Bradley L. Smith

Global Survey of Phosphotyrosine Signaling Identifies Oncogenic Kinases in Lung Cancer

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.

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

Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non–small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. Experimental Design: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. Results: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry–based DNA sequencing. Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.

Tissue microarray-based analysis shows phospho-β-catenin expression in malignant melanoma is associated with poor outcome

Beyond depth of invasion, there are very few prognostic markers to predict outcome in melanoma. It has been shown recently that the β‐catenin oncogene is mutated or shows altered subcellular localization suggesting that activation of β‐catenin mediated signaling plays a role in oncogenesis. We hypothesize that assessment of activated β‐catenin, as detected by a phospho‐specific antibody, may be useful to predict outcome in melanoma. We use immuno‐histochemical analysis of β‐catenin and phospho‐β‐catenin, first to verify the specificity of the phospho‐β‐catenin antibody and then to assay expression in a tissue microarray‐based study. The subcellular localization of β‐catenin is membranous in some cases and cytoplasmic and nuclear in others. We validate the specificity of a ser33/37/thr41 phospho‐β‐catenin antibody in transfected cells and show that the expression is almost exclusively localized to the nucleus in both cultured cells and human tissue. Evaluation of both total and phospho‐β‐catenin antibodies showed that cytoplasmic/nuclear staining was more common in primary lesions, whereas nuclear phospho‐β‐catenin was more common in metastatic lesions. High levels of nuclear phospho‐β‐catenin are associated with significantly worse overall survival (51% vs. 25% overall survival at 5 years, p = 0.046). These results suggest that phospho‐specific antibodies to β‐catenin define a unique subset of cases and that monitoring of phospho‐β‐catenin expression may be useful for assessing prognosis in malignant melanoma.

Antibody-Based Profiling of the Phosphoinositide 3-Kinase Pathway in Clinical Prostate Cancer

Purpose: As kinase inhibitors transition from the laboratory to patients, it is imperative to develop biomarkers that can be used in the clinic. The primary objectives are to identify patients most likely to benefit from molecularly targeted therapies and to document modulation of the drug target. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway and its downstream effectors, as a result of PTEN loss or by other mechanisms, occurs in a high proportion of prostate cancers, making it an ideal template for the design of clinical trials involving PI3K pathway inhibitors. Prostate cancers also present unique organ-specific challenges, in that tumors are heterogeneous and diagnostic tissue is extremely limited. Experimental Design: Working within these limitations, we have developed a set of immunohistochemical assays that define activation of the PI3K pathway in clinical samples. Results and Conclusions: Using both univariate and multivariate analyses, we show that loss of PTEN is highly correlated with the activation of AKT, and this, in turn, is associated with the phosphorylation of S6, one of its main effectors. These three antibodies are potentially able to define a molecular signature of PTEN loss and/or AKT pathway activation in prostate cancer.

Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell‐based measure of Bcr/Abl kinase activity

Stat5 1 (Signal Transducer and Activator of Transcription 5) is normally phosphorylated and activated by Janus kinases. In cells transformed with BCR/ABL, Stat5 is constitutively activated by promiscuous phosphorylation. Cytometry of intracellular antigens can be used to evaluate cell treatments affecting gene expression, because it precisely provides the fraction of affected cells and the quantitative change in expression. Here, we asked whether we could measure a phosphorylated epitope on Stat5 by cytometry, and whether that measurement would respond to Bcr/Abl inhibition.