WO2006054991A1

WO2006054991A1 - Magnetic enrichment of circulating cells, fragments and debris for enabling hts proteomics and genomics in disease detection

Info

Publication number: WO2006054991A1
Application number: PCT/US2004/038608
Authority: WO
Inventors: Gerald V. Doyle; Shawn Mark O'hara; Herman Rutner
Original assignee: Immunivest Corporation
Priority date: 2004-11-17
Filing date: 2004-11-17
Publication date: 2006-05-26
Also published as: CA2587765A1

Abstract

The methods and reagents described in this invention are used to analyze circulating tumor cells, clusters, fragments, and debris. Analysis is performed with a number of platforms, including flow cytometry, CellSpotterTM fluorescent microscopy imaging andmass spectrometric profiling. In addition to analyzing and enumerating morphologicaly classified cells by imaging, the presence of damaged cells and derived debris in magnetically enriched specimens has been shown to be an important “smoking gun” indicator of demised tumor cells. Described herein are improved methods to screen, diagnose and monitor disease based on intact circulating rare cells and associated fragments or debris. The present invention incorporates proteomic and genomic profiling of cellular debris in cancer cell analysis, either complementary to or independent of image cytometry. The technology provides diagnosis and prognostic tools in diseases such as cancer that incorporate not only detection and quantitation of intact circulating cells but also of cellular fragments and debris that bear generic cytoskeletal and/or tumor-specific markers. The presence of such markers reflects immune function in early disease and/or therapeutic effectiveness in later stages.

Description

Title. Magnetic Enrichment of Circulating Cells, Fragments and Debris for Enabling HTS Proteomics and Genomics in Disease Detection

Inventors: Gerald Doyle, Shawn Mark O'Hara, and Herman Rutner

PRIORITY INFORMATION

This application is a continuation-in-part of U.S. application 10/780,399, filed 23 August 2002 as PCT/US02/26861 , and US Provisional Applications

60/314,151 , filed 23 August 2001 , 60/369,628, filed 03 April 2002, and 60/524,918, filed 25 November 2003, under 35 USC §119(e). These applications are incorporated by reference herein.

Background

Field of the Invention This invention generally relates to the use of proteomic and mRNA transcript investigation as diagnostic tools as it relates to the fields of oncology and diagnostic testing. More specifically, the present invention relates to the use of proteomics and mRNA transcript profiling as a source of information in the analysis of tumor cells for early diagnosis of cancer and in predicting clinical outcomes.

Background Art

Most cancer deaths are not caused by the primary tumor. Instead, death results from metastases, i.e. multiple widespread tumor colonies established by malignant cells that detach themselves from the site of the original tumor and travel through the body to distant sites. If a primary tumor is detected early enough, it can often be eliminated by surgery, radiation, or chemotherapy or some combination of those treatments. Because of the difficulty in detection, metastatic colonies are harder to detect and eliminate and it is often impossible to treat all of them successfully. From a clinical perspective, metastasis is considered a conclusive event in the natural progression of cancer. Moreover, the ability to metastasize is the property that uniquely characterizes a malignant tumor. Cancer metastasis comprises a complex series of sequential events. These begin with the extension from the primary locus into surrounding tissues, penetration into body cavities and vessels, release of tumor cells for transport through the circulatory system to distant sites, reinvasion of tissue at the site of arrest, and adaptation to the new environment so as to promote tumor cell survival, vascularization and tumor growth.

Based on the complexity of cancer and cancer metastasis and the frustration in treating cancer patients over the years, many attempts have been made to develop diagnostic tests to guide treatment and monitor the effects of such treatment on metastasis or relapse. Such tests presumably could also be used for cancer screening, replacing relatively crude tests such as mammography for breast tumors or digital rectal exams for prostate cancers. Towards that goal, a number of tests have been developed over the last 20 years and their benefits evaluated. One of the first attempts was the formulation of an immunoassay for carcinoembryonic antigen [CEA]. This antigen appears on fetal cells and reappears on tumor cells in certain cancers. Extensive efforts have been made to evaluate the usefulness of testing for CEA as well as many other "tumor' antigens, such as PSA, CA 15.3, CA 125, PSMA, CA27.29. These efforts have proven to be somewhat futile as the appearance of such antigens in blood have not been generally predictive and are often detected when there is little hope for the patient. In the last few years, one test has proven to be useful in the early detection of cancer (i.e. prostate specific antigen [PSA] for prostate cancers. When used with follow-up physical examination and biopsy, the PSA test has played a remarkable role in the very early detection of prostate cancer, at a time when it is best treated.

Despite the success of PSA testing, the test is not totally acceptable. For example, high levels of PSA do not always correlate with cancer not do they appear to be an indication of the metastatic potential of the tumor. This may be due in part to the fact that PSA is a component of normal prostate tissue as well as other unknown factors. Moreover, it is becoming clear that a large percentage of prostate cancer patients will continue to have localized disease which is not life threatening. Based on the desire to obtain better concordance between those patients with cancers that will metastasize and those that won't, attempts have been made to determine whether or not prostate cells are in the circulation. When added to high PSA levels and biopsy data, the existence of circulating tumor cells (CTC) might give indications as to how vigorously the patient should be treated. The approach for determining the presence of circulating prostate tumor cells has been to test for the expression of messenger RNA of PSA in blood. This is being done through the laborious procedure of isolating all of the mRNA from a blood sample and performing reverse transcriptase PCR. As of this date, no good correlation exists between the presence of such cells in blood and the ability to predict which patients are in need of vigorous treatment (Gomella LG. J of Urology. 158:326-337 (1997)). It is noteworthy that PCR is difficult to perform quantitatively, i.e. determine the number of tumor cells per unit volume of biological sample. Additionally, false positives are often observed using this technique. There is an added drawback which is that there is a finite and practical limit to the sensitivity of this technique based on the sample size examined. Typically, the test is performed on 10⁵ to 10⁶ cells purified away from interfering red blood cells. This corresponds to a practical lower limit of sensitivity of one tumor cell per 0.1 ml of blood. Hence, there needs to be about 10 tumor cells in an ml of blood before a signal is detectable. As a further consideration, tumor cells are often genetically unstable. Accordingly, cancer cells having genetic rearrangements and sequence changes may be missed in a PCR assay as the requisite sequence complementation between PCR primers and target sequence can be lost.

A useful diagnostic test needs to be very sensitive and reliable. A blood test developed to detect the presence of a single tumor cell in one ml of blood (as described in US 6,365,362) corresponds to the detection of 3000 - 4000 total cells in circulation, a number that establishes tumors in inoculated animals. Further if 3000 - 4000 circulating cells represent 0.01 % of the total cells in a tumor, then it would contain about 4 x 10⁷ total cells. A tumor containing that number of cells would not be visible by any technique currently in existence. Hence, if tumor cells are shed in the early stages of cancer, a test with the sensitivity mentioned above would detect the cancer. If tumor cells are shed in some functional relationship with tumor size, then a quantitative test could assess tumor burden. The general view is that tumors are initially well confined and hence there are few if any circulating cells in early stages of disease.

Based on the above, a method for identifying those cells in circulation with metastatic potential prior to establishment of a secondary tumor is highly desirable, particularly early on in the cancer.

Many laboratory and clinical procedures employ biospecific 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 others.

Various methods are available for analyzing or separating the above- mentioned 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, such as by settling, or 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 biospecific affinity reactions (see, for example, US 4,554,088). Generally, any material which 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.

Consequently, immunomagnetic separation as described in US 6,365,362 has significant clinical ramifications for the diagnosis and treatment of cancer. These include the fact that tumor cells are present in the blood of patients who are considered to have clinically localized, primary tumors, that the number of tumor cells present in the circulation correlates with all stages of cancer from its inception to its terminal stages and that the changes in the number of tumor cells present in the circulation is indicative of disease progression. A decrease in the number of circulating tumor cells is indicative of an improvement in patient status or efficacy of treatment, whereas an increase indicates a worsening of the disease.

As shown in WO00/47998, US #6,190,870, and other publications, CTC can circulate as both live and dead cells, wherein "dead" comprises the full range of damaged and fragmented cells as well as CTC-derived debris. The tumor burden is probably best represented by the total of both intact CTC, including clusters, and damaged CTC, which bear morphological characteristics of cells, but are distinct from clumps and/or aggregates. However, some damaged cells, may have large pores allowing leakage of the liquid and particulate cytosolic contents resulting in a change in the buoyant densities from about 1.06-1.08 to greater than 1.12, or well above the densities of RBC (live and dead cells can be separated at the interface of gradients of d=1.12 and 1.16 according to a Pharmacia protocol). Conventional density gradients, as used in # WO00/47998 would lose such damaged CTC in the discarded RBC layer having a range in density of about 1.08 to 1.11. CTC debris that is positively stained for cytokeratin may also have densities falling in the RBC or higher ranges, since most intracellular components (with the possible exception of lipophilic membrane fragments that may be located near the plasma-buffy coat interface) have densities in the range of 1.15 to 1.3. Hence, a substantial portion of damaged CTC and CTC debris may be located outside the buffy coat layer, and would not be seen by the density gradient methods, such as those in WO00/47998. Some images of damaged or fragmented CTC are shown, but it is quite possible the damage occurred during cytospin or subsequent processing, and is thus artifactual. While the densities of most intact tumor cells may fall in the WBC region, it is quite likely that damaged CTC in patient samples have higher densities that may place them in the RBC layer; outside the reach of gradient techniques. US Patent Application 10/780,399 describes methods for binding fragments and debris to beads. That application describes analysis of the density of fragments and debris of interest. Upon centrifugation, the beads will be located in a layer above RBC, because of the pre-determined specific gravity (density) of the beads coupled to fragments and/or debris. However, this system is dependent on correctly binding fragments and debris to these beads. If any other sample component binds the beads, they may not appear in the desired location, and subsequently will not be subject to analysis.

Epithelial cells in their tissue of origin obey established growth and development "rules". Those rules include population control. This means that under normal circumstances the number and size of the cells remains constant and changes only when necessary for normal growth and development of the organism. Only the basal cells of the epithelium or immortal cells will divide and they will do so when it is necessary for the epithelium to perform its function, whatever it is depending in the nature and location of the epithelium. Under some abnormal but benign circumstances, cells will proliferate and the basal layer will divide more than usual, causing hyperplasia. Under some other abnormal but benign circumstances, cells may increase in size beyond what is normal for the particular tissue, causing cell gigantism, as in folic acid deficiency.

Epithelial tissue may increase in size or number of cells also due to pre- malignant or malignant lesions. In these cases, changes similar to those described above are accompanied by nuclear abnormalities ranging from mild . in low-grade intraepithelial lesions to severe in malignancies. It is believed that changes in these cells may affect portions of the thickness of the

_* epithelium and as they increase in severity will comprise a thicker portion of such epithelium. These cells do not obey restrictions of contact inhibition and continue growing without tissue controls. When the entire thickness of the epithelium is affected by malignant changes, the condition is recognized as a carcinoma in situ (CIS).

The malignant cells eventually are able to pass through the basement membrane and invade the stroma of the organ as their malignant potential increases. After invading the stroma, these cells are believed to have the potential for reaching the blood vessels. Once they infiltrate the blood vessels, cells find themselves in a completely different environment from the one they originated.

The cells may infiltrate the blood vessels as single cells or as clusters of two or more cells. A single cell of epithelial origin circulating through the circulatory system is destined to have one of two outcomes. It may die or it may survive. Single Cells: 1. The cell may die either through apoptosis due to internal changes or messages in the cell itself. These messages may have been in the cell before intravasation or they may be received while in the blood, or it may die due to the influence of the immune system of the host, which may recognize these cells as "alien" to this environment. The results of cellular death are identifiable in imaging systems as enucleated cells, speckled cells or amorphous cells. These cells do not have the potential for cell division or for establishing colonies or metastases.

• Enucleated cells are the result of nuclear disintegration and elimination (karyorrhexis and karyolysis). They are positive for cytokeratin, and negative for nucleic acid.

• The speckled cells are positive for cytokeratin and DAPI and show evidence of cellular degeneration and cytoplasmic disintegration.

These cells may represent response to therapy or to the host's immune system as the cytoskeletal proteins retract.

• Another dying tumor cell identifiable is the amorphous cell. These cells are probably damaged during the preparation process, a sign that these may be weaker, more delicate cells but may also be the result of apoptosis or immune attack.

2. A viable malignant epithelial cell may have the potential to survive the circulation and form colonies in distant organs. These "survivor cells" appear in as intact cells with high nuclear material/cytoplasmic material ratio. These cells are probably undifferentiated and can potentially divide in blood and form small clusters (Brandt et a/. "Isolation of prostate-derived single cells and cell clusters from human peripheral blood" Cancer Research 56, 4556-4561 , 1996) that may extravasate in a distant capillary, where the cell may establish a new colony, or it may remain as a single cell until it extravasates, dividing once it establishes itself in the new tissue, starting this way a new colony.

Once a new colony is established in a new organ, some malignant cells will continue replicating to form a new tumor. If they reach new capillaries, the metastasis story may be repeated and secondary metastasis occurs. Monitoring

By monitoring during treatment in patients with known carcinomas, a decrease in the number of tumor cells and/or some change in an appropriate index may represent a response to patient therapy. For example, the response index represents a measure of response to a patient therapy whereby

• Total tumor cells = Dying cells + Survivor cells (TTC = DC + SC).

• Response Index = dying cells / total tumor cells (Rl= DC / TTC). Thus, the higher the response index, the better the response to therapy. A low response index may indicate that the patient is not responding to the treatment and or that the pt's immune system is not able to handle the tumor load.

A patient who has 50 total tumor cells that were all survivor cells at pre- treatment visit (a Rl = 0/50 = 0) and has 50 TTC on follow-up (after treatment) visit may have different outcomes depending in the Rl. If all the TTC are SC (i.e. DC = 0), there was no response to therapy. If there are 50 cells but the response index is 40/50 = 0.8, then either the immune system or the therapy is having a negative effect on tumor load, therefore, is a positive patient response.

Follow-up

When a pap smear is diagnosed as having cells with atypia or low-grade intraepithelial lesions, there is always the possibility that these patients have a more severe abnormality, which cells were missed as a sampling error. These patients are biopsied and asked to return in three months for a repeat pap smear. If the atypical cells were concurrent with a small focal area of malignant cells that did not get sampled, the patient will wait 3 months before she gets any follow-up, opening the possibility of misdiagnosis. Using image analysis all patients with an abnormal pap (5-10% of the pap smears in the USA) are relatively easily and quickly tested for circulating epithelial cells. Patients with positive tests can be followed-up aggressively. This simplifies the decision making process for the physician and health professionals. Screening

Image cytometry analysis is useful for screening the general population. Identification of CTC in a patient could indicate that there is a primary malignancy that has started or is starting the process of metastasis. If these cells are identified as of the tissue of origin with new markers, then organ specific tests, like guided fine needle aspirations (FNA) can be used to verify the presence or absence of such malignancies. Patients where a primary cannot be identified can be followed-up with repeat tests after establishing an individual base line. All or some of the above-cited factors were found to contribute to debris and/or aggregate formation that have been observed to confound the detection of CTC by direct enrichment procedures from whole blood as disclosed in this invention. The number of intact CTC, damaged or suspect CTC as well as the degree of damage to the CTC, may further serve as diagnostically important indicators of the tumor burden, the proliferative ₎ potential of the tumor cells and/or the effectiveness of therapy. The present invention has a distinct advantage in that the methods and protocols of the prior art combine unavoidable in vivo damage to CTC with avoidable in vitro storage and processing damage, thus yielding erroneous information on CTC and tumor burdens in cancer patients. This relatively simple blood test described herein, which functions with a high degree of sensitivity and specificity, can be thought of as a "whole body biopsy".

Proteomics Incorporating a more global analysis of diagnosis, follow-up, and screening as related to protein expression is another embodiment of the present invention. Assessing global patterns of protein expression in individual cells, tissues, or body fluids, has been the basic foundation in proteomics and provide an improvement to current methods. Coupled with genetic information, protein expression in individual cells can take on several different forms based upon the nucleotide sequence, whether a splice variant occurs, or whether there is a post-translational modification. Thus, the transcription, translation, and post-translational modification of each protein define a specific biochemical function within a living cell. Proteomics looks at the transcripts of genomic DNA (messenger RNA) as they directly encode proteins, and that these proteins are further modified by mechanisms such as phosphorylation or glycosylation. As a consequence of this sequence of events, there are functional variations in protein expression. Thus, proteomics is a process of transcriptional profiling to determine which genes, or combination thereof, are transcribed in a particular cell type or disease state. To this end, protein profiling is examined by various techniques which include two-dimensional gel electrophoresis (2D-GeI) and mass spectroscopy (MS), co-immunoprecipation, affinity chromatography, protein binding analysis, overlay analysis, using yeast in protein-protein interaction, the analysis of signal transduction and other complex cellular process, three-dimensional structure modeling and large-scale protein folding, and the incorporation of bioinformatics with proteomic data.

Two-dimensional gel electrophoresis alone has several inherent problems, especially when applied in diagnosis. These include difficulties in the analysis of the gels, the insufficiency of the resolving power to separate various distinct proteins in a particular sample, and a lack of reproducibility from one gel sample to the next.

Methods are available for utilizing MS in the analysis of target polypeptides. Here, the polypeptides are solubilized in a solution or reagent system depending upon the properties of the polypeptide (i.e. organic or inorganic solvents) and the type of MS performed (WO 93/24834 by Chait et al.).

Mass spectrometer analysis includes ionization (I) techniques, including but not limited to matrix assisted laser desorption (MALDI), continuous or pulsed electrospray (ESI) and related methods (IONSPRAY or THERMOSPRAY), or massive cluster impact (Cl). These ion sources are matched with detection formats including linear or non-linear reflection time- off-light (TOF), single or multiple quadropole, single or multiple magnetic sector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, LC/MS, MS/MS, and combinations thereof.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) refers to the formation of a matrix with several small, acidic, light absorbing chemicals that is mixed in solution with the analyte in such a manner so that, upon drying on the probe element, the crystalline matrix- embedded analyte molecules are successfully desorbed (by laser irradiation) and ionized from the solid phase (crystals) into the gaseous or vapor phase and accelerated as intact molecular ions. In the MALDI process, the analyte is mixed with a freshly prepared solution of the chemical matrix and placed on the inert probe element surface to air dry just before the mass spectrometric analysis (see US 5,808,300).

Another general category, utilizing a sample presenting means, is Surfaces Enhanced for Laser Desorption/lonization (SELDI) and described in US 6,020,208, within which there are three (3) separate subcategories. The SELDI process is directed toward a sample presenting means (i.e., probe element surface) with surface-associated (or surface-bound) molecules to promote the attachment and subsequent detachment of analyte molecules in a light-dependent manner, wherein the surface-associated molecule(s) are selected from the group consisting of photoactive (photo labile) molecules that participate in the binding (docking, tethering, or^cross linking) of the analyte molecules to the sample presenting means (by covalent attachment mechanisms or otherwise).

Regardless of the MS method, the mass of the target polypeptideis then compared to the mass of a reference polypeptide of known identity.

MS based processes for detecting a particular nucleic acid sequence in a biological sample has been described in US 6,043,031. The process is used to diagnose a genetic disease or chromosomal abnormality, a predisposition to a disease or condition, infection by a pathogenic organism, or for determining heredity. Detection of the desired fragments is optimum between 7,000 to 20,000 Da obtained from tryptic digests.

The use of proteomics in diagnosing the existence or predicting the development and/or progression of abnormal physiological conditions based upon the presence of proteomic materials has been previously described (US 20020260420). Several recent publications and reviews by L. Anderson of The Plasma Proteome Institute (Washington, DC) also discuss the status of current MS methods in proteomics and the requirements for adapting highly sophisticated MS methods to practical clinical diagnostics, for example, detection of less than 20 relevant protein markers, cost per analysis of $2- $100 and assay time of about 15 minutes. After obtaining a patient sample containing proteomic materials, the patient sample is prepared by isolating proteomic material with characteristics identifiable for normal and abnormal physiological conditions or associated predictive endpoints, e.g down regulation or up regulation of proteins also present in healthy individuals. The proteomic materials are separated to permit analysis of one or more specific proteomic materials thereby enabling the diagnostician to characterize an individual's condition as being either positively or negatively indicative of one or more abnormal physiological conditions. While proteomics and current methods have been applied in cancer diagnostics, such methods lack simple and efficient S/N amplification or pre- enrichment methods that would improve the sensitivity and reduce the sample processing time and cost of analysis of clinical specimens.

Summary of the Invention

The present invention provides a tool for clinicians in the diagnosis and prognosis of disease states such as cardiovascular disorders and cancer, and provides a sensitive, simple, and efficient analysis of disease detection to complement other means of detection known in the art. The methods and reagents described in this invention are used to analyze circulating tumor cells, clusters, fragments, and debris. Analysis is performed with a number of platforms, including flow cytometry and imaging systems and mRNA transcript profiling. The examples show the importance of not only analyzing obvious or intact CTC, but suspect CTC or damaged fragments, clusters of CTC, and debris. Similar analysis is possible with endothelial cells. In this type analysis, assessing the damage that forms fragments and debris is easier. It is also possible to inhibit further damage of CTC between the blood draw and sample processing through the use of stabilizing agents. It has been shown herein that the ability to differentiate between in vitro damage, caused by specimen acquisition, transport, storage, processing, or analysis, and in vivo damage, caused by apoptosis, necrosis, or the patient's immune system. Indeed, it is desirable to confine, reduce, eliminate, or at least qualify in vitro damage to prevent it from interfering in analysis. After amplification by immunomagnetic enrichment of cells and/or fragments from a patient blood sample, an analysis of proteomic parameters alone or in combination with cytometric imaging of circulating debris or cells after immunomagnetic partitioning. Thus, 2-D Gel electrophoresis, MS, SELDI or microarray detection of cells and fragments would be used alone or in conjuction with image analysis on the enriched fraction of debris and/or cells, captured by positive selection of antibody-coupled magnetic particles. The present invention also includes any specific antibody-antigen, ligand- receptor, or labeling means. MS is accomplished directly on the captured ferrofluid particles or on the captured target materials after dissociation from the ferrofluid by a reversible binding reaction, such as by the dissociation of the bond between target-Mab-desthiobiotin and streptavidin on the ferrofluid with soluble biotin to liberate the Mab labeled target material. The direct mode is most suited for diagnostic correlation with cell counts, clinical diagnosis and the ability to differentiate target material from ferrofluid associated proteins, as well as potential utility as a complementary or independent modality to cell imaging.

A second approach is to limit analysis to only MS after immunomagnetic enrichment (or non-magnetic enrichment) from separate, unprocessed specimens such as whole blood, plasma or serum. This approach is without cell permeabilization, antibodies and staining reagents, incorporated with image analysis, to minimize the introduction of extraneous components that would interfere with MS analysis.

The one embodiment of the present invention is the enrichment of target specific cell fragments, debris, and non-particulate soluble protein. These include immune complexes which are normally present at low levels in the early stages of disease and increase as the disease progresses.

Incorporating proteomics in cancer detection, especially at early stages, provides additional information in the analysis of circulating rare cells if enrichment provides sufficient mass for MS detection. In addition to a substantial amount of CTC debris present during low CTC, capture of debris containing the same surface markers as the intact cells, followed by MS analysis provides a new platform for early cancer diagnosis. In addition with the use of monclonal antibodies as capture agents (i.e. CD 146, CD 105, CD 31 , CD 133, CD 106), the present invention considers diseases associated with circulating endothelial cells and their analysis. These diseases include those relating to cardiovascular disorders. Herein are described methods to diagnose, monitor, and screen disease based on circulating rare cells, including malignancy as determined by CTC, clusters, fragments, and debris. Also, proteomic and transcriptome analysis, especially with the enriched cell/cell debris/cell fragment components, are utilized in methodologies for diagnosing, monitoring and screening disease.

Brief Description of the Drawings

Figure 1: Models of tumor shedding and metastasis. 1a. shows possible stages of cells, clusters, and fragments. 1b. shows the same model with actual images from samples. Figure 2: Flow cytometric analysis of immunomagnetically enriched tumor cells from a 7.5ml blood of a metastatic prostate patient. Figure 3: Image cytometry analysis with 7.5ml blood sample from a metastatic prostate cancer patient that was immunomagnetically enriched for tumor cells. The lines of thumbnails correspond to the different dyes used in the staining process showing tumor candidates stained with cytokeratin PE and DAPI.

Figure 4: Classifications of tumor cells from a whole blood sample of a patient with metastatic prostate cancer stained with cytokeratin PE and DAPI. A: intact cells B: damaged tumor cells C: tumor cell fragments. Figure 5: A comparison of the number of obvious CTC and suspect CTC in 20 clinical samples.

Figure 6: Classification of paclitaxel treated LnCaP cells spiked into whole blood and isolated then stained with cytokeratin PE and DAPI. A: intact cells B: dying tumor cells C: tumor cell fragments Figure 7: Outline of one embodiment in a sample preparation for proteomic analysis. Detailed Description of the Invention

General Definitions

Proteomics refers to the study of proteins and their DNA messenger RNA transcripts that directly encode for them. These expressed proteins can be further modified by post-translational modification, e.g. such as phosphorylation and glycosylation that alter protein expression.

The term "rare cells" as used herein refers to a variety of cells, microorganisms, bacteria, and the like. Cells are characterized as rare in a sample because they are not present in normal samples of the same origin, and are several orders of magnitude lower in concentration that the typical cells in a normal sample. Embodiments of the present invention include circulating cancer cells, virally, infected cells, fetal cells in maternal circulation, or endothelial cells efficiently isolated from non-rare cells and other bioentities, using the methods and apparatus of the present invention in conjunction with previously described technology (US 6,365,362).

The term "analyte" refers to any atom and/or molecule; including their complexes and fragments ions. In the case of biological molecules/macromolecules or "biopolymers", such analytes include but are not limited to: proteins, peptides, DNA, RNA, carbohydrates, steroids, and lipids.

Detailed Description

Evidence that minimal residual disease in patients with carcinoma has clinical significance is mounting. To effectively monitor minimal residual disease, a qualitative and quantitative assessment is needed. As the frequency of carcinoma cells in blood or bone marrow is low, the laborious manual sample preparation methods involved in the preparation of samples for analysis often leads to erroneous results. To overcome these limitations a semi-automated sample preparation system was developed that minimize variability and provide more consistent results, as described in commonly- owned US Application No. 10/081 ,996 (filed 20 February 2002) which is incorporated by reference herein.

Various methods are available for analyzing or separating the above- mentioned 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, by centrifugation of finely divided particles or beads coupled to the target substance. 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. 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. Nanoparticles, also known as ferrofluids or ferrofluid-like materials, have many of the properties of classical ferrofluids, and 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 depends on many factors.

For example, 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, reflecting poorly on the efficiency.

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 as possible. Increasing the concentration of the larger particles does not markedly improve the ability to enumerate the cells of interest or to examine them. The preferred magnetic particles for use in the present invention are particles that behave as colloids. Such particles are characterized by their sub-micron particle size, which is generally less than about 200nm, and their stability to gravitational separation from solution for extended periods of time. In addition to the many other advantages, this size range makes individual particles essentially invisible to analytical techniques commonly applied to cell analysis. Particles within the range of 90-150nm and having between 70-90% magnetic mass are contemplated for use in the present invention. Suitable magnetic particles are composed of a crystalline core of superparamagnetic material surrounded by molecules which are bonded, e.g., physically absorbed or covalently attached, to the magnetic core and which confer stabilizing colloidal properties. The coating material should preferably be applied in an amount effective to prevent non-specific interactions between biological macromolecules found in the sample and the magnetic cores. Such biological macromolecules may include carbohydrates such as sialic acid residues on the surface of non-target cells, lectins, glycproteins, and other membrane components. In addition, the material should contain as much magnetic .mass per nanoparticle as possible. The size of the magnetic crystals comprising the core is sufficiently small that they do not contain a complete magnetic domain. The size of the nanoparticles is sufficiently small such that their Brownian energy exceeds their magnetic moment. As a consequence, magnetic alignment and subsequent mutual attraction/repulsion of these colloidal magnetic particles does not appear to occur even in moderately strong magnetic fields, contributing to solution stability. Finally, the magnetic particles are separated in high magnetic gradient external field separators, facilitating sample handling and providing economic advantages over the more complicated internal gradient columns loaded with ferromagnetic beads or steel wool. Magnetic particles having the above- described properties can be prepared by modification of base materials described in U.S. Patents 4,795,698, 5,597,531 , and 5,698,271 , each incorporated by reference herein.

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. Additionally for magnetic separations to be successful, the magnetic particles must be specific for epitopes that are not present on hematopoetic cells.

A large variety of analytical methods and criteria are used to identify tumor cells, and the first attempts are being undertaken to standardize criteria that define what constitutes a tumor cell by immunocytochemistry. In this study, blood samples from prostate cancer patients were immunomagnetically enriched for cells that expressed EpCAM. Tumor cells were identified by the expression of the cytoskeletal proteins cytokeratin (CK+), the absence of the common leukocyte antigen CD45 (CD45-) and the presence of nucleic acids (NA+) by multicolor fluorescence analysis. Rare events or rare cells can be immunophenotyped by both flowcytometry and fluorescence microscopy. Flowcytometric analysis excels in its ability to reproducibly quantify even low levels of fluorescence whereas microscopy has the better specificity as morphological features can aid in the classification of the immunophenotypically identified objects. Although there was a correlation between the number of CTC detected in blood of prostate cancer patients by flowcytometry and microscopy, microscopic examination of the CK+, CD45-, NA+ objects showed that only few of the objects appeared as intact cells. This observation agrees with other reports that showed apoptosis in a substantial portion of circulating tumor cells. The terms "biological specimen" or "biological sample" may be used interchangeably, and refer to a small potion of fluid or tissue taken from a human test subject that is suspected to contain cells of interest, and is to be analyzed. A biological specimen refers to the fluidic portion, the cellular portion, and the portion containing soluble material. Biological specimens or biological samples include, without limit bodily fluids, such as peripheral blood, tissue homogenates, nipple aspirates, colonic lavage, sputum, bronchial (alveolar) lavage, pleural fluids, peritoneal fluids, pericardial fluids, urine, and any other source of cells that is obtainable from a human test subject. An exemplary tissue homogenate may be obtained from the sentinel node in a breast cancer patient.

The term "rare cells" is defined herein as cells that are not normally present in biological specimens, but may be present as an indicator of an abnormal condition, such as infectious disease, chronic disease, injury, or pregnancy. Rare cells also refer to cells that may be normally present in biological specimens, but are present with a frequency several orders of magnitude less than cells typically present in a normal biological specimen.

The term "determinant", when used in reference to any of the foregoing target bioentities, refers broadly to chemical mosaics present on macromolecular antigens that often induce an immune response. Determinants may also be used interchangeably with "epitopes". A "biospecific ligand" or a "biospecific reagent," used interchangeably herein, may specifically bind determinants. A determinant refers to that portion of the target bioentity involved in, and responsible for, selective binding to a specific binding substance (such as a ligand or reagent), the presence of which is required for selective binding to occur. In fundamental terms, determinants are molecular contact regions on target bioentities that are recognized by agents, ligands and/or reagents having binding affinity therefor, in specific binding pair reactions. The term "specific binding pair" as used herein includes antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fc receptor or mouse IgG- protein A, avidin-biotin, streptavidin-biotin and virus-receptor interactions. The term "detectably label" is used to herein to refer to any substance whose detection or measurement, either directly or indirectly, by physical or chemical means, is indicative of the presence of the target bioentity in the test sample. Representative examples of useful detectable labels, include, but are not limited to the following: molecules or ions directly or indirectly detectable based on light absorbance, fluorescence, reflectance, light scatter, phosphorescence, or luminescence properties; molecules or ions detectable by their radioactive properties; molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties. Included among the group of molecules indirectly detectable based on light absorbance or fluorescence, for example, are various enzymes which cause appropriate substrates to convert (e.g., from non-light absorbing to light absorbing molecules, or from non-fluorescent to fluorescent molecules). Analysis can be performed using any of a number of commonly used platforms, including multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, image cytometry analysis, and other automated cell analysis.

The phrase "to the substantial exclusion of refers to the specificity of the binding reaction between the biospecific ligand or biospecific reagent and its corresponding target determinant. Biospecific ligands and reagents have specific binding activity for their target determinant yet may also exhibit a low level of non-specific binding to other sample components.

The phrase "early stage cancer" is used interchangeably herein with "Stage I" or "Stage II" cancer and refers to those cancers that have been clinically determined to be organ-confined. Also included are tumors too small to be detected by conventional methods such as mammography for breast cancer patients, or X-rays for lung cancer patients. While mammography can detect tumors having approximately 2 x 10⁸ cells, the methods of the present invention should enable detection of circulating cancer cells from tumors approximating this size or smaller.

The term "enrichment" as used herein refers to the process of substantially increasing the ratio of target bioentities (e.g., tumor cells) to non-target materials in the processed analytical sample compared to the ratio in the original biological sample. In cases where peripheral blood is used as the starting materials, red cells are not counted when assessing the extent of enrichment. Using the method of the present invention, circulating epithelial cells may be enriched relative to leucocytes to the extent of at least 2,500 fold, more preferably 5,000 fold and most preferably 10,000 fold.

The terms "anti-coagulant" or "anti-coagulating agent" may be used interchangeably, and refer to compositions that are added to biological specimens for the purpose of inhibiting any undesired natural or artificial coagulation. An example of coagulation is blood clotting and common anti- coagulants are chelating agents, exemplified by ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), 1 ,2- diaminocyclohexane tetraacetic acid (DCTA), ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA), or by complexing agents, such as heparin, and heparin species, such as heparin sulfate and low-molecular weight heparins. This may be further collectively defined as "clumping' or "clump formation". However, such clumps must be differentiated from "clusters" or aggregates of CTC that are counted as a single Intact CTC if they meet the classification criteria for Intact CTC.

Clusters of CTC are believed to have greater proliferative potential than single CTC and their presence is thus diagnostically highly significant. One possible cause for an increased propensity to establish secondary metastatic tumor sites may be the virtue of their adhesiveness. An even more likely cause is the actual size of a CTC cluster; larger clusters will become lodged in small diameter capillaries or pores in bone. Once there, the viability of the cells in the cluster would determine the chance of survivability at the new metastatic site.

The ideal "stabilizer" or "preservative" (herein used interchangeably) is defined as a composition capable of preserving target cells of interest present in a biological specimen, while minimizing the formation of interfering aggregates and cellular debris in the biological specimen, which in any way can impede the isolation, detection, and enumeration of targets cells, and their differentiation from non-target cells. In other words, when combined with an anti-coagulating agent, a stabilizing agent should not counteract the anti- coagulating agent's performance. Conversely, the anti-coagulating agent should not interfere with the performance of the stabilizing agent. Additionally, the disclosed stabilizers also serve a third function of fixing, and thereby stabilizing, permeabilized cells, wherein the expressions "permeabilized" or "permeabilization" and "fixing", "fixed" or "fixation" are used as conventionally defined in cell biology. The description of stabilizing agents herein implies using these agents at appropriate concentrations or amounts, which would be readily apparent to one skilled in cell biology, where the concentration or amount is effective to stabilize the target cells without causing damage. One using the compositions, methods, and apparatus of this invention for the purpose of preserving rare cells would obviously not use them in ways to damage or destroy these same rare cells, and would therefore inherently select appropriate concentrations or amounts. For example, the formaldehyde donor imidazolidinyl urea has been found to be effective at a preferred concentration of 0.1-10%, more preferably at 0.5-5% and most preferably at about 1-3% of the volume of said specimen. An additional agent, such as polyethylene glycol has also been found to be effective, when added at a preferred concentration of about 0.1% to about 5%, more preferably about 0.1 % to about 1 %, and most preferably about 0.1 % to about 0.5% of the specimen volume. Stabilizing agents are necessary to discriminate between in vivo tumor cell disintegration and disintegration due to in vitro sample degradation. Therefore, stabilizing agent compositions, as well as methods and apparatus for their use, are described in a co-pending application entitled "Stabilization of cells and biological specimens for analysis." That commonly owned application is incorporated by reference herein.

The terms "obvious cells" or "intact cells" may be used interchangeably, and refer to cells found during imaging analysis that contain nucleic acid and cytokeratin. These cells are usually visually round or oval, but may sometimes be polygonal or elongated, and appear as individual cells or clusters of cells. The nucleic acid area (i.e. labeled by nucleic acid dye) is smaller than the cytoplasmic area (i.e. labeled by anti-cytokeratin), and is surrounded by the cytoplasmic area.

The terms "suspicious cells", "suspect cells", or "fragments" may be used interchangeably, and refer to cells found during imaging analysis that resemble intact cells, but are not as visually distinct as intact cells. Based on imaging analysis, there are a number of possible types of suspect cells, including:

1. Enucleated cells, which are shaped like Obvious cells, are positively stained for cytokeratin, but negative for nucleic acid;

2. Speckled or punctate cells, which are positively stained for nucleic acid, but have irregularly-stained cytokeratin; and

3. Amorphic cells, which stain positively for cytokeratin and nucleic acid, but are irregular in shape, or unusually large. These suspicious cells are considered in the present invention because they give additional information to the nature of the CTC, as well as the patient's disease. The staining or image artifacts observed during analysis provide additional informaton. For example, enucleated cells sometimes appear to have a "ghost" region where the nucleus should have stained, but the corresponding region is nucleic acid negative. This may be caused by a number of external factors, including the labeling or imaging techniques. Also, cells have been observed with "detached" nuclei. While this may possibly indicate a cell releasing its nucleus, it is more likely that this appears due to an artifact of the imaging system. However, such "artifacts," when real, give valuable information about what may be happening to the intact cells. Therefore, the present invention considers suspicious cells as a component in the analysis.

Cell fragments are different than "debris" in that debris does not necessarily resemble a cell. The term debris as used herein, refers to unclassified objects that are specifically or non-specifically labeled during processing, and are visible as images during analysis, but are distinct from intact and/or suspect cells. For example, it has been observed that damaged cells will release nuclear material. During processing, this nuclear material may be non-specifically magnetically labeled, and subsequently labeled with the nucleic acid stain. During analysis, the magnetically labeled and stained nuclear material can be observed when it has cytokeratin still attached. There are other objects that are similarly magnetically selected and stained which appear during analysis that are classified as debris. The term "morphological analysis" as used herein, refers to visually observable characteristics for an object, such as size, shape, or the presence/absence of certain features. In order to visualize morphological features, an object is typically non-specifically stained. The term "epitopical analysis" as used herein, refers to observations made on objects that have been labeled for certain epitopes. In order to visualize epitopic features, an object is specifically stained or labeled. Morphological analysis may be combined with epitopical analysis to provide a more complete analysis of an object. Figure 1 is a model of various CTC stages, including shedding and metastasis. Figure 1a. shows these stages for cells, clusters, fragments, and debris. Figure 1b. shows actual images from samples at these same stages. The images of cells clusters, fragments, and debris were taken from patient samples after immunomagnetic enrichment and image cytometry. The images of tissue samples (Origin and Metastatic sites) were taken from elsewhere (Manual of Cytology, American Society of Clinical Pathologists Press. 1983).

Briefly, a single cell shed from a primary tumor into the blood either survives or dies in blood. If it survives, it may possibly divide in blood, or colonize at a secondary site. If the cell dies, depending on the method, the cell degrades into various types of fragments or debris. Another possibility is a cluster of cells is shed from a primary tumor into the blood, where it may dissociate into single cells, or remain intact, and colonize at a secondary site. If the cluster dissociates, it can behave similar to the single cell described above. If the cluster remains intact, it is more likely to for a secondary colony for the reasons described above, which includes the large diameter cluster becoming lodged in a small diameter capillary. Once lodged, if the cells are viable, the cluster would form a new tumor.

The presence of fragments and debris with very few intact cells suggests that there will be little chance of metastasis. Fragmented cells will not divide, and cannot form secondary tumors. Indeed, only intact CTC or possibly CTC clusters would be capable of colonizing secondary sites. Identification of antigens that play a role in the adhesion and penetration process may help. Follow up and assessment of metastatic sites of the patients with and without clusters will also provide further insight. Nuclear morphology is used to determine the activity status and abnormality of a cell. Chromatin clumping, the presence or absence of nucleoli, and hyperchromasia, are criteria used to determine whether a cell is benign or malignant, reacting to an immune response, or reacting to treatment. The cytoplasmic morphology is used to determine the level of differentiation (i.e. tissue of origin). For example, cytomplasmic morphology can classify cells as squamous versus glandular.

During blood draw and subsequent specimen processing, the surviving battered tumor cells present in the peripheral circulation may be further stressed and damaged by turbulence during blood draw into an evacuated tube and by specimen processing, e.g. transport of the blood tube and mixing prior to analysis. Such mechanical damage is additional to on-going immunological, apoptotic, and necrotic processes leading to destruction of CTC that occur in vitro in a time dependent manner. We have found that the longer the specimen is stored, the greater the loss of CTC, and the larger the amounts of interfering debris and/or aggregates. Indeed, data presented in this specification (Figures 2 and 3) show dramatic declines in CTC counts in several blood specimens stored at room temperature for 24 hrs or longer, indicating substantial in vitro destruction of CTC after blood draw. While the losses of hematopoietic cells are well known phenomena and the subject of above-cited patents by Streck Labs and by others, the occurrence of mechanical damage due to mixing or transport have to date not been recognized factors in the loss of CTC or rare cells. The formation of cellular debris and the interfering effects of accumulating debris and/or aggregates in the analysis of CTC or other rare cells have similarly been unrecognized to date. It appears to be most evident and problematic in highly sensitive enrichment assays requiring processing of relatively large blood volumes (5- 5OmL), and subsequent microscopic detection or imaging of target cells after volume reduction (less than 1mL). Such debris are either not normally seen, or do not interfere in conventional non-enrichment assays, for example, by flow cytometry or in enrichment by density gradients methods.

To explore if these damaged epithelial cells and epithelial cell fragments observed in patients could be caused by apoptosis of tumor cells induced by chemotherapy, a model to mimic tumor cell death was developed. Cells of the prostate cell line LnCaP were cultured with or without paclitaxel and spiked into blood of healthy donors. The immunomagnetically selected cells of the paclitaxel treated samples resembled those observed in the patient blood samples. Cells treated with paclitaxel displayed signs of apoptosis. The punctate cytokeratin staining pattern of the cells appear to correspond with a collapse of the cytoskeletal proteins. The initiating event in the sequence resulting from the microtubule stabilizing effects of paclitaxel which in turn may activate the pro-apoptotic gene Bim that senses cytoskeletal distress. Further evidence of caspase-cleaved cytokeratin resulting from apoptosis was obtained with the M30 Cytodeath antibody (Roche Applied Science, Mannheim, Germany) that recognizes an epitope of cytokeratin 18 that is only exposed following caspase cleavage in early apoptosis. Only the paclitaxel treated LnCaP cells stained with M30 and most of the dimmer cytokeratin cells stained with M30, which is consistent with cells undergoing apoptosis.

With the technological resolution power of proteomics in recent years, the use as tool for clinicians in diagnosis and prognosis is becoming practical. The present invention utilizes this approach to provide clues in the early diagnosis of cancer and in prediction of clinical outcomes. One of the biggest problems in the clinical use of this approach is the selective extraction or enrichment of the desired global target entities, which typically number fewer than 100, from highly complex samples containing millions of irrelevant entities.

Prior attempts have used partial fractionation and enrichment on 2-D electrophoresis. This is a highly tedious, inefficient and costly procedure, resulting in a complex array of protein zones. The zones frequently contain numerous components of identical size and charge/mass ratio, totally unrelated to their functionality or diagnostic relevance. The analysis of proteins in these zones becomes more completed after proteolytic digestion into smaller fragments by mass spectrometry (MS). As mentioned above, MS is a highly sensitive analytical tool for identification of mass fragments based on charge/mass ratios which allows reconstruction of the parent molecule, but identification can only occur following removal of unrelated components. To increase sensitivity and remove these unrelated components, Surfaces Enhanced for Laser Desorption/lonization Time-Of-Fight (SELDI-TOF) MS on multiplexed microarray, microchip, or biochip detection of specific target proteins or protein digests are used on enriched fractions to provide an analytical mechanism for diagnosis. A direct analysis of unpurified samples such as serum and tissues by SELDI-TOF have been used by others, but these require analysis of complex fragmentation profiles and associated large data filed due to the presence of huge amounts of non-target materials.

In the case of rare target materials, effective detection requires prior enrichment and current fractionation methods of two-dimensional electrophoresis. As a diagnostic tool, these steps are affected by cost and clinical utility. Using magnetic or non-magnetic enrichment as described in the present invention reduces these unwanted factors and provides a basis for developing MS as a diagnostic tool. Besides magnetic separation, non-magnetic affinity-based solid phase separation can also be used to selectively enrich specific targets or target populations (e.g. antibody coated particles or solid phases for capturing the target materials, followed by analysis of the enriched fraction without or with prior dissociation from the support). Magnetic separation with ferrofluid particles described in US 6,365,362 provides a means of enrichment that is inexpensive and simple. Further, these ferrofluid particles provide higher binding capacities than other larger particles or non-magnetic solid phase particles (e.g. gel particles).

Thus, instead of running multiple costly and time-consuming MS analysis on numerous non-target spots for two-dimensional electrophoresis, only one single MS analysis on essentially target-specific proteins will be needed per sample, thereby dramatically increasing throughput, sensitivity and specificity of detection.

The one embodiment of the present invention, in part, uses the procedure described in US 6, 365,362 to incorporate multiparametric image cytometry and morphological characterization of selectively stained tumor cells together with proteomic analysis in cancer diagnosis. As previously described for rare cell detection methods in cancer diagnosis and management, magnetic enrichment of rare target cells, along with associated cell fragments and debris are coupled with proteomics as an alternative means of cancer cell detection.

Magnetic enrichment of rare target cells can occur after pretreatment with or without preservative (U.S. application 10/780,399). After immunomagnetic (or alternatively non-magnetic) enrichment, pathological cells, cell fragments, debris, and soluble cell fractions from patient specimens are assessed by MS, SELDI, microchips, biochips, or multiplexed micro array analysis. The detection of cell fragments, debris and soluble cell fractions from patient specimens are found in large quantities in the blood or tissues of some cancer patients, allowing for MS analysis. The importance and potential diagnostic utility of cell debris detection has been the subject of pending U.S. application 10/780,399. Figure 6 shows a diagramatic representation of one method for isolating the debri/cell fraction. The components of the crude enriched whole blood fraction are separated by acidification to remove bovine serum ferrofluid (BSA-FF) and streptavidin, conjugated to a monoclonal antibody (streptavidin- Mab). White blood cells (WBC) and red cells in the remaining cell and cell debris are removed by negative selection. Lipids, such as found in the membrane, are removed by solvent extraction. Thus, the only remaining components are the rare cells of interest (i.e. tumor cells and/or endothelial cells) and serum protein/glycoproteins. These are enriched by N₂ evaporation.

When imaging and proteomics are combined in this format, the magnetically enriched fractions are retrieved from the viewing chamber after imaging by magnetic separation of the supernatant buffer and buffer components. The buffer is replaced by an enzyme-compatible saline solution and analyzed directly. Instead of direct analysis, reversible chemical dissociation or tryptic dissociation digestion into fragments prior to MS analysis are done within the chamber by adding a dissociating agent or enzyme solution to a suspension of the magnetic particles to separate the ferrofluid particles. Thus, the captured cell and/or proteins are dissociated from the ferrofluid particles with an optional digestion to peptide fragments prior to analysis by MS. The preferred size for MS detection after tryptic digestion is 7,000 to 20,000 Da. This is a range that is lower than the sizes of most soluble tumor markers, and much lower than the sizes reported for circulating tumor cell debris.

Another embodiment incorporates magnetic enrichment of the target cells and/or cell debris using a proteomic analysis system as the only platform. lmmunomagnetic enrichment provides a simple amplification method to improve the sensitivity to a level that allows for consistent diagnostic use.

As a specific example, the captured target cells or proteins, complexed with desthiobiotinylated monoclonal antibody-ferrofluid (Mab-FF), are assessed by MS either alone or in combination with image analysis. For MS analysis, the captured target cells or proteins are dissociated from the ferrofluid with biotin to generate and enriched sample fraction, free of proteins derived from the ferrofluid particles. For cell and/or proteins from epithelial- derived cell membranes of circulating tumor cells (and debris from damaged circulating tumor cells), epithelial cell adhesion molecule (EpCAM) MAb-FF captures most of the target entities in the enriched sample fraction while other gradient methods may lose a substantial portion of entities.

Both approaches yield tumor specific mass profiles that are subtracted from MS profiles for BSA, MAbs-FF, or other sample enriched components. These subtracted profiles can be compared for disease and/or disease state, yet without knowledge of the identity of the measured proteins. The two embodiments, mentioned above, allow for complementary confirmation of CTC obtained by imaging, or possibly earlier cancer diagnosis in MS analysis without associated imaging. Surprisingly, MS analysis can provide a means for early cancer diagnosis even before intact CTC are detectable by imaging from a small blood specimen. For example, Her2/neu levels in the low ng/ml range in plasma can be immunomagnetically enriched to provide debris levels sensitive enough for MS analysis.

Further with respect to immunomagnetic enrichment and multiparametric image cytometry, MS proteomic analysis would obviate the need for immediate analysis or stabilization of blood samples for later analysis, required in image analysis. In addition, controlled aggregation may be unnecessary when analyzing captured debris. These factors could provide an improved sensitivity to diseases such as early cancer detection. It should be noted that a number of different cell analysis platforms can be used to identify and enumerate cells in the enriched samples. Examples of such analytical platforms are described in US Patents 5,876,593; 5,985,153 and 6,136,182, each of which are incorporated by reference herein as disclosing the respective apparatus and methods for manual or automated quantitative and qualitative cell analysis.

Other analysis platforms include laser scanning Cytometry (Compucyte), bright field base image analysis (Chromavision), and capillary Volumetry (Biometric Imaging). The enumeration of circulating epithelial cells in blood using the methods and compositions of a preferred embodiment of the present invention is achieved by immunomagnetic selection (enrichment) of epithelial cells from blood followed by the analysis of the samples. The immunomagnetic sample preparation is important for reducing sample volume and obtaining as much as a 10⁴ fold enrichment of the target (epithelial) cells. The reagents used for the multi-parameter flow cytometric analysis are optimized such that epithelial cells are located in a unique position in the multidimensional space created by the listmode acquisition of two light scatter and three fluorescence parameters. These include 1. an antibody against the pan-leukocyte antigen, CD45 to identify leucocytes (non-tumor cells);

2. a cell type specific or nucleic acid dye which allows exclusion of residual red blood cells, platelets and other non-nucleated events; and

3. a biospecific reagent or antibody directed against cytokeratin or an antibody having specificity for an EpCAM epitope which differs from that used to immunomagnetically select the cells.

It will be recognized by those skilled in the art that the method of analysis of the enriched tumor cell population will depend on the intended use of the invention. For example, in screening for cancers or monitoring for recurrence of disease, as described hereinbelow, the numbers of circulating epithelial cells can be very low. Since there is some "normal" level of epithelial cells, (very likely introduced during venipuncture), a method of analysis that identifies epithelial cells as normal or tumor cells is desirable. In that case, microscopy based analyses may prove to be the most accurate. Such examination might also include examination of morphology, identification of known tumor diathesis associated molecules (e.g., oncogenes).

Patients Patients' age range was 47-91 year (mean 74), with initial diagnosis 2 to

10 years prior to study. Medical records were reviewed for therapy and stage. Patients and healthy volunteers signed an informed consent under an approved research study. Blood was drawn into 10ml EDTA Vacutainer™ tubes (Becton-Dickinson, NJ). Samples were kept at room temperature and processed within 6 hours after collection unless indicated otherwise.

Sample Preparation

Magnetic nanoparticles labeled with monoclonal antibodies identifying epithelial cell adhesion molecule (EpCAM) were used to label and separate by magnetic means epithelial cells from hematopoietic cells, as taught in commonly-owned US Patent #6,365,362, and US Patent Application 10/079,939, filed 19 February 2002, both of which are fully incorporated by reference herein. The magnetically captured cells resuspended in a volume of 200DI are fluorescently labeled to differentiate between hematopoietic and epithelial cells. A monoclonal antibody that recognizes keratins 4, 5, 6, 8, 10, 13, and 18, conjugated to Phycoerythrin (CK-PE) was used to identify epithelial cells and a monoclonal antibody that recognizes CD45 was used to identify leukocytes and identify hematopoietic cells that non-specifically bind to cytokeratin. For multicolor fluorescent microscopy analysis, CD45 was conjugated to

Allophycocyanin (CD45-APC, Caltag, CA) whereas for flow cytometric analysis peridinin chlorophyll protein conjugated CD45 (CD45-PerCP, BDIS San Jose, CA) was used. The nucleic acid specific dye DAPI (4,6-diamidino- 2-phenylindole) was used to identify and visualize the nucleus and the nucleic acid dye in the Procount system (BDIS, San Jose.CA) was used to identify cells by flow cytometry. After incubation, the excess staining reagents were aspirated and the captured cells were resuspended and transferred into a 12x75 mm tube for flow cytometric analysis or image cytoometry analysis (as described in US Application 10/074,900, filed 12 February 2002, incorporated by reference herein) contained within a magnetic yoke assembly that holds the chamber between two magnets (Captivate, Molecular Probes, OR).

Example 1 Sample Analysis via Flow Cytometry

Samples were analyzed on a FACSCalibur flow cytometer equipped with a 488nm Argon ion laser (BDIS, San Jose, CA). Data acquisition was performed with CellQuest (BDIS, San Jose, CA) using a threshold on the fluorescence of the nucleic acid dye. The acquisition was halted after 8000 beads or 80% of the sample was analyzed. Multiparameter data analysis was performed on the listmode data (Paint-A-Gate^Pr0, BDIS, San Jose, CA). Analysis criteria for CTC events included size defined by forward light scatter, granularity defined by orthogonal light scatter, positive staining with the PE-labeled anti- cytokeratin MAb and no staining with the PerCP-labeled anti-CD45 Mab. For each sample, the number of events present in the region typical for epithelial cells was multiplied by 1.25 to account for the sample volume not analyzed by flow cytometry.

Figure 2 Panels A, B and C shows flow cytometric analysis of a blood sample of a patient with metastatic prostate cancer. Two vertical lines in Panel B illustrate the low and high boundary of nucleic acid (NAD) content of leukocytes (red dots). CTC candidates express Cytokeratin (CK+), lack CD45 (CD45-) and contain nucleic acids (NAD+). CTC candidates having NAD equal or higher than leukocytes are considered cells and are depicted black. CK+, CD45- events with NAD content less than leukocytes were not considered target cells and depicted blue. The blue events were clearly smaller as compared with the black colored CTC as evident by the smaller forward light scatter signals. The threshold on the NAD staining intensity clearly excluded a large portion of CK+, CD45- events with even lower NAD staining intensity. In analysis of blood samples from healthy donors few such CK+, CD45- events are observed suggesting that this phenomenon is related to cancer. A typical example of an analysis of a blood from a healthy donor is shown in Figures 2D, 2E, and 2F. Example 2 Sample Analysis by Image Cytometry

The image cytometry system consists of a microscope with a Mercury Arc Lamp, a 1OX objective, a high resolution X, Y, Z stage and a four-filter cube changer. Excitation, dichroic and emission filters in each of four cubes were for DAPI 365nm/400nm/400nm, for DiOCI 6 480nm/ 495nm/ 510nm, for PE 546nm/ 560nm/ 580nm and for APC 620nm/ 660nm/ 700nm. Images were acquired with a digital camera connected to a digital frame grabber. The surface of the chamber is 80.2 mm² and 4 rows of 35 images for each of the 4 filters resulting in 560 images have to be acquired to cover the complete surface. The acquisition program automatically determines the region over which the images are to be acquired, the number of images to acquire, the position of each image and the microscope focus to use at each position. All the images from a sample are logged into a directory that is unique to the specific sample identification. An algorithm is applied on all of the images acquired from a sample to search for locations that stain for DAPI and CK-PE. If the staining area is consistent with that of a potential tumor cell (DAPI+, CK- PE+) the software stores the location of these areas in a database. The software displays thumbnails of each of the boxes and the user can confirm that the images represented in the row are consistent with tumor cells, or stain with the leukocyte marker CD45. The software tabulates the checked boxes for each sample and the information is stored in the database.

Figure 3 shows examples of image analysis of a blood sample from a patient with metastatic prostate cancer. Regions that potentially contain tumor cells are displayed in rows of thumbnails. The ruler in the left lower corner of the figure indicates the sizes of the thumbnails. From right to left these thumbnails represent nuclear (DAPI), cytoplasmic cytokeratin (CK-PE), control cells stained with a membrane dye (DiOCi₆(3)) and surface CD45 (CD45-APC) staining. The composite images shown at the left show a false color overlay of the purple nuclear (DAPI) and green cytoplasmic (CK-PE) staining. The check box beside the composite image allow the user to confirm that the images represented in the row are consistent with tumor cells and the check box beside the CD45-APC image is to confirm that a leukocyte or tumor cell stain non-specifically. In this patient sample, the software detected 2761 rows of thumbnails that demonstrated staining consistent with tumor cells. Eighteen of the 2761 rows are shown in the figure labeled 1631- 1640 and 1869-1876. Rows numbered 1631 , 1636, 1638, 1640, and 1873- 1876 are checked off and display features of CTC defined as a size greater than 4Dm, the presence of a nucleus surrounded by cytoplasmic cytokeratin staining and absence of DiOC-i₆(3) and CD45 staining. Note the difference in appearance of the tumor cells: the cell in row 1638 is large and the one in row 1640 is significantly smaller. The immunophenotype of the events in rows 1634 and 1869 are consistent with tumor cells but their morphology is not consistent with intact cells. The thumbnails in row 1869 shows a large nucleus and speckled cytoplasmic due to retraction of cytoskeletal proteins consistent with apoptosis of the cell. The thumbnail in row 1634 shows a damaged cell that appears to extrude its nucleus. The thumbnail shown in row 1632 shows a cell that stains both with cytokeratin as well as CD45 and is either a tumor cell non-specifically binding to CD45 or a leukocyte non specifically staining with cytokeratin. In this instance the morphology of the cell closely resembles that of a lymphocyte. The thumbnails shown in rows 1633, 1635, 1637, 1639, 1870 and 1872 shows cytokeratin staining objects that are larger that 4 Dm but have no resemblance to cells. The cytokeratin staining objects in thumbnails 1637, 1639 and 1872 are in close proximity of a leukocyte.

Based on observation of images of CTC candidates in several patient samples, CTC were classified into three categories: intact CTC, damaged CTC, and CTC fragments all not staining with CD45 and not appearing in the DiOC-|₆(3) filter. Figure 4 displays examples of the three categories of CTC isolated from a single tube of blood of a patient with metastatic prostate cancer undergoing therapy. Intact tumor cells shown in Figure 3A were defined as objects larger than 4mm with a relatively smooth cytoplasmic membrane, cytoskeletal proteins throughout the cytoplasm, and an intact nucleus encompassed within the nucleus. Damaged CTC shown in Figure 4B were defined as objects larger than 4mm with speckled cytokeratin staining or ragged cytoplasmic membrane, and a nucleus associated with the cytokeratin staining. Tumor cell fragments shown in Figure 4C were defined as round cytokeratin staining objects larger than 4mm with or without association of nuclear material that had no morphological resemblance to a cell.

Example 3 CTC in Prostate Cancer Patients

CTC were enumerated in 18 blood samples of prostate cancer patients and 27 samples from healthy individuals by both flow cytometry and image cytometry The results shown in Table 1 were sorted by increasing number of intact CTC detected.

Table 1 - Enumeration of CTC by image cytometry and flow cytometry in 18 blood samples of prostate cancer patients and 27 samples from healthy individuals.

# - number CTC in 7.5 ml blood % - percentage of all CTC detected by Image Cytometry The proportion of intact CTC clearly constituted the smallest fraction of CTC and ranged from 0% to 22% of all CTC (mean 4%). The proportion of damaged CTC ranged from 1% to 100% (mean 34%) and the CTC fragments constituted the largest portion of CTC ranging from 0% to 93% (mean 62%). The distribution of CTC over the three categories between the patients varied considerably as amplified by a lack of correlation between intact CTC and damaged CTC (R² = 0.20) and intact CTC and CTC fragments (R² = 0.42) and some correlation between damaged CTC and CTC fragments (R² = 0.88). Comparison of intact CTC by and CTC enumerated by flow cytometry showed no significant correlation (R² = 0.26) whereas significant correlations were found between the damaged CTC and CTC by flow cytometry (R² = 0.92) and CTC fragments and CTC by flow cytometry (R² = 0.93). Comparison of the CTC detected by flow cytometry and image cytometry suggests that CTC detected by flow cytometry encompass intact CTC as well as damaged CTC and to a certain extent, CTC fragments.

Example 4

Mimicking cell damage by in-vitro induction of apoptosis in LnCaP cells To investigate the effect of apoptosis induced by cytotoxic agents on flow cytometric and image cytometry on CTC, cells from the prostate cell line LnCaP were cultured in the presence or absence of 4OnM paclitaxel for 72 hours. Following incubation, untreated LnCaP cells demonstrated a viability of >95% by trypan blue exclusion and 33% for the paclitaxel treated cells. The treated and untreated LnCaP cells were spiked into blood of healthy donors, selected by the ferrofluid methods described above, and analyzed by the image cytometry. In experiments in which LnCaP cells were spiked into blood that were not treated with paclitaxel greater than 95% of the LnCaP cells were classified as intact tumor cells. The morphologic appearance of the paclitaxel treated LnCaP cells showed close resemblance to that of the CTC observed in the patient samples and are shown in Figure 6. Intact LnCaP cells that survived paclitaxel treatment are shown in Figure 6A, damaged LnCaP, of which the majority show speckled cytokeratin staining, are shown in Figure 6B, and tumor fragments are shown in Figure 6C. Normal blood samples spiked with paclitaxel treated and untreated LnCaP cells were also prepared for flow cytometric analysis. In Figures 2G, 2H, and 21, the flow cytometric analysis of a blood sample spiked with 501 LnCaP cells is shown. A predominantly bright cytokeratin positive population with a nucleic acid content greater than normal human leukocytes and relatively large size as illustrated by the large forward light scatter signals are shown and depicted black in the figure. Only few CK+, CD45- events with NAD content less than leukocytes and depicted blue are detected in the sample. Figures 2J, 2K, and 2L shows the flow cytometric analysis of paclitaxel treated LnCaP cells spiked in blood. In contrast to viable LnCaP cells, a wide distribution of cytokeratin staining was observed with a significant portion of the population demonstrating a decreased concentration of nucleic acid content. In addition, numerous small cytokeratin positive events with less nucleic acid content as leukocytes were observed. The pattern of the patient closely resembled that of the pattern of the paclitaxel treated LnCaP cells supporting the hypothesis that the CTC detected by flow cytometry represent intact CTC as well as a variety of disintegrating cells in blood of cancer patients.

The data shown above demonstrate that in the blood of patients with prostate cancer, CTC detected by both flow cytometry and image cytometry are comprised of intact cells and cells of cells at various stages of disintegration. The apoptosis induced in vitro by paclitaxel suggests that the detected CTC in patient blood samples are undergoing apoptosis, necrosis, or in vivo damage to a varying degree caused by the treatment or therapy, mechanical damage by passage through the vascular system, or by the immune system.

Another source of cell disintegration, caused in vitro could however, be introduced by the sample preparation or the lack of stabilization of CTC or other blood components after blood draw. To investigate the effect of sample aging, known to cause damage, blood samples drawn from 12 patients with prostate cancer were processed and analyzed by flow cytometry within two hours, after 24 hours, and after 6 and 18 hours if sufficient blood was available. In 8 of the 12 patient samples, CTC were detected at a level greater than the mean +3SD of that detected in normal donors. As shown in Table 2, a loss of CTC with sample aging was observed in all 8 samples.

Table 2 - Enumeration of CTC by flow cytometry in 8 blood samples of prostate cancer patients processed and analyzed at different time points after blood draw

hr = hours #CTC = number of CTC in 5 ml blood

Significant reductions in the number of CTC were detected when blood processing was delayed demonstrating the fragility of CTC, and making it necessary to process non-stabilized blood samples no later than six hours after blood draw to obtain accurate CTC counts. To reliably assess if clinically relevant information is contained within the different stages of tumor cell degradation, a blood preservative is needed that stabilizes CTC at the time of blood draw to obtain an accurate reflection of what is occurring inside the body. Furthermore, the sample preparation method for sensitive assays used to enrich for CTC requires that all classes of CTC are captured, and therefore excludes the use of traditional density gradient separation methods in the prior art.

Example 5 Obvious CTC and Suspect CTC are important indicators

It is important to be able to distinguish between in vivo and in vitro damage for sensitive assays, such as those described here. This is especially evident when the assay attempts to determine the effectiveness of treatments or therapies, which are known to cause in vivo cellular damage. If sample handling, processing, or analysis were to result in damaging the target cells, forming Suspect cells, fragments, or debris, the assay will not give meaningful results. An assay was used to directly detect CTC in 100Dl of blood without any enrichment method by flow cytometry. The 100DI assay detects only EpCAM positive cells and the sensitivity is very low. However, some advanced stage cancer patients with high CTC counts are expected to be observable. This assay should give a reliable confirmatory estimation of CTC because it is a direct assay that involves no manipulation. Data were generated with several patient samples using the assay to answer several questions.

The 100Dl assay categorizes cells based on properties such as size and staining intensity. Obvious CTC have bright nucleic acid staining (similar to leukocytes), positive EpCAM antigen staining and size similar to leukocytes or larger. Suspect CTC are any objects positive for EpCAM but not characterized as Obvious CTC (i.e. dim nucleic acid, size smaller than leukocytes). The assay identifies objects from both categories.

Figure 5 shows the presence of obvious and suspect CTC in blood as determined by the 100Dl assay. The Suspect CTC are not created during sample processing (in vitro damage) as the 100DI assay is a direct assay and does not involve any separation or wash steps. The data above also show there is a relationship between the number of Obvious and Suspect CTC. The number of Suspect CTC seems to increase as the number of Obvious CTC increases. When the numbers of Suspect versus Obvious CTC is plotted, the slope of 2.92 indicates the proportion of Suspect CTC present in sample when compared to Obvious CTC. The correlation coefficient of r² = 0.0.97 shows an excellent correlation between Obvious CTC and Suspect CTC for a number of clinical samples. In addition, Suspect CTC are also seen in the ferrofluid-selection assay, and have properties similar to Suspect CTC detected in the blood by the direct assay. It is important to include Suspect CTC in addition to Obvious CTC in total tumor cell count.

An important question is how the data from the 100DI assay compares with ferrof Iu id-selected CTC (enriched CTC). Does the ferrofluid assay quantitatively detect CTC? Another question is what is the recovery of CTC in the ferrofluid-selected assay if the flow assay data is correct. The three main factors determining the recovery of CTC in the assay are:

• EpCAM density,

• cytokeratin positivity, and • nucleus positivity.

The suspect CTC have lower EpCAM density compared to obvious CTC and significance of this is not yet well understood.

A comparison was made of obvious and suspect CTC by the 100 ml assay to the ferrofluid-selection assay using 7.5 ml of blood. This data was obtained from prostate patient samples and analyzed by flow cytometry. Both obvious and suspect CTC increased with storage time and the trend was similar to CTC detected in the ferrofluid-selection assay, thereby validating the 100DI assay. The recovery of CTC from the ferrofluid-selection assay was about 90% based on the CTC in 100 ml of blood. It was also known that MFI (Mean Fluorescence Intensity which correlates the EpCAM density) of CTC from this patient was high (MFI=300), and all EpCAM positive cells are cytokeratin positive. However, the recoveries of CTC from some other clinical samples have been as low as 20%. There may be several factors that contribute for a lower recovery, such as EpCAM positive/cytokeratin negative cells, cytokeratin dim cells, and mucin on the cell surface inhibiting the ability of ferrofluid to bind cells.

The assay described herein was performed on patients at two times. Response was measured by bi-dimensional imaging of the lesion. The Ratio (Ratio = Obvious CTC / Total CTC) is similar to the Response Index described earlier, and can be used as a numeric indicator of treatment success. The results are summarized in Table III. Ratios near 1.0 indicate the Total CTC are obvious CTC, and ratios near 0.0 indicate more suspect CTC or debris. Progressive indicates the lesion increasing in size, partial response indicates a response to treatment where the Ratio is relatively low, and Stabilized indicates no change, or reduction in lesion size. A positive change indicates an increase in the number of Intact CTC, corresponding to the progression of the disease. A negative change indicates a decrease in the number of Intact CTC, or a possible increase in the number of suspect CTC and/or debris, corresponding to a response to treatment.

These results show the importance of including suspect CTC and debris when analyzing response to treatment because the numbers of intact or obvious CTC alone would not provide as much information. Furthermore, such indicators are useful for short-term monitoring of treatments and therapies, or longer term monitoring for remission and/or relapse.

Table 3- Obvious CTC and Suspect CTC corresponding to treatment response

Example 6 Reduction in mRNA content after plasma washing.

Patients with >20 CTC/7.5 ml of blood were assayed for the quantity of CTC specific mRNA. Individual samples from patients (n=12) were matched by dividing a patient's sample into plasma wash and no plasma wash groups after addition of cell preservative (U.S. Appl. No. 10/780,349). Following EpCAM magnetic enrichment and centrifugation, PSA mRNA remaining in the enriched fraction was assessed by quantitative RT-PCR. The table below shows that plasma washings, either at the time of blood draw (0 hr EDTA) or 24 hr after blood draw, resulted in a significant loss of mRNA.

Table 4- Reduced CTC specific mRNA for PSA after plasma wash

Table 4 shows that plasma washing eliminates at least 3 fold mRNA. Because intact CTCs do not remain in the plasma following centrifugation at

800(g), RNA signals must come from a fraction of cell debris that does not partition from the plasma fraction and remains in the plasma, subsequently aspirated away with washing. Consequently, an even larger difference could result with the incorporation of rare cell debris, partitioned from the plasma after centrifugation.

Example 7

The presence of gene expression in enriched blood samples of patients with metastatic colorectal cancer devoid of intact CTC. Two 7.5 ml blood samples from 37 healthy donors and 49 patients with CRC were immunomagnetically enriched by targeting the EpCAM antigen. One sample was used to determine the number of intact CD45-, cytokeratin 8, 18 and/or 19 positive tumor ceils and the other sample was used to evaluate the expression of CK20, CEA, CK19, EGFR, GUC, EpCAM, VEGF, TS and Muc-1. Gene expression was evaluated by creating an aRNA library from the CTC enriched samples followed by RT-PCR of the individual genes.

No intact CTC and no RT-PCR signals for CK19, CK20, CEA, and EGFR were detected in the control group. RT-PCR for GUC, EpCAM, VEGF, TS and Mud showed positive signals in the control group and were not further evaluated in CRC. In 19 of 49 (39%) samples of CRC patients, 2 or more CTC were detected in 7.5 ml blood samples. The number and percentage of samples that scored positive for CK20, CEA₁ CK19, EGFR in the samples with and without intact CTC are listed in the table 5 below.

Table 5- Comparison of RT-PCR signals from enriched samples with/without intact CTC.

In 15 of 19 (79%) CRC samples with intact CTC one of the genes was expressed but surprisingly in 18 of 30 (60%) samples with no intact CTC scored positive for at least one of the genes from the gene panel.

Intact CTC and genes expressed in epithelial cells can be detected in blood samples of CRC patients enriched for EpCAM expression. The finding of RT-PCR positives in patients in which no intact CTC were detected may be due to carcinoma cells shed into the blood that have been damaged or destroyed.

Enumeration of tumor cell debris may prove more significant in cancer diagnostics and therapeutics than detection of large proliferative cell clusters. Since debris particles in the size range, probably about 1-3Dm (the size of platelets), have been observed to be present in much larger amounts than intact cells, they may constitute a separate, independent, and possibly more sensitive marker than intact tumor cells. The presence of damaged CTC may be particularly relevant in detecting early-stage cancer, when the immune system is intact and most active. Similarly, dramatic increases in debris during therapy may suggest breakdown of both circulating and tissue tumor cells (i.e. therapeutic effectiveness), paralleling the massive release of cellular components like calcium observed during tumor disintegration. Like soluble tumor markers, such debris may be detectable in blood without enrichment, or with minimal enrichment in the buffy coat layer and constitute an alternative, and potentially simpler diagnostic tool than intact cell enrichment/analysis. Since morphology is lost in CTC debris, detection could be done by flow cytometry as long as the debris is stained for the appropriate determinants, such as cytokeratin.

As previously discussed, damaged or fragmented CTC with or without DNA are theoretically to be expected, and therefore are not undesirable events in specimens from patients undergoing effective therapy and in untreated patients with strong immune systems. The ratio or percent of intact CTC to total detectable events may prove to be a more useful parameter to the clinician in assessing a patient's immune system or response to therapy. The normal immune defenses, especially activated neutrophils, also can damage or destroy CTC as foreign species by a process called "extracellular killing" even if the CTC are larger than the neutrophils. It does not seem surprising to find only a small percentage of the shed CTC as intact cells, unless the immune system is overwhelmed in the late stages of disease or therapy is ineffective.

Hence, there are a number of methods for in vitro cancer detection: conclusive detection of intact circulating cells/clusters, and inferential methods like circulating tumor debris (including total and tumor-specific RNA/DNA, and conventional soluble tumor markers. However, no method by itself may be sufficiently sensitive. Lower specificity of debris detection compared to CTC morphology may be a problem in screening that could be minimized (e.g. with triple labeling), but it may be a lesser problem in monitoring. Further statistical analysis and correlations on debris data relative to intact CTC and diagnostic stage in patients compared to normals appear worthwhile in assessing the sensitivity and specificity of debris analysis. This data would include sequence analysis of nucleic acid or protein components from rare cells in the enriched samples. Sequence analysis includes the quantification, and/or qualification of an individual sequence or groups of sequences associated with the disease of interest. For example, RNA sequence analysis is accomplished by multigene RNA profile analysis. Sequence quantification is accomplished by quantitative RT-PCR while sequence qualification is accomplished through array analysis. The present invention is not limited to this analysis, but includes all sequence analysis accepted by individuals in the field.

Examples of different types of cancer that may be detected using the compositions, methods and kits of the present invention include apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell and transitional cell reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, throphoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, antiokeratoma, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (Kaposi's, and mast-cell), neoplasms (e.g., bone, digestive system, colorectal, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis, and cervical dysplasia.

However, the present invention is not limited to the detection of circulating epithelial cells and/or clusters, fragments, or debris. For example, endothelial cells have been observed in the blood of patients having a myocardial infarction. Endothelial cells, myocardial cells, and virally infected cells, like epithelial cells, have cell type specific determinants that are recognized by available monoclonal antibodies. Accordingly, the methods and the kits of the invention may be adapted to detect such circulating endothelial cells. Additionally, the invention allows for the detection of bacterial cell load in the peripheral blood of patients with infectious disease, who may also be assessed using the compositions, methods and kits of the invention. It would be reasonable to expect that these rare cells will behave similarly in circulation, and that fragments and/or debris will be present in similar conditions as those described hereinabove. The preferred embodiments of the invention as herein disclosed, are also believed to enable the invention to be employed in fields and applications additional to cancer diagnosis. It will be apparent to those skilled in the art that the improved diagnostic modes of the invention are not to be limited by the foregoing descriptions of preferred embodiments. Finally, while certain embodiments presented above provide detailed descriptions, the following claims are not limited in scope by the detailed descriptions. Indeed, various modifications may be made thereto without departing from the spirit of the following claims.

While the preferred embodiments of the present invention as disclosed relate to cancer and cardiovascular diagnostics, those skilled in the art will recognize that the invention is enabling in other fields and applications. Thus, it is apparent that the improved diagnostic modes of the present invention are not to be limited by the foregoing descriptions. While certain embodiments presented above provide detailed descriptions, the following claims are not limited in scope by the detailed descriptions. Accordingly, various modifications may be made thereto without departing from the spirit of the following claims.

Claims

We claim:

1. An improvement to a method for diagnosing disease in a test subject by obtaining a body fluid specimen from a test subject, said specimen comprising a mixed cell population suspected of containing at least one member of a group consisting of intact rare cells and cell components, such as cell fragments and cellular debris derived from rare cells; preparing a magnetically-labeled sample wherein said specimen is mixed with magnetic particles coupled to a first biospecific ligand which reacts specifically with said group member to the substantial exclusion of other specimen components and analyzing said group member, the presence of said group member indicating the presence of disease, said improvement comprises analyzing for proteomic information in an immunomagnetically enriched body fluid sample.

2. The improvement as described in claim 1 where said magnetically-labeled specimen is contacted with at least one additional biospecific ligand which specifically labels said group member, to the substantial exclusion of other specimen components.

3. The improvement as described in claim 1 where said rare cell component is composed of members from a group consisting of nucleic acid, RNA, DNA, protein, and combinations thereof.

4. The improvement as described in claim 1 where said first biospecific ligand is a monoclonal antibody specific for EpCAM.

5. The improvement as described in claim 1 where said rare cells are from a group consisting of epithelial cells, endothelial cells, virally infected cells, bacterially infected cells, and any combination thereof.

6. The improvement as described in claim 5 where said endothelial cells are melanoma derived.

7. The improvement as described in claim 6 where said first biospecific ligand specifically interacts with melanoma antibody M330.

8. The improvement as described in claim 1 where said first biospecific ligand specifically interacts with antibody from a group consisting of CD

146, CD 105, and combinations thereof.

9. The improvement as described in claim 1 where said proteomic analysis includes a group consisting of ESI MS/MS, 2D gels, MALDI TOF/TOF, SELDI, and combinations thereof.

10.An improvement to a method for diagnosing disease in a test subject by obtaining a body fluid specimen from a test subject, said specimen comprising a mixed cell population suspected of containing at least one member of a group consisting of intact rare cells and cell components, such as cell fragments and cellular debris derived from rare cells; preparing a magnetically-labeled sample wherein said specimen is mixed with magnetic particles coupled to a first biospecific ligand which reacts specifically with said group member to the substantial exclusion of other specimen components and analyzing said group member, the presence of said group member indicating the presence of disease, said improvement comprises analyzing for the presence of rare cell components in said magnetically-labeled sample whereby said analysis is from a group consisting of sequence quantification, sequence qualification, and combinations thereof.

11. The improvement as described in claim 10 where said magnetically- labeled specimen is contacted with at least one additional biospecific ligand which specifically labels said group member, to the substantial exclusion of other specimen components.

12. The improvement as described in claim 10 where said rare cell component is from a group consisting of nucleic acid, RNA, DNA, protein, and combinations thereof.

13. The improvement as described in claim 10 where said first biospecific ligand is a monoclonal antibody specific for EpCAM.

14. The improvement as described in claim 10 where said analysis is multigene RNA profile analysis.

15. The improvement as described in claim 10 where said analysis is from a group consisting of quantitative RT-PCR, array analysis, and combinations thereof.

16. The improvement as described in claim 10 where said rare cells is from a group consisting of epithelial cells, endothelial cells, virally infected cells, bacterially infected cells, and any combination thereof.

17. The improvement as described in claim 16 where said endothelial cells are melanoma derived.

18. The improvement as described in claim 17 where said first biospecific ligand specifically interacts with melanoma antibody M330.

19. The improvement as described in claim 10 where said first biospecific ligand specifically interacts with antibody from a group consisting of CD 146, CD 105, and combinations thereof.