WO2006105319A2

WO2006105319A2 - Assessment of cardiac disease by cec gene profile analysis

Info

Publication number: WO2006105319A2
Application number: PCT/US2006/011691
Authority: WO
Inventors: Shawn Mark O'hara; Denis Smimov
Original assignee: Immunivest Corporation
Priority date: 2005-03-30
Filing date: 2006-03-30
Publication date: 2006-10-05
Also published as: WO2006105319A3

Abstract

bstract Elevated number of Circulating Endothelial Cells (CEC) have been implicated in disease conditions associated with the formation or destruction of blood vessels such as acute coronary syndrome, thrombocytopenic purpura, sickle cell disease, sepsis, lupus, nephrotic syndromes, rejection of organ transplants, surgical trauma and cancer. This invention provides a method for assessing the levels of CEC which vary between different studies using a sensitive enrichment, imaging, and enumberation analysis. Neuroligin, GATA binding protein 4, and fatty acid binding protein are found in CEC populations, yet are in trace amounts in the white blood cell population. This provides a method for diagnosing and finding the organ of origin in cardiovascular disease states.

Description

Title: Assessment of cardiac disease by CEC gene profile analysis.

Inventors: Mark O'Hara, Denis A. Smirnov

FIELD OF THE INVENTION

This invention relates to the fields of cardiology, oncology and diagnostic testing. The invention is a useful adjunct in clinical diagnosis and prognosis for diseases such as melanoma and cardiovascular disorders. The invention is also useful in the screening of therapeutic agents used in cardiovascular disorders, cancer or the like. More specifically, the present invention provides reagents, methods and test kits that facilitate genetic analysis and enumeration of rare circulating endothelial cells isolated from biological samples and provides a basis for diagnostic and/or prognostic evaluation in clinically relevant diseases.

BACKGROUND OF THE INVENTION

The American Heart Association estimates that approximately 600,000 patients undergo heart bypass surgery and 400,000 have stents implanted to open blocked arteries. Approximately 6 million patients visit the emergency rooms each year because of suspected heart attacks. In each of these situations, the patients may be treated with angiogenesis-stimulating drugs. Thus, critical analysis in assessing the efficacy of these drugs and/or the disease state would provide a valuable tool to the clinician. Endothelial cells are involved in the formation of blood vessels, or angiogenesis. This process is important for the growth of tumors and as a transport mechanism for circulating tumor cells (CTCs). Anti-angiogenesis drugs are in clinical development, either alone or in combination with traditional chemotherapeutic agents. Analysis of endothelial cells using CellTracks™ has applications in clinical trails as a tool to monitor efficacy as well as a tool for monitoring cancer patients receiving these drugs once they are marketed. Further, this type of analysis has applications in monitoring melanoma patients. Circulating endothelial cells have been implicated in cardiovascular (such as assessing cardiac risk), inflammatory, and infectious diseases.

Surface antigens of vascular endothelial cells has come from several lines of research, including studies of lymphocyte homing, inflammation, blood clotting, and tumor metastasis. Monoclonal antibodies (mAbs) have proven to be valuable tools for dissecting the antigenic structure of endothelial cells in different organs, tissues or segments of the vascular system, and the endothelial responses to inflammation, tissue damage, and tumor growth. Furthermore, mAbs have been used in the biochemical and molecular genetic characterization of endothelial antigens and in the functional analyses of endothelial molecules in vitro and in vivo.

Several categories of endothelial antigens have been distinguished, based on their distribution patterns in normal and lesion blood vessels. These include (i) antigens with wide distribution in the vascular system, such as Factor VII l-related antigen (FVIIIRA), PAL-E, and CD13, as per Schlingemann et al., Lab Invest. 52:71-76 (1985); Kuzu et al., J. Clin. Path. 45:143-148 (1992); (ii) antigens restricted to vessels in specific organs or tissues, or to unique histologic types of vessels, as illustrated by vascular addressins and GlyCAM-1 ; and (iii) inducible antigens, such as E-selectin, VCAM-1 , and ICAM-1 , that are not present or expressed at low levels in normal endothelium but are upregulated in inflamed tissues in vivo and/or induced or cultured endothelial cells by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1 ) (Pober et al., Lab. Invest. 64:301-305 (1991); Kuzu et al., Lab. Invest. 69:322-328 (1993)). supra).

Immuno-compromised patients, including cancer patients receiving high-dose chemotherapy, transplant patients, burn victims, and patients with AIDS, are at especially high risk for acquiring fungal infections. There are approximately Vz million cases of septicemia in the U.S. annually and microbiology and fungal cultures are used at least once for each of these cases. Approximately 200,000 cases progress to septic shock and approximately 100,000 die form this disease, of which 30,000 cases are attributable to fungal infections. In summary, a useful diagnostic test needs to be very sensitive and reliably quantitative. A blood test that detects a single or a few endothelial or fungal cells in 30ml of blood would provide a very sensitive and early detection mechanism in disease assessment.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting genetic information from rare CEC cells in a biological sample, which methods generally comprise:

a. obtaining a biological sample containing a mixed population of cells from an individual suspected of having target CEC cells;

b. fractionating said biological sample to obtain a fraction suspected of containing said rare cells;

c. assessing said fraction for target gene profile;

d. determining a second gene profile of said depleted fraction; and

e. subtracting said second gene profile from said first gene profile to obtain said genetic information from said rare cells.

In a preferred embodiment of the invention, the method involves selecting the rare cells from a group consisting of cancer cells, epithelial cells, endothelial cells, activated T-lymphocyte cells, dendritic cells and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Neuroligin 1 expression in heart. A Tissue RNA surveyed for NLGN1 by QRT-PCR. B: CEC RNA assayed for NLGN1 by QRT-PCR.

Figure 2: Fatty acid binding protein expression in heart. A: Tissue RNA surveyed for FABP3 by QRT-PCR. B: CEC RNA assayed for FABP3 by QRT- PCR. Figure 3: GATA binding protein 4 in heart. A: Tissue RNA surveyed for GATA4 by QRT-PCR. B: CEC RNA assayed for GATA4 by QRT-PCR.

Figure 4: IRX4 RNA levels. A: Tissue RNA surveyed for GATA4 by QRT-PCR. B: CEC RNA assayed for IRX4 by QRT-PCR.

Figure 5: NKX2-5 RNA levels. A: Tissue RNA surveyed for NKX2-5 by QRT- PCR. B: CEC RNA assayed for NKX2-5 by QRT-PCR.

Figure 6: TBX5 RNA levels. A: Tissue RNA surveyed for TBX5 by QRT-PCR.

B: CEC RNA assayed for TBX5 by QRT-PCR.

Figure 7: FABP RNA levels. A: Tissue RNA Surveyed for FABP1 liver by QRT-PCR. B: Tissue RNA surveyed for FABP3 heart by QRT-PCR. C: Tissue RNA surveyed for FABP7 brain by QRT-PCR.

DISCRIPTION

The present invention is based on several important discoveries which have significant diagnostic, prognostic, therapeutic, and drug discovery ramifications.

Once rare cells are identified in circulation, it is desirable to further characterize the isolated cells phenotypically and/or genotypically. Thus, particular molecules associated with patient disorders, such as nucleic acid molecules, proteins, or carbohydrates connected to the phenotypic or genotypic characteristics may be analyzed. Specifically, methods are provided for measuring the level of expression of predetermined molecules associated with the disorder or on endothelial cells identified in the circulation to assist the clinician in diagnosing melanoma and assessing the efficacy of chemotherapeutic intervention strategies.

CECs found in peripheral blood of patients with various heart conditions (acute coronary syndrome etc) originate from the damaged endothelial tissues of the heart and local arteries such as the coronary and aorta. In order to pinpoint the origin of circulating endothelial cells that are found in peripheral blood of heart patients, approximately 35 marker genes were selected because they were preferentially expressed in endothelial cells of the heart and coronary arteries. We have tested the expression of these selected genes in a collection of cDNA samples prepared from various regions of the heart and control tissues (Table I). We also tested expression of these genes in a few CEC enriched samples from patients with heart conditions, patients with metastatic cancer and normal donors. Several markers of CEC were specific to heart derived CEC as they show high expression in heart tissue, negligible expression in the white blood cells and are detectable from enriched CEC samples. NLGN1 (neuroligin 1) is expressed in the heart and shows minimal expression in white blood cells (Figure 1A). In CEC samples, NLGN1 expression was detected in heart and cancer patients, but not in normal donors (Figure 1 B). Also, FABP3 (fatty acid binding protein) is expressed in the heart and shows minimal expression in white blood cells (Figure 2A). In CEC samples, FABP3 expression was detected in heart and cancer patients (Figure 2B). FABP3 is known to be a promising serum protein marker released when the myocardium is injured and as such is a marker for the diagnosis of acute myocardial infarction. Also considered in the present application is the orgin specific nature of FABP's.

These fatty acids are expressed in specific regions/organs and it it contemplated to be used as an indicator of CEC origination. Consequently, fatty acid CEC expression is an indicator of organ specific disease state. Finally, GATA4 (GATA binding protein 4) is expressed in the heart and shows minimal expression in white blood cells (Figure 3A). In CEC samples, GATA4 expression was only detected in one of the tested heart patients, and not in cancer and normal donors (Figure 3B). The presence of GATA4 in CEC are indicative of cardiac hypertrophy and congenital heart defects. The expression 3 above markers are specifically or preferentially expressed in heart tissue, not expressed in blood cells and detectable in CEC. Thus, CEC genetic expression provides a sensitive and early method of detection for defined heart diseases such as an Acute Coronary Syndrome (ACS). Many laboratory and clinical procedures employ bio-specific affinity reactions for isolating rare cells from biological samples. Such reactions are commonly employed in diagnostic testing, or for the separation of a wide range of target substances, especially biological entities such as cells, proteins, bacteria, viruses, nucleic acid sequences, and the like.

Various methods are available for analyzing or separating target substances based upon complex formation between the substance of interest and another substance to which the target substance specifically binds. Separation of complexes from unbound material may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, US Patent No. 4,554,088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinburgh (1983). Generally, any material that facilitates magnetic or gravitational separation may be employed for this purpose. However, it has become clear that magnetic separation means are the method of choice. Magnetic particles can be classified on the basis of size; large (1.5 to about 50 microns), small (0.7-1.5 microns), or colloidal (<200nm), which are also referred to as nanoparticles. The third, which are also known as ferrofluids or ferrofluid-like materials and have many of the properties of classical ferrofluids, are sometimes referred to herein as colloidal, superparamagnetic particles.

Small magnetic particles of the type described above are quite useful in analyses involving bio-specific affinity reactions, as they are conveniently coated with biofunctional polymers (e.g., proteins), provide very high surface areas and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, US Patent Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunological reagents. The efficiency with which magnetic separations can be done and the recovery and purity of magnetically labeled cells will depend on multiple factors. If the level of non-specific binding of a system is substantially constant, as is usually the case, then as the target population decreases so will the purity.

As an example, a system with 0.8% NSB that recovers 80% of a population which is at 0.25% in the original mixture will have a purity of 25%. Whereas, if the initial population was at 0.01% (one target cell in 10⁶ bystander cells), and the NSB were 0.001%, then the purity would be 8%. Hence, a high the purity of the target material in the specimen mixture results in a more specific and effective collection of the target material. Extremely low non-specific binding is required or advantageous to facilitate detection and analysis of rare cells, such as epithelial derived tumor cells present in the circulation. Less obvious is the fact that the smaller the population of a targeted cell, the more difficult it will be to magnetically label and to recover. Furthermore, labeling and recovery will markedly depend on the nature of magnetic particle employed. For example, when cells are incubated with large magnetic particles, such as Dynal beads, cells are labeled through collisions created by mixing of the system, as the beads are too large to diffuse effectively. Thus, if a cell were present in a population at a frequency of 1 cell per ml of blood or even less, as may be the case for tumor cells in very early cancers, then the probability of labeling target cells will be related to the number of magnetic particles added to the system and the length of time of mixing. Since mixing of cells with such particles for substantial periods of time would be deleterious, it becomes necessary to increase particle concentration as much a possible. There is, however, a limit to the quantity of magnetic particle that can be added, as one can substitute a rare cell mixed in with other blood cells for a rare cell mixed in with large quantities of magnetic particles upon separation. The latter condition does not markedly improve the ability to enumerate the cells of interest or to examine them.

Based on the foregoing, high gradient magnetic separation with an external field device employing highly magnetic, low non-specific binding, colloidal magnetic particles is the method of choice for separating a cell subset of interest from a mixed population of eukaryotic cells, particularly if the subset of interest comprises but a small fraction of the entire population. Such materials, because of their diffusive properties, readily find and magnetically label rare events, such as tumor cells in blood. For magnetic separations for tumor cell analysis to be successful, the magnetic particles must be specific for epitopes that are not present on hematopoeitic cells.

Accordingly, it is to be appreciated that the foregoing preferred embodiments of the present invention are not intended to be limitative of its scope, and that one skilled in the art will be able to conceive of various variations and modifications of such particular embodiments, all of which should be considered to be within the scope of the invention, which is limited solely by the following claims.

Claims

What is claimed is:

1. A method for detecting the tissue of origin of CEC cells in a biological sample comprising:

a. obtaining a biological sample containing a mixed population of cells from an individual suspected of having CEC cells;

b. .fractionating said biological sample to obtain a fraction suspected of containing said CEC cells;

c. assessing said fraction for a gene profile; and

d. correlating gene profile with tissue of origin.

2. The method of claim 1 wherein said gene profile is in a group consisting of neuroligin, GATA binding protein 4, fatty acid binding protein, IRX4, NKX2-5, TBX5.

3. The method of claim 1 wherein said fractionating is by immunomagnetic enrichment.