Paul D. Wagner

α-Fetoprotein, Des-γ Carboxyprothrombin, and Lectin-Bound α-Fetoprotein in Early Hepatocellular Carcinoma

BACKGROUND & AIMS Alpha-fetoprotein (AFP) is widely used as a surveillance test for hepatocellular carcinoma (HCC) among patients with cirrhosis. Des-gamma carboxy-prothrombin (DCP) and lectin-bound AFP (AFP-L3%) are potential surveillance tests for HCC. The aims of this study were to determine performance of DCP and AFP-L3% for the diagnosis of early HCC; whether they complement AFP; and what factors affect DCP, AFP-L3%, or AFP levels. METHODS We conducted a large phase 2 biomarker case-control study in 7 academic medical centers in the United States. Controls were patients with compensated cirrhosis and cases were patients with HCC. AFP, DCP, and AFP-L3% levels were measured blinded to clinical data in a central reference laboratory. RESULTS A total of 836 patients were enrolled: 417 (50%) were cirrhosis controls and 419 (50%) were HCC cases, of which 208 (49.6%) had early stage HCC (n = 77 very early, n = 131 early). AFP had the best area under the receiver operating characteristic curve (0.80, 95% confidence interval [CI]: 0.77-0.84), followed by DCP (0.72, 95% CI: 0.68-0.77) and AFP-L3% (0.66, 95% CI: 0.62-0.70) for early stage HCC. The optimal AFP cutoff value was 10.9 ng/mL leading to a sensitivity of 66%. When only those with very early HCC were evaluated, the area under the receiver operating characteristic curve for AFP was 0.78 (95% CI: 0.72-0.85) leading to a sensitivity of 65% at the same cutoff. CONCLUSIONS AFP was more sensitive than DCP and AFP-L3% for the diagnosis of early and very early stage HCC at a new cutoff of 10.9 ng/mL.

Phosphorylation of ATP-Citrate Lyase by Nucleoside Diphosphate Kinase

Rat liver nucleoside diphosphate kinase (NDPK) and PC12 cell cytosol were used to determine whether NDPK could function as a protein kinase. NDPK was phosphorylated on its catalytic histidine using ATP, and the phosphorylated NDPK separated from [γ-32P]ATP. The addition of phosphorylated NDPK to dialyzed PC12 cell cytosol resulted in the phosphorylation of a protein with a subunit molecular mass of about 120 kDa. This phosphorylation appeared to occur by a direct transfer of a phosphoryl group from the catalytic histidine of NDPK to a histidine on the 120-kDa protein. The 120-kDa protein was partially purified and shown by peptide sequencing to be ATP-citrate lyase. ATP-citrate lyase is the primary source of cytosolic acetyl-CoA. NDPK phosphorylated the histidine at the catalytic site of ATP-citrate lyase. This histidine can also be phosphorylated by ATP, and its phosphorylation is the first step in the conversion of citrate and CoA to oxaloacetate and acetyl-CoA by ATP-citrate lyase. The level of phosphorylation of PC12 cell ATP-citrate lyase by phosphorylated NDPK was comparable with that by ATP. Thus, in addition to its nucleoside diphosphate kinase activity, NDPK can function as a protein kinase.

Anti-HER2 IgY antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells.

BackgroundNanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells.MethodsThe complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining.ResultsUsing a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously.ConclusionWe have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy.

Challenges for Biomarkers in Cancer Detection

Abstract: Cancer remains the leading cause of death in the United States. Biomarkers can be used to detect cancer in different stages, initiation, development, and progression. The desirable property and utility of a biomarker lie in its ability to provide an early indication of disease progression. Biomarkers should be easy to detect, measurable across populations, and useful for detection of cancer at an early stage, identification of high‐risk individuals, detection of recurrence, or monitoring endpoints in intervention studies. Recent technological advances have helped develop noninvasive, sensitive, and specific biomarkers to detect cancer at early stages of the disease.

Interaction of Phosphorylated Tryptophan Hydroxylase with 14-3-3 Proteins

Rabbit brain tryptophan hydroxylase (TPH) has been expressed in insect cells (Spodoptera frugiperda) as a histidine-tagged enzyme. The specific activity of the purified fusion enzyme is 80 nmol of 5-hydroxytryptophan/min/mg. Multifunctional regulatory 14-3-3 proteins were purified from fresh bovine brain. Phosphorylation and 14-3-3 proteins play important roles in the regulation of TPH activity. We have found that phosphorylation of TPH by cAMP-dependent protein kinase increased the activity of the hydroxylase by 25–30% and that 14-3-3 proteins increased the hydroxylase activity of phosphorylated TPH by ∼45%. Under these conditions, the 14-3-3 proteins were not phosphorylated, and unphosphorylated TPH was not activated by 14-3-3 proteins. Surface plasmon resonance analysis demonstrated that 14-3-3 proteins bind to phosphorylated TPH with an affinity constant (K a ) of 4.5 × 107 m −1. Binding studies using affinity chromatography also showed that 14-3-3 proteins interact with phosphorylated TPH. The dephosphorylation of TPH by protein phosphatase-1 was inhibited by 14-3-3 proteins. Our results demonstrate that 14-3-3 proteins form a complex with phosphorylated brain TPH, thereby increasing its enzymatic activity and inhibiting its dephosphorylation.

Performance of mitochondrial DNA mutations detecting early stage cancer

BackgroundMutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites.MethodsWe determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region.ResultsUsing this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors.ConclusionOur findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is unclear the biological relevance of these detected mitochondrial mutations. Whether the detection of tumor-specific mtDNA mutations in body fluidsy this method will be useful for diagnosis and monitoring applications requires further investigation.

Biomarkers in molecular medicine: cancer detection and diagnosis.

In spite of advances in diagnostics and therapeutics, cancer remains the second leading cause of death in the U.S. Successful cancer treatment depends not only on better therapies but also on improved methods to assess an individuals risk of developing cancer and to detect cancers at early stages when they can be more effectively treated. Current cancer diagnostic imaging methods are labor-intensive and expensive, especially for screening large asymptomatic populations. Effective screening strategies depend on methods that are noninvasive and detect cancers in their early stages of development. There is increasing interest and enthusiasm in molecular markers as tools for cancer detection and prognosis. It is hoped that newly discovered cancer biomarkers and advances in high-throughput technologies would revolutionize cancer therapies by improving cancer risk assessment, early detection, diagnosis, prognosis, and monitoring therapeutic response. These biomarkers will be used either as stand-alone tests or to complement existing imaging methods.

Quantitation of HER2 and telomerase biomarkers in solid tumors with IgY antibodies and nanocrystal detection.

In an effort to improve affinity biomarker validation in fixed patient tissue specimens, we have developed a novel quantum dot‐based bioimaging system that utilizes chicken IgY antibody for high sensitivity and specificity relative quantitation of cancer proteins. Monospecific, polyclonal IgYs were generated against human HER2 and telomerase, and analytically validated for specificity by western blot and immunohistochemistry on tumor and normal cells and for relative affinity by layered peptide array (LPA). IgYs bound desired targets in cell lines and fixed tissues and showed greater affinity than commercial mammalian antibodies for both HER2 and telomerase proteins. In tissue microarray experiments, HER2 quantitation with IgY antibody and quantum dot imaging correlated well with chromogenic in situ hybridization (CISH), whereas telomerase quantitation suggested a trend toward correlation with prostate cancer Gleason Grade and differentiation. Although patient numbers were small, these findings demonstrate the feasibility of relative quantitation of cancer biomarkers with IgY and quantum dot fluorophores, and show promise for rigorous clinical validation in large patient cohorts.

Mitochondrial Genome Deletion Aids in the Identification of False- and True-Negative Prostate Needle Core Biopsy Specimens

We report the usefulness of a 3.4-kb mitochondrial genome deletion (3.4 mtdelta) for molecular definition of benign, malignant, and proximal to malignant (PTM) prostate needle biopsy specimens. The 3.4 mtdelta was identified through long-extension polymerase chain reaction (PCR) analysis of frozen prostate cancer samples. A quantitative PCR assay was developed to measure the levels of the 3.4 mtdelta in clinical samples. For normalization, amplifications of a nuclear target and total mitochondrial DNA were included. Cycle threshold data from these targets were used to calculate a score for each biopsy sample. In a pilot study of 38 benign, 29 malignant, and 41 PTM biopsy specimens, the difference between benign and malignant core biopsy specimens was well differentiated (P & .0001), with PTM indistinguishable from malignant samples (P = .833). Results of a larger study were identical. In comparison with histopathologic examination for benign and malignant samples, the sensitivity and specificity were 80% and 71%, respectively, and the area under a receiver operating characteristic (ROC) curve was 0.83 for the deletion. In a blinded external validation study, the sensitivity and specificity were 83% and 79%, and the area under the ROC curve was 0.87. The 3.4 mtdelta may be useful in defining malignant, benign, and PTM prostate tissues.

14-3-3 proteins bind to histone and affect both histone phosphorylation and dephosphorylation

14‐3‐3 proteins appear to play a critical role in Ca2+‐stimulated secretion in permeabilized chromaffin cells. 14‐3‐3 proteins have been reported to be both stimulators and inhibitors of protein kinase C (PKC). We have found that 14‐3‐3 proteins, isolated on the basis of their ability to enhance secretory activity, stimulated histone phosphorylation by PKC, but they had no effect on myosin light chain phosphorylation by PKC. 14‐3‐3 proteins were also found to inhibit the rate of [32P]histone dephosphorylation but not the rate of [32P]myosin light chain dephosphorylation. Cross‐linking experiments and affinity chromatography demonstrated that 14‐3‐3 proteins bind to histones. These results suggest that at least some of the reported effects of 14‐3‐3 proteins on PKC activity may result from 14‐3‐3 proteins binding to histone.