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Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays
Abstract
Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. This article is part of a Special Issue entitled: Translational Proteomics.
... Among the vast number of different cancer circulating biomarkers, altered proteins during tumor formation and progression that are able to induce a humoral response in cancer patients provide an effective, reliable, and noninvasive tool for cancer screening and preclinical diagnosis [31][32][33][34][35] . Although the molecular mechanisms and the subsequent production of autoantibodies against self-proteins of the humoral response in cancer are far from being known, it is reported that proteins present alterations, including alternative splicings, punctual mutations, truncations, overactivation, aberrant glycosylations, overexpression, and/or aberrant degradation 31,32 . ...
... Among the vast number of different cancer circulating biomarkers, altered proteins during tumor formation and progression that are able to induce a humoral response in cancer patients provide an effective, reliable, and noninvasive tool for cancer screening and preclinical diagnosis [31][32][33][34][35] . Although the molecular mechanisms and the subsequent production of autoantibodies against self-proteins of the humoral response in cancer are far from being known, it is reported that proteins present alterations, including alternative splicings, punctual mutations, truncations, overactivation, aberrant glycosylations, overexpression, and/or aberrant degradation 31,32 . ...
The p53-family is tightly regulated at transcriptional level. Due to alternative splicing, up to 40 different theoretical proteoforms have been described for p73 and at least 20 and 10 for p53 and p63, respectively. However, only the canonical proteins have been evaluated as autoantibody targets in cancer patients for diagnosis. In this study, we have cloned and expressed in vitro the most upregulated proteoforms of p73, ΔNp73α and ΔNp73β, for the analysis of their seroreactivity by a developed luminescence based immunoassay test using 145 individual plasma from colorectal cancer, premalignant individuals and healthy controls. ∆Np73α seroreactivity showed the highest diagnostic ability to discriminate between groups. The combination of ∆Np73α, ∆Np73β and p73 proteoforms seroreactivity were able to improve their individual diagnostic ability. Competitive inhibition experiments further demonstrated the presence of unique specific epitopes in ΔNp73 isoforms not present in p73, with several colorectal patients showing unique and specific seroreactivity to the ΔNp73 proteoforms. Overall, we have increased the complexity of the humoral immune response to the p53-family in cancer patients, showing that the proteoforms derived from the alternative splicing of p73 possess a higher diagnostic ability than the canonical protein, which might be extensive for p53 and p63 proteins.
... The choice of the platform and/or the solid support used to probe the protein can also affect the final result. It has been recently published that autoantigens perform differently if the protein is coated (ELISA), immobilized on membranes (WB or protein microarrays), or highly unfolded as is the case for the hEx1 proteins (Murphy et al. 2012a). As each cancer patient can develop a different immune response to protein regions presented as discontinuous, conformational, or linear epitopes, diagnostic tests should contain multiple TAAs for a full coverage of cancer patients (Lam et al. 2011;Murphy et al. 2012a). ...
... It has been recently published that autoantigens perform differently if the protein is coated (ELISA), immobilized on membranes (WB or protein microarrays), or highly unfolded as is the case for the hEx1 proteins (Murphy et al. 2012a). As each cancer patient can develop a different immune response to protein regions presented as discontinuous, conformational, or linear epitopes, diagnostic tests should contain multiple TAAs for a full coverage of cancer patients (Lam et al. 2011;Murphy et al. 2012a). The use of multiplexed immunoassays (magnetic beads or multiplexed ELISA) (Ling et al. 2007) or low-density protein microarrays (Liu et al. 2009) could be useful for the development of diagnostic tests. ...
Circulating CRC markers might become useful tools for the massive screening of people since they satisfy a high degree of accuracy, noninvasiveness, reproducibility, and economy. Within circulating biomarkers, we will focus on the detection of autoantibodies in serum from cancer patients and their target tumor-associated antigens (TAAs). Although the sensitivity, specificity, and predictive value of individual TAAs present low scores, the combination of multiple TAAs shows improved values to discriminate between
CRC patients and controls. In this review, we outline the methodologies used to identify circulating autoantibodies and their target proteins and discuss the relevance of CRC autoantibodies for diagnosis at, particularly, early stages. An overview of the reported biomarkers is given, showing the large complexity of the autoantibody response in cancer. Different strategies to improve CRC diagnostic tests by combining autoantibodies from different studies will be discussed. Association of autoantibodies to prognosis, recurrence, and the survival of patients will be introduced. We conclude that there is a great potential for the use of autoantibodies as diagnostic CRC biomarkers in the near future.
... Very little data are currently available on the prevalence of anti-p53 antibodies in patients with epithelial ovarian cancer [16]. Murphy et al. [17] used ELISA to detect anti-p53 antibody in sera in normal heath individuals and ovarian cancer patients, however, the distribution of anti-p53 antibody in the two groups showed no significant difference. Additionally, Høgdall et al. [18] found that the frequency of anti-p53 antibodies in ovarian cancer patients increased somewhat with increasing clinical stage of disease, but the differences did not reach statistical significance. ...
Objectives:
The tests conducted were intended to analyze the concentration of p53 protein and anti-p53 autoantibodies in serum of women with ovarian tumours.
Material and methods:
The study included patients with diagnosed ovarian cancer: Cystadenoma serosum or Cystadenocarcinoma papillare serosum at IIIc stage (including 10 women who had G1, 14 women who had G2 and 30 women who had G3 staging). Concentrations of parameters were measured by ELISA.
Results:
The analysis of the obtained results showed statistical significance between the concentration of p53 protein depending on the degree of differentiation of G1 and G3 (p < 0.001) and anti-p53 autoantibodies depending on the degree of differentiation of G1 and G2 (p < 0.05) as well as G2 and G3 (p < 0.01). In addition, the determined p53/anti-p53 autoantibodies ratio was only significant between G1 and G2 (p < 0.05), as was the assessment of the percentage of the tested parameters in the immune complex.
Conclusions:
Immune system disorders involving the p53 protein and anti-p53 autoantibodies may be one of the immune mechanisms involved in the pathogenesis of ovarian serous cancer.
... Results from multiplex TAAB profiling using different platforms (e.g., ELISA, magnetic beads, protein arrays) should be compared to account for the possibility that techniqueassociated exposure, inaccessibility, or destruction of TAA epitopes influences the AAB response to a TAA panel in a particular cancer type. 16,17 Also, differences in the stringency of cut-off values used for exclusion or inclusion of a positive antibody reaction can significantly influence the frequency of detected TAABs as well as the sensitivity and specificity of a given TAAB panel. ...
Testing for autoantibodies (AABs) is becoming more and more relevant, not only for diagnosing autoimmune diseases (AIDs) but also for the differentiation of defined AID subtypes with different clinical manifestations, course and prognosis as well as the very early diagnosis for adequate management in the context of personalized medicine. A major challenge to improve diagnostic accuracy is to harmonize or even standardize AAB analyses. This review presents the results of the 12th Dresden Symposium on Autoantibodies that focused on several aspects of improving autoimmune diagnostics. Topics that are addressed include the International Consensus on ANA Patterns (ICAP) and the International Autoantibody Standardization (IAS) initiatives, the optimization of diagnostic algorithms, the description and evaluation of novel disease-specific AABs as well as the development and introduction of novel assays into routine diagnostics. This review also highlights important developments of recent years, most notably the improvement in diagnosing and predicting the course of rheumatoid arthritis, systemic sclerosis, idiopathic inflammatory myopathies, and of autoimmune neurological, gastrointestinal and liver diseases; the potential diagnostic role of anti-DFS70 antibodies and tumor-associated AABs. Furthermore, some hot topics in autoimmunity regarding disease pathogenesis and management are described.
... This may be detectable well in advance of clinically detected disease using current conventional diagnostic techniques. 7 AAbs to TAAs could represent novel biomarkers for cancer screening, diagnosis, prognosis, monitoring, and prediction of response to chemotherapy. The challenge is how to measure and interpret these changes among cancer specific AAbs and develop an assay and algorithm for an accurate, low-risk tool for the diagnosis of cancer. ...
In order to develop a new tool for diagnosis of breast cancer based on autoantibodies against a panel of biomarkers, a clinical trial including blood samples from 507 subjects was conducted. All subjects showed a breast abnormality on exam or breast imaging and final biopsy pathology of either breast cancer patients or healthy controls. Using an enzyme-linked immunosorbent assay, the samples were tested for autoantibodies against a predetermined number of biomarkers in various models that were used to determine a diagnosis, which was compared to the clinical status. Our new assay achieved a sensitivity of 95.2% [CI = 92.8-96.8%] at a fixed specificity of 49.5%. Receiver-operator characteristic curve analysis showed an area under the curve of 80.1% [CI = 72.6-87.6%]. These results suggest that a blood test which is based on models comprising several autoantibodies to specific biomarkers may be a new and novel tool for improving the diagnostic evaluation of breast cancer.
... In a recent review Murphy et al [46] have observed that due to the changing repertoire of the antigens as the tumor progresses, there is probably a constant qualitative and quantitative change in the autoantibody response to an antigen. In addition the autoantibody response will depend on the technique used to assess it since the immune response could be to a sequence in the linear protein or to an epitope presented as a consequence of the folding of the native protein [47]. These factors along with differential expression of an antigen, its post translational modifications and its relocation would provide a complex pattern which has to be deciphered to explain the autoantibody response profiles obtained for the different antigens. ...
Purpose:
Studies from our laboratory have reported 14 tumor antigens that elicit an autoantibody response in patients with cancer of the gingivobuccal complex (GBC) In this study, utility of the autoantibody response has been evaluated for prognosis of cancer of the GBC.
Experimental design:
Autoantibody response was evaluated using immunoproteomics and the prognostic significance was assessed by Kaplan-Meier survival and multivariate analysis.
Results:
Autoantibody response against α-enolase isoforms a, b, and c and Hsp70 was detected in 27, 53, 64, and 26% of the 78 patients, respectively. Patients positive for autoantibody response to α-ENO and Hsp70 individually and in combination, showed significantly reduced disease-free survival (DFS) compared to those who do not show autoantibody response to either of them. Further the patients, who exhibit autoantibody response to α-ENO and Hsp70 in combination with nodal involvement and/or differentiation status, have significantly lowered DFS. The relative risk of recurrence is 3.41 for patients who exhibit autoantibody response to both the antigens.
Conclusions and clinical relevance:
Autoantibody response against α-ENO and Hsp70 provides an additional parameter and may be utilized along with nodal involvement and differentiation status for better prognosis of cancer of GBC.
Introduction:
Despite advances in surgery and chemotherapy for ovarian cancer, 70% of women still succumb to the disease. Biomarkers have contributed to the management of ovarian cancer by monitoring response to treatment, detecting recurrence, distinguishing benign from malignant pelvic masses and attempting to detect disease at an earlier stage. Areas Covered: This review focuses on recent advances in biomarkers and imaging for management of ovarian cancer with particular emphasis on early detection. Relevant literature has been reviewed and analyzed. Expert commentary: Rising or persistent CA125 blood levels provide a highly specific biomarker for epithelial ovarian cancer, but not an optimally sensitive biomarker. Addition of HE4, CA 72.4, anti-TP53 autoantibodies and other biomarkers can increase sensitivity for detecting early stage or recurrent disease. Detecting disease recurrence will become more important as more effective therapy is developed. Early detection will require the development not only of biomarker panels, but also of more sensitive and specific imaging strategies. Effective biomarker strategies are already available for distinguishing benign from malignant pelvic masses, but their use in identifying and referring patients with probable ovarian cancer to gynecologic oncologists for cytoreductive operations must be encouraged.
Methods:
To detect TAAb we probed high-density programmable protein microarrays (NAPPA) containing 10,247 antigens with sera from patients with serous ovarian cancer (n=30 cases/30 healthy controls) and measured bound IgG. We identified 735 promising tumor antigens and evaluated these with an independent set of serous ovarian cancer sera (n=30 cases/30 benign disease controls/30 healthy controls). Thirty-nine potential tumor autoantigens were identified and evaluated using an orthogonal programmable ELISA platform against a total of 153 sera samples (n=63 cases/30 benign disease controls/60 healthy controls). Sensitivities at 95% specificity were calculated and a classifier for the detection of high-grade serous ovarian cancer was constructed.
Results:
We identified 11-TAAbs (ICAM3, CTAG2, p53, STYXL1, PVR, POMC, NUDT11, TRIM39, UHMK1, KSR1, and NXF3) that distinguished high-grade serous ovarian cancer cases from healthy controls with a combined 45% sensitivity at 98% specificity.
Conclusion:
These are potential circulating biomarkers for the detection of serous ovarian cancer, and warrant confirmation in larger clinical cohorts.
African American (AA) men suffer from a disproportionately high incidence and mortality of prostate cancer (PCa) compared to other racial/ethnic groups. Despite these disparities, African American men are underrepresented in clinical trials and in studies on PCa biology and biomarker discovery. We used immunoseroproteomics to profile anti-tumor autoantibody responses in AA and European American (EA) men with PCa, and explored differences in these responses. This minimally invasive approach detects autoantibodies to tumor-associated antigens that could serve as clinical biomarkers and immunotherapeutic agents. Sera from AA and EA men with PCa were probed by immunoblotting against PC3 cell proteins, with AA sera showing stronger immunoreactivity. Mass spectrometry analysis of immunoreactive protein spots revealed that several AA sera contained autoantibodies to a number of proteins associated with both the glycolysis and plasminogen pathways, particularly to alpha-enolase (ENO1). The proteomic data was deposited in ProteomeXchange with identifier PXD003968. Analysis of sera from 340 racially diverse men by enzyme-linked immunosorbent assays (ELISA) showed higher frequency of anti-ENO1 autoantibodies in PCa sera compared to control sera. We observed differences between AA-PCa and EA-PCa patients in their immunoreactivity against ENO1. While EA-PCa sera reacted with higher frequency against purified ENO1 in ELISA and recognized by immunoblotting the endogenous cellular ENO1 across a panel of prostate cell lines, AA-PCa sera reacted weakly against this protein by ELISA but recognized it by immunoblotting preferentially in metastatic cell lines. These race-related differences in immunoreactivity to ENO1 could not be accounted by differential autoantibody recognition of phosphoepitopes within this antigen. Proteomic analysis revealed differences in the posttranslational modification profiles of ENO1 variants differentially recognized by AA-PCa and EA-PCa sera. These intriguing results suggest the possibility of race-related differences in the anti-tumor autoantibody response in PCa, and have implications for defining novel biological determinants of PCa health disparities.
Purpose:
Mutations in TP53 induce autoantibody immune responses in a subset of cancer patients, which have been proposed as biomarkers for early detection. Here, we investigate the association of p53 specific autoantibodies with multiple tumor subtypes and determine the association with p53 mutation status and epitope specificity.
Experimental design:
IgG p53 autoantibodies (p53-AAb), were quantified in 412 serum samples using a programmable ELISA assay from patients with serous ovarian, pancreatic adenocarcinoma, and breast cancer. To determine if patients generated mutation specific autoantibodies we designed a panel of the most relevant 51 p53 point mutant proteins, to be displayed on custom programmable protein microarrays. To determine the epitope specificity we displayed 12 overlapping tiling fragments and 38 N- and C-terminal deletions spanning the length of the wild-type p53 protein.
Results:
We detected p53-AAb with sensitivities of 58.8% (ovarian), 22% (pancreatic), 32% (triple negative breast cancer), and 10.2% (HER2+ breast cancer) at 94% specificity. Sera with p53-AAb contained broadly-reactive autoantibodies to 51 displayed p53 mutant proteins, demonstrating a polyclonal response to common epitopes. All p53-AAb displayed broad polyclonal immune response to both continuous and discontinuous epitopes at the N- and C-terminus as well as the DNA binding domain.
Conclusion and clinical relevance:
In this comprehensive analysis, mutations in tumor p53 induce strong, polyclonal autoantibodies with broadly reactive epitope specificity. This article is protected by copyright. All rights reserved.
This systematic review aims to outline and summarize known tumor-associated antigens (TAAs) or anti-TAA autoantibodies and their diagnostic values in ovarian cancer (OC).
A systematic literature search was conducted in two databases. Data were independently extracted and reviewed by two junior investigators and the disagreement was further resolved by consulting one of the senior investigators.
Sixty publications reporting 113 different TAAs or anti-TAA autoantibodies were included. The majority of the studies were conducted in western countries. CA125, p53 and HE4 were the most frequently tested TAAs in OC. Meta-analysis showed that there was a significant association between serum anti-p53 autoantibody and increased risk of OC.
Serum TAAs or anti-TAA autoantibodies are promising diagnostic biomarkers in the detection of OC. A customized mini-array of multiple TAAs may enhance the detection of anti-TAA autoantibodies in OC. Additional studies with sufficient number of OC patients and carefully selected controls are needed to further verify the results.
Colorectal cancer is one of the most common cancers worldwide with almost 700,000 deaths every year. Detection of colorectal cancer at an early stage significantly improves patient survival. Cancer-specific autoantibodies found in sera of cancer patients can be used for pre-symptomatic detection of the disease. In this study we assess the zinc finger proteins ZNF346, ZNF638, ZNF700 and ZNF768 as capture antigens for the detection of autoantibodies in colorectal cancer. Sera from 96 patients with colorectal cancer and 35 control patients with no evidence of cancer on colonoscopy were analysed for the presence of ZNF-specific autoantibodies using an indirect ELISA. Autoantibodies to individual ZNF proteins were detected in 10-20% of colorectal cancer patients and in 0-5.7% of controls. A panel of all four ZNF proteins resulted in an assay specificity of 91.4% and sensitivity of 41.7% for the detection of cancer patients in a cohort of non-cancer controls and colorectal cancer patients. Clinicopathological and survival analysis revealed that ZNF autoantibodies were independent of disease stage and did not correlate with disease outcome. Since ZNF autoantibodies were shared between patients and corresponding ZNF proteins showed similarities in their zinc finger motifs, we performed an in silico epitope sequence analysis. Zinc finger proteins ZNF700 and ZNF768 showed the highest sequence similarity with a bl2seq score of 262 (E-value 1E-81) and their classical C2H2 ZNF motifs were identified as potential epitopes contributing to their elevated immunogenic potential. Our findings show an enhanced and specific immunogenicity to zinc finger proteins, thereby providing a multiplexed autoantibody assay for minimally invasive detection of colorectal cancer.
Ovarian cancer is the most lethal gynecological malignancy and survival of this disease has remained relatively unchanged over the past 30 years. A contributing factor to this has been the lack of reliable biomarkers for the clinical management of ovarian cancer. Rapid advances in high-throughput technologies over the past decade has allowed for new and exciting opportunities for biomarker discovery in the field of ovarian cancer, especially with respect to serum biomarkers that can be used for various clinical applications. This review highlights the major genomic and proteomic studies dedicated to ovarian cancer biomarker discovery over the past decade. An emphasis will be placed on the HE4, Risk of Malignancy Algorithm (ROMA) and OVA1™ serum-based tests/algorithms that have recently been approved by the US FDA as ovarian cancer biomarkers.
Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.
Antibody-based microarrays are a rapidly evolving affinity-proteomic methodology that recently has shown great promise in clinical applications. The resolution of these proteomic analyses is, however, directly related to the number of data-points, i.e. antibodies, included on the array. Currently, this is a key bottleneck because of limited availability of numerous highly characterized antibodies. Here, we present a conceptually new method, denoted global proteome survey, opening up the possibility to probe any proteome in a species-independent manner while still using a limited set of antibodies. We use context-independent-motif-specific antibodies directed against short amino acid motifs, where each motif is present in up to a few hundred different proteins. First, the digested proteome is exposed to these antibodies, whereby motif-containing peptides are enriched, which then are detected and identified by mass spectrometry. In this study, we profiled extracts from human colon tissue, yeast cells lysate, and mouse liver tissue to demonstrate proof-of-concept.
Autoantibodies, a hallmark of both autoimmunity and cancer, represent an easily accessible surrogate for measuring adaptive immune responses to cancer. Sera can now be assayed for reactivity against thousands of proteins using microarrays, but there is no agreed-upon standard to analyze results. We developed a set of tailored quality control and normalization procedures based on ELISA validation to allow patient comparisons and determination of individual cutoffs for specificity and sensitivity. Sera from 60 patients with pancreatic cancer, 51 patients with ovarian cancer, and 53 age-matched healthy donors were used to assess the binding of IgG antibodies against a panel of >8000 human antigens using protein microarrays and fluorescence detection. The resulting data interpretation led to the definition and ranking of proteins with preferred recognition by the sera from cancer patients in comparison with healthy donors, both by frequency and strength of signal. We found that 202 proteins were preferentially immunogenic in ovarian cancer sera compared to 29 in pancreatic cancer, with few overlaps. Correlates of autoantibody signatures with known tumor expression of corresponding antigens, functional pathways, clinical stage, and outcome were examined. Serological analysis of arrays displaying the complete human proteome (seromics) represents a new era in cancer immunology, opening the way to defining the repertoire of the humoral immune response to cancer.
CA125 is the gold standard tumor marker in ovarian cancer. Serum level of CA125 is used to monitor response to chemotherapy, relapse, and disease progression in ovarian cancer patients. Thus, it is reasonable to investigate whether CA125 may have utility as a prognostic indicator as well in ovarian cancer. A large number of epidemiological studies have been carried out to this effect. This review summarizes all available epidemiological literature on the association between CA125 levels and survival in ovarian cancer. To place these studies in context, we provide some background information on CA125 and its role in ovarian cancer.
Knowledge of mucins and their multiple roles in various normal and pathological processes has improved greatly in the past two decades. Mucins belong to a family of glycoproteins characterised by densely O-glycosylated repetitive domains and expressed by various surface epithelial cells. Altered expression of mucins is present in various diseases, including cancer. Ovarian cancer is the sixth most common cancer worldwide and the seventh leading cause of cancer-related deaths in women. The most common ovarian cancer is epithelial ovarian carcinoma, which is characterised by few early symptoms, widespread peritoneal dissemination, and ascites at advanced stages that result in poor prognosis. After diagnosis, 5 year survival is only 35-45%. Therefore, improved strategies for early diagnosis and treatment are needed. Because of the surface epithelial origin of epithelial ovarian cancer, mucins are obvious biomolecules for investigation as markers for early diagnosis and as therapeutic targets. We discuss the potential role and clinical usefulness of mucins in early diagnosis, prognosis, and treatment of ovarian cancer.
Alteration of the p53 gene is the most frequent genetic feature of human cancer and leads to overexpression of the altered protein in the tumor cell nucleus. Two diagnostic procedures are currently available to assess p53 mutations: (a) molecular analysis of the gene sequence; and (b) immunohistochemical analysis of p53 protein accumulation. We now report a third approach, serological analysis. Fifteen % of primary breast cancer patients were found to have circulating antibodies to p53 protein by immunoprecipitation or immunoblotting. We have found a close correlation between the presence of such antibodies and bad prognosis such as high histological grade and the absence of hormone receptors. Furthermore, we found that the B-cell response to p53 protein is induced by two immunodominant regions located at the carboxy and amino termini of the protein, outside the central mutational hot spot region. These findings suggest that serological analysis, combined with molecular and histochemical methods, may be suitable for assessing the state of the p53 gene in cancer patients.
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation
or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible
expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded
onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6 sequence of expressed proteins (RGS·His antibody, Qiagen) detected 20% of the library as putative expression clones. Two
example genes, GAPDH and HSP90α, were identified on high-density filters using DNA probes and antibodies against their proteins.
p53 antibodies (p53-Abs) were discovered 20 years ago during the course of tumor-associated antigens screening. The discovery of p53 mutation and accumulation of p53 in human tumors shed new light on the p53 humoral response. In the present review, we have compiled more than 130 papers published in this specific field since 1992. We demonstrate that p53-Abs are found predominantly in human cancer patients with a specificity of 96%. Such antibodies are predominantly associated with p53 gene missense mutations and p53 accumulation in the tumor, but the sensitivity of such detection is only 30%. It has been demonstrated that this immune response is due to a self-immunization process linked to the strong immunogenicity of the p53 protein. The clinical value of these antibodies remains subject to debate, but consistent results have been observed in breast, colon, oral, and gastric cancers, in which they have been associated with high-grade tumors and poor survival. The finding of p53-Abs in the sera of individuals who are at high risk of cancer, such as exposed workers or heavy smokers, indicates that they have promising potential in the early detection of cancer.
Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.
Objective Four breast cancer subtypes defined by immunohistochemistry (IHC) expression of estrogen receptor (ER) or progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) are ER/PR+,Her2+; ER/PR+,Her2-; ER/PR-,Her2+; and ER/PR-,Her2-. Methods A 7-year retrospective study of 1134 invasive breast cancer subjects. Clinical and pathologic features and survival of the four subtypes were compared. Results Using ER/PR+ and Her2- as a reference, ER/PR-,Her2- had worst overall survival (hazard ratio, 1.8; 95% confidence interval [CI], 1.06-3.2), and worst disease-free survival (hazard ratio, 1.5; 95% CI, 0.8-3.0). In ER/PR+,Her2-, chemotherapy conferred significant overall and disease-free survival advantages. Subtype comparison revealed statistically significant differences in outcomes. Conclusion Triple negative subtype having worst overall and disease free survival. Efforts should be directed at standardization of current testing methods and development of more reliable and reproducible testing.
Background
No large-scale studies have examined the use of serial measurements of serum p53 antibodies (s-p53Abs) combined with carcinoembryonic antigen (CEA) measurements during the follow-up of colorectal cancer (CRC) patients after curative resection.
Methods
A highly specific enzyme-linked immunosorbent assay was used to analyze s-p53Abs levels in 305 CRC patients before and after curative resection at a single institution. Agreement between recurrence and serial s-p53Ab and CEA measurements was evaluated by diagnostic accuracy odds ratio (DOR), kappa, and area under receiver operating characteristic curve (AUC).
Results
Among 305 patients, 76 (25%) patients had disease recurrence during follow-up. None of the 168 s-p53Ab seronegative patients (s-p53Ab < 10 U/μL) without recurrence had an abnormal s-p53Ab test during follow-up. Among the remaining low-level (10 U/μL ≤ s-p53Ab ≤ 76 U/μL, n = 103) and high-level (s-p53Ab titer > 76 U/μL, n = 34) seropositive patients, recurrence defined by s-p53Ab tests resulted in a DOR of 4.3 and ∞, a kappa of 0.35 and 1.00, and an AUC of 0.633 [95% confidence interval (CI), 0.495 to 0.772; P = 0.047], and 1.0 (95% CI, 1.000 to 1.000; P < 0.0001), respectively. Recurrence defined by CEA tests had an AUC of 0.781 (95% CI, 0.654 to 0.909) for low-level and 0.796 (95% CI, 0.611 to 0.982) for high-level seropositive patients.
Conclusions
Agreement between clinical recurrence and serial s-p53Ab test was dependent upon preoperative s-p53Ab level. Serial s-p53Ab testing outperformed CEA testing when predicting clinical recurrence in colorectal cancer patients with an abnormal preoperative s-p53Ab level.
The calcium ion (Ca(2+)) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca(2+) are mediated by intracellular -Ca(2+)-binding proteins. Calcium ions shuttle into and out of the cytosol, transported across membranes by channels, exchangers, and pumps that regulate flux across the ER, mitochondrial and plasma membranes. Calcium regulates both rapid events, such as cytoskeleton remodelling or release of vesicle contents, and slower ones, such as transcriptional changes. Moreover, sustained cytosolic calcium elevations can lead to unwanted cellular activation or apoptosis. Calmodulin represents the most significant of the Ca(2+)-binding proteins and is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile novel protein-protein interactions that calmodulin participates in, we probed a high-content recombinant human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human proteins expressed from a human brain cDNA library. We describe the identification of a high affinity interaction between calmodulin and the single-pass transmembrane proteins STIM1 and STIM2 that localise to the ER. Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store operated calcium entry in the cell.
Secretagogin is a hexa EF-hand Ca(2+)-binding protein expressed in neuroendocrine, pancreatic endocrine and retinal cells. The protein has been noted for its expression in specific neuronal subtypes in the support of hierarchical organizing principles in the mammalian brain. Secretagogin has previously been found to interact with SNAP25 involved in Ca(2+)-induced exocytosis. Here, the cellular interaction network of secretagogin has been expanded with nine proteins: SNAP-23, DOC2alpha, ARFGAP2, rootletin, KIF5B, β-tubulin, DDAH-2, ATP-synthase and myeloid leukemia factor 2, based on screening of a high content protein array and validation and quantification of binding with surface plasmon resonance and GST pulldown assays. All targets have association rate constants in the range 10(4)-10(6) M(-1) s(-1), dissociation rate constants in the range 10(-3)-10(-5) s(-1) and equilibrium dissociation constants in the 100 pM to 10 nM range. The novel target SNAP23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion. Complementary roles in vesicle trafficking are known for ARFGAP2 and DOC2alpha in regulating fusion of vesicles to membranes, kinesin 5B and tubulin for transport of vesicles in the cell, while rootletin builds up the rootlet believed to function as a scaffold for vesicles. The identification of a discrete network of interacting proteins that mediate secretion and vesicle trafficking suggests a regulatory role for secretagogin in these processes.
Mutations in the TP53 (p53) gene are present in a large fraction of human tumours, which frequently express mutant p53 proteins at high but heterogeneous levels. The clinical significance of this protein accumulation remains clouded. Mouse models bearing knock-in mutations of p53 have established that the mutant p53 proteins can drive tumour formation, invasion and metastasis through dominant negative inhibition of wild-type p53 as well as through gain of function or 'neomorphic' activities that can inhibit or activate the function of other proteins. These models have also shown that mutation alone does not confer stability, so the variable staining of mutant proteins seen in human cancers reflects tumour-specific activation of p53-stabilizing pathways. Blocking the accumulation and activity of mutant p53 proteins may thus provide novel cancer therapeutic and diagnostic targets, but their induction by chemotherapy may paradoxically limit the effectiveness of these treatments.
To compare the clinicopathologic features and survival in the four breast cancer subtypes defined by immunohistochemistry (IHC) expression of estrogen receptor (ER) or progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2): ER/PR+, Her2+; ER/PR+, Her2-; ER/PR-, Her2+; and ER/PR-, Her2-.
A 7-year retrospective study of 1134 invasive breast cancer subjects. Clinical and pathologic features and survival of the four subtypes were compared.
Using ER/PR+ and Her2- as a reference, ER/PR-, Her2- had the worst overall survival (hazard ratio, 1.8; 95% confidence interval [CI], 1.06-3.2) and the worst disease-free survival (hazard ratio, 1.5; 95% CI, 0.8-3.0). In ER/PR+, Her2-, chemotherapy conferred significant overall and disease-free survival advantages. Subtype comparison revealed statistically significant differences in outcomes.
The triple negative subtype has the worst overall and disease free survival. Efforts should be directed at standardization of current testing methods and development of more reliable and reproducible testing.
To address the hypothesis that type II ovarian carcinoma, mutation of p53 and plasma levels of particular cytokines are associated with the generation of p53-specific serum autoantibody (AAb) responses in patients.
Levels of CA125, 17 cytokines and AAbs to tumor-associated antigens including p53 were measured in plasma of 130 gynecologic tumor patients and 84 healthy controls. TP53 exons 4-9 were sequenced in tumor specimens.
p53 AAbs are associated with high grade, but not low grade ovarian carcinoma. Seropositivity for p53 AAb occurred only in those ovarian carcinoma patients whose tumors contained mutated TP53, regardless of the exon targeted. Higher p53 AAb levels were detected in ovarian carcinoma patients who had higher stage disease, but p53 AAb levels were not correlated with CA125 levels. Among high-grade carcinoma patients, there was no relationship between p53 AAb seropositivity and seropositivity to other tumor-associated antigens tested, CA125 level or survival outcome. Both high and low grade ovarian carcinoma patients exhibited elevated levels of IL6, IL8 and IL10 as compared to healthy volunteers, although increased levels of IL5, MCP1, MIP1 and TNFalpha were associated only with high grade and advanced disease. Higher levels of p53AAb responses were correlated with elevated circulating IL4 and IL12, but reduced IL8 levels.
Type II, but not type I, ovarian carcinoma patients had elevated serum levels of p53 AAb. P53 AAb is associated with mutation of TP53, higher plasma IL4 and IL12 but lower plasma IL8 levels and no survival advantage.
Despite substantial progress in the understanding of the pathogenesis of cancer, the development and implementation of strategies for early cancer detection have lagged behind. Harnessing the immune response to tumor antigens is particularly useful for early detection because the immune response occurs early during tumor development and affords signal amplification with the end product, namely reactive immunoglobulins, being released into the circulation allowing easy access through the blood. This article presents recent developments in autoantibody profiling with a focus on proteomic approaches and applications to lung cancer.
Ovarian carcinomas are a heterogeneous group of neoplasms and are traditionally subclassified based on type and degree of differentiation. Although current clinical management of ovarian carcinoma largely fails to take this heterogeneity into account, it is becoming evident that each major histological type has characteristic genetic defects that deregulate specific signaling pathways in the tumor cells. Moreover, within the most common histological types, the molecular pathogenesis of low-grade versus high-grade tumors appears to be largely distinct. Mouse models of ovarian carcinoma have been developed that recapitulate many of the morphological features, biological behavior, and gene-expression patterns of selected subtypes of ovarian cancer. Such models will likely prove useful for studying ovarian cancer biology and for preclinical testing of molecularly targeted therapeutics, which may ultimately lead to better clinical outcomes for women with ovarian cancer.
The display of a random peptide library on the surface of phage has been applied to the discovery of antigens specific to prostate cancer.
Because TP53 mutations can induce an immune response and can occur early in the carcinogenic process for some tumors, p53 autoantibodies may be useful biomarkers for risk of development of cancer. Using banked serum samples from an asbestosis cohort at high risk for cancer, we demonstrate for the first time a statistically significant relationship between p53 autoantibodies and the subsequent development of malignancy (hazard ratio [HR] = 5.5, 95% confidence interval [CI] = 2.8-10.9) with a positive predictive value of 0.76 and an average lead time to diagnosis of 3.5 years. p53 autoantibodies were also significantly associated with p53 alterations in the resultant tumors (kappa = 0.78, p = 0.01).
Autoantibodies are often detected in the patients with esophageal cancer. We applied serological analysis of recombinant cDNA expression libraries (SEREX) to a case of esophageal squamous cell carcinoma in order to identify tumor antigens. A cDNA library derived from an esophageal cancer cell line was bacterially expressed and screened for interaction with antibodies in five allogeneic sera of patients with esophageal squamous cell carcinoma. To examine the specific immunoreactivity of the antigens, sera from 16 more patients with esophageal squamous cell carcinoma, 16 patients with gastric cancer, 16 patients with colon cancer, 16 patients with breast cancer and 37 healthy volunteers were screened. We identified 11 independent cDNA clones that potentially encoded esophageal cancer tumor antigens. The identified cDNA clones were SURF1, HOOK2, CENP-F, ZIC2, hCLA-iso, Ki-1/57, enigma, HCA25a, SPK and two EST clones named LOC146223 and AGENCOURT_7565913. The sero-positive rates of antibodies against SURF1 (48%), LOC146223 (38%), HOOK2 (14%) and AGENCOURT_7565913 (14%) were significantly higher in esophageal cancer patients than in healthy controls. At least one of these antibodies was detected in 18 (86%) of 21 sera from esophageal cancer patients. A disease-specific humoral immune response against SURF1, LOC146223, HOOK2 or AGENCOURT_7565913 was observed in most patients with esophageal squamous cell carcinoma. Antibodies against these SEREX antigens may represent a pool of candidates for serum tumor markers of esophageal squamous cell carcinoma.
Patients with ovarian cancer usually present with advanced disease, and the disease is generally managed with surgical resection followed by platinum-based chemotherapy. Recent chemotherapeutic advances have led to improved survival, and a better understanding of genetic risk factors has permitted a tailored approach to preventive strategies, such as bilateral salpingo-oophorectomy in selected women. This review describes the clinical features of ovarian cancer and recent advances in postoperative management.
The release of proteins from tumors triggers an immune response in cancer patients. These tumor antigens arise from several mechanisms including tumor-specific alterations in protein expression, mutation, folding, degradation, or intracellular localization. Responses to most tumor antigens are rarely observed in healthy individuals, making the response itself a biomarker that betrays the presence of underlying cancer. Antibody immune responses show promise as clinical biomarkers because antibodies have long half-lives in serum, are easy to measure, and are stable in blood samples. However, our understanding of the specificity and the impact of the immune response in early stages of cancer is limited. The immune response to cancer, whether endogenous or driven by vaccines, involves highly specific T lymphocytes (which target tumor-derived peptides bound to self-MHC proteins) and B lymphocytes (which generate antibodies to tumor-derived proteins). T cell target antigens have been identified either by expression cloning from tumor cDNA libraries, or by prediction based on patterns of antigen expression ("reverse immunology"). B cell targets have been similarly identified using the antibodies in patient sera to screen cDNA libraries derived from tumor cell lines. This review focuses on the application of recent advances in proteomics for the identification of tumor antigens. These advances are opening the door for targeted vaccine development, and for using immune response signatures as biomarkers for cancer diagnosis and monitoring.
Breast cancer has been the most common malignant tumor among women in many large cities of China. The aim of this study was to clarify the prognostic significance of serum anti-p53 antibodies (p53 Abs) in Chinese patients of breast cancer. One hundred and forty-four patients with invasive ductal carcinoma of breast were involved in this study. The expressions of ER, PR, c-erbB-2 and p53 were immunostained in tumor tissues and serum p53 Abs were assayed using ELISA method. The correlations between p53 Abs and other clinical and biological markers were analyzed. Among 144 patients, 31 (21.5%) had positive p53 Abs, which was associated with several poor prognostic parameters including higher clinical stage (p = 0.0233), lymph nodes metastasis (p = 0.0033), negative ER expression (p = 0.0250) and positive c-erbB-2 status (p = 0.0227). There was also a strong correlation between p53 Abs and tumor p53 positivity (p < 0.0001). These results indicated that the presence of p53 Abs is probably triggered by the accumulation of tumor p53 protein, and it could be a useful marker to complement routine prognostic factors in breast cancer patients.
A variety of biomarkers have been developed to monitor growth of ovarian cancer and to detect disease at an early interval. CA125 (MUC16) has provided a useful serum tumor marker for monitoring response to chemotherapy, detecting disease recurrence, distinguishing malignant from benign pelvic masses, and potentially improving clinical trial design. A rapid fall in CA125 during chemotherapy predicts a favorable prognosis and could be used to redistribute patients on multiarmed randomized clinical trials. Several studies now document that CA125 can serve as a surrogate marker for response in phase II trials. Serial measurement of CA125 might also provide a useful marker for monitoring stabilization of disease with cytostatic targeted therapeutic agents. The greatest potential for serum markers may be in detecting ovarian cancer at an early stage. A rising CA125 can be used to trigger transvaginal sonography (TVS) in a small fraction of patients. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risk for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the UK that will test critically the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, as 20% of ovarian cancers have little or no expression of CA125, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125 by different investigators. Some of the most promising include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s), and soluble EGF receptor. Two proteomic approaches have been used: one examines the pattern of peaks on mass spectroscopy and the other uses proteomic analysis to identify a limited number of critical markers that can be assayed by more conventional methods. Both approaches are promising and require further development. Several groups are placing markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.
Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.
p53 mutations are found in 50% of human cancers. Molecular epidemiology has shown strong correlations between the spectrum of p53 mutations and exposure to exogenous carcinogens. This spectrum is influenced quantitatively and qualitatively by various upstream genetic filters that modulate carcinogen activation, detoxification, and/or DNA repair. In this review, we will discuss how other factors such as tissue specificity, SNP of genes associated with the p53 pathway, other genetic alterations, or p53 mutant heterogeneity can act as a second set of downstream filters that also have a profound impact on the spectrum of p53 mutations.
There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum; may exist in greater concentrations than their cognate antigens; and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.
The purpose of the present study was to screen the autoantibody signature of colon cancers to develop serum markers for colon cancer detection.
A phage cDNA expression library of colon cancer was built. The library was sequentially screened by a pool of 10 colon cancer sera, goat antihuman IgG, and a pool of two healthy sera to identify phage-expressed antigens recognized by tumor-associated antibodies. The clones picked out by these screening were subjected to a training set with 24 colon cancer sera and 24 healthy sera. The antigen combination, which got the most satisfactory classification, was tested by an independent set of 24 colon cancer sera with equal number of sera from normal donors. The carcinoembryonic antigen (CEA) level of these sera was detected for the additional classification analysis with or without the antigen combination.
A cDNA expression library consisting of 2 x 10(6) primary clones was prepared. After three turns of screening, 24 antigens recognized by tumor-associated antibodies were picked out for serum marker identification. The training set showed that a six-marker combination got the most satisfactory classification in a logistic regression model; leave-one-out validation achieved 91.7% sensitivity and 91.7% specificity. In a testing set with this marker panel, we correctly predicted 85% of the samples. Although according to CEA level alone, we correctly predicted 75% of the samples with 42% of cancer patients misclassified. When CEA was combined with the six markers, the sensitivity and specificity increased to 91.7% and 95.8%, respectively. The six antigen sequences in the phage display system are relatively short peptides. Only two of them showed homology to known protein sequences.
Autoantibodies against phage-expressed antigens derived from colon cancer tissues could be used as serum markers for the detection of colon cancer.
A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library 0 1 2 ) 4 6 6 8 – 4 6 7 5 r[19] Lueking A Profiling of alopecia areata autoantigens based on protein microarray technology
- K Bussow
- D Cahill
- W Nietfeld
- D Bancroft
- E Scherzinger
- H Lehrach
Bussow K, Cahill D, Nietfeld W, Bancroft D, Scherzinger E, Lehrach H, et al. A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library. Nucleic Acids Res 1998;26:5007–8. 4674 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 4 6 6 8 – 4 6 7 5 r[19] Lueking A, Huber O, Wirths C, Schulte K, Stieler KM, Blume-Peytavi U, et al. Profiling of alopecia areata autoantigens based on protein microarray technology. Mol Cell Proteomics 2005;4:1382–90.
p53 autoantibodies, cytokine levels and ovarian carcinogenesis United States
- M Tsai-Turton
- A Santillan
- D Lu
- Re Bristow
- Shih Kc Chan
- Iem
Tsai-Turton M, Santillan A, Lu D, Bristow RE, Chan KC, Shih IeM, et al. p53 autoantibodies, cytokine levels and ovarian carcinogenesis. Gynecol Oncol 2009:12–7 United States. 4675 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 4 6 6 8 – 4 6 7 5
The immune response to p53 in breast cancer patients is directed against immunodominant epitopes unrelated to the mutational hot spot
- B Schlichtholz
- Y Legros
- D Gillet
- C Gaillard
- Marty M Lane
Schlichtholz B, Legros Y, Gillet D, Gaillard C, Marty M, Lane D,
et al. The immune response to p53 in breast cancer patients is
directed against immunodominant epitopes unrelated to the
mutational hot spot. Cancer Res 1992;52:6380–4.