Leaders in Pharmaceutical Business Intelligence (LPBI) Group

New methods for Study of Cellular Replication, Growth, and Regulation

Writer and Curator: Larry H Bernstein, MD, FCAP

Introduction:

This article is the first in a series on genomics, epigenomics and cancer.  It is necessary here to introduce the large advancement in the technological advances that have followed the Human Genome Project and its thrust into the domain of genome expression.  While the genome is the code that is passed on from generation to generation in a family chain, and while there now is an ability to trace genes that have existed and are traceable by evolutionary history to early eukaryotic species, that portion of the cell line is defining and only modified in time.  It is only a beginning in the unraveling of the question – What is life?

This article has the following structure:

1.1.1       Gene Amplification

1.1.2       Protein-binding Receptors

1.1.3 Advanced Proteomic Technologies for Cancer Biomarker Discoveries

1.1.3.1 State of the art technologies

1.1.3.1.1 2D difference gel electrophoresis (2DIGE)

1.1.3.1.2 MALDI imaging technology (see also 1.1.5)

1.1.3.1.3 Electron Transfer Dissociation

1.1.3.1.4 Reverse-phase Protein Array (RPA)

1.1.3.2 Principles of Protein Microarrays

1.1.3.3 Disposable reagentless electrochemical immunosensor array based on a polymer/sol/gel membrane for simultaneous measurement of several tumor markers

1.1.4 p16INK4a Expression Correlates with Degree of Cervical Neoplasia: A Comparison with Ki-67 Expression and Detection of High-Risk HPV Types

1.1.5 Quantitative real-time detection of magnetic nanoparticles by their nonlinear magnetization

1.1.6 Proteomics and biomarkers

1.1.6.1 Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine

1.1.6.2 Multiplexed proteomic analysis of oxidation and concentrations of CSF proteins in Alzheimer’s disease

1.1.6.3 The Brain Injury Biomarker VLP-1 Is Increased in the Cerebrospinal Fluid of Alzheimer Disease Patients

1.1.6.4 Determination of non-α1-antichymotrypsin-complexed PSA as an indirect measurement of free PSA: analytical performance and diagnostic accuracy

1.1.6.5 Ultrasensitive densitometry detection of cytokines with nanoparticle-modified aptamers

1.1.6.6 Protein profiling of microdissected pancreas carcinoma and identification of HSP27 as a potential serum marker

1.1.7 Mass Spectrometry Methods

1.1.7.1 LC-MS/MS quantification of Zn-α2 glycoprotein: A potential serum biomarker for prostate cancer

1.1.7.2 A novel, high-throughput workflow for discovery and identification of serum carrier protein-bound peptide biomarker candidates in ovarian cancer samples

1.1.7.3 Mass Spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications

1.1.7.4 Current state and future directions of neurochemical biomarkers for Alzheimer’s disease

1.1.7.5 Use of SELDI-TOF mass spectrometry for identification of new biomarkers: potential and limitations

1.1.1 Gene Amplification

An increase in the number of copies of a gene. There may also be an increase in the RNA and protein made from that gene. Gene amplification is common in cancer cells, and some amplified genes may cause cancer cells to grow or become resistant to anticancer drugs. Genes may also be amplified in the laboratory for research purposes.

http://www.cancer.gov/dictionary?cdrid=650175

Actinin-4 gene Amplification in Ovarian Cancer: A Candidate Oncogene Associated with Poor Patient Prognosis and Tumor Chemoresistance

Yamamoto S, Tsuda H, Kazufumi H, Onozato K, …, Matsubar O

Medscape 6/18/2009

http://medscape.com/viewarticle/704105

Actinin-4, an isoform of non-muscular α-actinin, enhances cell motility by bundling the actin cytoskeleton. We previously reported a prognostic implication of high histochemical expression of actinin-4 protein in ovarian cancers.  Chromosomal gain or amplification of the 19q 12-q13 region has been reported in ovarian cancer. We hypothesized that the actinin-4 (ACTN4) gene might be a target of the 19q 12-q13 amplicon and play an essential role in ovarian cancer progression. In total, we investigated 136 advanced-stage ovarian cancers the copy number of the ACTN4 gene on chromosome 19q3, and used fluorescence in situ hybridization to determine the correlation of the ACTN4 copy number with actinin-4 protein immunoreactivity and major clinicopathological factors. We detected a higher copy number ( > 4) of  of the ACTN4 gene in 29 (21%) cases and it was associated with the intensity of the actinin-4 immunoreactivity (p < 0.0001), a high histological tumor grade (p < 0.030), a clear-cell adenocarcinoma histology (p = 0.012), resistancxe to first- line chemotherapies (p = 0.028), and poor patient outcome (p=0.0011). Uni-
variate analyses using the Cox regression model showed that a higher ACTN4 gene copy numberwas predicted patient outcome more accurately than high actinin-4 immunoreactivity (relative risk: 2.48 vs 1.55). Multivariate analysis indicated that a higher copy number of the ACTN4 gene may be a targetof the 19q amplicon, acting as a candidate oncogene, and serve as a predictor of poor outcome and tumor chemoresistance in patients with advanced-stage ovarian cancers (from Modern Pathology).

1.1.2       Protein-binding Receptors

Customizing the Targeting of IGF-1 Receptor

Renato Baserga
Medscape 5/6/2009. From Future Oncology
http://medscape.com/viewarticle/589011

The type 1 IGF receptor (IGF—IR) is activated by two ligands, IGF-1 and IGF-2, and by insulin at supraphysiological concentrations. It plays a significant role in the growth of normal and abnormal cells, and antibodies against the IGF-IR are now in clinical trials. Targeting of the IGF-IR in cancer cells (by antibodies or other means) can be improved by the appropriate selection of responsive tumors.

The prominence of IGF-IR has increased considerably in the past few years, progressing from a redundant insulin receptor to one that is important in cell and body growth, cell survival and malignant transformation. The IGF-IR can send either a mitogenic or a differentiation signal depending on substrate availability. In many cell types (fibroblasts, epithelial cells, etc.) the IGF-IR sends an unambiguous mitogenic, antiapoptotic signal. In other cell types, such as myeloid cells, neuronal cells and others, activation of IGF-IR induces differentiation. When cells do not express or express very low levels of IRS-1, a substrate of both the IGF_IR and the insulin receptor (InR), IGF-1 induces differentiation. This is the case with 32D myeloid precursor cells that do’t express IRS-1 and are induced to differentiate by IGF-1. Ectopic expression of IRS-1 in 32D cells abrogates differentiation; the cells become transformed and even form tumors in mice. IRS-1 activates the P13K pathway, which is the main mitogenic pathway originating from the IGF-IR. However, the shc-Ras-ERKs pathway also plays a role in the mitogenic signal of the IGF-IR, with the two converging at GSK3-β. The demonstration that knockout mouse embryo cells for the IGF-IR receptor were refractory to transformation by viruses, oncogenes and overexpressed growth factor receptors clearly demonstrated the major role played by the IGF-IR in cellular transformation.

Methods for Targeting the IGF-1 Receptor

Summary of IRS-1

The ability of IRS-1 to cause cell transformation, and the tendency to lose the transformed phenotype in cells in which IRS-1 is low or has been downregulated, suggests that the importance of the IGF-IR in cancer may be dependent on IRS-1 as much as on the receptor itself. When IRS-1 is activated directly, for instance by v-src, the IGF-IR is no longer a requiremeny for malignant transformation.  Metastases are very susceptible to IGF-IR therapy.

IGF-IR Targeting Summary

• In the absence of IRS-1, the IGF1R sends a differentiation signal, which becomes mitogenic with IRS1 expression, and targeting IGF1R in cells that do not express IRS-1 may be counterproductive.
• In colon cancer liver metastases the cancer cells of awash in IGF-1.
• In Ewing’s sarcoma, a tumor sensitive to IGF1r targeting in clinical trials, there is an autocrine mechanism that may make the cancer cells IGF-1 dependent, but an oncogene/IRS-1 interaction may also make these cells incapable of switching to other growth factors.
• IGF-1R sends a potent anti-apoptotic signal, independent of its mitogenicity. This property could be exploited to increase chemo- or radio- toxicity.
• IGF-1R expression is required for anchorage independence.

1.1.3 Advanced Proteomic Technologies for Cancer Biomarker Discoveries

1.1.3.1 State of the art technologies

Wong SCC, Chan CML, Ma BBY,…,Chan ATC.

Medscape 6/10/2009. From Expert Review of Proteomics.
http://medscape.com/viewarticle/703566

Proteomic technologies have experienced major improvements in recent years. Such advances have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis, prognosis and treatment monitoring of cancer patients. The topic of discussion is four state of the art technologies: 2D difference gel electrophoresis, MALDI imaging mass spectrometry, electron transfer dissociation mass spectrometry and reverse-phase protein array.  These have contributed to large advances in proteomic technologies from 1997-2008.

1.1.3.1.1 2D difference gel electrophoresis (2DIGE)

The 2D DIGE method is an improved 2GE technique. Two different protein samples (e.g., control and disease) and, optionally, one reference sample (e.g., control and disease together) are labeled with one of three different fluorophore: cyanine (cy)2, 3 or 5. These fluorophores have the same charge, similar molecular weight and distinct fluorescent properties, allowing their discrimination during scanning using appropriate optical filters.Two types of cyanine dyes are available: CyDye^TM  DIGE Fluor minimal dyse and CyDye DIGE Fluor saturation dye (GE Healthcare, Uppsala, Sweden).The minimal dye labelks a small percentage of lysine residues with minimal change in the electrophoretic mobility pattern of the protein, whereas the saturation dye labels all available cysteine residues and is, therefore, more sensitive, but causes EP mobility shift of labelled proteins.  Different types of protein sample may be used.Labeled sample pairs are mixed and  run in a single gel.The same pooled reference sample is used for all gels within an experiment.The gel is scanned at three wavelengths for Cy2 (488 nm), Cy3 (532 nm) and Cy5 (633 nm), and a gel image for each of the samples is obtained.Variation between the gels is minimized. Correct matching of protein spots is improved.Normalization and quantitation of the spots is most accurate.The linear dynamic range is four orders of magnitude and it is fully compatible for quantitation with MS. The technique is mainly used for the discovery of novel biomarkers.

1.1.3.1.2 MALDI imaging technology (see also 1.1.5)

MALDI Imaging Mass Spectrometry gives a deeper understanding of biochemical processes in the tumor cells and tissues. Immunohistochemistry is limitated, but the MALDI technique is high-throughput.MALDI IMS was developed to allow researchers to analyze proteomic expression profiles directly from patient tissue sections.The tissue is first mounted, then MALDI matrix is applied onto the tissue sample and MALDI MS is applied to obtain mass spectra from predefined locations across the tissue section.All acquired spectra are then compiled into a composite 2D map for the tissue sample.The expression profiles of numerous proteins can be obtained without the need for antibodies. It is also possible to correlate the mapping with tissue histology.

Post-translational modifications have a role in structure and function of proteins: protein folding, protein localization, regulation of activity and mediation of protein-protein interaction. Two common forms of PTM have been implicated in cancer neoplasia: phosphorylation and glycosylation.  Phosphoproteomic studies led to identification of novel tyrosine kinase substrates in breast cancer, and to discovery of novel therapeutic targets for brain cancer, and to increased understanding of signaling pathways in lung cancer.  The identification of novel therapeutic targets for ovarian cancer resulted from identification of abnormally glycosylated proteins – mucins.

1.1.3.1.3 Electron Transfer Dissociation

Electron Transfer Dissociation is a recently developed technique for the analysis of peptides by MS, utilizing radiofrequency quadrupole ion traps such as 2D linear IT, spherical IT and Orbitrap^TM (Thermo Fisher,MA).Peptides are fragmented by transfer of electrons from anions to induce cleavage of CαN bonds along the peptide backbone, producing c- and z-type ions. In contrast to CID, ETD preserves the localization of labile PTM and provides peptide-sequence information, but it fails to fragment peptide bonds adjacent to proline.CID and ETD should be used to complement each other. An advantage of the TED is that in the analysis of phosphopeptides a near complete series of c- and z-ions is observed without the loss of phosphoric acid. The method has provided for proteomic researchers a tool for comprehensive analysis of peptides and their PTMs.

1.1.3.1.4 Reverse-phase Protein Array (RPA)

Then there is the Reverse-phase Protein array, which has the advantage that it identifies changes associated with the development of cancer. The identification of such proteins can be used as biomarkers for diagnosis, prognosis, treatment decisions and therapeutic monitoring. Still, patient samples pose a challenge:

• Proteomic patterns differ among cell types;
• Protein expression changes dynamically over time;
• Proteins have a broad dynamic range of expression levels spanning several orders of magnitude;
• Proteins can be present in multiple forms, such as polymorphysms and splice variants;
• Traditional proteomic methods, such as, @DE, require larger amounts of protein than those obtained from biopsy samples;
• Many existing proteomic technologies cannot ber used to study protein-protein interactions.

The method of RPA is simple and requires the spotting of patient samples in an array format onto a nitrocellulose support.Each array is incubated with a particular antibody, and signal intensity is proportional to the amount of analyte. Signal detection is by fluorescence, chemiluminescence or colorimetric methods.  The results are qwuantified by scanning and analyzed by softwares such as P-SCAN and ProteinScan.

Main advantages of RPA are:

• Various types of biological samples;
• Investigation of PTMs;
• Protein-protein interactions;
• Labeling of samples with fluorescent dyes or mass tags;
• Quantitation within the linear range of detection;
• Direct measurement of target proteins by spotting reference standards.

Key Issues

• 2DE couple with MS has been a mainstay for discovery of novel biomarkewrs;
• 2D DIGE has improved quantification accuracy;
• MALDI imaging MS allows detedtion and comparison with histopathology;
• ETD-MS has opened up the possibility of identifying the structure and localization of PTM and the peptide/protein.
• RPA is a powerful tool for high-throughput validation across hundreds of samples.

1.1.3.2  Principles of Protein Microarrays

Preface, Foreward and Chapter 1: In Protein Microarrays, Ed. Mark Schena
Mark Schena, Joseph L. Hackett and Emanuel F. Petricoin
Jones and Bartlett Publ. 2002, ON, CA

What is true inside the cell cannot always be recapitulated outside the cell.  The year is 1986 and the second year of graduate school of UCSF. With cloned receptor in hand (just isolated by Roger Miesfeld), I set out to test whether glucocorticoid receptor function could be recapitulated in yeast cells. This might allow us to test evolutionary nconservation in eukaryotes.  Remarkably, the rat receptor sprsang to life on the first attempt, producing a diagnostic blue colot change in yeast cells expressing a β-galactosidase fusion and a broad smile on the face of a young scientist. Receptor experiments in yeast necessarily required grinding up yeast cells, fractionating the proteins by denaturing polyacrylamide gel electrophoresis, transferring the proteins onto nitrocellulose, probing the immobilized proteins with a monoclonal antibody, and examining the filter to confirm the presence of the expressed rat protein.

Protein-ntibody interactions on protein chips are determined by complex associations between epitopes on the target protein and the antigen-binding site on the detection molecule. Individual protein-ligand pairs can possess widely different affinities.  Proteomic microarrays require capture and detection molecules with high affinities and low dissociation rates . For these and other reasons protein chips are more challenging than DNA chips. Antibodies, aptamers, recombinant proteins, peptides, phage, evem small molecular weight chemicals/drugs can be used as a bait molecule and/or detection reagent. The molecule may be an antibody or the cellular lysate itself, which are immobilized onto the substratum and act as a bait molecule.  Each spot contains one type of immobilized antibody or bait protein. The first problem is the vast range of concentrations to be detected (up to a factor of 10¹⁰ .  Adequate sensitivity must be achieved (at least femtomolar range), and the amplification chemistry must be tolerant to the large dynamic range of the analytes.

Microarrays are analytical devices that possess four distinct characteristics:

1. Microscopic target elements or spot;
2. Planar substrates;
3. Rows and columns of elements; and
4. Specific binding between microarray target elements on the substrate and probe molecules in solution.

The scope of microarray research includes:

1. Gene expression
2. Signal transduction
3. Genome mismatch scanning
4. Inflammation
5. Cancer
6. Cell cycle
7. DNA replication
8. Oxidative stress
9. Hormone action
10. Apoptosis
11. Neurodegenerative disease
12. Infectious disease
13. Cytoskeleton, and
14. Protein trafficking.

The proliferation of microarrays beyond the realm of DNA and gene expression was inevitable, and the idea of making and using microarrays of proteins, lipids, carbohydrates, and small molecules was an obvious extension of the original DNA microarray format. This exciting technology area provides the foundation for the book, Protein Microarrays.  Proteins, not mRNAs are the true functional components of cells. They mediate gene regulation, enzyme catalysis, cellular metabolism, DNA replication, and cell division and confer cell shape and mobility and the capacity to communicate within and between cells. a hypothetical 400 amino acid protein would have a molecular weight of 54 kDa.
Many cellular proteins fall in the molecular weight range of 10-125 kDa, and nearly every human protein weighs < 500 kDa.

The 20 amino acids are chemically diverse and correspondingly confer to the proteins their structural and functional diversity and impart their catalytic specificity and binding properties. The amino acids are bound in the protein by the amino acid side chains of the polypeptide. The nh-CO peptide unit has a partial double-bond character due to the amide bond, and its conformations are restricted by that structure. In addition, proteins have secondary, tertiary and quaternary structure. Hydrophobic amino acids are in the interior, and hydrophilic amino acid residues are on the exterior. The hydrophilic exterior allows for water solubility.

The following are microarray assay formats used in expression profiling:

1. Protein expression
2. Serum-based diagnostics
3. Protein-protein binding
4. Drug-target binding
5. Receptor-epitope binding.

1.1.3.3  Disposable reagentless electrochemical immunosensor array based on a polymer/sol/gel membrane for simultaneous measurement of several tumor markers

Wu J, Yan F, Zhang X, Yan Y, Tang J, Ju H.
Clin Chem 2008; 54(9):1481-1489.
http://dx.doi.org:/10.1373/clinchem.2007.102350

Background: A reagentless sensor array for simultaneous multianalyte testing (SMAT) may enable accurate diagnosis and be applicable for point-of-care testing. We developed a disposable reagentless immunosensor array for simple immunoassay of panels of tumor markers. Methods: We carried out SMAT with a direct capture format, in which colloidal gold nanoparticles with bound horseradish peroxidase (HRP)-labeled antibodies were immobilized on screen-printed carbon electrodes with biopolymer/sol-gel to trap their corresponding antigens from sample solution. Upon formation of immunocomplex, the direct electrochemical signal of the HRP decreased owing to increasing spatial blocking, and the analytes could be simultaneously determined by monitoring the signal changes.
Results: The proposed reagentless immunosensor array allowed simultaneous detection of carcinoma antigen 153, carcinoma antigen 125, carbohydrate antigen 199, and carcinoembryonic antigen in clinical serum samples in the ranges of 0.4–140 kU/L, 0.5–330 kU/L, 0.8–190 kU/L, and 0.1–44 μg/L, respectively, with detection limits of 0.2 kU/L, 0.5 kU/L, 0.3 kU/L, and 0.1 μg/L corresponding to the signals 3 SD above the mean of a zero standard. The interassay imprecision of the arrays was <9.5%, and they were stable for 35 days. The positivity detection rate of panels of tumor markers was >95.5% for 95 cases of cancer-positive sera. Conclusions: The immunosensor array provides a SMAT with short analytical time, small sampling volume, no need for substrate, and, no between-electrode cross-talk. This method not only proved the capability of the array in point-of-care testing, but also allowed simultaneous testing of several tumor markers.

Cancer is one of the leading causes of mortality, and early clinical diagnosis is crucial for successful treatment of the disease. Many immunosensors and immunoassay methods have been developed for the determination of a single tumor marker, whose concentration in human serum is associated with the stages of tumors (1)(2)(3)(4). Because many cancers express 1 marker [e.g., breast cancer is associated with carcinoma antigens 153 and 125 (CA 153 and CA 125)1 and carcinoembryonic antigen (CEA)], and concentrations of several tumor markers often increase in the serum of a patient, accurate simultaneous multianalyte test (SMAT) of combinations of tumor markers may improve the diagnosis of certain types of tumor (5)(6)(7)(8).

SMAT may offer a shorter analytical time, higher sample throughput, lower sampling volume, and lower cost per assay compared with traditional single-analyte tests (9)(10). Thus, multilabel assays and spatially resolved assay systems have been developed as the main modes to perform SMAT (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Application of the multilabel assays has been limited by difficulty in accurate quantification due to different optimal assay conditions and the signal overlap of different labels (11)(12)(13). Although a set of substrate zone-resolved techniques have been proposed to overcome these drawbacks (14)(15), the restriction in the number of available labels still greatly limits their application.

Spatially resolved assays with a single label seem well suited for performing SMAT, and optical SMAT, relying on fluorescence emission and optical reflectance, has been developed into mature technology. Optical SMAT, however, often needs an expensive array detector, such as a charge-coupled device camera (16)(17). Electrochemical array, which is distinguished by its convenient miniaturization for high-throughput systems, low assay cost, and absence of sophisticated and expensive array detectors, shows promising application in cancer screening (18)(19). Electrochemical cross-talk caused by diffusion of the detectable enzymatic products is the main problem in the fabrication of electrochemical array. To solve this problem, many approaches have been followed. One approach, for example, is to ensure that the distance between adjacent electrodes is larger than the diffusion distance of enzymatic product (20)(21)(22)(23), but such an approach conflicts with the goal of miniaturization. Another simple method to completely avoid the electrochemical cross-talk can be achieved by immobilizing the electron-transfer mediator on an individual immunosensor to shuttle electrons (24)(25), but this approach requires the addition of hydrogen peroxide, leading to limited practical application.

A reagentless electrochemical immunosensor is an attractive strategy (26). In our previous work, we prepared several reagentless immunosensors by using sol-gel matrix to immobilize immunoreagents and detected the direct electron transfer of labeled enzyme, horseradish peroxidase (HRP) (27)(28)(29)(30)(31). To achieve SMAT, this work further fabricated a reagentless immunosensor array by individually embedding 4 kinds of HRP-labeled antibody-modified gold nanoparticles in a newly designed biopolymer/sol-gel matrix formed on screen-printed carbon electrodes (SPCEs), where the HRP-Ab-Au nanoparticles were limited in the holes of the biopolymer/sol-gel film. Chitosan, a biopolymer with excellent film-forming ability, biocompatibility, nontoxicity, and high mechanical strength, acted as the adhesion frame in the synthesis of the sol-gel and made the electrical communication between redox sites of the enzyme and sensing surface easier due to the cooperative effort of chitosan and sol-gel matrix. The presence of gold nanoparticles accelerated the electron transfer between immobilized HRP and the electrode and increased the hole size for improving the permeability of the sol-gel matrix so that the antigens in solution could easily penetrate into the sol-gel film for immunoreaction. Upon formation of immunocomplexes, the electrochemical responses decreased due to increasing spatial blocking, leading to the reagentless immunosensing to corresponding antigens without cross-talk. The proposed electrochemical immunosensor array had high analyte throughput, showed acceptable comparability to conventional methods for measuring several tumor markers, could be fabricated with mass production techniques, and thus provided the potential for application in point-of-care testing (POCT).

Schematic diagrams of immunosensors array and multianalyte electrochemical immunoassay system.

Figure 1.

Schematic diagrams of immunosensors array and multianalyte electrochemical immunoassay system.

(a), Nylon sheet, (b), silver ink, (c), graphite auxiliary electrode, (d), Ag/AgCl reference electrode, (e), graphite working electrode, (f), insulating dielectric.

The DPV curves of both the bare and biopolymer/sol-gel modified SPCEs in 0.2 mol/L PBS, pH 6.9, did not show any detectable signal in the applied potential window (Fig. 3⇓ ). After we embedded 0.17 μL of 50 mg/L HRP-anti-CA 153 in the biopolymer/sol-gel, the modified SPCEs displayed a sensitive peak around −540 mV (vs Ag/AgCl) (curve d, Fig. 3⇓ ), which was close to the reduction peak potential of HRP/biopolymer/sol-gel prepared with 0.17 μL of 2.0 mg/L HRP (curve c, Fig. 3⇓ ), indicating the direct electron transfer between electrode and the labeled HRP with regard to Fe(III)-Fe(II) conversion. The small difference of peak potentials between HRP-anti-CA 153 and HRP resulted from the change of microenvironment around HRP molecules because of the presence of antibody. In the presence of gold nanoparticles in the biopolymer/sol-gel, the reduction peak of the equal amount of HRP-antibody conjugate increased 2.1-fold (curve e, Fig. 3⇓ ). The cyclic voltammetric experiments at different gold electrodes showed the same appearance, and upon incorporation of gold nanoparticles into the biopolymer/sol-gel at SPCEs, the reduction peak current at the same scan rate increased 1.98-fold (see Supplemental Fig. 1 in the Data Supplement that accompanies the online version of this article at  http://www.clinchem.org/content/vol54/issue9). Thus the Au nanoparticles could accelerate the direct electrochemistry of HRP to further amplify the detectable signal. This peak was also 2.7 times higher than that of HRP-anti-CA 153-Au nanoparticles/sol-gel modified SPCE (curve f, Fig. 3⇓ ), indicating the positive effects of chitosan with good biocompatibility and hydrophilicity (35), which enhanced water uptake and swelling of the film and led to better permeability of the film for the transfer of counter ions to neutralize the charge change during the redox process and a favorable microenvironment for electron hopping or electron self-exchange between immobilized HRP molecules (36). Thus electron transfer kinetics and direct electrochemical signal increased. After the modified SPCE was incubated with CA 153, the direct electrochemical signal decreased markedly due to the increased barrier that resulted from the formation of immunocomplex (curve g, Fig. 3⇓ ), leading to a reagentless immunosensing method for antigen detection.

DPVs of bare SPCE

DPVs of bare SPCE (a), biopolymer/sol-gel (b), HRP/biopolymer/sol-gel (c), HRP-anti-CA 153/biopolymer/sol-gel (d), HRP-anti-CA 153-Au nanoparticles/biopolymer/sol-gel (e), HRP-anti-CA 153-Au nanoparticles/sol-gel modified SPCE in pH 6.9 PBS (f), and panel e in pH 6.9 PBS after incubation in 20 μL of 50 kU/L CA 153 at room temperature for 40 min (g).

The formation of immunocomplex depended on the incubation temperature and time. For the sake of convenient manipulation, the incubation step was performed with 20 μL antigen solution or the mixture of antigens for SMAT at room temperature, after which the DPV response of the labeled HRP decreased with increasing incubation time and reached a relatively stable value at 30–40 min (Fig. 4A⇓ ), indicating saturated formation of immunocomplex in the membrane. Thus, 40 min was chosen as the optimal incubation time for SMAT.

Dependences of DPV responses of immunosensors on incubation time

Dependences of DPV responses of immunosensors on incubation time (A) and pH of detection solution (B) for CA 153, CA 125, CA 199, and CEA.

In comparison with previous reports (24)(25), this array avoids the addition of mediator to shuttle electrons, and thus can exclude the electrochemical cross-talk at the electrode dimensions used here. Furthermore, the measurement of the direct electrochemical signal of HRP labeled to immunoreagents also avoids the need for other reagents in the detection process. Although the measurements show acceptable results, adding sulfite in the detection solution is not the best solution for the removal of oxygen. Thus, a system has been developed for POCT to exclude oxygen from the detection solution (see Supplemental Fig. 3 in the online Data Supplement).

1.1.3.4  p16^INK4a Expression Correlates with Degree of Cervical Neoplasia: A Comparison with Ki-67 Expression and Detection of High-Risk HPV Types

S Nicholas Agoff, Patricia Lin, Janice Morihara, Constance Mao, Nancy B Kiviat and Laura A Koutsky
Mod Pathol 2003;16(7):665–673

Although recent studies have suggested that p16^INK4a may be a useful surrogate biomarker of cervical neoplasia, Ki-67 and human papillomavirus testing have also been shown to be useful in detecting neoplasia. To help delineate the utility of p16^INK4a, biopsy samples (n = 569: negative, 133; reactive, 75; atypical, 39; low grade, 76; moderate, 80; and severe intraepithelial neoplasia, 113; also, squamous cell carcinoma, 46; adenocarcinoma, 7) were analyzed by immunohistochemistry for expression of p16^INK4a and Ki-67 (n = 432), as well as by in situhybridization for human papillomavirus Type 16 (n = 219). Testing for high-risk human papillomavirus types by polymerase chain reaction and HybridCapture2 was performed on concurrent cervical swab specimens. Recuts of the original blocks were reexamined (n = 198). Endometrial biopsies (n = 10) were also analyzed for p16^INK4a expression. Degree of p16^INK4a and Ki-67 expression correlated with degree of cervical neoplasia (P < .001) and with presence of high-risk human papillomavirus types (P < .001). There was no relationship between p16^INK4a overexpression and inflammation or hormonal status. Ki-67 expression correlated with inflammation (P = 0.003) and was expressed in more reactive and atypical lesions than p16^INK4a (P = 0.008). Probes for human papillomavirus 16 stained 54% of cervical neoplastic lesions; the degree of staining correlated significantly with degree of neoplasia (P < .001) and p16^INK4astaining (P < .001). Interobserver reproducibility was substantial for p16^INK4a and Ki-67 interpretation (weighted : 0.74 and 0.70, respectively). Expression of p16^INK4a was observed in all endometrial biopsies. Compared with Ki-67 expression and detection of high-risk human papillomavirus, p16^INK4a was less likely to be positive in samples from women with negative, reactive, and atypical biopsies. Although expression of p16^INK4ain endometrial epithelium may be problematic in terms of screening, the potential of p16^INK4a as a screening test warrants investigation.

The screening of women by Pap smear has led to a remarkable decline in the mortality from cervical cancer; however, secondary to subjective criteria, interpretation of Pap smears is subject to marked inter- and intraobserver variability as well as having a relatively low sensitivity for cervical neoplasia on a single sample (as low as 66% sensitivity for biopsy-proven high-grade squamous intraepithelial lesions [HSIL]) (1, 2). Recently, histology, which is thought of as the gold standard for the diagnosis of cervical neoplasia, has also been found to suffer from marked intra- and interobserver variability, and testing for high-risk human papillomavirus (HPV) by Hybrid Capture 2, which has been shown to be very sensitive in the detection of cervical neoplasia and useful in the triaging of ASCUS smears, has a low specificity for cervical neoplasia (1,3). Thus, new biomarkers that are more sensitive and specific in the detection of cervical neoplasia and more reproducible than cervical cytology are needed.

Human papillomaviruses (HPV) are known to be a major causative agent in cervical neoplasia and invasive cervical carcinoma. Many different HPV types associated with cervical neoplasia have been discovered, and they have been subdivided into high- and low-risk categories based on their association with invasive cervical carcinoma (4). This association is based, in part, on the relative affinity that the HPV-type specific oncoproteins E6 and E7 bind to cellular regulatory proteins, specifically, the p53 tumor suppressor protein and the retinoblastoma protein (Rb) (5). Inactivation of these factors, either by degradation (p53) or functional inactivation (Rb), leads to disruption of the cell cycle and increased proliferation, thought to ultimately give rise to carcinoma.

p16^INK4a is a cyclin-dependent kinase inhibitor that regulates the activity of cyclin-dependent kinases 4 and 6 and is often inactivated in many cancers by genetic deletion or hypermethylation (6). In non-HPV–associated tumors, this inactivation leads to increased cyclin-dependent kinase activity and inactivation of Rb. However, in HPV-associated tumors, inactivation of Rb by E7 leads to markedly increased levels of p16^INK4a. Recent studies have documented overexpression of p16^INK4a not only in cervical intraepithelial neoplasia (CIN) but in cervical cancer as well (6, 7, 8, 9, 10).

For p16^INK4a, the results were reported in semiquantitative fashion (negative, or 1+ to 3+) based on none, 5–25%, 25–75%, and >75% of cells immunostained in a lesion. Strong nuclear as well as cytoplasmic staining was considered a positive reaction. Wispy weak cytoplasmic staining present in rare cells (<5%) was considered , and for analysis was grouped into the negative category. For Ki-67, the results were also reported in a semiquantitative fashion as cells in the lower 1/3 of the epithelium staining (i.e., usually basilar cell staining), cells in the middle 1/3–2/3 staining, or cells in the upper 1/3 staining (14). Strong nuclear staining was considered a positive reaction. Stains were analyzed by two authors (SNA and JM) for reproducibility; each was blinded to the other’s result.

The degree of p16^INK4a expression correlated well with the degree of cervical neoplasia, and this correlation improved slightly when compared with the recut slide diagnosis (P < .001; Fig. 1; Tables 1 and 2). There was very little expression in negative and reactive lesions, with only 11% to 12% showing 1+ staining (24 of 208 -original diagnosis, 12 of 112 recut diagnosis). 57% of the CIN I cases had 1+ expression, compared with 75% of CIN II lesions and 91% of CIN III lesions. On the recut diagnosis, 97% of CIN III lesions stained 1+. There were 10 (9%) CIN III original diagnosis that did not stain for p16^INK4a, but on review, the majority of these were secondary to the lesion being cut through and not present on the immunohistochemistry (IHC) slide. For the recut diagnosis, there was only 1 (3%) case that did not stain with p16^INK4a, and on review, two of three pathologists agreed that this represented CIN III, whereas the third felt it represented atypical squamous metaplasia. p16^INK4a expression of 1+ or greater was present in 89%(47/53) of the invasive carcinomas. Review of negative cases confirmed the carcinoma diagnosis.

p16INK4a and Ki-67 expression in normal, low-grade squamous dysplasia, and high grade squamous dysplasia

p16^INK4a and Ki-67 expression in normal cervical squamous mucosa (A, H&E stain; B, p16^INK4a; C, Ki-67), low-grade squamous dysplasia (CIN I; D, H&E stain; E, p16^INK4a; F, Ki-67), and high grade squamous dysplasia (CIN III; G, H&E stain; H, p16^INK4a, I, Ki-67).

1.1.3.5  Quantitative real-time detection of magnetic nanoparticles by their nonlinear magnetization

1.1.6  Proteomics and biomarkers

1.1.6.1 Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine

Kageyama S, Isono Y, Iwaka H,…, Yoshiki T.
Clin Chem 2004; 50(5):857-866.
http://dx.doi.org:/10.1373/clinchem.2003.027425
How are we going to discover new cancer biomarkers? A proteomic approach for bladder cancer.
Editorial. Eftherios P. Diamandis
Clin Chem 2003; 50(5):794-795.
http://dx.doi.org:/10.1373.2004.032177

BACKGROUND: New methods for detection of bladder cancer are needed because cystoscopy is both invasive and expensive and urine cytology has low sensitivity. We screened proteins as tumor markers for bladder cancer by proteomic analysis of cancerous and healthy tissues and investigated the diagnostic accuracy of one such marker in urine. METHODS: Three specimens of bladder cancer and healthy urothelium, respectively, were used for proteome differential display using narrow-pH-range two-dimensional electrophoresis. To evaluate the presence of calreticulin (CRT) as detected by Western blotting, we obtained 22 cancerous and 10 noncancerous surgical specimens from transurethral resection or radical cystectomy. To evaluate urinary CRT, we collected 70 and 181 urine samples from patients with and without bladder cancer, respectively. Anti-CRT COOH-terminus antibody was used to detect CRT in tissue and urine. RESULTS: Proteomic analysis revealed increased CRT (55 kDa; pI 4.3) in cancer tissue. Quantitative Western blot analysis showed that CRT was increased in cancer tissue (P = 0.0003). Urinary CRT had a sensitivity of 73% (95% confidence interval, 62-83%) at a specificity of 86% (80-91%) for bladder cancer in the samples tested. CONCLUSIONS: Proteomic analysis is useful in searching for candidate proteins as biomarkers and led to the identification of urinary CRT. The diagnostic accuracy of urinary CRT for bladder cancer appears comparable to that of Food and Drug Administration-cleared urinary markers, but further studies are needed to determine its diagnostic role.

A handful of cancer biomarkers are currently used routinely for population screening, disease diagnosis, prognosis, monitoring of therapy, and prediction of therapeutic response. Unfortunately, most of these biomarkers suffer from low sensitivity, specificity, and predictive value, particularly when applied to rare diseases in population screening programs. Thus, for the classic cancer biomarkers much is left to be desired in terms of clinical applicability. We need new cancer biomarkers that will further enhance our ability to diagnose, prognose, and predict therapeutic response in many cancer types. Because biomarkers can be analyzed relatively noninvasively and economically, it is worth investing in discovering more biomarkers in the future. The completion of the Human Genome Project has raised expectations that the knowledge of all genes and proteins will lead to identification of many candidate biomarkers for cancer and other diseases. These predictions still need to be realized. The prevailing view among specialists is that the most powerful single cancer biomarkers may have already been discovered. Likely, in the future we will discover biomarkers that are less sensitive or specific but could be used in panels, in combination with powerful bioinformatic tools, to devise diagnostic algorithms with improved sensitivity and specificity. These efforts are currently in progress¹.

1. Stephan C, Vogel B, Cammann H, Lein M, Klevecka V, Sinha P, et al. [An artificial neural network as a tool in risk evaluation of prostate cancer. Indication for biopsy with the PSA range of 2–20 microg/l]. Urologe A 2003; 42:1221–9.

In this issue of Clinical Chemistry, Kageyama et al. propose proteomic analysis of urine as a new way to identify bladder cancer biomarkers. Previously, Celis et al. ² used two-dimensional gel electrophoresis and developed a comprehensive database for bladder cancer profiles of both transitional and squamous cell carcinomas. Through their studies, Kageyama et al. were able to identify a potential tumor marker, calreticulin, which is found in the urine of patients with bladder carcinoma. The authors used a differential display method of bladder cancer vs healthy urothelial tissue and mass spectrometry to identify proteins that are increased in cancer tissue. In addition to calreticulin, an endoplasmic reticulum chaperone, they found nine other candidate proteins that could constitute new biomarkers for bladder carcinoma. The authors confirmed their data with quantitative Western blot analysis, immunoprecipitation, and immunohistochemistry. Their reported sensitivity and specificity were 73% and 86%, respectively, similar to the values reported for other biochemical bladder markers. However, the diagnostic accuracy of their test was vulnerable to urinary tract infections³.

³ Positive correlations were found among the appearance of adenylate kinase activity in the urine and the existence of bacteriuria, the Fairley test, and other criteria of urinary infection. Since the adenylate kinase isozymes of human tissues are organ specific and can be distinguished from one another, the appearance of adenylate kinase isozymes in urine was used in this study to identify the existence of infection in bladder or kidney. The findings suggest the usefulness of measuring the appearance of urinary adenylate kinase isozymes for the purpose of detection and differential diagnoses of urinary infections, particularly since adenylate kinase is absent or found in low concentrations in urine and serum under normal conditions.

Currently, potential bladder tumor markers can be used in various clinical scenarios, including⁴:

• Serial testing for earlier detection of recurrence;
  • Complementary testing to urine cytology to improve the detection rate;
  • Providing a less expensive and more objective alternative

to the urine cytology test; and
• Directing the cytoscopic evaluation of patient followup.

The gold standard for the detection of urothelial neoplasia is cytologic examination of urothelial cells from voided urine, urinary bladder washings, and urinary tract brushing specimens in combination with cystoscopic examination^5,6.

2. Celis A, Rasmussen HH, Celis P, Basse B, Lauridsen JB, Ratz G, et al. Short-term culturing of low-grade superficial bladder transitional cell carcinomas leads to changes in the expression levels of several proteins involved in key cellular activities. Electrophoresis 1999;20:355–61.
3. Bernstein LH, Horenstein JM, and Russell PJ. Urinary adenylate kinase and urinary infections. J Clin Microbiol. 1983 Sep; 18(3): 578–584
4. Fritsche HA. Bladder cancer and urine tumor marker tests. In: Diamandis EP, Fritsche HA, Lilja H, Chan DW, Schwartz MK. Tumor markers: physiology,pathobiology, technology and clinical applications. Washington: AACC Press, 2002; 281–6.
5. Bailey MJ. Urinary markers in bladder cancer. BJU Int 2003; 91:772–3
6. Eissa S, Kassim S, El-Ahmady O. Detection of bladder tumours: role of cytology, morphology-based assays, biochemical and molecular markers. Curr Opin Obstet Gynecol 2003;15:395–403

Current guidelines suggest that low-risk patients should be surveyed once a year with cystoscopy and high-risk patients at 3-month intervals. Currently, cystoscopy is always combined with VUC. Because, as mentioned earlier, new urinary bladder tests such as BTA or NMP22 could detect lower-grade disease recurrence with higher sensitivity than VUC, it could be worthwhile to consider including one or more of these tests in the routine follow-up of patients with bladder carcinoma. However, large prospective studies will be necessary to test the clinical utility of these assays against cytology.

1.1.6.2  Multiplexed proteomic analysis of oxidation and concentrations of CSF proteins in Alzheimer’s disease

Korolainen MA, Nyman TA, Nyyssonen P, Hartikainen ES, Pirttila Y.
Clin Chem 2007; 53(4):657-665.
http://dx.doi.org:/10.1373/clinchem.2006.078014

Carbonylation is an irreversible oxidative modification of proteins that has been linked to various conditions of oxidative stress, aging, physiological disorders, and disease. Increased oxidative stress is thus also considered to play a role in the pathogenesis of age-related neurodegenerative disorders such as Alzheimer disease (AD). In addition, it has recently become evident that the response mechanisms to increased oxidative stress may depend on sex. Several oxidized carbonylated proteins have been identified in plasma and brain of AD patients by use of 2-dimensional oxyblotting.

Signals for beta-trace, lambda chain, and transthyretins were decreased in probable AD patients compared with controls. The only identified protein exhibiting an increased degree of carbonylation in AD patients was lambda chain. The concentrations of proteins did not generally differ between men and women; however, vitamin D-binding protein, apolipoprotein A-I, and alpha-1-antitrypsin exhibited higher extents of carbonylation in men.

None of the brain-specific proteins exhibited carbonylation changes in probable AD patients compared with age-matched neurological controls showing no cognitive decline. The carbonylation status of proteins differed between women and men. Two-dimensional multiplexed oxyblotting is applicable to study both the concentrations and carbonylation of cerebrospinal fluid proteins.

1.1.6.3  The Brain Injury Biomarker VLP-1 Is Increased in the Cerebrospinal Fluid of Alzheimer Disease Patients

Jin-Moo Lee, Kaj Blennow, Niels Andreasen, Omar Laterza, Vijay Modur, Jitka Olander, Feng Gao, Matt Ohlendorf, and Jack H. Ladenson
Clinical Chemistry  2008; 54:10 1617–1623
http://dx.doi.org:/10.1373/clinchem.2008.104497

BACKGROUND: Definitive diagnosis of Alzheimer disease (AD) can be made only by histopathological examination of brain tissue, prompting the search for premortem disease biomarkers. We sought to determine if the novel brain injury biomarker, visinin-like protein 1 (VLP-1), is altered in the CSF of AD patients compared with controls, and to compare its values to the other well-studied CSF biomarkers 42-amino acid amyloid- peptide (A1–42), total Tau (tTau), and hyperphosphorylated Tau (pTau). METHODS: Using ELISA, we measured concentrations of A1–42, tTau, pTau, and VLP-1 in CSF samples from 33 AD patients and 24 controls. We compared the diagnostic performance of these biomarkers using ROC curves. RESULTS: CSF VLP-1 concentrations were significantly higher in AD patients [median (interquartile range) 365 (166) ng/L] compared with controls [244 (142.5) ng/L]. Although the diagnostic performance of VLP-1 alone was comparable to that of A, tTau, or pTau alone, the combination of the 4 biomarkers demonstrated better performance than each individually. VLP-1 concentrations were higher in AD subjects with APOE 4/4 genotype [599 (240) ng/L] compared with 3/4 [376 (127) ng/L] and 3/3 [280 (115.5) ng/L] genotypes. Furthermore, VLP-1 values demonstrated a high degree of correlation with pTau (r 0.809) and tTau (r 0.635) but not A1–42 (r 0.233). VLP-1 was the only biomarker that correlated with MMSE score (r 0.384, P 0.030). CONCLUSIONS: These results suggest that neuronal injury markers such as VLP-1 may have utility as biomarkers for AD.

The diagnosis of Alzheimer disease (AD),6 the most common form of dementia in Western countries, is largely based on historical and clinical criteria. Although many studies report a reasonably high degree of diagnostic accuracy (80%–90%), these studies often include patients with advanced disease evaluated at specialized centers (1 ). At present, postmortem examination of brain tissue is the only tool for definitive diagnosis. Therefore, the development of a biomarker for AD would aid greatly in the diagnosis of this disease. In addition, such a marker could potentially be used to measure efficacy in future therapeutic trials. Most studies of AD biomarkers have focused on known pathological substrates for the disease. Amyloid plaques and neurofibrillary tangles are pathological hallmarks of AD (2 ) and primarily comprise abnormally aggregated endogenous proteins. Amyloid plaques (extracellular proteinaceous aggregates) are principally composed of the amyloid- peptide (A), a 38 – to 42–amino acid peptide fragment of the amyloid precursor protein (APP). The major species, the 42– amino acid peptide (A1–42) (3, 4 ), is significantly decreased in the cerebrospinal fluid (CSF) of patients with AD (5– 8 ). Neurofibrillary tangles are intraneuronal protein aggregates found mainly in neurites and primarily composed of hyperphosphorylated Tau (pTau), a microtubule-associated protein.

Fig. 1. CSF VLP-1 values in AD patients and controls. Scatter plot of CSF VLP-1 values in control vs AD patients. The line within the box represents the median value, the box encompasses 25th to 75th percentiles, and the error bars encompass the 10th to 90th percentiles. A significant difference was found in control vs AD patients (P 0.001, Student t-test).

To see if VLP-1 provides utility to the diagnosis of AD beyond the contribution of A, tTau, or pTau alone, we performed a ROC analysisfor each individual biomarker alone compared to the combination of all biomarkers. The AUCs for VLP-1, A, tTau, pTau, and an optimum linear combination of all biomarkers are shown in Fig. 2. AUCs were similar between all biomarkers individually; however, the linear combination of all biomarkers resulted in an approximately 5% improvement (Fig. 2).

To examine possible relationships between CSF VLP-1 values and patient characteristics, we performed correlation analyses between VLP-1 and age, disease duration, MMSE, and the number of APOE 4 alleles. VLP-1 correlated with MMSE and the number of APOE 4 alleles (Fig. 3A). None of the other biomarkers correlated with MMSE in this patient population (A1–42, r 0.350, P 0.497; tTau, r 0.295, P 0.100; pTau, r 0.202, P 0.264). To further examine the relationship between APOE genotype and CSF VLP-1 concentrations, we calculated mean CSF VLP-1 values by different genotypes. APOE 4/4 individuals had the highest concentrations, followed by 3/4 and 3/3 individuals (Fig. 3B).

To examine if VLP-1 concentrations in the CSF were related to values of the other biomarkers studied, we performed correlations between VLP-1 and tTau, pTau, or A1–42 using data from both AD patients and controls (Fig. 4). VLP-1 and pTau showed the greatest correlation (r 0.809) (Fig. 4C), whereas A1–42 did not correlate with VLP-1 (Fig. 4A, r 0.233). Individual correlations for AD patients analyzed separately from controls were also performed, and revealed results similar to that of the total patient population: VLP-1 vs A1–42 was not statistically significant (r 0.29671 and 0.1698 in AD and controls, respectively), whereas VLP-1 vs tTau (r 0.6221 and 0.7247 in AD and controls) and pTau (r 0.8747 and 0.6227 in AD and controls) were significantly correlated in the AD and control populations analyzed separately.

Dementia severity appears to correlate with the number of neurofibrillary tangles, but not to the degree of plaque deposition (13 ). The close correlation between VLP-1 and pTau concentrations in the CSF of AD patients is consistent with these findings, as is the lack of correlation with A. There are several limitations to this study. First, the number of patients in both control and disease groups is limited. Further studies will be needed to confirm our findings in larger, more well-characterized populations. Second, because the diagnosis of AD was made by clinical criteria, there will undoubtedly be a small but significant group of patients that were misdiagnosed (10%–20%) (1 ). This may account for some of the overlap in values for CSF biomarkers. ApoE genotyping in the control group might help with this diagnostic uncertainty. A much more rigorous study would require autopsy confirmation of diagnosis. Third, our study is limited to a comparison of VLP-1 concentrationsin AD patients vs controls, a situation thatis unlikely to occur clinically. A more relevant comparison should be made across patients carrying the differential diagnosis of dementia. Finally, our CSF samples represent a single snapshot in AD pathogenesis; further studies will be required to understand the time course or biomarker evolution with disease pathogenesis.

1.1.6.4 Determination of non-α₁-antichymotrypsin-complexed PSA as an indirect measurement of free PSA: analytical performance and diagnostic accuracy.

Wesselin S, Dtephan C, Semjonow A,…, Jung K.
Clin Chem 2003;49(6):887-894.
http://dx.doi.org:/10.1373/49.6.887

Background: A new assay measures prostate-specific antigen (PSA) not complexed to α1-antichymotrypsin (nACT-PSA) after removing PSA complexed to ACT by use of anti-ACT antibodies. We evaluated nACT-PSA and its ratio to total PSA (tPSA) as alternatives to free PSA (fPSA) and its ratio to tPSA in differentiating prostate cancer (PCa) and benign prostatic hyperplasia (BPH) in patients with tPSA of 2–20 μg/L. Methods: PSA in serum of 183 untreated patients with PCa and 132 patients with BPH was measured retrospectively on the chemiluminescence immunoassay analyzer LIAISON® (Byk-Sangtec Diagnostica) with the LIAISON tPSA and LIAISON fPSA assays. The nACT-PSA fraction was determined with a prototype assay measuring the residual PSA after precipitation of ACT-PSA with an ACT-precipitating reagent.
Results: nACT-PSA was higher than fPSA in samples with fPSA concentrations <1 μg/L but lower in samples with >1 μg/L fPSA. The median ratios of fPSA/tPSA and of nACT-PSA/tPSA were significantly different between patients with BPH and PCa (19.4% vs 12.2% and 17.4% vs 13.0%, respectively). Within the tPSA ranges tested (2–20, 2–10, and 4–10 μg/L), areas under the ROC curves for the fPSA/tPSA ratios were significantly larger than those for nACT-PSA/tPSA. In the tPSA ranges <10 μg/L, the areas under the ROC curves for fPSA/tPSA were significantly larger than those for tPSA, whereas the areas for nACT-PSA/tPSA were not. At decision limits for 95% sensitivity and specificity, both ratios significantly increased specificity and sensitivity, respectively, compared with tPSA, but the fPSA/tPSA ratio showed higher values. Conclusions: nACT-PSA and its ratio to tPSA provide lower diagnostic sensitivity and specificity than fPSA/tPSA. The fPSA/tPSA ratio represents the state-of-the-art method for differentiating between PCa and BPH.

1.1.6.5 Ultrasensitive densitometry detection of cytokines with nanoparticle-modified aptamers

Li yuan-Yuan, Zhang C, Li Bo-Sheng, …, Xu Shun-Quing
Clin Chem 2007; 53(6):1061-1066
http://dx.doi.org:/10.1373/clinchem.2006.082271

Background: Aptamers mimic properties of antibodies and sometimes turn out to be even better than antibodies as reagents for assays. We describe the establishment of an ultrasensitive densitometry method for cytokine detection by nanoparticle (NP)-modified aptamers. Methods: The assay simultaneously uses a gold NP–modified aptamer and a biotin-modified aptamer to bind to the target protein, forming a sandwich complex. The absorbance signal generated by the aptamer-protein complex is amplified and detected with a microplate reader. Results: The assay for platelet-derived growth factor B-chain homodimer (PDGF-BB) was linear from 1 fmol/L to 100 pmol/L (R2 = 0.9869). The analytical detection limit was 83 amol/L. The intraassay and interassay imprecision (CVs) was ≤7.5%. Serum concentrations of PDGF-BB determined with the gold NP–modified aptamer assay and with ELISA were not significantly different. Conclusions: The gold NP–modified aptamer assay provides a fast, convenient method for cytokine detection and improves the detection range and the detection limit compared with ELISA.

1.1.6.6  Protein profiling of microdissected pancreas carcinoma and identification of HSP27 as a potential serum marker.

Melle C, Ernst G, Escher N, Hartmann D,…, von Eggeling F.
Clin Chem 2007; 53(4):629-635.
http://dx.doi.org:/10.1373/clinchem.2006.079194

Background: Patients with pancreatic adenocarcinomas have a poor prognosis because of late clinical manifestation and the tumor’s aggressive nature. We used proteomic techniques to search for markers of pancreatic carcinoma. Methods: We performed protein profiling of microdissected cryostat sections of 9 pancreatic adenocarcinomas and 10 healthy pancreatic tissue samples using ProteinChip technology (surface-enhanced laser desorption/ionization). We identified proteins by use of 2-dimensional gel electrophoresis, peptide fingerprint mapping, and immunodepletion and used immunohistochemistry for in situ localization of the proteins found. We used ELISA to quantify these proteins in preoperative serum samples from 35 patients with pancreatic cancer and 37 healthy individuals. Results: From among the differentially expressed signals that were detected by ProteinChip technology, we identified 2 proteins, DJ-1 and heat shock protein 27 (HSP27). We then detected HSP27 in sera of patients by use of ELISA, indicating a sensitivity of 100% and a specificity of 84% for the recognition of pancreatic cancer. Conclusions: The detection of DJ-1 and HSP27 in pure defined tissue and the retrieval of HSP27 in serum by antibody-based methods identifies a potential marker for pancreatic cancer.

1.1.7  Mass Spectrometry Methods

1.1.7.1 LC-MS/MS quantification of Zn-α2 glycoprotein: A potential serum biomarker for prostate cancer

Bondar OP, Barnidge DR, KKlee EW, Davis BJ, Klee GG
Clin Chem 2007; 53(4):673-678 http://dx.doi.org:/10.1373/clinchem.2006.079681

LC-MS/MS – tandem mass spectrometry

Background: Zn-α2 glycoprotein (ZAG) is a relatively abundant glycoprotein that has potential as a biomarker for prostate cancer. We present a high-flow liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for measuring serum ZAG concentrations by proteolytic cleavage of the protein and quantification of a unique peptide. Methods: We selected the ZAG tryptic peptide 147EIPAWVPEDPAAQITK162 as the intact protein for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Standards using recombinant ZAG in bovine serum albumin, 50 g/L, and a pilot series of patient sera were denatured, reduced, alkylated, and digested with trypsin. The concentration of ZAG was calculated from a dose–response curve of the ratio of the relative abundance of the ZAG tryptic peptide to internal standard. Results: The limit of detection for ZAG in serum was 0.08 mg/L, and the limit of quantification was 0.32 mg/L with a linear dynamic range of 0.32 to 10.2 mg/L. Replicate digests from pooled sera run during a period of 3 consecutive days showed intraassay imprecision (CV) of 5.0% to 6.3% and interassay imprecision of 4.4% to 5.9%. Mean (SD) ZAG was higher in 25 men with prostate cancer [7.59 (2.45) mg/L] than in 20 men with nonmalignant prostate disease [6.21 (1.65) mg/L, P = 0.037] and 6 healthy men [3.65 (0.71) mg/L, P = 0.0007]. Conclusions: The LC-MS/MS assay can be used to evaluate the clinical utility of ZAG as a cancer biomarker.

1.1.7.2 A novel, high-throughput workflow for discovery and identification of serum carrier protein-bound peptide biomarker candidates in ovarian cancer samples.

Lopez MF, Mikulskis A, Kuzdzal S, Golenko E,…, Fishman D.
Clin Chem 2007; 53(6):1067-1074.
http://dx.doi.org:/10.1373/clinchem.2006.080721

MALDI-TOF MS

Background: Most cases of ovarian cancer are detected at later stages when the 5-year survival is ∼15%, but 5-year survival approaches 90% when the cancer is detected early (stage I). To use mass spectrometry (MS) of serum proteins for early detection, a seamless workflow is needed that provides an opportunity for rapid profiling along with direct identification of the underpinning ions. Methods: We used carrier protein–bound affinity enrichment of serum samples directly coupled with MALDI orthagonal TOF MS profiling to rapidly search for potential ion signatures that contained discriminatory power. These ions were subsequently directly subjected to tandem MS for sequence identification. Results: We discovered several biomarker panels that enabled differentiation of stage I ovarian cancer from unaffected (age-matched) patients with no evidence of ovarian cancer, with positive results in >93% of samples from patients with disease-negative results and in 97% of disease-free controls. The carrier protein–based approach identified additional protein fragments, many from low-abundance proteins or proteins not previously seen in serum. Conclusions: This workflow system using a highly reproducible, high-resolution MALDI-TOF platform enables rapid enrichment and profiling of large numbers of clinical samples for discovery of ion signatures and integration of direct sequencing and identification of the ions without need for additional offline, time-consuming purification strategies.

1.1.7.3  Mass Spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications.

Kemna EHJM, Tjalsma H, Podust VN, Swinkels DW.
Clin Chem 2007; 53(4):620-628.
http://DX.DOI.ORG:/10.1373/clinchem.2006.079186

SELDI-TOF MS

Background: Discovery of the central role of hepcidin in body iron regulation has shed new light on the pathophysiology of iron disorders. Information is lacking on newer analytical approaches to measure hepcidin in serum and urine. Recent reports on the measurement of urine and serum hepcidin by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) necessitate analytical and clinical evaluation of MS-based methodologies. Methods: We used SELDI-TOF MS, immunocapture, and tandem MS to identify and characterize hepcidin in serum and urine. In addition to diagnostic application, we investigated analytical reproducibility and biological and preanalytical variation for both serum and urine on Normal Phase 20 and Immobilized Metal Affinity Capture 30 ProteinChip arrays. We obtained samples from healthy controls and patients with documented iron-deficiency anemia, inflammation-induced anemia, thalassemia major, and hereditary hemochromatosis. Results: Proteomic techniques showed that hepcidin-20, -22, and -25 isoforms are present in urine. Hepcidin-25 in serum had the same amino acid sequence as hepcidin-25 in urine, whereas hepcidin-22 was not detected in serum. The interarray CV was 15% to 27%, and interspot CV was 11% to 13%. Preliminary studies showed that hepcidin-25 differentiated disorders of iron metabolism. Urine hepcidin is more affected by multiple freeze-thaw cycles and storage conditions, but less influenced by diurnal variation, than is serum hepcidin. Conclusion: SELDI-TOF MS can be used to measure hepcidin in both serum and urine, but serum requires a standardized sampling protocol.

1.1.7.4  Current state and future directions of neurochemical biomarkers for Alzheimer’s disease.

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