growth factor alpha (TGFα) are considered high-affinity ligands, whereas amphiregulin (AREG), epiregulin (EREG), and epigen (EPI) are considered low-affinity ligands. EGFR overexpression in tumors is associated with higher risk of recurrence, metastasis, poorer survival and resistance to chemotherapy. Therefore, this pathway is a compelling target for the development of anti-EGFR therapeutics. Using Merrimack's Network Biology approach, we performed a computational systems analysis to identify an optimal strategy to inhibit the EGFR-ERK signaling network, which is characterized by robust signal amplification from the receptor to downstream effectors. As a result, we developed MM-151, an oligoclonal therapeutic composed of three fully human monoclonal antibodies targeted to distinct EGFR epitopes. MM-151 inhibits EGFR pathway activation by the dual mechanism of EGFR ligand-blocking and enhancement of receptor downregulation. Our preclinical in vitro studies revealed greater potency for MM-151 versus existing monoclonal antibodies (e.g. cetuximab, panitumumab and nimotuzumab). Importantly, MM-151 was shown to inhibit in vitro ERK signaling and cell proliferation induced by both high- and low-affinity EGFR ligands, unlike existing monoclonal therapeutics, which only block low-affinity ligand-induced signaling and cell proliferation. In in vitro cell proliferation assays, several cell line models that were responsive to anti-EGFR monoclonals in the presence of the low-affinity ligand AREG became increasingly unresponsive to treatment upon titrating in increasing amounts of the high-affinity ligand EGF. Conversely, cells remained responsive to MM-151 even in the presence of high-affinity EGF ligand burden. In preclinical non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) EGFR-wildtype cell line models, overexpression of autocrine AREG is positively correlated with increased sensitivity to the anti-EGFR monoclonal cetuximab or tyrosine kinase inhibitor gefitinib (K. Yonesaka et al., Clin. Cancer Res., 2008, 14: 6963–6973). In the clinic, metastatic colon cancer patients with wild type K-ras tumors highly expressing the low-affinity ligands AREG and EREG are more likely to exhibit disease control on cetuximab treatment (J. B. Baker et al., Br. J. Cancer, 2011, 104: 488–495). Our preclinical data suggest that elevated high-affinity ligand expression would likely correlate with decreased patient response to anti-EGFR monoclonals, and that patients whose tumors are driven by high-affinity EGFR ligands might instead benefit from MM-151 treatment. Together, these data suggest that MM-151, capable of blocking both high and low affinity ligand-driven EGFR signaling, may have the potential to more broadly benefit lung and colon cancer patients as compared to existing EGFR-directed therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A144.

Abstract A185: Identification of drug-associated proteins in NSCLC xenograft models by reverse-phase-protein-arrays.

Abstract At present, most non-small cell lung cancer (NSCLC) treatments are not adapted to the individual response of a patient. The stratification of patients for the most efficient response to conventional chemotherapeutics and targeted therapies will improve established therapy schemes and patient's perspectives. Our project aims at unraveling the influence of specific signaling molecules on the response to common NSCLC drugs. We searched for predictive markers for the response of NSCLC tumors to certain drugs and novel combinations of treatments, which may be useful for therapy decisions towards a more efficient personalized treatment. Expression of 77 proteins described to be involved in EGFR signaling (e.g. MAPK, JAK/STAT, PI3K/AKT pathways) and NSCLC disease in general were quantified in 53 patient derived NSCLC xenograft models using the reverse-phase protein array technology (RPPA). The tumor models are characterized by different response rates upon treatment with different chemotherapeutics and EGFR-targeted therapies. The response rates were obtained directly in the xenograft models. After RPPA analysis we associated the protein expression with the response to established chemotherapeutics (e.g. Vinorelbine, Paclitaxel, Carboplatin) and EGFR-targeted therapies (Cetuximab, Erlotinib). Another goal is to analyze the protein expression changes after treatment in dependence on the response rate. Therefore, protein profiles were raised in NSCLC xenograft models before and after treatment with Cetuximab, a chimeric (mouse/human) monoclonal antibody that inhibits EGFR. The major goal in this experimental subset is to find links between the activities of specific proteins and the response to Cetuximab treatment. A similar experiment with Erlotinib (an EGFR inhibitor) treated samples is ongoing. Statistical analysis indicated significant associations between the expression of distinct proteins and the response rate to certain drugs. Proteins of the ErbB signaling pathway were differentially expressed in Carboplatin responders and non-responders. MEK1 upregulation was observed upon Cetuximab treatment in responders. We revealed an association between higher P-SRC expression and increased Gemcitabine response rates. This result fits to observations that describe coherence between SRC inhibition and reduced suppression of cell growth and survival in the presence of Gemcitabine in cancer cell lines. We are now strengthening our results in various validation experiments. First, we selected seven target proteins based on RPPA analysis. Predictive targets for drug sensitivity are being analyzed to investigate the dependency between target protein activity, downstream signaling and response to distinct drugs in tumor cell lines in a pharmacogenomic approach. Therefore, we knocked down target genes in several NSCLC cell lines and unraveled changes in the drug sensitivity. Microarray and protein analyzes were carried out to study the mechanisms of target-drug association by investigating the downstream signaling upon target knockdown. In summary, our study suggests predictive markers for the response of NSCLC tumors to certain drugs, which may be useful for therapy decisions towards a more efficient personalized treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A185.

KRAS and BRAF Mutation Analysis in Metastatic Colorectal Cancer: A Cost-effectiveness Analysis from a Swiss Perspective

Monoclonal antibodies against the epidermal growth factor receptor (EGFR), such as cetuximab, have led to significant clinical benefits for metastatic colorectal cancer (mCRC) patients but have also increased treatment costs considerably. Recent evidence associates KRAS and BRAF mutations with resistance to EGFR antibodies. We assessed the cost-effectiveness of predictive testing for KRAS and BRAF mutations, prior to cetuximab treatment of chemorefractory mCRC patients. A life-long Markov simulation model was used to estimate direct medical costs (€) and clinical effectiveness [quality-adjusted life-years (QALY)] of the following strategies: KRAS testing, KRAS testing with subsequent BRAF testing of KRAS wild-types (KRAS/BRAF), cetuximab treatment without testing. Comparison was against no cetuximab treatment (reference strategy). In the testing strategies, cetuximab treatment was initiated if no mutations were detected. Best supportive care was given to all patients. Survival times/utilities were derived from published randomized clinical trials. Costs were assessed from the perspective of the Swiss health system. Average remaining lifetime costs ranged from €3,983 (no cetuximab) to €38,662 (no testing). Cetuximab treatment guided by KRAS/BRAF achieved gains of 0.491 QALYs compared with the reference strategy. The KRAS testing strategy achieved an additional gain of 0.002 QALYs compared with KRAS/BRAF. KRAS/BRAF testing was the most cost-effective approach when compared with the reference strategy (incremental cost-effectiveness ratio: €62,653/QALY). New predictive tests for KRAS and BRAF status are currently being introduced in pathology. Despite substantial costs of predictive testing, it is economically favorable to identify patients with KRAS and BRAF wild-type status.

NSC 74859‐mediated inhibition of STAT3 enhances the anti‐proliferative activity of cetuximab in hepatocellular carcinoma

Cetuximab [an epidermal growth factor receptor (EGFR) inhibitor], which was shown to be effective in rectal and non-small cell lung cancers (NSCLCs), was only modestly effective in clinical trials of hepatocellular carcinoma (HCC). STAT3, which is thought to be a determinant of HCC sensitivity to antitumour drugs, may be involved. To evaluate the efficacy of combination therapy using cetuximab and NSC 74859 (a novel STAT3 inhibitor) in EGFR and STAT3 overexpressing hepatoma cells. Hepatoma cell lines were treated with cetuximab, NSC 74859 or a combination of both drugs. Efficacy of treatment was evaluated by determining cell viability using MTT assays and proliferation by cell counting. Expression and activation of STAT3 were determined using Western blot analysis. We evaluated the role of STAT3 in single and combination therapy using siRNA-mediated knock-down of STAT3 or STAT3 overexpression strategies. HepG2 and Huh-7 cells, which had lower levels of pSTAT3 than SK-HEP1 cells, were more sensitive to cetuximab treatment when compared with SK-HEP1 cells. Although none of these cell lines was sensitive to NSC 74859 alone, NSC 74859 potentiated the antiproliferative effect of cetuximab in all three cell lines. siRNA knock-down of STAT3 increased the sensitivity of these cell lines to cetuximab, whereas STAT3 overexpression antagonized these effects. Enhanced growth inhibition in hepatoma cells treated with both NSC 74859 and cetuximab suggests that cetuximab resistance is probably mediated via STAT3. Combination therapy using both inhibitors of EGFR and STAT3 signalling warrants further investigation under in vivo condition.

Pneumatosis Intestinalis After Cetuximab-containing Chemotherapy for Colorectal Cancer

Cetuximab, a chimeric monoclonal antibody to epidermal growth factor receptor, is an effective chemotherapeutic agent for patients with metastatic colorectal cancer. Whereas several specific adverse reactions to cetuximab such as skin rash and nail toxicity have been reported, there have been few reports of pneumatosis intestinalis related to cetuximab-containing chemotherapy. We describe here three patients with colorectal cancer who developed pneumatosis intestinalis during treatment with cetuximab-containing chemotherapy, which developed after 7, 19 and 47 weeks of cetuximab treatment, and discovered on routine follow-up computed tomographic scans for response evaluations. None of these patients complained of abdominal pain, showed signs of peritoneal irritation on physical examination or had elevated serum concentrations of acute inflammatory markers. Following cessation of cetuximab and conservative medical treatment, all three patients showed complete resolution of pneumatosis intestinalis on abdominal pelvic computed tomographic scans.

Interferon/STAT1 and neuregulin signaling pathways are exploratory biomarkers of cetuximab (Erbitux�) efficacy in KRAS wild-type squamous carcinomas: A pathway-based analysis of whole human-genome microarray data from cetuximab-adapted tumor cell-line models

KRAS mutation status is being used as the sole biomarker to predict therapeutic efficacy of cetuximab in metastatic colorectal cancer (mCRC). A significant number of mCRC patients with KRAS wild-type (WT) tumors, however, do not benefit from cetuximab. We are also lacking efficacy predictors in head and neck squamous cell carcinomas with an intact KRAS signaling and in non-small cell lung cancer in which KRAS mutations do not predict cetuximab efficacy. We recently established pre-clinical models of EGFR gene-amplified KRAS WT A431 squamous carcinoma cells chronically adapted to grow in the presence of cetuximab. We employed the ingenuity pathway analysis software to functionally interpret data from Agilent's whole human genome arrays in the context of biological processes, networks, and pathways. Cetuximab-induced activation of the interferon (IFN)/STAT1 appeared to switch from 'growth inhibitory' in acutely-treated cells to 'pro-survival' in chronically-adapted cells. Cetuximab treatment appeared to negatively select initially dominant IFN-sensitive clones and promoted selection of IFN- and cetuximab-refractory tumor clones constitutively bearing an up-regulated IFN/STAT1 signaling. High-levels of mRNAs coding for the EGFR ligands amphiregulin (AREG), epiregulin (EREG), and neuregulin-1/heregulin (NRG1) predicted for acute cetuximab's functioning. Chronic cetuximab, however, appeared to negatively select initially dominant AREG/EREG/NRG1-positive clones to promote selection of cetuximab-refractory clones exhibiting a knocked-down neuregulin signaling. Our current evolutionary mapping of the transcriptomic changes that occur during cetuximab-induced chronic blockade of EGFR/KRAS WT signaling strongly suggests that mRNAs coding for IFN/STAT1- and EGFR ligands-related genes can be evaluated as novel predictors of efficacy in KRAS WT squamous cancer patients being treated with cetuximab.

Comparison of methodologies for KRAS mutation detection in metastatic colorectal cancer

Cetuximab and panitumumab are two monoclonal antibodies targeting the epidermal growth factor receptor that have been approved for treatment of metastatic colorectal cancer. Recent clinical trials found an association between KRAS mutation status and resistance to anti-epidermal growth factor receptor therapy, leading to the recommendation to perform KRAS mutation analysis before cetuximab or panitumumab treatment. This study was designed to compare and evaluate the efficacy of four different methodologies--high resolution melting, Sanger sequencing, DxS kit, and SNaPshot--for KRAS mutation detection in a clinical setting. In total, 372 samples from patients with metastatic colorectal cancer were analyzed by high resolution melting and SNaPshot, with 184 of those being further analyzed by Sanger sequencing and 188 with the DxS kit. Sensitivities were compared after consensus findings were determined by the presence of the same result in two of the three methodologies used in each case. The frequency of KRAS codon 12 and 13 mutations in our population was 43.5%, and a discordant finding was observed in 22 samples. Comparing to Sanger sequencing, significantly more consensus mutations were detected by the DxS kit (P=0.0139), high resolution melting (P=0.0004), and SNaPshot (P=0.00001), but no statistically significant differences were found among the three methodologies with higher sensitivity.

Ultrasound Therapy Boosts Drug Toxicity In Vitro and In Vivo

Objective: Ultrasound-mediated therapy (UST) utilizes mechanical vibration of microbubble contrast agent to enhance cell permeability and anti-cancer drug delivery. Here we determine if UST can improve cisplatin or cetuximab treatment of HNSCC in vitro and in vivo. Method: Cell permeability was assessed after UST in four HNSCC cell lines (SCC1, SCC5, Cal27, and FaDu). Combination of UST with cisplatin or cetuximab was assessed by cell survival in vitro. In vivo effects on flank tumors were measured in immune-suppressed mice. Results: Cell permeability was 48.2 ± 4.8% greater in cells treated with UST compared to controls ( P in vitro, UST alone had no effect on cell survival ( P = .59), but combination treatment with cetuximab ( P = .002) or cisplatin ( P in vivo) ( P = .81), but combination with cetuximab ( P = .04) orcisplatin ( P = .01) enhanced their antitumor effects. TUNEL analysis of flank tumors demonstrated increased apoptosis in UST+ cetuximab (55% vs 44%, P = .05), and UST+ cisplatin (84% vs 77%, P = .24) compared with monotherapy with either drug. Conclusion: The antitumor effects of cetuximab and cisplatin in HNSCC are enhanced by concurrent treatment with ultrasound-mediated therapy in vitro and in vivo.

Anti-cetuximab IgE ELISA for identification of patients at a high risk of cetuximab-induced anaphylaxis

Cetuximab, a chimeric mouse-human IgG1 monoclonal antibody against the epidermal growth factor receptor, has proven effective in the treatment of metastatic colorectal cancer and squamous cell carcinoma of the head and neck. However, a high incidence of immediate hypersensitivity reactions (HSR) to cetuximab after the first infusion has been observed. We have developed a test for identification of patients likely to show treatment-related HSR to cetuximab. An enzyme-linked immunosorbent assay (ELISA) for detecting anti-cetuximab IgEs was developed and tested on serum samples collected from cancer patients before start of cetuximab treatment, and from healthy blood donors. Similar levels of anti-cetuximab IgE were detected in pre-treatment patient sera (24/92, 26.1%) and sera from healthy blood donors (33/117, 28.2%). HSR were observed in 14 out of the 92 patients (15.2%), and 8 of these (57.1%) were grade 3-4. Anti-cetuximab IgEs were detected in 7/8 of the patients (87.5%) with severe HSRs as compared with 14/78 patients (17.9%) with no HSR (p=0.0002). Predictive value of the anti-cetuximab IgE test for HSR events of grades 3-4 was calculated using Receiver Operating Characteristics analysis. With a cut-off value of 29 arbitrary units for the anti-cetuximab IgE, the ELISA test showed a sensitivity of 87.5%, specificity of 82.1%, positive predictive value of 33.3% and negative predictive value of 98.5%. Anti-cetuximab IgE ELISA detection could be a valuable tool to help the physician anticipate an anaphylaxis episode following cetuximab infusion and opt for a suitable alternative treatment.

Diverse effects of combined radiotherapy and EGFR inhibition with antibodies or TK inhibitors on local tumour control and correlation with EGFR gene expression

PurposeTo compare functional effects of combined irradiation and EGFR inhibition in different HNSCC tumour models in vivo with the results of molecular evaluations, aiming to set a basis for the development of potential biomarkers for local tumour control. Material and methodsIn five HNSCC tumour models, all wild-type for EGFR and KRAS, the effect of radiotherapy alone (30 fractions/6weeks) and with simultaneous cetuximab or erlotinib treatment on local tumour control were evaluated and compared with molecular data on western blot, immunohistochemistry and fluorescence-in situ-hybridisation (FISH). ResultsErlotinib and cetuximab alone significantly prolonged tumour growth time in 4/5 tumour models. Combined irradiation and cetuximab treatment significantly improved local tumour control in 3/5 tumour models, whereas erlotinib did not alter local tumour control in any of the tumour models. The amount of the cetuximab-effect on local tumour control significantly correlated with the EGFR/CEP-7 ratios obtained by FISH. ConclusionBoth drugs prolonged growth time in most tumour models, but only application of cetuximab during irradiation significantly improved local tumour control in 3/5 tumour models. The significant correlation of this curative effect with the genetic EGFR expression measured by FISH will be further validated in preclinical and clinical studies.

Inhibition of STAT-3 results in greater cetuximab sensitivity in head and neck squamous cell carcinoma

ObjectiveThe inhibition of epidermal growth factor receptor (EGFr) with the monoclonal antibody cetuximab reduces cell proliferation and survival which correlates with increased DNA damage. Since the signal transducer and activator of transcription-3 (STAT-3) is involved in the EGFr-induced signaling pathway, we hypothesized that depletion of STAT-3 may augment cetuximab-induced processes in human head and neck cancer cells. Materials and methodsHuman head and neck squamous carcinoma cells (UM-SCC-5) were transfected with short hairpin RNA (shRNA) against STAT-3 (STAT3-2.4 and 2.9 cells). A mutated form of this shRNA was transfected for a control (NEG4.17 cells). Radiosensitivity was assessed by a standard colony formation assay. Proliferation was assessed by daily cell counts following treatment and apoptosis was assessed by an annexin V-FITC assay. The alkaline comet assay was used to assess DNA damage. ResultsThe STAT-3 knockdown cells (STAT3-2.4 and STAT3-2.9 cells) demonstrated enhanced radiosensitivity compared to control NEG4.17 cells, which correlated with increased apoptosis. Also, the STAT-3 knockdown cells demonstrated decreased proliferation with cetuximab treatments compared to control cells (NEG4.17). The increased cetuximab sensitivity of the STAT-3 knockdown cells correlated with increased apoptosis and DNA damage compared to control cells (NEG4.17). ConclusionThese studies revealed that the greater anti-proliferative effects and increased cytotoxicity of cetuximab in the STAT3-2.4 and STAT3-2.9 cells compared to control NEG4.17 cells, may be a result of STAT3-mediated effects on cellular apoptosis and DNA damage.

FCGR2A H131R and FCGR3A V158F polymorphism status in mCRC patients treated with single-agent cetuximab (IMCL 0144 and CA225045) or with second-line irinotecan plus cetuximab (EPIC): A pooled statistical analysis.

e14025 Background: Beyond K-RAS mutation status, additional positive predictive markers are needed to identify patients who will benefit from cetuximab treatment. Recent studies demonstrate antibody-dependent cell-mediated cytotoxicity (ADCC) may be one of the modes of action for cetuximab. Previous data suggest two Fc gamma receptor polymorphisms (FCGR2A H131R/FCGR3A V158F) may predict efficacy of cetuximab. Here we present analyses of these 2 polymorphisms in 3 phase II trials of mCRC patients treated with either single agent cetuximab (IMCL0144 and CA225045 [045]) or with second-line irinotecan plus cetuximab (EPIC). Methods: A total of 317 patients (133 from IMCL 0144; 84 from EPIC; 100 from 045) had been tested for FCGR2A H131 R and FCGR3A V158F polymorphisms. FCGR2A/3A polymorphisms were tested by PCR-RFLP or allele-specific PCR technique. Results: In a pooled analysis of IMCL 0144 and EPIC, we found patients with FCGR2A 131 HH genotype had shorter PFS (median PFS=1.3 months (95%C.I: 1.2-2.4) compared to those with HR/RR genotypes. (median PFS= 2.5 months [95%CI: 1.5-3.0] p=0.036, log-rank test). No statistically significant associations were observed between FCGR2A/3A polymorphisms and tumor response rate in either all evaluable patients or in the wild-type KRAS subgroup. (p>0.05, Fisher’s exact test). There are no associations between FCGR3A polymorphisms and OS, PFS. In a separate analysis of 045, no statistically significant associations were observed between FCGR2A/3A genotype and the assessed endpoints (response rate or PFS). Conclusions: Our pooled analysis suggested that the FCGR2A polymorphism may be associated with PFS in patients with mCRC, however FCGR3A polymorphism was not associated with efficacy of cetuximab-based treatment. Further larger statistically powered studies to evaluate the significance of FCGR polymorphisms are needed, and international collaborations are necessary to address the need for larger studies to validate molecular markers in the future.

Analysis of the anti-tumor effect of cetuximab using protein kinetics and mouse xenograft models

BackgroundThe binding of EGFR and its ligands leads to autophosphorylation of receptor tyrosine kinase as well as subsequent activation of signal transduction pathways that are involved in regulating cellular proliferation, differentiation, and survival. An EGFR inhibitor, cetuximab binds to EGFR and consequently blocks a variety of cellular processes. KRAS/BRAF mutations are known to be associated with a low response rate to cetuximab. In the present study, to clarify the anti-tumor mechanisms of cetuximab, we evaluated the KRAS/BRAF status, phosphorylation level of the EGFR pathway, and the tumor suppression effect in vivo, using a human colon cancer cell line HT29, which exhibited the highest EGFR expression in response to the cetuximab therapy among the 6 colorectal cancer cell lines tested.FindingsThe conventional growth suppression assay did not work efficiently with cetuximab. EGF, TGF-α, and IGF activated the EGFR/MAPK cell signaling pathway by initiating the phosphorylation of EGFR. Cetuximab partially inhibited the EGFR/MAPK pathway induced by EGF, TGF-α, and IGF. However, cetuximab exposure induced the EGFR, MEK, and ERK1/2 phosphorylation by itself. Mouse xenograft tumor growth was significantly inhibited by cetuximab and both cetuximab-treated and -untreated xenograft specimens exhibited phosphorylations of the EGFR pathway proteins.ConclusionsWe have confirmed that cetuximab inhibited the EGFR/MAPK pathway and reduced tumor growth in the xenografts while the remaining tumor showed EGFR pathway activation. These results suggest that: ( i ) The effect of cetuximab in growth signaling is not sufficient to induce complete growth suppression in vitro; ( ii ) time-course monitoring may be necessary to evaluate the effect of cetuximab because EGFR signaling is transmitted in a minute order; and ( iii ) cetuximab treatment may have cells acquired resistant selectively survived in the heterogeneous cancer population.

Cutaneous Toxicity Associated With Cetuximab Treatment in Metastatic Colorectal Cancer

ObjectiveTo evaluate the impact and type of side-effects in patients treated with cetuximab and provide a description of the general measures and treatment. MethodsRetrospective safety study. We included all patients that received cetuximab from January to December 2009. All information was obtained from the Pharmacy and Oncology Department's Access databases and reviewed the patient's medical history. All data was registered in an Excel workbook. Skin toxicity was graded by the current National Cancer Institute-Common Toxicity Criteria (NCI-CTC). ResultsDuring the study period 43 patients received treatment with cetuximab. Acneiform eruption was present in 30 of the cases (69.8%): 14 patients with grade 1 (48.3%), 13 with grade 2 (44.8%) and 3 with grade 3 (10.3%). These adverse effects appeared in a median of seven (4–28) days. In a median of 40 (20–56) days, ten patients (23.3%) presented xerosis, and three (7%) suffered painful fissures in hands and feet after a median of 28 (21–35) days. Paronychia was present in two patients after a median of 42 (35–49) days. Finally, an alteration in hair growth was observed in two patients with overgrowth of facial hair and one patient with overgrowth of the eyelashes. Five patients presented important conjunctivitis. Three infusion reactions occurred.A grade-based treatment algorithm was used for all patients that presented cutaneous toxicity. ConclusionsA considerable number of patients treated with cetuximab develop dermatological side-effects which left untreated could represent a threat to the efficacy of the therapy. Therefore effective management is mandatory, patient education and immediate treatment based on a grade-based algorithm to alleviate symptoms is necessary, so that patient compliance is guaranteed.

Epidermal growth factor receptor (EGFR) gene promoter methylation and cetuximab treatment in colorectal cancer patients

Background:Epidermal growth factor receptor (EGFR) promoter methylation may be responsible for the loss of EGFR expression in neoplastic cells. The primary aim of our study was to verify a possible correlation between EGFR gene promoter methylation and clinical outcome in metastatic colorectal cancer patients receiving chemotherapy with irinotecan and cetuximab.Methods:Colorectal samples from patients treated with irinotecan–cetuximab were analysed for EGFR promoter methylation and EGFR immunohistochemistry.Results:Fifty-two patients were analysed. Thirty patients (58%) showed EGFR promoter hypermethylation. In EGFR promoter methylated and EGFR promoter unmethylated patients, we observed a partial response in 3 (10%) and 13 (59%) patients, respectively (P=0.03), progressive disease was obtained in 19 (63%) and 2 (9%) patients, respectively, with EGFR promoter methylated and EGFR promoter unmethylated tumours (P=0.0001). Median progression-free survival was 2.4 months in patients showing EGFR promoter methylated tumours and 7.4 months for those who had EGFR promoter unmethylated tumours (P<0.0001; Figure 1). Median overall survival was 6.1 months in patients showing EGFR promoter methylated tumours and 17.8 months for those who had EGFR promoter unmethylated tumours (P<0.0001; Figure 2). CONCLUSION: EGFR promoter hypermethylation, after confirmation in larger data set, may represent a valuable asset in further studies investigating EGFR as a therapeutic target in colorectal cancer.

Overall and KRAS-specific results of combined cetuximab treatment and chemotherapy for metastatic colorectal cancer: a meta-analysis

This study compared the efficacy and toxicities of cetuximab combined with chemotherapy versus chemotherapy for patients with metastatic colorectal cancer (mCRC). The influence of KRAS mutation status on the outcomes was also investigated. Literature retrieval, trials selection and assessment, data collection, and statistical analysis were performed according to the Cochrane Handbook 5.0.2. The outcome measures were tumor response rate, progression-free survival, overall survival, and adverse effects. Four randomized controlled trials, comprising totally 2,912 patients, were included. Meta-analysis showed higher response rate (RR 1.93; 95% CI, 1.14-3.26) and significant improvement in progression-free survival (PFS; HR 0.80; 95% CI, 0.67-0.95) in cetuximab-chemotherapy groups versus chemotherapy groups. There was no significant difference between the two treatment groups regarding overall survival (OS; HR 0.95; 95% CI, 0.87-1.05). In wild-type KRAS patients, treatment with cetuximab plus chemotherapy significantly increased response rate (RR 1.44; 95% CI, 1.20-1.73) and improved PFS (HR 0.64; 95% CI, 0.50-0.84), as compared with chemotherapy groups, but not for OS (HR 0.84; 95% CI, 0.64-1.11). In mutant KRAS patients, there was no significant difference between those treated with cetuximab plus chemotherapy and those with chemotherapy alone regarding response rate (RR 0.81; 95% CI, 0.61-1.08), PFS (HR 1.37; 95% CI, 0.81-2.31), and OS (HR 1.03; 95% CI, 0.74-1.44). The risk of grade 3/4 rash, diarrhea, neutropenia, and fatigue was significantly increased in cetuximab combination groups as compared with chemotherapy groups. The use of cetuximab in addition to chemotherapy was a valid alternative for patients with mCRC. Benefit of cetuximab was only limited to patients with wild-type KRAS tumors.

Abstract 726: Erlotinib overcomes acquired resistance to cetuximab

Abstract The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of proliferation and progression in many human epithelial tumors. Following more than 20 years of preclinical development, five EGFR inhibitors, two monoclonal antibodies and three small molecule tyrosine kinase inhibitors (TKIs), have gained FDA approval in oncology (panitumumab, cetuximab, erlotinib, gefitinib and lapatinib). Both strategies of EGFR inhibition have demonstrated clinical successes. However, many tumors do not show response to EGFR inhibition and some of the responders eventually manifest resistance to the treatment. The underlying mechanisms of intrinsic and acquired resistance to EGFR inhibitors remain an intense area of scientific and clinical investigation. To explore potential molecular mechanisms of acquired resistance to cetuximab we established a series of cetuximab-resistant clones by chronically exposing the NCI-H226 NSCLC cell line to cetuximab. Cetuximab-resistant clones exhibited a dramatic increase in the steady-state expression of EGFR, due to alterations in trafficking and endocytosis as compared to parental controls. This increase in steady-state expression of the EGFR led to constitutive activation of HER2 and HER3 and downstream signals mediated by MAPK and AKT. Cells with acquired resistance to cetuximab remained dependent on EGFR signaling as shown by RNAi studies. This finding prompted investigation on whether or not cells with acquired resistance to cetuximab would be sensitive to erlotinib, an EGFR directed TKI. In vitro, erlotinib was able to decrease AKT and MAPK activation and subsequent cell proliferation measured by BrdU incorporation. Additionally, erlotinib treatment induced apoptosis in cetuximab resistant cells as measured by Annexin V analysis. To determine if erlotinib could have therapeutic benefit in vivo, we treated cetuximab sensitive parental lines, H226, with cetuximab in mouse xenograft studies. Mice harboring tumors were treated for approximately 70-150 days before resistant tumors manifested. At this time a series of mice with resistant tumors were treated with erlotinib for 15-30 days. Mice treated with erlotinib exhibited either a regression or tumor growth delay as compared to controls maintained on cetuximab. Analysis of the erlotinib treated tumors demonstrated a decrease in cell proliferation as measured by Ki67 and higher rates of apoptosis, as measured by TUNEL assays when compared to tumors maintained on cetuximab treatment. The work presented herein suggest that 1) cells with acquired resistance to cetuximab maintain their dependence on EGFR signaling via the MAPK and AKT pathways and 2) tumors developing resistance to cetuximab can be treated with erlotinib. This work may provide data strengthening the rationale for the use of erlotinib to treat patients with cetuximab resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2011-726

Abstract LB-317: BRAF and PIK3CA mutations in addition to KRAS, improve prediction of resistance to cetuximab in a large panel of colon carcinoma xenografts

Abstract Accurate prediction of response to therapy is a prerequisite for individualized approaches to colorectal cancer treatment. Testing of various cancer drugs on the individual patient tumor can support selection of effective therapies. To this end, we have established 149 stably passageable NOD/SCID mouse tumor xenografts from patients of all four Dukes stages within the framework of the prospective multicenter clinical study. All patients gave written informed consent prior to surgery. In a pharmacological study, 75 xenograft tumor models underwent single-agent treatment with oxaliplatin (OX), cetuximab (CE), or bevacizumab (BE). Each of the 75 tumors was transplanted onto 20 mice (5 controls and 5 for each drug). Models with a treated-to-control tumor growth ratio of 20% or lower were defined as responders. This resulted in response rates of 7% for OX, 24% for CE, and 4% for BE. Mutation analysis was conducted on 71 xenografts by allele-specific RT-PCR for the following hotspots: KRAS: 34G&amp;gt;A, 34G&amp;gt;T, 34G&amp;gt;C, 35G&amp;gt;A, 35G&amp;gt;T, 35G&amp;gt;C, 38 G&amp;gt;A and 436 G&amp;gt;A; BRAF: 1799 T&amp;gt;A, and PIK3CA: 1624G&amp;gt;A, 1633G&amp;gt;A, 3140A&amp;gt;G. KRAS mutations were found in 46%, BRAF in 10%, and PIK3CA in 15% of the xenografts. 59% of the xenografts carry at least one mutation. KRAS mutations were never observed together with BRAF-mutations while it combined with PIK3CA in 8 cases (11%). The corresponding primary tumors showed the same mutation profile. We examined mutation status with response to therapy to CE and OX. 3/5 OX responders were KRAS wildtype (wt) (Sensitivity (S+) 66%) while 31/66 OX non-responders were KRAS mutated (Specificity (S-) 46%). If also mutations in BRAF and PIK3CA are considered 40/60 OX non-responders were identified (S-66%). Of the 18 CE responders, all are wt for BRAF and PIK3CA. Three show mutations of KRAS, two of them in codon 13, which seems to confirm a recently published clinical observation by De Roock et al. (JAMA 2010, 304:1812) that this mutation may be related to an improved response under CE treatment. Wt status in all three genes was observed in 83% of the responders. KRAS mutations were seen in 57% of the non-responders. If mutations in BRAF and PIK3CA are also considered, the fraction of recognized non-responders increases to 73%. We also investigated how well response can be predicted from the mutation data. A machine learning approach was used to estimate unbiased prospective prediction rates for response to CE based on the mutation profile. In each of a large number of bootstrap loops, a new predictor was trained on a new randomly selected subset of the tumors and applied to the remaining tumors. KRAS, BRAF and PIK3CAmutations achieve a S+ of 84% (95% CI: 59% and 96%) and a S- of 68% (95% CI: 54% and 80%). KRAS, BRAF, and PIK3CA mutation status combined has potential clinical utility higher than KRAS alone to individualize CE treatment, also in the adjuvant setting, for patients with Dukes B and Dukes C. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2011-LB-317