Elizabeth B. Gottlin

Panel of Serum Biomarkers for the Diagnosis of Lung Cancer

PURPOSE Currently, a blood test for lung cancer does not exist. Serum biomarkers that could aid clinicians in making case management decisions would be enormously valuable. We used two proteomic platforms and a literature search to select candidate serum markers for the diagnosis of lung cancer. METHODS We initially assayed six serum proteins, four discovered by proteomics and two previously known to be cancer associated, on a training set of sera from 100 patients (50 with a new diagnosis of lung cancer and 50 age- and sex-matched controls). Classification and Regression Tree (CART) analysis selected a panel of four markers that most efficiently predicted which patients had lung cancer. An independent, blinded validation set of sera from 97 patients (49 lung cancer patients and 48 matched controls) determined the accuracy of the four markers to predict which patients had lung cancer. RESULTS Four serum proteins-carcinoembryonic antigen, retinol binding protein, alpha1-antitrypsin, and squamous cell carcinoma antigen-were collectively found to correctly classify the majority of lung cancer and control patients in the training set (sensitivity, 89.3%; specificity, 84.7%). These markers also accurately classified patients in the independent validation set (sensitivity, 77.8%; specificity, 75.4%). Remarkably, 90% of patients who fell into any one of three groupings in the CART analysis had lung cancer. CONCLUSION This panel of four serum proteins is valuable in suggesting the diagnosis of lung cancer. These data may be useful for treating patients with an indeterminate pulmonary lesion, and potentially in predicting individuals at high risk for lung cancer.

Kinetic Analysis of the Catalytic Domain of Human Cdc25B

The Cdc25 cell cycle regulator is a member of the dual-specificity class of protein-tyrosine phosphatases that hydrolyze phosphotyrosine- and phosphothreonine-containing substrates. To study the mechanism of Cdc25B, we have overexpressed and purified the catalytic domain of human Cdc25B (Xu, X., and Burke, S. P. (1996) J. Biol. Chem. 271, 5118-5124). In the present work, we have analyzed the kinetic properties of the Cdc25B catalytic domain using the artificial substrate 3-O-methylfluorescein phosphate (OMFP). Steady-state kinetic analysis indicated that the kcat/Km for OMFP hydrolysis is almost 3 orders of magnitude greater than that for p-nitrophenyl phosphate hydrolysis. Like other dual-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechanism, characterized by formation and breakdown of a phosphoenzyme intermediate. Pre-steady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is ∼20 times faster than subsequent phosphoenzyme breakdown. The resulting burst pattern of product formation allowed us to derive rate constants for enzyme phosphorylation (26 s−1) and dephosphorylation (1.5 s−1) as well as the dissociation constant for OMFP (0.3 mM). Calculations suggest that OMFP binds with higher affinity and reacts faster with Cdc25B than does p-nitrophenyl phosphate. OMFP is a highly efficient substrate for the dual-specificity protein-tyrosine phosphatases VHR and rVH6, but not for two protein-tyrosine phosphatases, PTP1 and YOP. The ability to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic residues in Cdc25 and other dual-specificity phosphatases.

Haptoglobin and Posttranslational Glycan-Modified Derivatives as Serum Biomarkers for the Diagnosis of Nonsmall Cell Lung Cancer

The purpose was to evaluate the clinical utility of serum haptoglobin (Hp) and posttranslational glycan modifications of Hp for the diagnosis of nonsmall cell lung cancer (NSCLC).

Complement Factor H Autoantibodies are Associated with Early Stage NSCLC

Purpose: To discover diagnostic biomarkers associated with early-stage non–small cell lung cancer (NSCLC), we searched for autoantibodies preferentially present in stage I patients compared with patients with advanced-stage disease. Here we describe an autoantibody against complement factor H (CFH) and this autoantibodys association with early-stage NSCLC. Experimental Design: Immunoblots were used to detect autoantibodies in the sera of stage I NSCLC patients. An autoantibody recognizing a 150 kDa protein was discovered, and the protein was identified by mass spectrometry. The association of the autoantibody with early-stage disease was suggested by the results of immunoblot analysis with sera from 28 stage I patients and 28 stage III/IV patients. This association was confirmed by protein microarray of sera from 125 NSCLC patients of all stages as well as 125 controls matched by age, gender, and smoking history. Results: The immunoreactive protein was identified as CFH. By immunoblot analysis, anti-CFH autoantibody was found in 50% of stage I NSCLC patients and 11% of late-stage NSCLC patients (P = 0.003). By protein microarray analysis, patients with stage I NSCLC had a significantly higher incidence of anti-CFH antibody than those with late-stage NSCLC (P = 0.0051). The percentage of sera with a positive level of CFH autoantibody was 30.4% in stage I, 21.1% in stage II, 12.5% in stage III, 7.4% in stage IV, and 8.0% in the control group. Conclusions: These findings suggest that in patients with NSCLC, CFH autoantibody is a molecular marker associated with early-stage disease. Clin Cancer Res; 16(12); 3226–31. ©2010 AACR.

Intracellular Expression of Peptide Fusions for Demonstration of Protein Essentiality in Bacteria

ABSTRACT We describe a “protein knockout” technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.

High-Throughput Screen for Inhibitors of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase by Surrogate Ligand Competition

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function. (Journal of Biomolecular Screening 2003:332-339)

Isolation of novel EGFR-specific VHH domains.

Epidermal growth factor receptor (EGFR) is overexpressed or mutated in a high percentage of tumors. EGFR has long been considered a promising target for cancer diagnostic and therapeutic applications. However, monoclonal antibodies and other large antibody constructs diffuse into tumors slowly, limiting their efficacy. To develop lower molecular weight probes for EGFR and other tumor cell receptors, the authors immunized a llama with the extracellular domains (ECDs) of EGFR and an oncogenic mutant receptor, EGFRvIII, and with extracts of tumor cell lines. From the immune repertoire of the llama, the authors constructed a heavy chain variable domain (VHH domain)—phage library. At ~16 kDa, the VHH domain is a tenth of the size of a monoclonal antibody and is the smallest antibody fragment that retains specificity. By affinity selection from this library, the authors isolated many VHH domains with specificity for EGFR. The VHH domains bind to whole cells expressing the receptor but not to control cells lacking the receptor and can immunoprecipitate EGFR from cell lysates. Some VHH domains have cross-specificity with existing anti-EGFR monoclonal antibodies and have reasonably high (nM) affinities. The llama-VHH domain library is also potentially a rich source of targeting agents directed toward other tumor cell receptors. (Journal of Biomolecular Screening 2009:77-85)

Biomarkers to help guide management of patients with pulmonary nodules.

RATIONALE Indeterminate pulmonary nodules are a common radiographic finding and require further evaluation because of the concern for lung cancer. OBJECTIVES We developed an algorithm to assign patients to a low- or high-risk category for lung cancer, based on a combination of serum biomarker levels and nodule size. METHODS For the serum biomarker assay, we determined levels of carcinoembryonic antigen, α1-antitrypsin, and squamous cell carcinoma antigen. Serum data and nodule size from a training set of 509 patients with (n = 298) and without (n = 211) lung cancer were subjected to classification and regression tree and logistic regression analyses. Multiple models were developed and tested in an independent, masked validation set for their ability to categorize patients with (n = 203) or without (n = 196) lung cancer as being low- or high-risk for lung cancer. MEASUREMENTS AND MAIN RESULTS In all models, a large percentage of individuals in the validation study with small nodules (<1 cm) were assigned to the low-risk group, and a large percentage of individuals with large nodules (≥3 cm) were assigned to the high-risk group. In the validation study, the classification and regression tree algorithm had overall sensitivity, specificity, and positive and negative predictive values for determining lung cancer of 88%, 82%, 84%, and 87%, respectively. The logistic regression model had overall sensitivity, specificity, and positive and negative predictive values of 80%, 89%, 89%, and 81%, respectively. CONCLUSION Integration of biomarkers with lung nodule size has the potential to help guide the management of patients with indeterminate pulmonary nodules.

Identification of potential prognostic biomarkers in patients with untreated, advanced pancreatic cancer from a phase 3 trial (Cancer and Leukemia Group B 80303).

Patients with advanced stage adenocarcinoma of the pancreas have a poor prognosis. The identification of prognostic and/or predictive biomarkers may help stratify patients so that therapy can be individualized.

The Association of Intratumoral Germinal Centers with Early-Stage Non-small Cell Lung Cancer

Introduction: Lung cancers can display immune cell infiltration although the role of an adaptive immune response in disease pathogenesis is unknown. To investigate the possibility of a functional humoral response to the tumor, we surveyed histologic sections from non-small cell lung cancer (NSCLC) tumors for germinal centers (GCs) and assessed whether there was an association between the presence of GCs and tumor stage. Methods: Tumor sections from 91 patients with all stages of NSCLC were examined by a pathologist blinded to clinical data. GCs were identified by hematoxylin and eosin staining patterns and confirmed by immunohistochemical staining for B-cell markers, BCL-6 and CD21. The distribution of GCs within the tumor or the tumor margin was recorded. Statistical analysis was performed to evaluate the association between stage and presence of GCs. Results: Thirty-five percent of all tumors evaluated contained GCs, and sections evaluated by immunohistochemistry showed positive staining for both B-cell markers. GCs were seen both within the tumor and the tumor margin, consistent with an immune response to antigen stimulation. Patients with stage I NSCLC had a higher prevalence of intratumoral GCs than patients with stages II to IV (Cochran-Armitage Trend Test p = 0.02011). There was no association of stage with GCs in the tumor margin. Conclusions: Intratumoral GCs are associated with early-stage NSCLC. Further characterization of intratumoral GCs may lead to new diagnostic and therapeutic strategies based on manipulation of the adaptive immune response.