Supplementary Data from Mutation-Specific Antibodies for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer

Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.

B2 Background: NSCLC patients carrying the somatic mutation of epidermal growth factor receptor (EGFR) has been shown to be hyperresponsive to the EGFR tyrosine kinase inhibitor Gefitinib and Erlotinib. The most common NSCLC associated EGFR mutations are the in-frame deletion in exon 19 (E746_A750del) and the point mutation in exon 21 (L858R), accounting for 85-90% EGFR mutations. The ability to detect mutated gene products in cancer cells can identify patients most likely benefit from such therapies. Methods: We generated rabbit monoclonal antibodies (RmAb) against EGFR with E746-A750 deletions and L858R point mutation. We tested the antibodies by Western blot, Immunofluorescence (IF) and Immunohistochemistry (IHC). In addition, we tested these antibodies on 40 molecularly pre-typed tumors by IHC. Finally, we used a panel of four antibodies (two mutant antibodies, a total EGFR antibody, and a pan-keratin antibody) to screen 340 cases of tumor samples with unknown genotype by IHC. Results: The results from Western blot, IF, and IHC confirmed that these antibodies could specifically detect the mutant EGFR proteins. IHC data from 40 molecular pre-typed samples perfectly matched the result from DNA sequencing. In those 340 cases of tumor samples, 28 cases were stained positive by L858R antibody and 24 cases were stained positive by EGFR deletion antibody. The positive rate by both antibodies is 15.3%. The DNA from all IHC positive samples and negative adenocarcinoma samples were sent for DNA sequencing. The sensitivity of these mutant specific antibodies is 92.5% and the specificity is 95.8%. In addition, some tumor samples positive for EGFR mutation by IHC and Mass Spec based DNA sequencing were negative by direct DNA sequencing due to low percentage of cancer cells. Conclusions: The IHC combined mutant EGFR specific antibodies and total EGFR antibody can be used to detect the EGFR mutations and measure the expression level of total EGFR protein in tumors. In addition, this assay enables us to examine paraffin blocks from small biopsy samples, which are difficult to extract enough high quality DNA for sequencing. This IHC assay has the potential to be developed for use to screen lung cancer patients for the treatment with EGFR kinase inhibitors in a clinical setting.

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.

Educators in effective schools know that a trusting school environment contributes to the success of teachers and students. According to analysis of the 2006 North Carolina Teacher Working Conditions Survey data, establishing an atmosphere of trust and mutual respect was strongly correlated with overall student performance at the elementary, middle and high school levels. Trust in the school environment was strongly correlated with teachers’ employment decisions as well. Consider the following:

Despite the success of tyrosine kinase-based cancer therapeutics, for most solid tumors the tyrosine kinases that drive disease remain unknown, limiting our ability to identify drug targets and predict response. Here we present the first large-scale survey of tyrosine kinase activity in lung cancer. Using a phosphoproteomic approach, we characterize tyrosine kinase signaling across 41 non-small cell lung cancer (NSCLC) cell lines and over 150 NSCLC tumors. Profiles of phosphotyrosine signaling are generated and analyzed to identify known oncogenic kinases such as EGFR and c-Met as well as novel ALK and ROS fusion proteins. Other activated tyrosine kinases such as PDGFRalpha and DDR1 not previously implicated in the genesis of NSCLC are also identified. By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels and can be applied broadly across all human cancers.

We previously reported the isolation of a 2.5 Mb tumor-suppressing subchromosomal transferable fragment (STF) from Ilpl5.5 and the iden tification of nine known genes and four novel genes within this STF. We now report the isolation of a fifth novel cDNA, tumor-suppressing STF cDNA 5, designated TSSC5, located within the STF. TSSC5 encodes a predicted protein of 424 amino acids. Sequence analysis suggests that TSSC5 is a membrane protein with 10 transmembrane segments, and it is located between two imprinted genes, p57KII>2and TSSC3. Northern blot hybridization revealed a 1.6-kb transcript in multiple adult tissues and in fetal liver and kidney, consistent with a potential role in embryonal tumors. We also found that TSSC5 is imprinted with preferential expres sion from the maternal chromosome. Reverse transcription-PCR analysis of TSSCS revealed frequent occurrence of aberrant RNA splicing, which deleted exons 4, 5. and 6 in Wilms' tumors. Mutational analysis of TSSCS by direct DNA sequencing of exons revealed a base substitution of G1120A in a Wilms' tumor, matched normal kidney, and the patient's mother, changed Arg at codon 309 to Gin. The G1120A substitution thus repre sents either a rare polymorphism or a tumor-predisposing mutation, because the mutant alÃ-elewas of maternal origin and preferentially ex pressed in the patient's tissue. A second base substitution, C892T, was found in a lung cancer, changing Ser at codon 233 to Phe. This substitu tion was absent from the matched normal tissue and thus represented a somatic mutation. We also found loss of heterozygosity in the lung cancer, suggesting that TSSCS may be a conventional tumor suppressor gene in the adult human lung and an imprinted tumor suppressor gene in the fetal kidney.

The 8p11 myeloproliferative syndrome (EMS) is associated with translocations that disrupt the FGFR1 gene. To date, 8 fusion partners of FGFR1 have been identified. However, no primary leukemia cell lines were identified that contain any of these fusions. Here, we screened more than 40 acute myeloid leukemia cell lines for constitutive phosphorylation of STAT5 and applied an immunoaffinity profiling strategy to identify tyrosine-phosphorylated proteins in the KG-1 cell line. Mass spectrometry analysis of KG-1 cells revealed aberrant tyrosine phosphorylation of FGFR1. Subsequent analysis led to the identification of a fusion of the FGFR1OP2 gene to the FGFR1 gene. Small interfering RNA (siRNA) against FGFR1 specifically inhibited the growth and induced apoptosis of KG-1 cells. Thus, the KG-1 cell line provides an in vitro model for the study of FGFR1 fusions associated with leukemia and for the analysis of small molecule inhibitors against FGFR1 fusions.