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1 Lowe Center for Thoracic Oncology and 2 Department of Medical Oncology, Dana Farber Cancer Institute; 3 Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School; 4 Shannon McCormack Advanced Molecular Diagnostic Laboratory, Dana Farber Cancer Institute; and Departments of 5 Pathology, 6 Biological Chemistry, and 7 Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts
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ABSTRACT |
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Arg), is also found in NSCLC cell line H3255, which is very sensitive to gefitinib treatment. We characterized nine NSCLC cell lines (three isolated from patients with bronchioloalveolar carcinoma and six isolated from patients with adenocarcinoma) for their in vitro sensitivity to gefitinib. Of these, only H3255 (EGFRL858R) and H1666 (EGFRWT) are sensitive to gefitinib with IC50 values of 40 nmol/L and 2 µmol/L, respectively. We examined the effects of gefitinib on H3255 and cell lines containing wild-type EGFR that are either sensitive (H1666) or resistant (A549 and H441) to gefitinib exposure in vitro. Gefitinib treatment (1 µmol/L) leads to significant apoptosis accompanied by increased poly(ADP-ribose) polymerase cleavage only in the H3255 cell line, leads to G1-S arrest in H1666, and has no effects in the A549 and H441 cell lines. Although EGFR and AKT are constitutively phosphorylated in H3255, H1666, and H441 cell lines, AKT is completely inhibited by gefitinib treatment only in the H3255 cell line. These findings further characterize a mechanism by which gefitinib treatment of NSCLC harboring EGFRL858R leads to a dramatic response to gefitinib. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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We and others have recently uncovered somatic mutations in the tyrosine kinase domain of EGFR in patients who achieve significant clinical responses to gefitinib (5
, 6)
. These mutations include both small deletions and missense mutations. One of these, L858R (LeuArg), occurs in a highly conserved amino acid among protein kinases and is found both in patients who develop significant clinical regressions to gefitinib and in adenocarcinoma cell line H3255, which is hypersensitive to gefitinib in vitro. The mechanism through which gefitinib treatment of EGFRL858R leads to tumor regression is presently unknown.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Drugs.
Gefitinib was a gift of AstraZeneca. Stock solutions were prepared in dimethyl sulfoxide and stored at –20°C. The drugs were diluted in fresh media before each experiment, and the final dimethyl sulfoxide concentration was <0.1%. Epidermal growth factor (EGF) was purchased from Biosource International Inc. (Camarillo, CA).
Growth Inhibition Assay.
Growth inhibition was assessed by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt) assay (Promega, Madison, WI). These studies were performed according to methods established previously in our laboratory (5)
. The number of cells per well used in these experiments for the cell lines were as follows: H1781, 15,000; H1666, 1,500; H441, 2500; H3255, 7500; H23, 3000; H2347, 7500; H3122, 5000; H358, 7500; and A549, 1500.
Antibodies and Western Blotting.
Cells were lysed in buffer containing proteinase inhibitors, and proteins were separated by gel electrophoresis on 5% to 12% polyacrylamide gels selected depending on the molecular weight of the target, transferred to nitrocellulose membranes and detected by immunoblotting using an enhanced chemiluminescence system (5)
. EGFR (SC-03) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The phospho-specific EGFR (pY1068), extracellular signal-regulated kinase (ERK) 1/2, and phospho-ERK (pT185/pY187) antibodies were purchased from Biosource International Inc. The phospho-specific Akt (pS473) and total Akt and poly(ADP-ribose) polymerase (PARP) antibodies were obtained from Cell Signaling Technology (Beverley, MA). 
-Tubulin antibody was purchased from Sigma-Aldrich (St. Louis, MO).
Cell Cycle Analysis.
Cells were plated at a density of 0.5 to 2 x 105 cells per plate in 10-cm2 plates (Becton Dickinson, Franklin Lakes, NJ). Gefitinib was added to the medium after 24 hours, and the cells were incubated for another 72 hours, after which the cells were analyzed as described previously (8)
. The percentage of apoptosis was estimated from the sub-G1 cell fraction.
Epidermal Growth Factor Receptor and K-ras Sequencing.
Total RNA was isolated from all NSCLC cell lines using Trizol (Invitrogen, Carlsbad, CA) and purified using RNeasy mini-elute cleanup kit (Qiagen, Valencia, CA). Complementary DNA was transcribed from 2 µg of total RNA with Superscript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA). The cDNA was used as template for subsequent polymerase chain reaction (PCR) amplifications of EGFR. The details of the PCR conditions and the primers have been published previously (5)
.
K-ras was sequenced from the A549, H1666, and H3255 cell lines using cDNA and genomic DNA. The sequencing was designed to cover codons 12, 13, and 61. The PCR conditions and primer sequences are available on request. The PCR products were sequenced according to the manufacturer’s recommendations and as described previously (5) . H441 is known to contain a codon 12 K-ras mutation.
Transforming Growth Factor Detection Assay.
Transforming growth factor (TGF)- determination in the media of NSCLC cells was performed similarly to a previously described method for detecting hepatocyte growth factor (9)
. For the determination of TGF-, enzyme-linked immunosorbent assays were performed according to the manufacturer’s recommended procedures (human TGF- immunoassay; Quantikine; R&D Systems, Minneapolis, MN). All samples were run in triplicate.
Quantitative Polymerase Chain Reaction.
Quantitative genomic PCR was used to determine EGFR copy number. DNA was prepared from each of the nine cell lines and from the peripheral blood of five normal volunteers using Qiagen genomic tips. All quantitative PCR reactions were carried out in an ABI 7700 thermal cycler using Taqman Universal PCR Master Mix (Applied Biosystems, Foster City, CA). The PCR conditions are available on request. The amount of template calculated using the EGFR probe was divided by the average amount of template calculated from seven reference genes distributed throughout the genome (CD20, 11q13; FANCA, 16q24; GART, 21q22; MAPK4, 18q12-q21; TSN, 2q21; VEST1, 8q13; and FLT3, 13q12) to generate the relative EGFR copy number. The average relative EGFR copy number for the five normal specimens was 2.54 (range, 2.12–2.92). For the nine tumor samples, the average relative EGFR copy number was 5.14 (range, 1.68–22.98). The PCR primers for EGFR were derived from exon 16 and are available for all genes on request.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. We sequenced EGFR in all of these cell lines; four of which have been published previously (Table 1
; ref. 5
). Only H3255 contained a mutation in EGFR (L858R). The strategy of enriching the group of cell lines under study to mirror the clinical population most responsive to gefitinib identified two sensitive cell lines. Both of the sensitive cell lines are also derived from women who are nonsmokers. This frequency is similar to the radiographic response rate observed after gefitinib treatment in this clinically enriched patient population (3)
. We chose to further examine four cell lines: A549, H441 (both EGFRWT and gefitinib resistant), H1666 (EGFRWT; gefitinib sensitive), and H3255 (EGFRL858R; gefitinib sensitive). A549 was chosen because its response to gefitinib has been extensively characterized (10)
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with anti-EGFR antibodies demonstrated a stronger signal from H3255 than from the other cell lines and suggested that EGFR may be amplified in this cell line. To determine EGFR copy number, we performed quantitative PCR for EGFR and seven reference genes distributed throughout the genome. The relative EGFR copy number for each cell line is shown in Table 1
. EGFR is amplified approximately 11-fold in H3255, whereas there is no significant copy number change in the remaining cell lines. On analysis of the sequencing peaks from this cell line at bp 2573 (site of the L858R mutation; CTGCGG), there was very little wild-type allele (T) compared with the mutant allele (G; data not shown; ref. 5
). This suggests that the L858R allele is preferentially amplified in H3255. Gene amplification of EGFR has been noted using fluorescence in situ hybridization in 9% of NSCLC but does not appear to correlate with any particular histologic subtype (11)
. It is presently not known whether EGFR amplification correlates with tumor regression in patients with NSCLC receiving gefitinib, although EGFR expression as detected by immunohistochemistry does not correlate with antitumor response to gefitinib (4)
. EGFR amplifications have not been previously correlated with EGFR mutations in NSCLC tumor specimens. In breast cancer in which Her2/neu (erbB2) is frequently amplified, the antitumor response to trastuzumab, a Her2/neu-specific antibody, is limited to patients whose tumors have 3+ amplification by fluorescence in situ hybridization (12
, 13)
. The epidermoid carcinoma cell line A431, which is very sensitive to gefitinib in vitro, is known to contain an EGFR amplification but is not known to contain a mutation in EGFR (14)
. Thus it is possible that EGFR amplification in addition to mutation in EGFR in H3255 contributes to its sensitivity to gefitinib. In a recent study, Lynch et al. (6)
found that gefitinib treatment caused greater growth inhibition in Cos-7 cells containing EGFRL858R expression constructs than in those expressing EGFRWT, suggesting that the L858R mutation contributes at least in part to the sensitivity to gefitinib.
Gefitinib Induces Apoptosis in H3255 Cell Lines.
To examine the mechanism of growth inhibition of gefitinib in H1666 and H3255 cell lines, we performed cell cycle analyses. All cell lines were treated for 72 hours in the presence and absence of 1 µmol/L gefitinib. We chose 1 µmol/L because this is a concentration of gefitinib that can be achieved in serum in patients being treated with gefitinib and also one used by other investigators, allowing us to compare our findings (10
, 15)
. As can be seen, only in the H3255 cell line does gefitinib induce a substantial fraction of apoptosis (Fig. 2A)
. The mean percentage of apoptosis increases from 2.14 ± 0.91% (untreated) to 24.73 ± 1.85% (1 µmol/L gefitinib). These differences are statistically significant (P = 0.034; paired t test). A similar degree of apoptosis was also observed in the H3255 cell line even with 100 nmol/L gefitinib treatment (data not shown). The H1666 cell line undergoes cell cycle arrest at the G1-S boundary after gefitinib treatment [absolute change (mean ± SD) in G0-G1 and S fractions, 14.2 ± 3.55% and –9.81 ± 2.43%, respectively], consistent with multiple prior observations using EGFR inhibitors across tumor types including lung cancer (10
, 16
, 17)
. There was no significant effect on cell cycle progression in A549 or H441 cells treated with gefitinib (data not shown), consistent with previously published studies (10)
. No apoptosis was observed in H1666, A549, or H441, even with 10 µmol/L gefitinib treatment (data not shown). We also performed Western blotting for PARP cleavage, an indicator of caspase-mediated apoptosis, after gefitinib treatment. The cleaved 89-kDa fragment increases substantially only in the H3255 cell line after gefitinib treatment (Fig. 2B)
. These findings suggest a critical role for the mutant EGFR receptor for cell survival in H3255 cells. Furthermore, they are consistent with the observations that only a minority of patients with NSCLC, those with EGFR mutations, treated with gefitinib have substantial antitumor responses. H1666, although it contains EGFRWT, undergoes cell cycle arrest after gefitinib treatment. This observation may be an in vitro equivalent of stable disease also observed in a substantial fraction of patients who are treated with EGFR inhibitors (2)
. Additional studies including sequencing of EGFR from tumors of such patients will need to be performed to validate this hypothesis.
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. In contrast, there is no EGFR phosphorylation in A549 cells (EGFRWT), except with the addition of EGF. Gefitinib treatment for 2 hours substantially decreases the constitutive EGFR phosphorylation in H3255 cells and completely prevents EGF-mediated EGFR phosphorylation in A549 cells \?\(Fig. 3
.). In H1666 and H441, there is also constitutive EGFR phosphorylation, which can be further increased with EGF stimulation but is only minimally effected by gefitinib treatment. These studies were all performed with 1 µmol/L gefitinib, which is below the IC50 for H1666 and H441; with higher (10 µmol/L) gefitinib concentrations, EGFR phosphorylation is also inhibited in these cells, as we have shown previously (5)
. To determine whether EGFR may be constitutively phosphorylated in H1666 through an autocrine activation mechanism, we examined the media for the presence of TGF-. In both H1666 and H441, we detect a small amount of TGF- (mean, 19.8 and 29.3 pg/mL, respectively), but none was detected in H3255 or A549. This suggests that the constitutive activation of EGFR in H1666 and H441 may be due to an autocrine mechanism.
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AKT but not ERK 1/2 Is Constitutively Active in H3255 Cells.
There is constitutive EGFR activation in H3255 cells. We wished to determine whether pathways downstream of EGFR were also constitutively active. AKT, but not ERK 1/2, is also constitutively phosphorylated in H3255 but not in A549 (Fig. 3)
. With EGF stimulation, phosphorylation of AKT increases substantially in A549 but does not increase and even decreases slightly in H3255. We have observed the latter finding multiple times, which may be due to activation of a negative feedback mechanism, and this is presently being explored. These findings suggest that the AKT pathway is preferentially activated by EGFRL858R. In contrast to AKT, EGF stimulation increases ERK 1/2 phosphorylation in both H3255 and A549 cells. In both H1666 and H441, AKT is also constitutively phosphorylated but is minimally effected by either gefitinib or EGF and thus may be activated by other growth factor-dependent or -independent pathways. In H1666, there is constitutive phosphorylation of ERK 1/2 that can be further phosphorylated with the addition of EGF, findings that are similar in H3255, A549, and H441. Unlike AKT, phosphorylation of ERK 1/2 is completely inhibited by gefitinib treatment in H1666 cells, consistent with the effect of the drug on proliferation and cell cycle arrest in this cell line. There is no effect on ERK 1/2 phosphorylation by gefitinib in H441. To determine whether the constitutive ERK 1/2 phosphorylation is due to K-ras activation, we sequenced K-ras from H3255, H1666, and A549. No mutations in K-ras were found in either H3255 or H1666, but a codon 12 mutation was present in A549, consistent with prior observations (20)
. H441 is known to contain a K-ras codon 12 mutation.
Our findings have several potential clinical implications. If the preferential activation of the AKT pathway takes place consistently, then this suggests that targeted agents developed to inhibit this pathway may be therapeutically more effective than those designed to inhibit the ERK 1/2 pathway in patients whose tumors contain EGFRL858R. Constitutive AKT activation has also been associated with resistance to chemotherapy and radiation in NSCLC cell lines (21) . We have also demonstrated previously that the H3255 cell line is resistant to paclitaxel and docetaxel in vitro (7) . It is thus possible that chemotherapy will be less effective in patients whose tumors contain EGFRL858R than in those with a wild-type EGFR. Sequencing of EGFR from tumor specimens obtained from patients participating in large randomized trials of chemotherapy with gefitinib or placebo would be useful to help answer this hypothesis (22) . Furthermore, combinations of chemotherapy with gefitinib or inhibitors of the AKT pathway should be tested in vitro in cell lines and in vivo in patients with EGFRL858R mutations to determine whether these may be additive or synergistic.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: S. Tracy and T. Mukohara contributed equally to this work.
Requests for reprints: Bruce E. Johnson, Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, D1234; 44 Binney Street, Boston, MA 02115. Phone: 617-632-4790; Fax: 617-632-5786; E-mail: bejohnson{at}partners.org
Received 6/ 9/04. Revised 8/11/04. Accepted 8/23/04.
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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S. Van Schaeybroeck, J. Kyula, D. M. Kelly, A. Karaiskou-McCaul, S. A. Stokesberry, E. Van Cutsem, D. B. Longley, and P. G. Johnston Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells Mol. Cancer Ther., May 1, 2006; 5(5): 1154 - 1165. [Abstract] [Full Text] [PDF]
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S.-W. Han, T.-Y. Kim, Y. K. Jeon, P. G. Hwang, S.-A. Im, K.-H. Lee, J. H. Kim, D.-W. Kim, D. S. Heo, N. K. Kim, et al. Optimization of Patient Selection for Gefitinib in Non-Small Cell Lung Cancer by Combined Analysis of Epidermal Growth Factor Receptor Mutation, K-ras Mutation, and Akt Phosphorylation Clin. Cancer Res., April 15, 2006; 12(8): 2538 - 2544. [Abstract] [Full Text] [PDF]
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J. V. Alvarez, H. Greulich, W. R. Sellers, M. Meyerson, and D. A. Frank Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung cancer-associated mutations of the epidermal growth factor receptor. Cancer Res., March 15, 2006; 66(6): 3162 - 3168. [Abstract] [Full Text] [PDF]
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 S. M Dougherty, W. Mazhawidza, A. R Bohn, K. A Robinson, K. A Mattingly, K. A Blankenship, M. O Huff, W. G McGregor, and C. M Klinge Gender difference in the activity but not expression of estrogen receptors {alpha} and {beta} in human lung adenocarcinoma cells. Endocr. Relat. Cancer, March 1, 2006; 13(1): 113 - 134. [Abstract] [Full Text] [PDF]
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G. A. Smolen, R. Sordella, B. Muir, G. Mohapatra, A. Barmettler, H. Archibald, W. J. Kim, R. A. Okimoto, D. W. Bell, D. C. Sgroi, et al. Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752 PNAS, February 14, 2006; 103(7): 2316 - 2321. [Abstract] [Full Text] [PDF]
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E. Conde, B. Angulo, M. Tang, M. Morente, J. Torres-Lanzas, A. Lopez-Encuentra, F. Lopez-Rios, and M. Sanchez-Cespedes Molecular Context of the EGFR Mutations: Evidence for the Activation of mTOR/S6K Signaling Clin. Cancer Res., February 1, 2006; 12(3): 710 - 717. [Abstract] [Full Text] [PDF]
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P. A. Janne, A. M. Borras, Y. Kuang, A. M. Rogers, V. A. Joshi, H. Liyanage, N. Lindeman, J. C. Lee, B. Halmos, E. A. Maher, et al. A Rapid and Sensitive Enzymatic Method for Epidermal Growth Factor Receptor Mutation Screening Clin. Cancer Res., February 1, 2006; 12(3): 751 - 758. [Abstract] [Full Text] [PDF]
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R. L. Yauch, T. Januario, D. A. Eberhard, G. Cavet, W. Zhu, L. Fu, T. Q. Pham, R. Soriano, J. Stinson, S. Seshagiri, et al. Epithelial versus Mesenchymal Phenotype Determines In vitro Sensitivity and Predicts Clinical Activity of Erlotinib in Lung Cancer Patients Clin. Cancer Res., December 15, 2005; 11(24): 8686 - 8698. [Abstract] [Full Text] [PDF]
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E. B. Haura, Z. Zheng, L. Song, A. Cantor, and G. Bepler Activated Epidermal Growth Factor Receptor-Stat-3 Signaling Promotes Tumor Survival In vivo in Non-Small Cell Lung Cancer Clin. Cancer Res., December 1, 2005; 11(23): 8288 - 8294. [Abstract] [Full Text] [PDF]
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T. Mukohara, G. Civiello, I. J. Davis, M. L. Taffaro, J. Christensen, D. E. Fisher, B. E. Johnson, and P. A. Janne Inhibition of the Met Receptor in Mesothelioma Clin. Cancer Res., November 15, 2005; 11(22): 8122 - 8130. [Abstract] [Full Text] [PDF]
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D. W. Bell, T. J. Lynch, S. M. Haserlat, P. L. Harris, R. A. Okimoto, B. W. Brannigan, D. C. Sgroi, B. Muir, M. J. Riemenschneider, R. B. Iacona, et al. Epidermal Growth Factor Receptor Mutations and Gene Amplification in Non-Small-Cell Lung Cancer: Molecular Analysis of the IDEAL/INTACT Gefitinib Trials J. Clin. Oncol., November 1, 2005; 23(31): 8081 - 8092. [Abstract] [Full Text] [PDF]
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J. Jiang, H. Greulich, P. A. Janne, W. R. Sellers, M. Meyerson, and J. D. Griffin Epidermal Growth Factor-Independent Transformation of Ba/F3 Cells with Cancer-Derived Epidermal Growth Factor Receptor Mutants Induces Gefitinib-Sensitive Cell Cycle Progression Cancer Res., October 1, 2005; 65(19): 8968 - 8974. [Abstract] [Full Text] [PDF]
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B. E. Johnson and P. A. Janne Selecting Patients for Epidermal Growth Factor Receptor Inhibitor Treatment: A FISH Story or a Tale of Mutations? J. Clin. Oncol., October 1, 2005; 23(28): 6813 - 6816. [Full Text] [PDF]
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B. E. Johnson and P. A. Janne Epidermal Growth Factor Receptor Mutations in Patients with Non-Small Cell Lung Cancer Cancer Res., September 1, 2005; 65(17): 7525 - 7529. [Abstract] [Full Text] [PDF]
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D. R. Gandara and P. H. Gumerlock Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors Plus Chemotherapy: Case Closed or Is the Jury Still Out? J. Clin. Oncol., September 1, 2005; 23(25): 5856 - 5858. [Full Text] [PDF]
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 J. D. Minna, M. J. Peyton, and A. F. Gazdar Gefitinib Versus Cetuximab in Lung Cancer: Round One J Natl Cancer Inst, August 17, 2005; 97(16): 1168 - 1169. [Full Text] [PDF]
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T. Mukohara, J. A. Engelman, N. H. Hanna, B. Y. Yeap, S. Kobayashi, N. Lindeman, B. Halmos, J. Pearlberg, Z. Tsuchihashi, L. C. Cantley, et al. Differential Effects of Gefitinib and Cetuximab on Non-small-cell Lung Cancers Bearing Epidermal Growth Factor Receptor Mutations J Natl Cancer Inst, August 17, 2005; 97(16): 1185 - 1194. [Abstract] [Full Text] [PDF]
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M. Taron, Y. Ichinose, R. Rosell, T. Mok, B. Massuti, L. Zamora, J. L. Mate, C. Manegold, M. Ono, C. Queralt, et al. Activating Mutations in the Tyrosine Kinase Domain of the Epidermal Growth Factor Receptor Are Associated with Improved Survival in Gefitinib-Treated Chemorefractory Lung Adenocarcinomas Clin. Cancer Res., August 15, 2005; 11(16): 5878 - 5885. [Abstract] [Full Text] [PDF]
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T. Shimamura, A. M. Lowell, J. A. Engelman, and G. I. Shapiro Epidermal Growth Factor Receptors Harboring Kinase Domain Mutations Associate with the Heat Shock Protein 90 Chaperone and Are Destabilized following Exposure to Geldanamycins Cancer Res., July 15, 2005; 65(14): 6401 - 6408. [Abstract] [Full Text] [PDF]
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E. L. Kwak, R. Sordella, D. W. Bell, N. Godin-Heymann, R. A. Okimoto, B. W. Brannigan, P. L. Harris, D. R. Driscoll, P. Fidias, T. J. Lynch, et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib PNAS, May 24, 2005; 102(21): 7665 - 7670. [Abstract] [Full Text] [PDF]
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P. A. Janne, J. A. Engelman, and B. E. Johnson Epidermal Growth Factor Receptor Mutations in Non-Small-Cell Lung Cancer: Implications for Treatment and Tumor Biology J. Clin. Oncol., May 10, 2005; 23(14): 3227 - 3234. [Abstract] [Full Text] [PDF]
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F. J. Kaye A Curious Link Between Epidermal Growth Factor Receptor Amplification and Survival: Effect of "Allele Dilution" on Gefitinib Sensitivity? J Natl Cancer Inst, May 4, 2005; 97(9): 621 - 623. [Full Text] [PDF]
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F. Cappuzzo, F. R. Hirsch, E. Rossi, S. Bartolini, G. L. Ceresoli, L. Bemis, J. Haney, S. Witta, K. Danenberg, I. Domenichini, et al. Epidermal Growth Factor Receptor Gene and Protein and Gefitinib Sensitivity in Non-Small-Cell Lung Cancer J Natl Cancer Inst, May 4, 2005; 97(9): 643 - 655. [Abstract] [Full Text] [PDF]
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G. J. Kelloff, J. M. Hoffman, B. Johnson, H. I. Scher, B. A. Siegel, E. Y. Cheng, B. D. Cheson, J. O'Shaughnessy, K. Z. Guyton, D. A. Mankoff, et al. Progress and Promise of FDG-PET Imaging for Cancer Patient Management and Oncologic Drug Development Clin. Cancer Res., April 15, 2005; 11(8): 2785 - 2808. [Abstract] [Full Text] [PDF]
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W. Pao and V. A. Miller Epidermal Growth Factor Receptor Mutations, Small-Molecule Kinase Inhibitors, and Non-Small-Cell Lung Cancer: Current Knowledge and Future Directions J. Clin. Oncol., April 10, 2005; 23(11): 2556 - 2568. [Abstract] [Full Text] [PDF]
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J. A. Engelman, P. A. Janne, C. Mermel, J. Pearlberg, T. Mukohara, C. Fleet, K. Cichowski, B. E. Johnson, and L. C. Cantley ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines PNAS, March 8, 2005; 102(10): 3788 - 3793. [Abstract] [Full Text] [PDF]
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H. Shigematsu, T. Takahashi, M. Nomura, K. Majmudar, M. Suzuki, H. Lee, I. I. Wistuba, K. M. Fong, S. Toyooka, N. Shimizu, et al. Somatic Mutations of the HER2 Kinase Domain in Lung Adenocarcinomas Cancer Res., March 1, 2005; 65(5): 1642 - 1646. [Abstract] [Full Text] [PDF]
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 H. VARMUS, W. PAO, K. POLITI, K. PODSYPANINA, and Y.-C.N. DU Oncogenes Come of Age Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 1 - 9. [Abstract] [PDF]
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R.K. THOMAS, H. GREULICH, Y. YUZA, J.C. LEE, T. TENGS, W. FENG, T.-H. CHEN, E. NICKERSON, J. SIMONS, M. EGHOLM, et al. Detection of Oncogenic Mutations in the EGFR Gene in Lung Adenocarcinoma with Differential Sensitivity to EGFR Tyrosine Kinase Inhibitors Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 73 - 81. [Abstract] [PDF]
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