Autoantibody signatures as biomarkers to distinguish prostate cancer from benign prostatic hyperplasia in patients with increased serum prostate specific antigen.

BACKGROUND: Serum prostate specific antigen (PSA) concentrations lack the specificity to differentiate prostate cancer from benign prostate hyperplasia (BPH), resulting in unnecessary biopsies. We identified 5 autoantibody signatures to specific cancer targets which might be able to differentiate prostate cancer from BPH in patients with increased serum PSA. METHODS: To identify autoantibody signatures as biomarkers, a native antigen reverse capture microarray platform was used. Briefly, well-characterized monoclonal antibodies were arrayed onto nanoparticle slides to capture native antigens from prostate cancer cells. Prostate cancer patient serum samples (n=41) and BPH patient samples (collected starting at the time of initial diagnosis) with a mean follow-up of 6.56 y without the diagnosis of cancer (n=39) were obtained. One hundred micrograms of IgGs were purified and labeled with a Cy3 dye and incubated on the arrays. The arrays were scanned for fluorescence and the intensity was quantified. Receiver operating characteristic curves were produced and the area under the curve (AUC) was determined. RESULTS: Using our microarray platform, we identified autoantibody signatures capable of distinguishing between prostate cancer and BPH. The top 5 autoantibody signatures were TARDBP, TLN1, PARK7, LEDGF/PSIP1, and CALD1. Combining these signatures resulted in an AUC of 0.95 (sensitivity of 95% at 80% specificity) compared to AUC of 0.5 for serum concentration PSA (sensitivity of 12.2% at 80% specificity). CONCLUSION: Our preliminary results showed that we were able to identify specific autoantibody signatures that can differentiate prostate cancer from BPH, and may result in the reduction of unnecessary biopsies in patients with increased serum PSA.

Antibody profiling with protein antigen microarrays in early stage cancer.

INTRODUCTION: Proteins not present in normal cells, that is, cancer cells, may elicit a host immune response that leads to the generation of antibodies that might react with these tumor-associated proteins. In recent years, a growing number of reports have showed that autoantibody profiling may provide an alternative approach for the detection of cancer. However, most studies of antigen-autoantibody reactivity have relied on recombinant proteins. Recombinant proteins lack the proper post-translational modifications present in native proteins. Because of this limitation, native or natural protein antigen microarrays are gaining popularity for profiling antibody responses. AREAS COVERED: i) To illustrate some examples of autoantibodies as signatures for early stage cancer; ii) to briefly outline the various protein antigen microarray platforms; iii) to illustrate the use of native or natural protein microarrays in the discovery of potential biomarkers and iv) to discuss the advantages of native protein antigen microarrays over other approaches. EXPERT OPINION: The nature of protein microarray platforms is conducive to multiplexing, which amplifies the potential for uncovering effective biomarkers for many significant diseases. However, the major challenge will be in integrating microarray platforms into multiplexed clinical diagnostic tools, as the main drawback is the reproducibility and coefficient of variation of the results from array to array, and the transportability of the array platform to a more automatable platform.

Native antigen fractionation protein microarrays for biomarker discovery.

In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.

CA125 immune complexes in ovarian cancer patients with low CA125 concentrations.

BACKGROUND: About 20% of women with ovarian cancer have low concentrations of serum cancer antigen 125 (CA125), and this important tumor marker cannot be used to monitor their disease. The measured concentration for mucin 1 (MUC1), or CA15-3, another tumor marker, can be lowered in breast and ovarian cancer patients when circulating immune complexes (CICs) containing antibodies bound to the free antigen are present. Because CA125 and MUC1 are related members of the mucin family, we sought to determine whether CICs might also exist for CA125 and interfere with its clinical assay. METHODS: We developed an antigen capture-based assay to determine the presence of CICs for CA125. We spotted mouse antibodies to CA125 onto nanoparticle slides, incubated them with patient serum, and added Cy5-tagged goat antihuman IgG antibodies. Fluorescence intensities were read and normalized to the intensities for glutathione S-transferase A1 as a control. RESULTS: Assay results for 23 ovarian cancer cases with high CA125 concentrations, 43 cases with low CA125 concentrations, and 19 controls (mean CA125 concentrations, 2706, 23, and 11 kilounits/L, respectively) revealed mean fluorescence intensities for CA125 CIC of 2.30, 2.72, and 1.99 intensity units (iu), respectively. A generalized linear model adjusted for batch and age showed higher CA125 CIC fluorescence intensities in low-CA125 cases than in high-CA125 cases (P = 0.03) and controls (P = 0.0005). Four ovarian cancer patients who had recurrent disease and always had low CA125 values had a mean CA125 CIC value of 3.06 iu (95% CI, 2.34-4.01 iu). CONCLUSIONS: These preliminary results suggest the existence of CICs involving CA125, which may help explain some ovarian cancer cases with low CA125 concentrations.

Autoantibody profiling to identify biomarkers of key pathogenic pathways in mucinous ovarian cancer.

Mucinous epithelial ovarian cancers are clinically and morphologically distinct from the other histopathologic subtypes of ovarian cancer. Unlike other ovarian subtypes, epidemiologic studies have indicated that tobacco exposure is a significant risk factor for developing mucinous ovarian cancer. Detection of autoantibody reactivity is useful in biomarker discovery and for explaining the role of important pathophysiologic pathways in disease. In order to study if there are specific antibody biomarkers in the plasma samples of mucinous ovarian cancer patients, we have initiated a screen by employing a 'reverse capture antibody microarray' platform that uses native host antigens derived from mucinous ovarian tissues as 'baits' for the capture of differentially labelled patient and control autoantibodies. Thirty-five autoantibodies that were significantly elevated in the cancer plasma samples compared with healthy controls, and six autoantibodies that segregated smoking and non-smoking patients were identified. Functional annotation of the antibody targets has identified nine target antigens involved in integrin and Wnt signalling pathways. Immunohistochemistry of archived ovarian specimens showed significant overexpression of eight of the nine target antigens in mucinous ovarian tumour tissues, suggesting that plasma autoantibodies from mucinous ovarian cancer patients might have heightened reactivities with epitopes presented by these overexpressed antigens. Autoantibody profiling may have an unexpected utility in uncovering key signalling pathways that are dysregulated in the system of interest.

Microelectrical sensors as emerging platforms for protein biomarker detection in point-of-care diagnostics.

Current methods used to measure protein expression on microarrays, such as labeled fluorescent imaging, are not well suited for real-time, diagnostic measurements at the point of care. Studies have shown that microelectrical sensors utilizing silica nanowire, impedimetric, surface acoustic wave, magnetic nanoparticle and microantenna technologies have the potential to impact disease diagnosis by offering sensing characteristics that rival conventional sensing techniques. Their ability to transduce protein binding events into electrical signals may prove essential for the development of next-generation point-of-care devices for molecular diagnostics, where they could be easily integrated with microarray, microfluidic and telemetry technologies. However, common limitations associated with the microelectrical sensors, including problems with sensor fabrication and sensitivity, must first be resolved. This review describes governing technical concepts and provides examples demonstrating the use of various microelectrical sensors in the diagnosis of disease via protein biomarkers.

Protein microarrays as an application for disease biomarkers.

Protein microarrays are an increasingly powerful technology in the hunt for new and novel diagnostic and prognostic biomarkers. Lending credit to the highly established DNA microarray, protein microarrays are versatile tools that utilize a variety of formats to facilitate the discovery of new biomarkers and our understanding of disease pathways. The aims of this review are: to detail a variety of protein microarray technologies currently used, including forward-phase technologies and reverse-phase technologies useful in both the discovery and validation of candidate biomarkers; to explore the strengths and weaknesses of various proteomic microarray platforms; to explain how bioinformatics helps compare data between microarray data sets; and to discuss the downstream applications of such technologies as they relate to the development of a highly personalized approach to medicine.

Identifying autoantigens as theranostic targets: antigen arrays and immunoproteomics approaches.

The development of theranostics holds great potential for the advancement of personalized medical treatments for human diseases. The theranostic approach combines diagnostic testing with predicted treatment outcomes in order to specifically tailor medical treatments for individual patients. As such, theranostic tests have the potential to revolutionize the diagnosis, treatment and monitoring of human diseases. Currently, the search is underway to identify novel targets for use in the development of theranostic approaches. The aims of this review are: to briefly describe the current capabilities of antigen-autoantibody microarrays, including our native antigen 'reverse-capture' autoantibody microarray platform to identify theranostic targets; to detail other immunoproteomics approaches for the identification of theranostic targets, including serological analysis of antigens by recombinant complementary (c)DNA expression cloning, serological proteome analysis, and autoantibody-mediated identification of antigens; and to provide examples of their use in theranostic discoveries.

The "reverse capture" autoantibody microarray : an innovative approach to profiling the autoantibody response to tissue-derived native antigens.

Recently, we reported the development and use of a "reverse capture" antibody microarray for the purpose of investigating antigen-autoantibody profiling. This platform was developed to allow researchers to characterize and compare the autoantibody profiles of normal and diseased patients. Our "reverse capture" protocol is based on the dual-antibody sandwich immunoassay of enzyme-linked immunosorbent assay (ELISA), and we have previously reported its use to detect autoimmunity to epitopes found on native antigens derived from tumor cell lines. In this protocol, we used ovarian cancer as a model system to adapt the "reverse capture" procedure for use with native antigens derived from frozen tissue samples. The use of this platform in studies of autoimmunity is valuable because it allows for the detection of autoantibody reactivity with epitopes found on the post-translational modifications (PTMs) of native antigens, a feature not present with other protein array platforms. In the first step in the "reverse capture" process, tissue-derived native antigens are immobilized onto the 500 monoclonal antibodies that are spotted in duplicate on the array surface. Using the captured antigens as "baits," we then incubate the array with labeled IgG from test and control samples, and perform a two-slide dye-swap to account for any dye effects. Here, we present a detailed description of the "reverse capture" autoantibody microarray for use with tissue-derived native antigens.

Immunoregulomics : a serum autoantibody-based platform for transcription factor profiling.

Gene expression is regulated by a group of proteins known as transcription factors (TFs). By binding to specific DNA cis-elements, each TF contributes a different functional role in gene expression. Panomics has developed a TranSignal TF-TF Interaction Array, which enables the user to determine TF complexes of interest with multiple other TFs. The process works by immunoprecipitating cis-elements bound to native cell nuclear extract TFs using specific antibodies to the TFs, and hybridizing the corresponding cis-elements to a membrane array spotted with different consensus sequences. In this protocol, we adapt this technology to characterize and compare autoantibody reactivity to TFs between patients with and without disease. Using Panomics combination DNA/protein arrays with over 300 different cis-elements spotted on the membrane, we can monitor the differences in autoimmune-targeted regulatory TFs, a process we termed immunoregulomics. This method allows for a qualitative analysis of the interactions with some quantifiable data. The findings can then be verified with the use of gel-shift experiments. Autoantibodies; interactome microarrays; transcription factors.

A comprehensive approach toward novel serum biomarkers for benign prostatic hyperplasia: the MPSA Consortium.

PURPOSE: Clinical benign prostatic hyperplasia is primarily diagnosed based on a diverse array of progressive lower urinary tract symptoms and is likely distinct from histological benign prostatic hyperplasia, which is detected by the presence of nonmalignant proliferation of prostate cells but may or may not be associated with symptoms. Pharmacological management of lower urinary tract symptoms has emerged as an effective initial treatment for clinical benign prostatic hyperplasia due to the introduction of new drug therapies shown to be effective in recent large clinical trials. Despite advances in symptom management and research into disease pathology, diagnostic strategies for the prediction of benign prostatic hyperplasia progression and response to drug modalities are lacking, and questions remain as to the molecular differences underlying clinical (symptomatic) vs histological (nonsymptomatic) benign prostatic hyperplasia. MATERIALS AND METHODS: As part of the Medical Therapy of Prostatic Symptoms (MTOPS) clinical trial, which demonstrated the effectiveness of combination drug therapy in slowing benign prostatic hyperplasia progression, an archive of biological specimens linked to clinical data was collected for future profiling of disease pathology and changes associated with response to drug therapy. The MTOPS Prostatic Samples Analysis (MPSA) Consortium was established to identify and validate molecular markers that may better define benign prostatic hyperplasia related pathologies, identify risk of progression of lower urinary tract symptoms, and predict response to drug therapy using the MTOPS archive. The cooperating MPSA Biomarker Discovery Sites and Pathology Coordinating Center use diverse methodologies and scientific approaches as well as unique expertise to address the goals of the Consortium. RESULTS: To date the MPSA has identified a number of promising biomarkers as well as other molecular and cellular changes associated with benign prostatic hyperplasia. CONCLUSIONS: These findings and ongoing Consortium discovery efforts have the potential to provide a greater understanding of the defects underlying disease pathology, and may lead to the development of early and more effective pharmacological treatment strategies for benign prostatic hyperplasia.

The zinc finger protein ras-responsive element binding protein-1 is a coregulator of the androgen receptor: implications for the role of the Ras pathway in enhancing androgenic signaling in prostate cancer.

Androgen receptor (AR) plays an important role in normal prostate function as well as in the etiology of prostate cancer. Activation of AR is dictated by hormone binding and by interactions with coregulators. Several of these coregulators are known targets of Ras-related signals. Recent evidence suggests that Ras activation may play a causal role in the progression of prostate cancer toward a more malignant and hormone-insensitive phenotype. In the present study, we used a transcription factor-transcription factor interaction array method to identify the zinc finger protein Ras-responsive element binding protein (RREB-1) as a partner and coregulator of AR. In LNCaP prostate cancer cells, RREB-1 was found to be present in a complex with endogenous AR as determined by coimmunoprecipitation, glutathione S-transferase pull down, and immunofluorescence analyses. RREB-1 bound to the prostate-specific antigen (PSA) promoter as assessed by chromatin immunoprecipitation. Transient expression of RREB-1 down-regulated AR-mediated promoter activity and suppressed expression of PSA protein. The repressor activity of RREB-1 was significantly attenuated by cotransfection of activated Ras. Moreover, expression of the dominant-negative N-17-Ras or, alternatively, use of the MAPK kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] abolished the effect of Ras in attenuating RREB-1-mediated repression. Furthermore, inhibition of RREB-1 expression by RNA interference enhanced the effect of Ras on PSA promoter activity and PSA expression. In addition, activation of the Ras pathway depleted AR from the RREB-1/AR complex. Collectively, our data for the first time identify RREB-1 as a repressor of AR and further implicate the Ras/MAPK kinase pathway as a likely antagonist of the inhibitory effects of RREB-1 on androgenic signaling.

Autoantibody microarrays for biomarker discovery.

Identification of autoantigens and the detection of autoantibody reactivity are useful in biomarker discovery and for explaining the role of important biochemical pathways in disease. Despite all of their potential advantages, the main challenge to working with autoantibodies is their sensitivity. Nevertheless, proteomics may hold the key to overcoming this limitation by providing the means to multiplex. Clearly, the ability to detect multiple autoantigens using a platform such as a high-density antigen microarray would improve sensitivity and specificity of detection for autoantibody profiling. The aims of this review are to: briefly describe the current status of antigen-autoantibody microarrays; provide examples of their use in biomarker discoveries; address current limitations; and provide examples and strategies to facilitate their implementation in the clinical setting.

A preliminary study of JM-27: a serum marker that can specifically identify men with symptomatic benign prostatic hyperplasia.

PURPOSE: Benign prostatic hyperplasia is a common disease in men that until recently was considered a single disease with varying symptoms. Our recent analysis has revealed that a molecular marker, JM-27, is able to distinguish at the tissue level between highly symptomatic individuals and those with histological disease. The goal of these studies was to determine if a serum based assay to detect JM-27 could distinguish men with different forms of benign prostatic hyperplasia. MATERIALS AND METHODS: A serum based enzyme-linked immunosorbent assay was developed using a novel anti-JM-27 monoclonal antibody. The assay was sensitive, detecting JM-27 at the low ng/ml level within the serum. A quantitative measurement of serum JM-27 levels was performed in 68 patients. The patients consisted of 3 groups of 29 patients with asymptomatic benign prostatic hyperplasia (American Urological Association symptom score of 15 or less), 39 with symptomatic benign prostatic hyperplasia (American Urological Association symptom score 16 to 32) and 17 with confirmed prostate cancer. The assay cutoff was determined after a pilot run of samples and applied prospectively. RESULTS: Using the determined cutoff, serum levels of JM-27 can distinguish between symptomatic and asymptomatic patient sets. The sensitivity and specificity of the assay are 90% and 77%, respectively. The presence of prostate cancer in these men does not appear to alter the marker levels. CONCLUSIONS: The present study is believed to represent the first characterization of a serum based marker for severe benign prostatic hyperplasia.

A native antigen "reverse capture" microarray platform for autoantibody profiling of prostate cancer sera.

Cancer sera contain antibodies that react with autologous cellular antigens. Here, we report the use of a novel native antigen-based platform, the "reverse capture" autoantibody microarray, for identification of autoantigens against which autoantibody expression may be used to differentiate between patients with prostate cancer and benign prostate hyperplasia (BPH). Serum samples were collected at our institution from patients with BPH and patients with prostate carcinoma with similar blood prostate-specific antigen levels. IgG was purified from individual prostate cancer sera, differentially labeled with fluorescent dyes, and competitively hybridized against purified IgG from a group of well-characterized BPH control patients on our reverse capture microarray platform. For each experiment, we performed a two-slide dye-swap. Using this platform, we identified 28 unique antigen-autoantibody reactivities that have the potential to discriminate prostate cancer from BPH. These autoantigens, with p-values ≤0.01, can be placed into the following general categories: protein kinases, cell-cycle regulators, cancer-growth factors, apoptosis mediators, and transcription factors. In addition, only 1 of the 28 autoantigens remained differentially targeted by autoantibodies in post-surgical prostate cancer patients after a minimum 1-year follow-up. Autoantibody profiling using this platform may be a useful tool for differentiating between malignant and benign diseases of the prostate.

Unraveling androgen receptor interactomes by an array-based method: discovery of proto-oncoprotein c-Rel as a negative regulator of androgen receptor.

The androgen receptor (AR) plays a key role in the development and function of male reproductive organs. Using a high-throughput transcription factor-transcription factor (TF-TF) interaction array method, we captured the AR interactomes in androgen-responsive LNCaP cells. Several known and unknown partners of AR, including AP-2, Pax 3/5 (BSAP), c-Rel, RREB-1, LIII BP, and NPAS2 were identified. We investigated one unreported AR-associated transcription factor, the proto-oncoprotein c-Rel, in detail. C-Rel belongs to the NF-kB/Rel families and is persistently active in a number of diseases, including cancer. The presence of c-Rel transcript, protein, and its in vitro and in vivo association with AR was determined. Co-localization of c-Rel with AR both in cytoplasm and nucleus was confirmed by indirect immunofluorescence analysis. Chromatin immunoprecipitation data indicated that c-Rel, like AR, is a part of the nucleoprotein complex regulating the androgen-responsive prostate-specific antigen (PSA) promoter. Overexpression of c-Rel downregulated the promoter activity of both PSA and GRE4-TATA-Luc plasmids in LNCaP and COS cells. Analysis of AR and c-Rel protein levels indicated that the promoter downregulation was not due to reciprocal decrease in the amounts of AR or c-Rel. In summary, we have identified several new partners of AR by using the TF-TF array method and have provided the first evidence of a functional role for c-Rel in androgen-responsive human prostate cancer cells.

Proteomics approaches to urologic diseases.

Biomarkers are greatly needed for several urologic diseases, such as interstitial cystitis, the symptomatic and clinical progression of benign prostate hyperplasia, as well as the specific detection of urologic cancers, including prostate and bladder cancer. This review aims to: briefly describe the need for biomarkers in the field and biomarkers that are currently available for clinicians; address the limitations and roadblocks to effective biomarker discovery; and provide examples and strategies for implementing biomarkers in clinical practice and/or drug discovery.

Development of a "reverse capture" autoantibody microarray for studies of antigen-autoantibody profiling.

Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.

The 'reverse capture' autoantibody microarray: a native antigen-based platform for autoantibody profiling.

We have previously reported the development and the use of a 'reverse capture' autoantibody microarray for studies of antigen-autoantibody profiling. We developed the 'reverse capture' autoantibody microarray to allow the user to characterize and to compare autoantibody profiles. Based on the dual-antibody sandwich immunoassay of ELISA, our 'reverse capture' protocol facilitates the detection of autoimmunity to native host antigens. Our method has the advantage over traditional protein arrays of being able to detect autoimmunity to epitopes found on the post-translational modifications (PTMs) of native antigens. The first step of this method is to immobilize native antigens onto the monoclonal antibodies spotted on the array surface. Using the antigens captured by the microarray as 'baits,' we then incubate the array with differentially labeled IgG from test and control samples, and perform a two-slide dye-swap to normalize for dye effects. In this protocol we present a detailed description of the 'reverse capture' autoantibody microarray, a method that can be completed in 9-10 h over 1-2 d.