Discovery and validation of cancer biomarkers using a protein analysis methodology to analyze specimens

Abstract

Methods are provided for the analysis, including the serial analysis, of very small samples of tissue. The methods utilize a nanofluidic proteomic immunoassay (NIA) to quantify total and low-abundance protein isoforms in a small amount of lysate. NIA detection accurately measure oncoprotein expression and activation in limited clinical specimens, including isoforms that differ in post-translational modifications, such as phosphorylation, and the like. The NIA detection method combines isoelectric protein focusing and antibody detection in a nanofluidic system.

Description

BACKGROUND OF THE INVENTION[0001]The recent explosion of information in the fields of genomics and proteomics has provided a rich ground for the discovery of molecular targets against which therapeutic and / or diagnostic agents can be directed. Tissues for potential target discovery may include tumors and other malignant growths, or infected or inflamed tissues. For example, methods have been described for gene expression profiling of tumor cells (see any one of Ono et al. (2000) Cancer Res. 60(18):5007-11; Svaren et al. (2000) J Biol Chem.; or Forozan et al. (2000) Cancer Res. 60(16):4519-25 for examples). Similarly, proteomics has been used to profile the protein expression in tumor samples (see Minowa et al. (2000) Electrophoresis 21(9):1782-6; Cole et al. (2000) Electrophoresis 21(9):1772-81; Simpson et al. (2000) Electrophoresis 21(9):1707-32); etc.[0002]Cancer is caused by multiple genetic events that result in the activation of proto-oncogenes and / or the inactivation of tumor ...

AI Technical Summary

Benefits of technology

[0042]In one embodiment of the invention, the NIA is used to guide selection of patient appropriate agents for therapy. A particular advantage of the invention is the ability to provide individualized diagnosis, taking advantage of small sample size to assess cancer patterns of expression over time.

[0043]The information obtained from NIA is used to monitor treatment, modify therapeutic regimens, and to further optimize the selection of therapeutic agents. With this approach, therapeutic and / or diagnostic regimens can be individualized and tailored according to the data obtained at different times over the course of treatment.DEFINITIONS

[0044]It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0045]As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of such compounds and reference to “the agent” includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.Post-Translational Modification

[0046]Glycosylation. Among the post-translational modifications that can be probed, are protein specific glycoslyation. Membrane associated carbohydrate is exclusively in the form of oliogsaccharides covalently attached to proteins forming glycoproteins, and to a lesser extent covalently attached to lipid forming the glycolipids. Glycoproteins consist of proteins covalently linked to carbohydrate. The predominant sugars found in glycoproteins are glucose, galactose, mannose, fucose, GalNAc, GlcNAc and NANA. The distinction between proteoglycans and glycoproteins resides in the level and types of carbohydrate modification. The carbohydrate modifications found in glycoproteins are rarely complex: carbohydrates are linked to the protein component through either O-glycosidic or N-glycosidic bonds. The N-glycosidic linkage is through the amide group of asparagine. The O-glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine. The linkage of carbohydrate to hydroxylysine is generally found only in the collagens. The linkage of carbohydrate to 5-hydroxylysine is either the single sugar galactose or the disaccharide glucosylgalactose. In ser- and thr-type O-linked glycoproteins, the carbohydrate directly attached to the protein is GalNAc. In N-linked glycoproteins, it is GlcNAc.

[0047]The predominant carbohydrate attachment in glycoproteins of mammalian cells is via N-glycosidic linkage. N-linked glycoproteins all contain a common core of carbohydrate attached to the polypeptide. This core consists of three mannose residues and two GlcNAc. A variety of other sugars are attached to this core and comprise three major N-linked families: High-mannose type contains all mannose outside the core in varying amounts; hybrid type contains various sugars and amino sugars; complex type is similar to the hybrid type, but in addition, contains sialic acids to varying degrees.

Problems solved by technology

Once the disease has progressed to locally advanced cancer or metastatic disease, these therapies are less successful.

With over 2000 human genes predicted to code for kinases and the potential for each kinase to act on multiple targets, signaling networks are immensely complex.

Imatinib treatment results in tumor cell signaling changes in vitro, leading to cell death.

Current methods of protein detection are insensitive to detecting subtle changes in oncoprotein activation that underlie key cancer signaling processes.

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