Antibody microarray profiling reveals individual and combined serum proteins associated with pancreatic cancer.

We used antibody microarrays to probe the associations of multiple serum proteins with pancreatic cancer and to explore the use of combined measurements for sample classification. Serum samples from pancreatic cancer patients (n = 61), patients with benign pancreatic disease (n = 31), and healthy control subjects (n = 50) were probed in replicate experiment sets by two-color, rolling circle amplification on microarrays containing 92 antibodies and control proteins. The antibodies that had reproducibly different binding levels between the patient classes revealed different types of alterations, reflecting inflammation (high C-reactive protein, alpha-1-antitrypsin, and serum amyloid A), immune response (high IgA), leakage of cell breakdown products (low plasma gelsolin), and possibly altered vitamin K usage or glucose regulation (high protein-induced vitamin K antagonist-II). The accuracy of the most significant antibody microarray measurements was confirmed through immunoblot and antigen dilution experiments. A logistic-regression algorithm distinguished the cancer samples from the healthy control samples with a 90% and 93% sensitivity and a 90% and 94% specificity in duplicate experiment sets. The cancer samples were distinguished from the benign disease samples with a 95% and 92% sensitivity and an 88% and 74% specificity in duplicate experiment sets. The classification accuracies were significantly improved over those achieved using individual antibodies. This study furthered the development of antibody microarrays for molecular profiling, provided insights into the nature of serum-protein alterations in pancreatic cancer patients, and showed the potential of combined measurements to improve sample classification accuracy.

[1]  Ruo-Pan Huang,et al.  Enhanced Protein Profiling Arrays with ELISA-Based Amplification for High-Throughput Molecular Changes of Tumor Patients’ Plasma , 2004, Clinical Cancer Research.

[2]  W. Steinberg.  The clinical utility of the CA 19-9 tumor-associated antigen. , 1990, The American journal of gastroenterology.

[3]  P. Lizardi,et al.  Mutation detection and single-molecule counting using isothermal rolling-circle amplification , 1998, Nature Genetics.

[4]  S. Ippolito,et al.  Lysosomal Aspartic and Cysteine Proteinases Serum Levels in Patients with Pancreatic Cancer or Pancreatitis , 1997, Pancreas.

[5]  J. Potter,et al.  A data-analytic strategy for protein biomarker discovery: profiling of high-dimensional proteomic data for cancer detection. , 2003, Biostatistics.

[6]  D. Ransohoff.  Bias as a threat to the validity of cancer molecular-marker research , 2005, Nature reviews. Cancer.

[7]  R. Huang,et al.  Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system. , 2001, Analytical biochemistry.

[8]  H. Miyake,et al.  Prediction of the extent of prostate cancer by the combined use of systematic biopsy and serum level of cathepsin D , 2003, International journal of urology : official journal of the Japanese Urological Association.

[9]  H. Yamamoto,et al.  Depression of plasma gelsolin level during acute liver injury. , 1992, Gastroenterology.

[10]  K. Sakamoto,et al.  Low vitamin K intake effects on glucose tolerance in rats. , 1999, International journal for vitamin and nutrition research. Internationale Zeitschrift fur Vitamin- und Ernahrungsforschung. Journal international de vitaminologie et de nutrition.

[11]  Andrea Tannapfel,et al.  Identification of novel proteins associated with hepatocellular carcinomas using protein microarrays , 2003, The Journal of pathology.

[12]  Paul Lizardi,et al.  Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements , 2004, Genome Biology.

[13]  S. Libutti,et al.  Advances in the early detection, diagnosis, and staging of pancreatic cancer. , 1997, Surgical oncology.

[14]  Troels Z. Kristiansen,et al.  Comprehensive proteomic analysis of human pancreatic juice. , 2004, Journal of proteome research.

[15]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Ziding Feng,et al.  Optimized Normalization for Antibody Microarrays and Application to Serum-Protein Profiling*S , 2005, Molecular & Cellular Proteomics.

[17]  Yoav Freund,et al.  A decision-theoretic generalization of on-line learning and an application to boosting , 1997, EuroCOLT.

[18]  E. B. Butler,et al.  Antibody microarray profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers , 2003, Proteomics.

[19]  Yoav Freund,et al.  A decision-theoretic generalization of on-line learning and an application to boosting , 1995, EuroCOLT.

[20]  S. Goodman,et al.  Pancreaticoduodenectomy for Cancer of the Head of the Pancreas 201 Patients , 1995, Annals of surgery.

[21]  A. Paradiso,et al.  Release of the aspartyl protease cathepsin D is associated with and facilitates human breast cancer cell invasion , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[22]  T. Barrette,et al.  Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins. , 2001, Cancer research.

[23]  S. Kingsmore,et al.  Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[24]  H. Liebman,et al.  Des‐γ‐carboxy (abnormal) prothrombin and hepatocellular carcinoma: A critical review , 1993 .

[25]  E. Nilsson,et al.  Synthesis of gamma-carboxylated polypeptides by alpha-cells of the pancreatic islets. , 2001, Biochemical and biophysical research communications.

[26]  Varshal K. Davé,et al.  Signal amplification by rolling circle amplification on DNA microarrays. , 2001, Nucleic acids research.

[27]  S. Kingsmore,et al.  Multiplexed protein profiling on microarrays by rolling-circle amplification , 2002, Nature Biotechnology.

[28]  K. Fearon,et al.  Fish oil-enriched nutritional supplement attenuates progression of the acute-phase response in weight-losing patients with advanced pancreatic cancer. , 1999, The Journal of nutrition.

[29]  P. Brown,et al.  Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions , 2001, Genome Biology.

[30]  K. Fearon,et al.  Cytokine regulation of constitutive production of interleukin-8 and -6 by human pancreatic cancer cell lines and serum cytokine concentrations in patients with pancreatic cancer. , 2002, International journal of oncology.

[31]  G. Ferland,et al.  Dietary induced subclinical vitamin K deficiency in normal human subjects. , 1993, The Journal of clinical investigation.

[32]  Ruo-Pan Huang,et al.  Detection of Multiple Cytokines by Protein Arrays from Cell Lysate and Tissue Lysate , 2003, Clinical chemistry and laboratory medicine.

[33]  Y. Freund,et al.  Discussion of the Paper \additive Logistic Regression: a Statistical View of Boosting" By , 2000 .

[34]  K. Gajl-Peczalska,et al.  A Study of Secretory Proteins, Cytology and Tumor Site in Pancreatic Cancer , 1979, Annals of Surgery.

[35]  L. Liotta,et al.  Proteomic profiling of the cancer microenvironment by antibody arrays , 2001, Proteomics.

[36]  E. Kubista,et al.  Use of High-Throughput Protein Array for Profiling of Differentially Expressed Proteins in Normal and Malignant Breast Tissue , 2004, Breast Cancer Research and Treatment.

[37]  S. Ippolito,et al.  Cathepsin D, B and L circulating levels as prognostic markers of malignant progression. , 1996, Anticancer research.

[38]  D. McMillan,et al.  Pancreatic Cancer as a Model: Inflammatory Mediators, Acute-phase Response, and Cancer Cachexia , 1999, World Journal of Surgery.