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Epidermal Growth Factor Receptor (EGFR) High Gene Copy Number and Activating Mutations in Lung Adenocarcinomas Are Not Consistently Accompanied by Positivity for EGFR Protein by Standard Immunohistochemistry

The purpose of this study was to investigate whether detectable protein biomarker overexpression is a prerequisite for the presence of increased gene copy number or activating mutations and responsiveness to the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib in patients with lung adenocarcinomas. EGFR status was prospectively analyzed in tumor biopsy samples by three methods: protein expression (n = 117) by standardized immunohistochemistry (IHC), gene copy number (n = 97) by fluorescent in situ hybridization (FISH), and mutation analysis by sequencing (n = 126). Fifty-nine percent of the samples were positive by IHC, 40% were positive by FISH, and 13.5% contained activating kinase domain mutations. Thirty-four percent of the FISH-positive and 27% of the mutant samples were also IHC-negative. All EGFR mutant patients had major clinical responses (five complete response and five partial response) to gefitinib or erlotinib treatment, although three of these tumors were IHC-negative and four were FISH-negative. In a retrospective analysis of samples from nine patients with excellent therapeutic responses (three complete response, five partial response, one stable disease) to erlotinib or gefitinib, mutations were identified in eight cases, but IHC was negative in four of these tumors. These results indicate that molecular diagnostic methods appear to be most important for the identification of lung adenocarcinoma patients who may benefit from EGFR inhibitor treatments.

Major partial response to crizotinib, a dual MET/ALK inhibitor, in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement

The initial radiotherapy of a 73 years old Caucasian male patient with advanced squamous cell lung carcinoma was terminated due to severe pericarditis. Subsequently, the tumor sample was analyzed for possible targets with comprehensive molecular diagnostics. EGFR, KRAS and PIK3CA genes were wild type, ALK and ROS1 were negative for rearrangement, but c-MET was amplified by fluorescent in situ hybridization. The kinase inhibitor crizotinib is already in clinical use for the treatment of ALK positive non-small cell lung cancers, but it is also known to be a potent c-MET inhibitor. The patient was treated with the standard dose of twice a day 250 mg crizotinib as a monotherapy. Major partial response to therapy was confirmed by chest CT and PET/CT after 8 weeks on therapy. C-MET expression is associated with poor prognosis and resistance to EGFR inhibitors. This case may indicate that c-MET tyrosine kinase inhibitors can be an effective targeted treatment option for squamous cell carcinoma patients, and future clinical trials should be expanded for this patient group as well.

Comparison of Elisa-Based Tyrosine Kinase Assays for Screening EGFR Inhibitors

Abstract Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu, Tyr)1:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.

Biochemical Assay-Based Selectivity Profiling of Clinically Relevant Kinase Inhibitors on Mutant Forms of EGF Receptor

Gefitinib and erlotinib are potent EGFR tyrosine kinase inhibitors (potentially) useful for the treatment of non-small-cell lung cancer (NSCLC). Clinical responses, however, in NSCLC patients have been linked to the presence of certain activating mutations of EGFR. We used an ELISA-based biochemical assay to confirm the selective inhibitory efficacy of gefitinib and erlotinib on the activated mutant receptor. Our results are in line with the clinical observations providing evidence for the predictive power of the kinase assay. Four additional compounds were also investigated: CI-1033 and EKB-569 had dramatic inhibitory effects on all EGFR forms, whereas PD153035 and AG1478 were active on wild-type and activating mutant protein. In docking simulations with wild-type EGFR, our inhibitory data are in good agreement with the binding scores. These data confirm that anilinoquinazolines are good starting structures for the next generation of selective drugs against mutant EGFR, whereas CI-1033 and EKB-569 may represent advances for patients with both wild-type and anilinoquinazoline-resistant mutant tumors.

Synthesis and Characterization of Novel Quinazoline Type Inhibitors for Mutant and Wild-Type EGFR and RICK Kinases

The development of selective protein kinase inhibitors has become an important area of drug discovery for the treatment of different diseases. We report the synthesis and characterization of a series of novel quinazoline derivatives against three therapeutically important and pharmacologically related kinases: 1) epidermal growth factor receptor (EGFR; wild type and mutant) in the field of cancer, 2) receptor-interacting caspase-like apoptosis-regulatory kinase (RICK) in the field of inflammation, and 3) pUL97 of human cytomegalovirus (HCMV). For reference purpose we have synthesized the four clinically relevant quinazolines, including the lead compounds, which we previously identified for RICK and pUL97. A total of 52 quinazoline derivatives were synthesized and tested on the basis of these leads to specifically target the hydrophobic pocket of the ATP-binding site. Selected compounds were tested on wild-type and mutant forms of EGFR, RICK, and pUL97 kinases; their logP and logS values for assessing suitability as drugs were calculated and hit or lead compounds identified.

Molecular subtype specific efficacy of MEK inhibitors in pancreatic cancers

Pancreatic cancer is an increasing cause of cancer related death worldwide. KRAS is the dominant oncogene in this cancer type and molecular rationale would indicate, that inhibitors of the downstream target MEK could be appropriate targeted agents, but clinical trials have failed so far to achieve statistically significant benefit in unselected patients. We aimed to identify predictive molecular biomarkers that can help to define subgroups where MEK inhibitors might be beneficial alone or in combination. Next-generation sequencing data of 50 genes in three pancreatic cancer cell lines (MiaPaCa2, BxPC3 and Panc1) were analyzed and compared to the molecular profile of 138 clinical pancreatic cancer samples to identify the molecular subtypes of pancreatic cancer these cell lines represent. Luminescent cell viability assay was used to determine the sensitivity of cell lines to kinase inhibitors. Western blot was used to analyze the pathway activity of the examined cell lines. According to our cell viability and pathway activity data on these model cell lines only cells harboring the rare G12C KRAS mutation and low EGFR expression are sensitive to single MEK inhibitor (trametinib) treatment. The common G12D KRAS mutation leads to elevated baseline Akt activity, thus treatment with single MEK inhibitors fails. However, combination of MEK and Akt inhibitors are synergistic in this case. In case of wild-type KRAS and high EGFR expression MEK inhibitor induced Akt phosphorylation leads to trametinib resistance which necessitates for MEK and EGFR or Akt inhibitor combination treatment. In all we provide strong preclinical rational and possible molecular mechanism to revisit MEK inhibitor therapy in pancreatic cancer in both monotherapy and combination, based on molecular profile analysis of pancreatic cancer samples and cell lines. According to our most remarkable finding, a small subgroup of patients with G12C KRAS mutation may still benefit from MEK inhibitor monotherapy.