| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cancer Therapy: Preclinical |
Authors' Affiliation: ImClone Systems Incorporated, New York, New York
Requests for reprints: Daniel J. Hicklin, Experimental Therapeutics, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014. Phone: 212-645-1405; Fax: 212-645-2054; E-mail: Dan.Hicklin{at}imclone.com.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Experimental Design: NSCLC cell lines were grown s.c. in nude mice. Dose-dependent efficacy was established for cetuximab. To determine whether combination therapy produces tumor regressions, cetuximab was dosed at half-maximal efficacy with chemotherapy used at maximum tolerated dose.
Results: Cetuximab showed antitumor activity in wt (A549, NCI-H358, NCI-H292) and mutated [HCC-827 (delE746-A750), NCI-H1975 (L858R, T790M)] EGFR-expressing xenografts. In the H292 model, cetuximab and docetaxel combination therapy was more potent to inhibit tumor growth than cetuximab or docetaxel alone. Cisplatin augmented efficacy of cetuximab to produce 6 of 10 regressions, whereas 1 of 10 regressions was found with cetuximab and no regression was found with cisplatin. Using H1975 xenografts, gemcitabine increased efficacy of cetuximab resulting in 12 of 12 regressions. Docetaxel with cetuximab was more efficacious with seven of nine regressions compared with single treatments. Cetuximab inhibited autophosphorylation of EGFR in both H292 and H1975 tumor lysates. Exploring the underlying mechanism for combination effects in the H1975 xenograft model, docetaxel in combination with cetuximab added to the antiproliferative effects of cetuximab but was the main component in this drug combination to induce apoptosis.
Conclusions: Cetuximab showed antitumor activity in NSCLC models expressing wt and mutated EGFR. Combination treatments increased the efficacy of cetuximab, which may be important for the management of patients with chemorefractory NSCLC.
Non–small cell lung cancer (NSCLC) is characterized by high incidence and mortality rates and rapid emergence of resistance against commonly used chemotherapeutics. Because EGFR is overexpressed in 65% to 100% of NSCLC cases (7), novel targeted therapeutics were developed that block activation of EGFR (8). Cetuximab is a chimeric IgG1 monoclonal antibody that blocks ligand binding to EGFR, leading to a decrease in receptor dimerization, autophosphorylation, and activation of signaling pathways (9, 10). In preclinical mouse models, cetuximab showed antitumor effects in renal (11), pancreatic (12, 13), colorectal (14), and lung (15–17) tumors. Clinical use of cetuximab is approved for late-stage chemorefractory colorectal cancer and for squamous cell carcinoma of the head and neck. Gefitinib and erlotinib are small-molecule inhibitors of EGFR. Both drugs compete for the ATP-binding site in the kinase domain of EGFR and are used against pancreatic cancer and NSCLC (8, 18).
Several somatic mutations in EGFR have been identified in a subset of NSCLC patients (10-15%), which were characterized by adenocarcinoma histology, oriental origin, female gender, and never-smoker status. Importantly, the patients with mutated EGFR responded better to treatment with gefitinib than those without mutations (19–21). The clinically identified changes included short amino acid deletions and point mutations in the ATP-binding pocket of the tyrosine kinase domain encoded by exons 18 to 21. These findings suggested that the observed correlation between expression of EGFR mutants and better response rate to gefitinib is due to hypersensitivity of the EGFR mutants to inhibitors of the kinase domain. The tumor cells depend on the continued activity of the EGFR pathway to maintain their malignant phenotype, also called "oncogene addiction" (22). Subsequently, acquired resistance to gefitinib and erlotinib has been studied in patient tumor samples and was attributed to a secondary point mutation (T790M) in the kinase domain of EGFR (23, 24). The genetics of resistance is recapitulated in the human NSCLC cell line NCI-H1975, which contains both the gefitinib-sensitizing L858R and the resistant T790M point mutations (24) and is resistant to gefitinib and erlotinib in vitro (24–26).
Demonstration of preclinical efficacy of cetuximab in NSCLC xenograft mouse models is limited to two studies using NCI-H226 (15) and NCI-H292 cells (16). Furthermore, cetuximab inhibited lung tumor growth in transgenic mice overexpressing an activating allele of EGFR (17). To better understand the potential of cetuximab in NSCLC, we tested cetuximab in a panel of NSCLC xenografts expressing wild-type (wt) EGFR and found antitumor effects in A549, NCI-H358, and H292 tumors. In addition, HCC-827 cells, which contain an activating exon 19 deletion mutation in EGFR (delE746-A750) and which were sensitive to cetuximab in vitro (27), were highly sensitive to cetuximab in our in vivo studies. The effects of cetuximab on gefitinib-resistant H1975 cells (L858R and T790M), either in vitro or in vivo, have not been described previously. We show that H1975 xenografts retained dose-dependent sensitivity to cetuximab. Cetuximab was tested in combination with the cytidine analogue gemcitabine (Gemzar), the microtubule-stabilizing agent docetaxel (Taxotere), platinum-based alkylating agents cisplatin (Platinol) and carboplatin (Paraplatin), and the antifolate pemetrexed (Alimta). Strong effects were found in both H292 and H1975 mouse models with cetuximab in combination with gemcitabine or docetaxel. Finally, we found that docetaxel augmented the antiproliferative effects of cetuximab but was mostly responsible for the induction of apoptosis in H1975 tumor extracts.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Sequencing of EGFR. Exons 18 to 21 of EGFR were sequenced from genomic DNA isolated from H292 cells using methods and sequencing primers described previously (19, 20).
S.c. NSCLC xenograft models. All experiments and procedures involving mice were done according to the U.S. Department of Agriculture, Department of Health and Human Services, and NIH policies regarding the humane care and use of laboratory animals. Eight-week-old female athymic (nu/nu) mice from Charles River Laboratories (Wilmington, MA) were housed under pathogen-free conditions in microisolator cages with laboratory chow and water ad libitum. NSCLC xenografts were established by injecting s.c. into the left flanks 2 x 107 (A549, NCI-H358, HCC-827), 5 x 106 (Calu-6, NCI-H292), 3 x 105 (NCI-H460), or 1 x 107 (NCI-H1975) cells per mouse mixed 1:1 in Matrigel (100% Matrigel for A549; Collaborative Research Biochemicals, Bedford, MA). Tumors were allowed to reach 200 to 400 mm3 for efficacy studies (10-12 mice per group) or 350 to 450 mm3 for mechanism of action studies (6 mice per group) before randomization.
Treatments and tumor measurements. Cetuximab was produced by ImClone System manufacturing facility (Branchburg, NJ). Human IgG antibodies were from Sigma and diluted in 0.9% USP saline. Gemcitabine was from SynChem OHG (Kassel, Germany) and prepared in 0.9% USP saline containing 12.5 mg/mL mannitol and 0.781 mg/mL sodium acetate. Docetaxel was from Fluka (Buchs, Switzerland) and prepared in 0.9% USP saline containing 4% ethanol and 10% polysorbate 80. Carboplatin and cisplatin were from Sigma and prepared in 0.9% USP saline. Pemetrexed was purchased from Briarwood Pharmacy (Jamaica, NY) and dissolved in 0.9% USP saline. Cetuximab followed by chemotherapeutics was administered i.p. at 20 µL per gram of body weight. Caliper measurements were used to calculate tumor volumes using the formula V = 
/6 (length x width x width). Antitumor effects are expressed as %T/C (treated versus control), dividing the tumor volumes from treatment groups with the control groups and multiplied by 100. For mechanism studies, docetaxel was injected up to 12 h before cetuximab.
Statistical analysis. Tumor volumes were analyzed using repeated measures ANOVA in the JMP Statistical Discovery package (v.5.1; SAS Institute Inc., Cary, NC). Frequencies of tumor regressions across four treatment groups were analyzed by 
2 test. Statistical significance between band intensities from immunoblots and absorbance units from histology and ELISA analysis was determined by Student t test (one-tailed distribution). *P = 0.05 to 0.01 (significant), **P = 0.01 to 0.001 (very significant), and ***P < 0.001 (extremely significant).
Antibodies for Western blotting. Antibodies against phospho-EGFR Tyr1068 and Tyr845 and phosphorylated mitogen-activated protein kinase (phospho-MAPK) were from Cell Signaling Technology (Beverly, MA). Antibodies against phospho-EGFR Tyr1086 and Tyr1173 were from BioSource International (Camarillo, CA). Anti-EGFR antibody was from Upstate (Lake Placid, NY). Anti–ß-actin antibody was from Sigma. Anti-mouse IgG peroxidase-linked whole antibody and anti-rabbit IgG peroxidase-linked species-specific whole antibody were from Amersham (Piscataway, NJ).
Tumor harvesting and Western blotting. Xenograft tumors were excised from euthanized mice and snap frozen in liquid nitrogen. Lysates were prepared in lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Triton X-100, 100 mmol/L sodium orthovanadate, 1 mmol/L EDTA, Complete Protease Inhibitor Cocktail Tablets; Roche Applied Science, Indianapolis, IN) using a dounce homogenizer. Protein concentration was determined using BCA Protein Assay Reagent (Pierce, Rockford, IL). Lysates were run at 50 µg per lane on 4% to 12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). Western blot band intensity quantification was done using MultiGauge software v2.3 (FujiFilm Medical Systems, Stanford, CT). To account for differences in protein loading, all band intensities were corrected for ß-actin.
Histologic analysis of phospho-EGFR and apoptosis markers. Tumors were fixed in 10% neutral-buffered formalin, incubated in 30% sucrose, and embedded in paraffin for sectioning and immunohistochemical staining of phospho–Tyr1086 of human EGFR (Invitrogen) and cleaved caspase-3 (Cell Signaling). DNA fragmentation (apoptosis) was detected with ApopTag Plus peroxidase in situ apoptosis kit (Chemicon, Temecula, CA). Quantification of phospho-EGFR, cleaved caspase-3, and ApopTag was done with a Zeiss Axiocam mounted on a Zeiss universal microscope (Carl Zeiss, Thornwood, NY). Morphometric analysis of digital images was done with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) to determine the average mean density of phospho-EGFR staining or the percentage of cells positive for colored reaction product (ApopTag or caspase-3) in 100 randomly selected tumor cells per section using five sections per tumor.
Phospho–histone H3 and cleaved caspase-3 assays. Lysates were prepared in the same buffer used for Western blotting. Fifty micrograms of extracts were analyzed in the PathScan phospho–histone H3 (Ser10) sandwich ELISA (Cell Signaling). Two hundred micrograms were used for the PathScan cleaved caspase-3 sandwich ELISA (Cell Signaling) following manufacturer's instructions. In brief, extracts were mixed with 100 µL of sample diluent and incubated in antibody-coated microwell strips. One hundred microliters of biotinylated phospho–histone H3 or cleaved caspase-3 detection antibodies were added to each well. Binding was detected with 100 µL of horseradish peroxidase–linked streptavidin antibody and 100 µL of TMB substrate solution. The colored reaction product was measured in a standard ELISA plate reader at 450 nm.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
0.4 mg/kg (Fig. 1D). All five cell lines express wt EGFR (28), which we confirmed for H292 cells by sequencing exons 18 to 21 from genomic DNA (data not shown). EGFR protein was readily detectable by immunoblotting in A549 and H292 cells, whereas H460 and Calu-6 cells expressed moderate levels of EGFR. Surprisingly, the cetuximab-sensitive H358 line expressed very low levels of EGFR (data not shown). Small deletions and point mutations in exons 19 to 21 of EGFR were reported in human NSCLC tumor samples, which correlated with clinical benefit to treatment with small-molecule inhibitors of EGFR (19–21). The adenocarcinoma cell line HCC-827 harbors such a deletion in EGFR (delE746-A750). Dosing of HCC-827 xenografts with 4 or 40 mg/kg of cetuximab (twice per week) completely inhibited tumor growth. A dose-response was established by lowering cetuximab to 0.4 mg/kg (Fig. 1E). Last, the gefitinib-resistant adenocarcinoma cell line H1975 (L858R and T790M) was established as a xenograft mouse model. Treatment of H1975 tumors with cetuximab (twice per week) produced dose-dependent growth inhibition. Cetuximab at 0.4 mg/kg was identified as an approximate IC50 in the H1975 xenograft model because of its half-maximal antitumor effects. Higher doses completely inhibited tumor growth for 2 weeks followed by moderate growth inhibition (Fig. 1F).
|
17 days with a tumor inhibition rate of 65% T/C after 42 days of treatment (Fig. 2A and E
). Gemcitabine at 125 mg/kg (once per week) had no effect on tumor growth but when combined with cetuximab a strong inhibition of tumor growth was observed up to day 42 with 22% T/C (Fig. 2A). Statistical analysis by repeated measures ANOVA revealed that the combination protocol added a significant benefit over monotherapy treatments (Fig. 2E). Furthermore, the combination treatment led to tumor regressions in 4 of 11 tumors, whereas cetuximab caused 1 of 10 regression. No regression was observed in either the saline or the gemcitabine group (Fig. 2E). The statistical significance of the regression frequencies across all four treatment groups was analyzed by a 2 test and found to be significant for the combination group (Fig. 2E). The combination group was followed in the absence of treatment after day 42 to determine whether the tumor reductions constituted tumor cures or simply tumor growth inhibition. The size of the tumors increased without treatment, indicating relapse of tumor growth (Fig. 2A).
|
Chemotherapy increases efficacy of cetuximab in NSCLC xenografts with mutations in EGFR. To extend our findings on antitumor efficacy with cetuximab and chemotherapy in a wt EGFR tumor model, we tested the same combinations in the H1975 xenograft model (L858R and T790M double mutation). Cetuximab was dosed at a concentration close to the IC50, 1 mg/kg (twice per week), in combination with chemotherapies used at their maximum tolerated dose (once per week). Combination effects of cetuximab with gemcitabine were evaluated after 22 days when the saline group was terminated due to ulceration of tumors. The combination treatment showed a lasting antitumor effect of 7% T/C (Fig. 3A and E
). Cetuximab had a half-maximal effect of 49% T/C, whereas gemcitabine at 125 mg/kg was slightly more effective with 26% T/C. Statistical analysis by repeated measures ANOVA documented the benefit of combination over monotherapies (Fig. 3E). Furthermore, cetuximab with gemcitabine produced 12 of 12 regressions, whereas fewer regressions were observed in the monotherapy treatments and no regression in the controls. Frequencies of regressions were analyzed by 2 test and found to be highly significant (Fig. 3E). No toxicity was associated with these treatments based on the absence of body weight loss. Treatment was stopped after day 33 to assess cure rate versus growth inhibition. All 12 tumors in the cetuximab and gemcitabine combination group resumed growth by day 60, indicating relapse of tumor growth (data not shown).
|
Cetuximab inhibits phosphorylation of EGFR in NSCLC xenografts with wt and mutated EGFR. The mechanism of action of cetuximab has been studied in pancreatic (12), epidermoid (29), and colon (30) xenograft tumor models and involved inhibition of phosphorylation of EGFR, MAPK, and AKT. To elucidate how cetuximab inhibited H292 tumor growth, we determined the change in phosphorylation of four EGFR tyrosine residues in xenograft extracts. Quantitative immunoblot analysis showed that cetuximab at 40 mg/kg reduced phosphorylation of Tyr845 and Tyr1173 by 57% and 34% after 3 days of treatment (Fig. 4A and C ). Both effects reached statistical significance by Student t test. Phosphorylation of Tyr1068 and Tyr1086 was reduced by a smaller degree. Furthermore, we found equal levels of total EGFR in saline- and cetuximab-treated tumor samples (Fig. 4E), indicating that cetuximab inhibited H292 tumor growth by reducing EGFR kinase activity and not through degradation of EGFR.
|
Docetaxel enhances inhibitory effects of cetuximab on EGFR and MAPK pathways in NSCLC xenografts. Our efficacy studies showed that docetaxel enhanced the antitumor effects of cetuximab in H292 and H1975 xenograft models. We wondered whether the combination of cetuximab and docetaxel would lead to a stronger decrease in phospho-EGFR compared with cetuximab alone. H1975 xenografts were chosen because phosphorylation of all four tyrosine residues was reduced by cetuximab (see Fig. 4B). Specifically, changes in Tyr1086 were analyzed because this site regulates ras/MAPK and phosphatidylinositol 3-kinase/AKT signaling pathways. Immunohistochemical analysis of tumor sections from saline-treated H1975 xenografts revealed strong Tyr1086 staining localized to the cell membranes of tumor cells (Fig. 5A ). Cetuximab at 10 mg/kg caused a reduction in membrane staining after 3 days, whereas intracellular staining for Tyr1086 increased (Fig. 5A). This supports the notion that cetuximab leads to internalization followed by inactivation of mutated EGFR in H1975 tumors. Docetaxel at 30 mg/kg had no effect but enhanced the activity of cetuximab in the combination leading to elimination of cell membrane and cytoplasmic staining of phospho-EGFR Tyr1086 (Fig. 5A). These observations were confirmed by quantitative morphometric measurements of the mean phosphorylation intensity of Tyr1086 in tumor sections (Fig. 5B). Statistical analysis by Student's t test showed a significant effect of cetuximab versus saline, whereas the combination treatment was significantly different from saline and both monotherapies.
|
Cetuximab does not increase the effects of docetaxel on apoptosis in NSCLC xenografts. Staining of H1975 tumor sections treated with cetuximab and docetaxel showed degenerative tumor cells with shrunken and dissociated nuclei (see Fig. 5A). Such pyknotic nuclei contain condensed chromatin characteristic of programmed cell death. We asked whether the combination of cetuximab and docetaxel was more potent to induce apoptosis than monotherapy alone. Immunohistochemical staining of tumor sections with an ApopTag apoptosis kit revealed a low incidence of cells with fragmented DNA in saline-treated H1975 xenografts. Morphometric measurement of the percentage of cells positive for ApopTag showed that cetuximab at 10 mg/kg produced a 2.2-fold increase in apoptosis after 3 days of treatment, whereas 30 mg/kg docetaxel increased cell death by 3.4-fold (Fig. 6A and B ). The combination of cetuximab and docetaxel was only slightly more potent than docetaxel without reaching statistical significance (Fig. 6A). This indicated that induction of apoptosis in xenograft tumors treated with the combination is mediated by the chemotherapy without any additional benefits from cetuximab.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
0.4 mg/kg. This compares favorably with small-molecule inhibitors of EGFR, such as gefitinib or erlotinib, which have published IC50 concentrations between 8 and 100 mg/kg depending on the wt-EGFR–expressing tumor model (15, 28, 31, 32). Our results complement previous reports describing activity of cetuximab at 1 mg/mouse (40 mg/kg) in H292 xenografts (16) and half-maximal efficacy at 0.2 mg/mouse (8 mg/kg) in a H226 tumor model (15). Our work supports the notion that EGFR expression levels in NSCLC do not predict responsiveness of tumors to cetuximab. H358 cells displayed low levels of EGFR but responded well to cetuximab. Conversely, H460 and Calu-6 cells expressed EGFR at moderate levels but were resistant to cetuximab. It was reported that signaling via ErbB2 and ErbB3 associated with resistance to gefitinib but not with resistance to cetuximab in head and neck squamous cell carcinoma cells (33). We analyzed ErbB2 expression levels by immunoblotting in NSCLC cell lines and found the highest levels of ErbB2 in the cetuximab-sensitive lines H292, HCC-827, and H1975 (data not shown). It should be noted that the presence of ErbB2 or ErbB3 is not expected to influence the responsiveness of cells to cetuximab because Erb family members require heterodimerization with EGFR for activation (6). Because cetuximab blocks ligand-mediated heterodimerization of EGFR with ErbB2 or ErbB3, expression of these family members would not be expected to result in decreased sensitivity. It is possible that mutations in signaling molecules downstream of EGFR such as ras, raf, or PTEN might modulate efficacy of cetuximab. The present study does not answer the contributions of ras because mutations in ras were present in A549 and NCI-H358 cell lines but absent in NCI-H292 cells. Nevertheless, an antitumor response to cetuximab was observed in all three lines grown as xenograft tumors. Although NSCLC patients have few treatment options and the response rate is generally low, a subset of patients with activating mutations in EGFR can achieve remarkable responses with gefitinib and erlotinib. Clinical benefits can last 2 to 3 years until patients develop resistance. The molecular determinants in responding patients are small exon 19 deletions and exon 21 point mutations in the ATP-binding pocket of EGFR. The NSCLC cell line HCC-827 contains an activating exon 19 deletion mutation and displays in vitro sensitivity to gefitinib, erlotinib, and cetuximab (27). We grew HCC-827 cells as xenografts and established an IC50 concentration for cetuximab of 0.4 mg/kg. Furthermore, cetuximab, as well as erlotinib, showed efficacy against lung tumors in transgenic mice overexpressing an activated allele of EGFR (17). Hence, our findings show that preclinical models of NSCLC with activating EGFR mutations are as sensitive to cetuximab as they are to small-molecule inhibitors. A second point mutation in exon 20 (T790M) prevents small-molecule inhibitors of EGFR from binding to the ATP-binding cleft, thereby creating resistance (34, 35). Knowing the existence of clinically relevant resistance mechanisms against gefitinib and erlotinib, cetuximab was reported to have limited in vitro activity in Ba/F3 cells overexpressing a gefitinib-resistant allele of EGFR containing the L858R and T790M double mutation (26). However, in our in vivo xenograft tumor studies using the gefitinib- and erlotinib-resistant NSCLC cell line H1975 (L858R and T790M), cetuximab was clearly efficacious with dose-response studies defining an approximate IC50 value for cetuximab of 0.4 mg/kg. In conclusion, the response to cetuximab between xenograft mouse models that express wt or mutant EGFR is similar based on IC50 values presented in this study.
Combined antitumor activity of cetuximab with standard chemotherapy is well documented in colorectal (14), pancreatic (12), and lung (16) mouse models. In addition, gefitinib worked synergistically with gemcitabine in a head and neck carcinoma mouse model (36). Combinations with gefitinib and paclitaxel showed antitumor effects in a renal mouse model (37), whereas sequence-specific dosing of gefitinib and docetaxel was analyzed in a bladder cancer model (38). These data, together with data presented in this study, indicate that inhibition of EGFR together with blockade of cellular processes that are involved in proliferation, cell division, or cell cycle control resulted in increased antitumor activity. A notable exception was the lack of better efficacy of the combination of cetuximab with platinum-based agents in H1975 xenografts. It is currently unknown whether this is caused by mutant EGFR or downstream signaling networks that are differentially affected by mutant EGFR. A clinical analysis of NSCLC patients with wt or mutated EGFR did not uncover differential response rates to platinum agents (39). Our preclinical combination data are thus far in agreement with human clinical trial data. Several phase II trials have reported antitumor effects with cetuximab as monotherapy (40) or in combination with gemcitabine, docetaxel, cisplatin, and carboplatin in chemotherapy-naïve and chemotherapy–refractory/resistant NSCLC patients (8, 41, 42). An exploratory study using 38 tumor specimens showed that two of three patients with mutant EGFR experienced stable disease in a cetuximab monotherapy trial enrolling pretreated NSCLC patients (40, 43). It remains to be determined whether clinical response rates to cetuximab combination treatments in NSCLC are influenced by EGFR mutations.
Basal levels of EGFR kinase activity were higher in H1975 xenografts compared with H292 tumors, whereas total EGFR levels were comparable. Higher activity in H1975 cells was most likely caused by the constitutively active kinase domain of EGFR. Cetuximab reduced kinase activity in both H292 and H1975 tumor extracts. The reduction in H1975 cells is noteworthy because EGFR is activated in a ligand-independent fashion through mutations in the kinase domain. We conclude that mutant EGFR kinase activity in H1975 xenografts was induced by circulating ligand. Because H1975 cells are heterozygous for mutated EGFR, cetuximab might inhibit EGFR expressed from the wt allele. This interaction could inhibit mutated EGFR through impaired heterodimerization. However, such an explanation seems unlikely because the copy number of the mutated allele is in excess over the wt allele in H1975 cells (24). We noticed a 30% reduction in total EGFR in H1975 tumors treated with cetuximab. Although the effect missed statistical significance, it points out a difference in the response of wt and mutant EGFR to cetuximab. Internalization and degradation of the receptor could explain why constitutively activated, ligand-independent mutant EGFR was susceptible to cetuximab in H1975 xenografts. Such a model is supported by immunohistochemical analysis, which revealed a transition of Tyr1086 staining from the cell membrane to the cytoplasm after cetuximab treatment. The addition of docetaxel enhanced the cetuximab-mediated decrease in phospho-EGFR although docetaxel did not affect phosphorylation of EGFR. Inhibition of the EGFR pathway by combination therapy also caused lowered levels of phospho-MAPK and ultimately a reduction in phosphorylated histone H3. Conversely, our studies looking at markers of apoptosis did not support a similar statement about combined activity of cetuximab and docetaxel. In H1975 tumors, docetaxel is the sole driver of apoptosis even in the presence of cetuximab. Altogether, antitumor efficacy of the combination of cetuximab and docetaxel was mediated by a decrease in proliferation with a concomitant increase in apoptosis. This treatment protocol highlights the benefits of combining two agents with different mechanisms of action.
Acquisition of drug resistance to the reversible inhibitors gefitinib and erlotinib appears upon somatic mutation of tyrosine T790M positioned in the kinase domain of EGFR. In fact, inherited susceptibility to lung cancer has been described in humans and is associated with germ line transmission of the T790M mutation (44). One additional patient-associated EGFR mutation resistant to gefitinib is an insertion mutation in exon 20 (45). Our research suggests that cetuximab might be a clinically viable option for NSCLC patients with the L858R/T790M double mutation or the T790M germ line mutation. The effects of cetuximab on the exon 20 insertion mutation have not been tested yet. Alternative approaches to overcome gefitinib resistance have been reported and involve irreversible small-molecule inhibitors of EGFR, such as HKI-272 or EKB-569 (25, 46) and CL-387,785 (23, 26, 45). To prevent the emergence of the T790M mutation, it has been suggested to combine gefitinib and cetuximab as an anti-EGFR strategy. Such an approach is partly based on lessons learned from the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors with imatinib (47). A common resistance mechanism to imatinib is the T315I mutation in the Abl tyrosine kinase domain of Bcr-Abl (48, 49). This mutation leads to a structural change in Abl, which is similar to the one in EGFR caused by the T790M mutation. Combining imatinib with a reagent that inhibits the T315I mutation in newly diagnosed chronic myeloid leukemia would not only inhibit Bcr-Abl but also prevent the clonal selection and spread of tumor cells that have acquired the resistance mutation. This treatment paradigm can be applied to NSCLC patients that are diagnosed with gefitinib-sensitizing mutations in exons 19 or 21. In fact, combinations of gefitinib and cetuximab were tested in preclinical lung and epidermoid carcinoma mouse models and showed superior and more durable efficacy over monotherapy (15, 29).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received 7/31/06; revised 12/ 4/06; accepted 12/14/06.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. J. Freeman, T. Bush, S. Ogbagabriel, B. Belmontes, T. Juan, C. Plewa, G. Van, C. Johnson, and R. Radinsky Activity of panitumumab alone or with chemotherapy in non-small cell lung carcinoma cell lines expressing mutant epidermal growth factor receptor Mol. Cancer Ther., June 1, 2009; 8(6): 1536 - 1546. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. N. Naumov, M. B. Nilsson, T. Cascone, A. Briggs, O. Straume, L. A. Akslen, E. Lifshits, L. A. Byers, L. Xu, H.-k. Wu, et al. Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance Clin. Cancer Res., May 15, 2009; 15(10): 3484 - 3494. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Nikolova, I. A. Asangani, L. D. Nelson, D. P.M. Hughes, D. R. Siwak, G. B. Mills, A. Harms, E. Buchholz, L. R. Pilz, C. Manegold, et al. Cetuximab Attenuates Metastasis and u-PAR Expression in Non-Small Cell Lung Cancer: u-PAR and E-Cadherin are Novel Biomarkers of Cetuximab Sensitivity Cancer Res., March 15, 2009; 69(6): 2461 - 2470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Fichtner, J. Rolff, R. Soong, J. Hoffmann, S. Hammer, A. Sommer, M. Becker, and J. Merk Establishment of Patient-Derived Non-Small Cell Lung Cancer Xenografts as Models for the Identification of Predictive Biomarkers Clin. Cancer Res., October 15, 2008; 14(20): 6456 - 6468. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Ciardiello and G. Tortora EGFR Antagonists in Cancer Treatment N. Engl. J. Med., March 13, 2008; 358(11): 1160 - 1174. [Full Text] [PDF]
|
|
|
|
|
|
H.-P. Kim, S.-W. Han, S.-H. Kim, S.-A. Im, D.-Y. Oh, Y.-J. Bang, and T.-Y. Kim Combined lapatinib and cetuximab enhance cytotoxicity against gefitinib-resistant lung cancer cells Mol. Cancer Ther., March 1, 2008; 7(3): 607 - 615. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Rosell, G. Robinet, A. Szczesna, R. Ramlau, M. Constenla, B. C. Mennecier, W. Pfeifer, K. J. O'Byrne, T. Welte, R. Kolb, et al. Randomized phase II study of cetuximab plus cisplatin/vinorelbine compared with cisplatin/vinorelbine alone as first-line therapy in EGFR-expressing advanced non-small-cell lung cancer Ann. Onc., February 1, 2008; 19(2): 362 - 369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Butts, D. Bodkin, E. L. Middleman, C. W. Englund, D. Ellison, Y. Alam, H. Kreisman, P. Graze, J. Maher, H. J. Ross, et al. Randomized Phase II Study of Gemcitabine Plus Cisplatin or Carboplatin, With or Without Cetuximab, As First-Line Therapy for Patients With Advanced or Metastatic Non-Small-Cell Lung Cancer J. Clin. Oncol., December 20, 2007; 25(36): 5777 - 5784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Doody, Y. Wang, S. N. Patel, C. Joynes, S. P. Lee, J. Gerlak, R. L. Rolser, Y. Li, P. Steiner, R. Bassi, et al. Inhibitory activity of cetuximab on epidermal growth factor receptor mutations in non small cell lung cancers Mol. Cancer Ther., October 1, 2007; 6(10): 2642 - 2651. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
