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EP1226439A1 - Method for the detection of mammalian carcinomas - Google Patents

Method for the detection of mammalian carcinomas

Info

Publication number
EP1226439A1
EP1226439A1 EP00984990A EP00984990A EP1226439A1 EP 1226439 A1 EP1226439 A1 EP 1226439A1 EP 00984990 A EP00984990 A EP 00984990A EP 00984990 A EP00984990 A EP 00984990A EP 1226439 A1 EP1226439 A1 EP 1226439A1
Authority
EP
European Patent Office
Prior art keywords
spatial distribution
sample
cancer
specific component
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00984990A
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German (de)
French (fr)
Inventor
Roeland Universiteit van Amsterdam VAN DRIEL
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Universiteit Van Amsterdam
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Universiteit Van Amsterdam
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Publication date
Application filed by Universiteit Van Amsterdam filed Critical Universiteit Van Amsterdam
Priority to EP00984990A priority Critical patent/EP1226439A1/en
Publication of EP1226439A1 publication Critical patent/EP1226439A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention is in the field of molecular and cell biology and diagnostics, and relates in particular to a new and efficient method for the early detection of certain carcinomas in humans.
  • cancer cells deviate from this normal behaviour. They are no longer susceptible to normal controls on proliferation, but follow a more independent way of reproduction.
  • a normal cell When a normal cell is transformed into such a genetically modified cell, it produces too much of the same type of cells.
  • the cancer cells invade neighbouring tissues, thereby displacing the surrounding tissue and disturbing the interdependent relations.
  • the most threatening property is the ability to move to other places from the site where they began and to invade other tissues and organs, thereby deregulating and disturbing the normal functions of these organs.
  • this pattern of cancer development continues unnoticed for a long period, the treatment will become more and more difficult or even impossible. Therefore, a reliable method for detecting abnormal growth and spreading of suspicious cells in the human body at a very early stage is crucial to adequate treatment.
  • cancer cells Other methods for detecting cancer cells are based on specific differences between cancer cells and normal cells that have emerged from fundamental research. For example, extensive research to detect differences between normal cells and cancer cells have revealed specific cellular components of cancer cells which are recognised by very specific antibodies which recognize tumor markers. These tumor markers in cancer cells differ in their presence from normal cells by their overproduction, persistent production, and/or their derangement of secretion (see, for example, G.I. Abelev & S. Sell, 1999, Seminars in Cancer Biology 9:61-65). Many of these tumor markers are used commercially.
  • US 5,310,653 discloses a method to purify the p65 tumor- associated protein and its use as a tumor marker for diagnosis of steroid- associated cancers.
  • US 5,272,256 discloses the isolation and use of a nuclear autoantigen for the detection of autoimmune disorders.
  • US 4,885,236 discloses a method for the identification and characterization of cells in a biopsy or sample of a body fluid which is based on the isolation and analysis of the components of a specific subcellular protein fraction referred to as "nuclear matrix".
  • the nuclear matrix includes proteins and nuclear matrix associated DNA specific to different cell types. These proteins and nucleic acids are altered or new ones expressed as a result of viral infection, genetic defects or malignancy.
  • PML promyelocytic protein
  • 5,599,919 discloses the use of detection of the CENP-F at the genomic and protein level in cells to obtain information about their proliferation state.
  • Dos Santos et al., Human Molecular Genetics (1997) 6:1549-1558 describe the nuclear localization pattern of two proteins.
  • LeBrun et al., Oncogene (1997) 15:2059-2067 disclose the nuclear distribution of two proteins.
  • Everett et al., J. Virol. (1998) 72:6581-6591 describe observations on the inetraction between viral gene products and PML bodies.
  • Duprez ef al., J. Cell Sci. (1999) 112:381-393 disclose the SUMO-modification of the PML protein in relation to the nuclear distribution of this protein.
  • a new method for detecting and grading a cell proliferation disorder in an individual which comprises: (a) determining the spatial distribution of at least one specific component in the cell nucleus of a sample from said individual to be analyzed;
  • the proliferation disorder to be investigated is in the gastro-intestinal tract.
  • said at least one specific component in the cell nucleus is selected from the group consisting of the antigens p80-coilin, PML, SC35, acetylated histones, CstF64, hnRNP-l, and NuMA protein.
  • the spatial distribution of said at least one specific component in the cell nucleus is determined, by immuno- fluorescence or other techniques.
  • Figures 1 and 2 show monolabeled nuclei in normal and tumor intestinal epithelial tissue sections, respectively.
  • Coiled bodies in the nuclei are immuno- labeled by monoclonal antibody 204/5 (prepared against p80-coilin).
  • the nuclei of normal cells contain less coiled bodies than tumor cells.
  • the tissue organisation in the tumor is disordered.
  • Figure 3 shows confocal sections of mono-labeled nuclei from normal
  • CstF 64 displayed a nucleoplasmic granular labeling pattern in epithelial nuclei of normal tissue section.
  • CstF 64 displayed a complex, interconnected domain-like pattern in epithelial nuclei of tumor tissue section.
  • Figure 4 shows confocal sections of mono-labeled nuclei from normal (A & B) and tumor (C & D) epthelium in tissue sections with the antibody 7G12 [donor no. 2].
  • a & B 7G12 strongly stained few bright foci in nuclei of healthy epithelium.
  • C & D hnRNPI displayed a weak and puntated labeling pattern in nuclei of tumor epithelium.
  • Figure 5 shows fluorescence microscopy images of monolabeled nuclei containing antigen p80-coilin, in normal (A) and tumor (B) tissue section, mag x25.
  • the term "individual” generally refers to any mammalian, both human beings and animals, such as pets, but the term is in particular used in connection with human beings.
  • sample is not limited to tissue sample, but encompasses any type of biological source which usually is investigated by pathologists (and others) for the screening of proliferation disorders such as various types of cancer.
  • the biological sources include also blood, lymphatic fluid, urine, stool, etc.
  • reference sample refers to essentially the same type of sample as the sample to be investigated, since spatial distribution patterns generally are tissue-specific.
  • the reference sample is usually derived from normal cells of the same or similar source, usually the same tissue as the sample to be analyzed, from the same or a different individual.
  • the reference sample is a statistic mean of spatial distribution patterns of the same or similar source, usually the same type of tissue, as the sample to be analyzed, derived from a relevant number of individuals, and selected by people skilled in the art, e.g. one or more pathologists or oncologists.
  • the present invention is based on the understanding that the spatial distribution of specific components, such as proteins and other molecules, in a mammalian cell nucleus allows one to establish whether a cell is in its normal state or has undergone transformation to a tumorogenous state. If transformation has occurred, such spatial distribution is different from normal cells and the extent of the different spatial distribution allows grading of the tumor.
  • the specific components in particular proteins (antigens), are identified by suitable detection techniques, e.g. fluorescence microscopy using specific antibodies against these antigens.
  • the cell nucleus is a highly dynamic and compartmentalized organelle, where many nuclear processes, including DNA replication, DNA repair, RNA synthesis, processing and transport, take place.
  • the cell nucleus consists of many readily identifiable nuclear structures including the nucleolus, heterochromatin domains, domains representing local high concentrations of functional nuclear machineries and a variety of nuclear bodies, e.g. coiled bodies, cleavage bodies and PML bodies. These nuclear structures are functionally organized in a specific pattern in the normal diploid mammalian cell nucleus.
  • the present invention is, in one embodiment, more specifically based on the correlation of the transformed phenotype of epithelial cells in colorectal cancer with an altered spatial organisation of nuclear components.
  • the spatial distribution of various nuclear components is investigated in epithelial cell nuclei of the large bowel of normal and adenomatous, carcinomatous tissues.
  • neoplastic epithelial cell nuclei various striking differences can be observed as compared with normal cell nuclei: (i) an increased number of nuclear bodies, such as PML bodies and coiled bodies, (ii) for some nuclear components (splicing factors, cleavage factors, hnRNP-AI, hnRNP-l, mitotic apparatus protein), a change in spatial distribution.
  • nuclear bodies such as PML bodies and coiled bodies
  • the present invention provides in one aspect a method for detecting and grading cell proliferating disorders in individuals by determining the spatial distribution of at least one specific component in the cell nucleus of a sample from said individual to be analyzed, comparing said spatial distribution with the spatial distribution of the same at least one specific component in a reference sample, and characterizing said at least one component of said sample to be analyzed based upon comparison with said at least one component in said reference sample.
  • the reference sample is usually derived from normal cells of essentially the same source, usually the same tissue, as the sample to be investigated, from the same or a different individual.
  • the sample to be investigated and the reference sample are derived from the same individual.
  • the sample to be investigated and the reference sample are derived from essentially the same tissue from the same individual.
  • the reference sample is a statistic mean of spatial distribution patterns from the same or similar source, usually the same type of tissue, as the sample to be analyzed, derived from a relevant number of individuals, and selected by people skilled in the art, e.g. one or more pathologists or oncologists.
  • selected spatial distribution patterns are conveniently stored in a data base or on a carrier. This alternative embodiment offers an excellent opportunity for large scale diagnosis and automation of results.
  • the detection and characterisation of specific components from the cell nucleus is known in the art and can be performed by any suitable means which are useful for obtaining clear distribution patterns of the various aimed components to enable adequate and preferably unambiguous comparisons.
  • a panel of available antibodies each recognizing one or more nuclear components, can be used to select antibodies that recognize one or more differences in spatial distribution of nuclear components between normaland tumorogenous tissues.
  • specific nuclear components that are suspected to be differently distributed in tumorogenous cells and normal cells can be isolated using standard procedures and used to prepare specific antibodies that are useful in discriminating between normal and tumorogenous cells.
  • Proteins as described herein are useful as immunogens for the preparation of antibodies when these antibodies are conjugated with colorimetric, immunological, fluorescent or radioactive labels.
  • Antibodies useful for employing in the detection and characterization of the spatial distribution patterns of the components mentioned above are any type of antibodies, whether monoclonal or polyclonal, which result in detectable and reproducible labelling patterns.
  • Suitable antibodies for the purpose of this invention include, for example, the monoclonal antibody 5E10, that recognizes nuclear matrix-associated nuclear bodies (Stuurman et al., J. Cell Sci. (1992) 101:773-784). The method of preparing said antibody described in this reference is generally applicable for the preparation of antibodies against any antigens of interest.
  • a variety of other components from the cell nucleus can be used as a source of antibodies that are suitable for the purpose of the present invention.
  • This source can originate e.g. from DNA, RNA, proteins, protein complexes with carbohydrates, and combinations thereof.
  • Antibodies thus obtained, and certain commercially available antibodies of choice, are useful in determining the spatial distribution of components of interest in cell nuclei and hence detecting the presence of tumor or viral antigens, abnormal proteins or the absence thereof, etc.
  • Antibodies labeled with radioactive or fluorescent material are particularly useful e.g. for diagnostic imaging or monitoring any progress of treatment of various diseases.
  • the detection and grading method of the present invention is suitable for investigating any type of cells, whether or not tumorous, since it is based on the spatial distribution of specific components of interest.
  • the method is particularly suitable for the early detection of a wide variety of proliferation disorders, in particular cancers, such as colon cancer, lung cancer, breast cancer, cervix cancer, ovary cancer, prostate cancer, skin cancer, Hodgkin and non- Hodgkin disease, etc.
  • cancers which can be detected using the method of the invention, see e.g. Scientific American, September 1996.
  • the cells to be investigated are usually taken from tissues for the detection of cancers such as those mentioned above, or from biological sample material, for example blood (for the detection of e.g. leukemia), lymphatic fluid (e.g. lymph node cancer), urine (e.g. bladder cancer) or stool (e.g. colon cancer).
  • a suitable data processing system for analyzing cytological material for the presence of cell proliferation disorders in an individual according to the present invention comprises the following steps:
  • monitoring device e.g. video camera
  • second storage means containing data from reference cytological material comprising the spatial distribution of the same at least one specific component in various types of cancer, the grade of cancer development, etc.
  • second processor means for comparing data from first storage means with second storage means, and determining if there are cell proliferation disorders present in the cytological material, and assessing the type of proliferation disorder and the grading of the disorder.
  • Tissue samples of the large bowel were obtained from patients supplied by a hospital in Amsterdam with a clinical diagnosis. From each donor, samples from healthy and adenocarcinoma tissues were taken. The donors are described in
  • Table 1 5 females and 5 males, ranging in age from 52 to 89 years with a mean age of 72 years.
  • Samples were stored at -70°C. Cryostat sectioning was performed with a microtome at -20°C. The sections (5 ⁇ m thick) were deposited on organosilane coated glass slides (MENZEL, Superfrost, 76 x 26 mm) and dried for 1-2 hours at room temperature. For immunofluorescence, the tissue sections were fixed with 4% (w/v) paraformaldehyde diluted in phosphate-buffered saline (PBS) for 10 minutes at 4°C.
  • PBS phosphate-buffered saline
  • Tumor samples were graded after tissue processing and histological staining according to their morphologic features at macroscopic and microscopic level (anaplasia) and staged according to the Duke' s classification.
  • tissue sections were rinsed twice with PBS and cells were permeabilised with 0.5 % Triton-X 100 (Sigma, Chemical Co, St Louis, MO) in PBS for 5 min. Tissue sections were subsequently rinsed twice in PBS and incubated in PBS containing 100 mM glycine (Sigma) for 10 min to inactivate remaining free aldehyde groups and blocked 10 min in PBG (PBS containing 0.5% BSA (Sigma) and 0.05% gelatine (Sigma)). For indirect immunofluorescent labeling fixed tissue sections were incubated overnight at 4°C with primary antibodies diluted in PBG. The primary antibodies used are listed in the following Table 2.
  • Tissue sections were subsequently washed 4 x 5 min in PBG and incubated with secondary antibodies diluted in PBG for 1-1.5 h. If biotin tagged secondary antibodies were used, tissue sections were rinsed again for 4 x 5 min with PBG and incubated for 30 min with streptavidin coupled to Cy3 or FITC [Jackson Immuno Research Laboratories, PA, USA] diluted with PBG.
  • the following secondary antibodies were used: Donkey-anti Mouse IgG coupled to either FITC or Cy3 [Jackson], Donkey- anti Mouse IgM coupled to FITC or Cy3 [Jackson], Donkey antiRabbit IgG coupled to FITC or Cy3 [Jackson].
  • tissue sections were washed 2 x 5 min in PBG and 2 x 5 min in PBS followed by incubation in PBS containing: 0.4 ⁇ g/ml Hoechst 33258 [Sigma] for 5 min, or Sytox Green diluted 1/100,000, or 1 mg/ml Propidium Iodide
  • PI diluted 1 :40.
  • the glass slides were mounted onto cover slips with mounting medium Vectashield [Vector Laboratories, USA], as an antifade agent.
  • Controls consisted of replacement of primary antibodies by preimmune serum or PBG; these controls were consistently negative or revealed that the tissue matrix (especially in tumor tissue) autofluoresces.
  • a Leica fluorescence microscope equipped with a CCD-camera was used to collect microscopic images.
  • Images of labeled tissue sections were also collected on a Leica Confocal Laser Scanning Microscope [CLSM] with 25x, 40x, 100x magnification oil immersion objectives.
  • a dual wavelength argon-ion laser was used to excite green (FITC or Sytox Green) and red (Cy3 or PI) fluorochromes, simultaneously at 488 nm and 514 nm, respectively.
  • the fluorescence signals were detected using a 525DF10 bandpass filter for FITC and Sytox-, Green and a 550 nm longpass filter for Cy3 or PI.
  • the fluorescence signals from both fluorochromes were recorded simultaneously. Images were recorded as single optical sections or as 512 x 512 x y voxel images (y depending on the number of optical sections per stack needed).
  • the image analysis software used was Metamorph and Image Pro-Plus. The digital images were then processed for presentation.
  • image analysis was performed using the software package Scillmage (Van Balen et al., 1994).
  • a distinct specific labeling was obtained with the following antibodies: 5E10, ⁇ -p80-coilin, ⁇ -CstF 64, ⁇ -CPSF 100, ⁇ -CPSF 160, SC-35, 4G3, 4B10, 4DII, 7G12, 2D3, R232, R252.
  • the signal could be easily detected, both with CCD camera and CLSM.
  • Figures 1 and 2 and the following Table 3 show typical examples in accordance with the present invention of the correlation of the spatial distribution of various nuclear components from nuclear proteins of human colorectal tumors and the untransformed phenotype. These results show that factors involved in RNA processing (cleavage factors, hnRNP, splicing factors, coiled and PML bodies), and acetylated histone distributions are altered in the nuclei of colorectal cancer tissue.

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Abstract

A method is provided for detecting a cell proliferation disorder in an individual which is based on the spatial distribution of specific components, such as proteins and other molecules, in a mammalian cell nucleus allowing one to establish whether a cell is in its normal state or has undergone transformation to a tumorogenous state. If transformation has occurred, such spatial distribution allows detection and grading of the tumor. The method comprises: a) determining the spatial distribution of at least one specific component in the cell nucleus of a sample from said individual to be analyzed; b) comparing said spatial distribution with the spatial distribution of the same at least one specific component in a reference sample; and c) characterizing said at least one specific component of said sample to be analyzed based upon comparison with said same at least one component in said reference sample. The method is useful in diagnostics as an efficient method for the (early) detection, and monitoring treatment, of proliferation disorders, in particular carcinomas, especially in humans.

Description

Method for the detection of mammalian carcinomas
Field of the Invention The present invention is in the field of molecular and cell biology and diagnostics, and relates in particular to a new and efficient method for the early detection of certain carcinomas in humans.
Background of the Invention and Prior Art Normal cells in a healthy body live in a complex, interdependent situation, whereby each cell is able to perform its special function. In order to maintain this complex situation, the cells control each others proliferation. Indeed, normal cells reproduce only when instructed to do so by special signals produced by cells in their vicinity.
In contrast, cancer cells deviate from this normal behaviour. They are no longer susceptible to normal controls on proliferation, but follow a more independent way of reproduction. When a normal cell is transformed into such a genetically modified cell, it produces too much of the same type of cells. Eventually the cancer cells invade neighbouring tissues, thereby displacing the surrounding tissue and disturbing the interdependent relations. The most threatening property is the ability to move to other places from the site where they began and to invade other tissues and organs, thereby deregulating and disturbing the normal functions of these organs. When this pattern of cancer development continues unnoticed for a long period, the treatment will become more and more difficult or even impossible. Therefore, a reliable method for detecting abnormal growth and spreading of suspicious cells in the human body at a very early stage is crucial to adequate treatment.
Fundamental research has revealed more than 100 forms of cancer so far. A large number of genes have been discovered which are involved in aberrant control of cell division (see, for example, R.A. Weinberg, Scientific American, September 1996). Mutations in genes may cause gene products to be overactive (in the case of oncogenes) or the normal inhibitory activity to be destroyed (in the case of tumor suppressor genes). Many other genes involved in the complex mechanisms of cell regulation have not yet been identified. A variety of methods have been explored for the screening of cancer cells. The traditional method still applied by professional pathologists comprises staining of cells contained in a small tissue sample and examining these cells by visual inspection by trained personnel. Aberrant growth is indicative of the presence of cancer cells and the stage of cancer development. This method is very slow and laborious and requires specially trained personnel. Moreover, only relatively advanced cancer growth is generally detected and a relatively large number of false positive and false negative results is associated with this method.
Several methods are based on the morphology of the cell nucleus as a parameter for the detection of malignant cells. For example, M. Derenzini et al., Amer. J. Path (1998) 152:1291-1297 studied the relationship between nucleolar function and size and cell doubling time in cancer cells. It was concluded that in cancer cells rRNA transcriptional activity and nuclear size are inversely related to cell doubling time. Quantitative distribution of nuclear structures within the cell represents a cytohistological parameter of the rapidity of cell proliferation.
P. Kronqvist et al., Anal. Cell. Path. (1997) 15:47-59 investigated the reproducibility of nuclear morphometric measurements in invasive breast carcinoma.
A more advanced development of this method is the ThinPrep2000 system developed by Cytyc Corporation, Boxborough, USA, wherein the screening of exfoliated cervical cells on the presence of cancer cells in the Papanicolaou (Pap) test is performed by digital cameras. It has met limited success since it is laborious, with no improvement in deviating results (see, for example, M.T. Fahey et al., Amer. J. Epidem. (1995) 141:680-689). No further developments in this area have been reported.
Other methods for detecting cancer cells are based on specific differences between cancer cells and normal cells that have emerged from fundamental research. For example, extensive research to detect differences between normal cells and cancer cells have revealed specific cellular components of cancer cells which are recognised by very specific antibodies which recognize tumor markers. These tumor markers in cancer cells differ in their presence from normal cells by their overproduction, persistent production, and/or their derangement of secretion (see, for example, G.I. Abelev & S. Sell, 1999, Seminars in Cancer Biology 9:61-65). Many of these tumor markers are used commercially.
Furthermore, US 5,310,653 discloses a method to purify the p65 tumor- associated protein and its use as a tumor marker for diagnosis of steroid- associated cancers. US 5,272,256 discloses the isolation and use of a nuclear autoantigen for the detection of autoimmune disorders.
Other methods rely on the genetic differences between cancer cells and normal cells. Recent developments have revealed that both large and very small differences in the genetic material of genes may be involved in the mechanisms of control of cell division or in genes which are strongly associated with inherited forms of cancer like certain types of breast cancer (BRCA1 , BRCA2), colon cancer (MSH2, MLH1 , APC), melanoma (CDK4), retinoblastoma (RB). For example, EP-A-0 819 767 discloses large chromosomal changes in detecting breast cancer cells. US 5,587,833 discloses a sensitive method for detecting single nucleotide changes in the DNA of cancer cells by a fluorescent microscopic method (FISH).
US 4,885,236 discloses a method for the identification and characterization of cells in a biopsy or sample of a body fluid which is based on the isolation and analysis of the components of a specific subcellular protein fraction referred to as "nuclear matrix". The nuclear matrix includes proteins and nuclear matrix associated DNA specific to different cell types. These proteins and nucleic acids are altered or new ones expressed as a result of viral infection, genetic defects or malignancy. Terris ef al., Cancer Research (1995) 55:1590-1597 studied the promyelocytic protein (PML) expression in several normal, inflammatory, and neoplastic human tissues. It was disclosed that the level of PML was low in normal tissues, but the expression of PML increased considerably during inflammation and in tumorous states. Moreover, in liver cancer, the overexpression of PML was accompanied by a delocalization into the cytoplasm. It was concluded that PML is overexpressed in distinct pathological situations that are associated with stimulated transcription and cell hyperactivity. Ochs ef al., J. Cell Sci (1994) 107:385-399 disclose the presence of coiled bodies in the nucleolus of breast cancer cells. In U.S. 5,264,343 differences in cancer cells and normal tissue are described based on a difference in accessibility of nuclear DNA. U.S. 5,891 ,857 discloses the distribution of a specific gene product (protein) in the cell. U.S. 5,599,919 discloses the use of detection of the CENP-F at the genomic and protein level in cells to obtain information about their proliferation state. Dos Santos et al., Human Molecular Genetics (1997) 6:1549-1558 describe the nuclear localization pattern of two proteins. LeBrun et al., Oncogene (1997) 15:2059-2067 disclose the nuclear distribution of two proteins. Everett et al., J. Virol. (1998) 72:6581-6591 describe observations on the inetraction between viral gene products and PML bodies. Duprez ef al., J. Cell Sci. (1999) 112:381-393 disclose the SUMO-modification of the PML protein in relation to the nuclear distribution of this protein. Everett et al., J. Cell Sci. (1999) 112:3443-3454 describe the interaction between PML body proteins and centromeres. Stuurman et al., J. Cell Sci. (1992) 101:773-784 describe a monoclonal antibody that detects an antigen that later was identified as PML and is widely used as a marker for PML bodies. Rafki-Beljebbar et al., Analytical Cellular Pathology (1999) 18:175-181 describe a change in spatial distribution of the nuclear protein NuMA in relation to the development of MDR in cells. None of the methods mentioned above employ differences in the spatial distribution of nuclear components between normal and malignant cells for clinical use.
It is an object of the present invention to provide a detection method based on differences in the spatial distribution of nuclear components between normal and malignant cells which is useful and advantageous for the early detection and diagnosis of cancer in mammalians, and in particular humans.
Summary of the Invention In accordance with the present invention, a new method is provided for detecting and grading a cell proliferation disorder in an individual, which comprises: (a) determining the spatial distribution of at least one specific component in the cell nucleus of a sample from said individual to be analyzed;
(b) comparing said spatial distribution with the spatial distribution of the same at least one specific component in a reference sample; and (c) characterizing said at least one specific component of said sample to be analyzed based upon comparison with said same at least one specific component in said reference sample.
In one aspect of the invention, the proliferation disorder to be investigated is in the gastro-intestinal tract. In another aspect of the invention said at least one specific component in the cell nucleus is selected from the group consisting of the antigens p80-coilin, PML, SC35, acetylated histones, CstF64, hnRNP-l, and NuMA protein.
In a further aspect of the invention, the spatial distribution of said at least one specific component in the cell nucleus is determined, by immuno- fluorescence or other techniques.
These and other aspects of the present invention will be explained in more detail in the following description and appended figures.
Brief Description of the Drawings Figures 1 and 2 show monolabeled nuclei in normal and tumor intestinal epithelial tissue sections, respectively. Coiled bodies in the nuclei are immuno- labeled by monoclonal antibody 204/5 (prepared against p80-coilin). The nuclei of normal cells contain less coiled bodies than tumor cells. In addition, the tissue organisation in the tumor is disordered. Figure 3 shows confocal sections of mono-labeled nuclei from normal
(A & B) and tumor (C & D) epithelium in tissue sections with the antibody against CstF 64 [donor no. 2]. (A & B) CstF 64 displayed a nucleoplasmic granular labeling pattern in epithelial nuclei of normal tissue section. (C & D): CstF 64 displayed a complex, interconnected domain-like pattern in epithelial nuclei of tumor tissue section.
Figure 4 shows confocal sections of mono-labeled nuclei from normal (A & B) and tumor (C & D) epthelium in tissue sections with the antibody 7G12 [donor no. 2]. (A & B): 7G12 strongly stained few bright foci in nuclei of healthy epithelium. (C & D): hnRNPI displayed a weak and puntated labeling pattern in nuclei of tumor epithelium.
Figure 5 shows fluorescence microscopy images of monolabeled nuclei containing antigen p80-coilin, in normal (A) and tumor (B) tissue section, mag x25.
Detailed Description of the Invention As used herein, the term "individual" generally refers to any mammalian, both human beings and animals, such as pets, but the term is in particular used in connection with human beings.
The term "sample" is not limited to tissue sample, but encompasses any type of biological source which usually is investigated by pathologists (and others) for the screening of proliferation disorders such as various types of cancer. The biological sources include also blood, lymphatic fluid, urine, stool, etc. The term "reference sample", as used herein, refers to essentially the same type of sample as the sample to be investigated, since spatial distribution patterns generally are tissue-specific. The reference sample is usually derived from normal cells of the same or similar source, usually the same tissue as the sample to be analyzed, from the same or a different individual. Alternatively, the reference sample is a statistic mean of spatial distribution patterns of the same or similar source, usually the same type of tissue, as the sample to be analyzed, derived from a relevant number of individuals, and selected by people skilled in the art, e.g. one or more pathologists or oncologists.
The present invention is based on the understanding that the spatial distribution of specific components, such as proteins and other molecules, in a mammalian cell nucleus allows one to establish whether a cell is in its normal state or has undergone transformation to a tumorogenous state. If transformation has occurred, such spatial distribution is different from normal cells and the extent of the different spatial distribution allows grading of the tumor. The specific components, in particular proteins (antigens), are identified by suitable detection techniques, e.g. fluorescence microscopy using specific antibodies against these antigens. The cell nucleus is a highly dynamic and compartmentalized organelle, where many nuclear processes, including DNA replication, DNA repair, RNA synthesis, processing and transport, take place. The cell nucleus consists of many readily identifiable nuclear structures including the nucleolus, heterochromatin domains, domains representing local high concentrations of functional nuclear machineries and a variety of nuclear bodies, e.g. coiled bodies, cleavage bodies and PML bodies. These nuclear structures are functionally organized in a specific pattern in the normal diploid mammalian cell nucleus.
Using colorectal cancer as a model, because the tumor grade and stage can be defined relatively easily, the present invention is, in one embodiment, more specifically based on the correlation of the transformed phenotype of epithelial cells in colorectal cancer with an altered spatial organisation of nuclear components. To this end, the spatial distribution of various nuclear components is investigated in epithelial cell nuclei of the large bowel of normal and adenomatous, carcinomatous tissues. In neoplastic epithelial cell nuclei, various striking differences can be observed as compared with normal cell nuclei: (i) an increased number of nuclear bodies, such as PML bodies and coiled bodies, (ii) for some nuclear components (splicing factors, cleavage factors, hnRNP-AI, hnRNP-l, mitotic apparatus protein), a change in spatial distribution. These findings in colorectal cancer cell nuclei show that carcinogenisis induces alterations in the spatial organisation of a number of nuclear components. Based on these principles, the present invention provides in one aspect a method for detecting and grading cell proliferating disorders in individuals by determining the spatial distribution of at least one specific component in the cell nucleus of a sample from said individual to be analyzed, comparing said spatial distribution with the spatial distribution of the same at least one specific component in a reference sample, and characterizing said at least one component of said sample to be analyzed based upon comparison with said at least one component in said reference sample. As mentioned above, the reference sample is usually derived from normal cells of essentially the same source, usually the same tissue, as the sample to be investigated, from the same or a different individual. Preferably, the sample to be investigated and the reference sample are derived from the same individual. More preferably, the sample to be investigated and the reference sample are derived from essentially the same tissue from the same individual. Alternatively, the reference sample is a statistic mean of spatial distribution patterns from the same or similar source, usually the same type of tissue, as the sample to be analyzed, derived from a relevant number of individuals, and selected by people skilled in the art, e.g. one or more pathologists or oncologists. Such selected spatial distribution patterns are conveniently stored in a data base or on a carrier. This alternative embodiment offers an excellent opportunity for large scale diagnosis and automation of results.
The detection and characterisation of specific components from the cell nucleus, such as nuclear proteins, is known in the art and can be performed by any suitable means which are useful for obtaining clear distribution patterns of the various aimed components to enable adequate and preferably unambiguous comparisons.
For example, a panel of available antibodies, each recognizing one or more nuclear components, can be used to select antibodies that recognize one or more differences in spatial distribution of nuclear components between normaland tumorogenous tissues. Alternatively, specific nuclear components that are suspected to be differently distributed in tumorogenous cells and normal cells can be isolated using standard procedures and used to prepare specific antibodies that are useful in discriminating between normal and tumorogenous cells.
Proteins as described herein are useful as immunogens for the preparation of antibodies when these antibodies are conjugated with colorimetric, immunological, fluorescent or radioactive labels. Antibodies useful for employing in the detection and characterization of the spatial distribution patterns of the components mentioned above are any type of antibodies, whether monoclonal or polyclonal, which result in detectable and reproducible labelling patterns. Suitable antibodies for the purpose of this invention include, for example, the monoclonal antibody 5E10, that recognizes nuclear matrix-associated nuclear bodies (Stuurman et al., J. Cell Sci. (1992) 101:773-784). The method of preparing said antibody described in this reference is generally applicable for the preparation of antibodies against any antigens of interest. A variety of other components from the cell nucleus can be used as a source of antibodies that are suitable for the purpose of the present invention. This source can originate e.g. from DNA, RNA, proteins, protein complexes with carbohydrates, and combinations thereof. Antibodies thus obtained, and certain commercially available antibodies of choice, are useful in determining the spatial distribution of components of interest in cell nuclei and hence detecting the presence of tumor or viral antigens, abnormal proteins or the absence thereof, etc. Antibodies labeled with radioactive or fluorescent material are particularly useful e.g. for diagnostic imaging or monitoring any progress of treatment of various diseases.
The detection and grading method of the present invention is suitable for investigating any type of cells, whether or not tumorous, since it is based on the spatial distribution of specific components of interest. However, the method is particularly suitable for the early detection of a wide variety of proliferation disorders, in particular cancers, such as colon cancer, lung cancer, breast cancer, cervix cancer, ovary cancer, prostate cancer, skin cancer, Hodgkin and non- Hodgkin disease, etc. For a survey of cancers which can be detected using the method of the invention, see e.g. Scientific American, September 1996. As already indicated above, the cells to be investigated are usually taken from tissues for the detection of cancers such as those mentioned above, or from biological sample material, for example blood (for the detection of e.g. leukemia), lymphatic fluid (e.g. lymph node cancer), urine (e.g. bladder cancer) or stool (e.g. colon cancer).
Reference is also made to Gordon et al., J. Cellular Biochem. (2000) 77:30-43, which was published after the priority date of the present patent application, disclosing interrelationships of modifications in nuclear morphology with the in situ localization of regulatory factors associated with other non- promyelolytic acute leukemias. This disclosure which is incorporated herein by reference corroborates the principles of the present invention, in particular in the field of acute leukemia. The information obtained from the method according to the present invention can be processed in several ways, varying from e.g. analyzing and processing data manually by a medical expert (e.g. a pathologist or oncologist) to electronic data processing. A suitable data processing system for analyzing cytological material for the presence of cell proliferation disorders in an individual according to the present invention comprises the following steps:
(a) monitoring device (e.g. video camera) generating data obtained from the spatial distribution of at least one specific component in the cell nucleus of a sample from said individual;
(b) first computer processor means for processing data obtained from the monitoring device;
(c) first storage means for storing data from the first computer on a first storage medium;
(d) second storage means containing data from reference cytological material comprising the spatial distribution of the same at least one specific component in various types of cancer, the grade of cancer development, etc.
(e) second processor means for comparing data from first storage means with second storage means, and determining if there are cell proliferation disorders present in the cytological material, and assessing the type of proliferation disorder and the grading of the disorder.
The following experimental part illustrates certain aspects of the present invention, however, without intending to limit the invention in any respect.
MATERIAL AND METHODS Tissue specimens
Tissue samples of the large bowel were obtained from patients supplied by a hospital in Amsterdam with a clinical diagnosis. From each donor, samples from healthy and adenocarcinoma tissues were taken. The donors are described in
Table 1 ; 5 females and 5 males, ranging in age from 52 to 89 years with a mean age of 72 years.
Table 1
Samples were stored at -70°C. Cryostat sectioning was performed with a microtome at -20°C. The sections (5 μm thick) were deposited on organosilane coated glass slides (MENZEL, Superfrost, 76 x 26 mm) and dried for 1-2 hours at room temperature. For immunofluorescence, the tissue sections were fixed with 4% (w/v) paraformaldehyde diluted in phosphate-buffered saline (PBS) for 10 minutes at 4°C.
Tumor samples were graded after tissue processing and histological staining according to their morphologic features at macroscopic and microscopic level (anaplasia) and staged according to the Duke' s classification.
Immunofluorescent labeling
All steps were performed at room temperature unless stated otherwise After fixation, tissue sections were rinsed twice with PBS and cells were permeabilised with 0.5 % Triton-X 100 (Sigma, Chemical Co, St Louis, MO) in PBS for 5 min. Tissue sections were subsequently rinsed twice in PBS and incubated in PBS containing 100 mM glycine (Sigma) for 10 min to inactivate remaining free aldehyde groups and blocked 10 min in PBG (PBS containing 0.5% BSA (Sigma) and 0.05% gelatine (Sigma)). For indirect immunofluorescent labeling fixed tissue sections were incubated overnight at 4°C with primary antibodies diluted in PBG. The primary antibodies used are listed in the following Table 2.
Table 2
1. N. Stuurman et al., J. Cell Sci. (1992) 101 :773-784. 2. W. Schul et al., Mol. Biol. Cell (1998) 9:1025-1036.
3. W. Schul et al., EMBO J. (1996) 15:2883-2892.
4. M.A. Grande et al., J. Cell Sci. (1997) 110:1781-1791.
5. K.A. Mattern ef al., Exp. Cell Res. (1999) 246.
Tissue sections were subsequently washed 4 x 5 min in PBG and incubated with secondary antibodies diluted in PBG for 1-1.5 h. If biotin tagged secondary antibodies were used, tissue sections were rinsed again for 4 x 5 min with PBG and incubated for 30 min with streptavidin coupled to Cy3 or FITC [Jackson Immuno Research Laboratories, PA, USA] diluted with PBG. For single and double labeling, the following secondary antibodies were used: Donkey-anti Mouse IgG coupled to either FITC or Cy3 [Jackson], Donkey- anti Mouse IgM coupled to FITC or Cy3 [Jackson], Donkey antiRabbit IgG coupled to FITC or Cy3 [Jackson].
After the labeling, tissue sections were washed 2 x 5 min in PBG and 2 x 5 min in PBS followed by incubation in PBS containing: 0.4 μg/ml Hoechst 33258 [Sigma] for 5 min, or Sytox Green diluted 1/100,000, or 1 mg/ml Propidium Iodide
(PI) diluted 1 :40. The glass slides were mounted onto cover slips with mounting medium Vectashield [Vector Laboratories, USA], as an antifade agent.
Controls consisted of replacement of primary antibodies by preimmune serum or PBG; these controls were consistently negative or revealed that the tissue matrix (especially in tumor tissue) autofluoresces.
Microscopy
A Leica fluorescence microscope equipped with a CCD-camera was used to collect microscopic images.
Confocal Laser Scanning Microscopy
Images of labeled tissue sections were also collected on a Leica Confocal Laser Scanning Microscope [CLSM] with 25x, 40x, 100x magnification oil immersion objectives. A dual wavelength argon-ion laser was used to excite green (FITC or Sytox Green) and red (Cy3 or PI) fluorochromes, simultaneously at 488 nm and 514 nm, respectively. The fluorescence signals were detected using a 525DF10 bandpass filter for FITC and Sytox-, Green and a 550 nm longpass filter for Cy3 or PI. The fluorescence signals from both fluorochromes were recorded simultaneously. Images were recorded as single optical sections or as 512 x 512 x y voxel images (y depending on the number of optical sections per stack needed).
Image analysis
For the CCD camera, the image analysis software used was Metamorph and Image Pro-Plus. The digital images were then processed for presentation. For the CLSM, image analysis was performed using the software package Scillmage (Van Balen et al., 1994). RESULTS
Labeling procedure with frozen material
A distinct specific labeling was obtained with the following antibodies: 5E10, α-p80-coilin, α-CstF 64, α-CPSF 100, α-CPSF 160, SC-35, 4G3, 4B10, 4DII, 7G12, 2D3, R232, R252. The signal could be easily detected, both with CCD camera and CLSM.
Immunofluorescent labeling
Figures 1 and 2 and the following Table 3 show typical examples in accordance with the present invention of the correlation of the spatial distribution of various nuclear components from nuclear proteins of human colorectal tumors and the untransformed phenotype. These results show that factors involved in RNA processing (cleavage factors, hnRNP, splicing factors, coiled and PML bodies), and acetylated histone distributions are altered in the nuclei of colorectal cancer tissue.
Table 3
Striking differences in nuclear distribution of specific antigens in human colorectal epithelial tissue
1. W. Schul et al. , J. Cell. Biochem. (1998) 70: 159- 171.
2. G.G. Maul, BioEssays (1998) 20:660-667.
3. T. Misteli and D.L. Spector, Current Opinion in Cell Biology (1998) 10:323- 331.
4. M.H. Kuo and CD. Allis, BioEssays (1998) 20:615-626. 5. G. Varani and K. Nagai, Annual Review of Biophysics and Biomolecular Structure (1998) 27:407-445.

Claims

Claims
1. A method for detecting and grading a cell proliferation disorder or monitoring the treatment of such cell proliferation disorder in an individual, which comprises:
(a) determining the spatial distribution of at least one specific component in the cell nucleus of a sample from said individual to be analyzed;
(b) comparing said spatial distribution with the spatial distribution of the same at least one specific component in a reference sample; and (c) characterizing said at least one component of said sample to be analyzed based upon comparison with said at least one specific component in said reference sample.
2. The method as claimed in claim 1 , wherein the sample is derived from a biological source selected from the group consisting of tissue, blood, lymphatic fluid, urine and stool.
3. The method as claimed in claim 1 or 2, wherein the reference sample is derived from normal cells of the same or similar source as the sample to be analyzed, and preferably from the same individual.
4. The method as claimed in claim 1 or 2, wherein the reference sample is a statistic mean of spatial distribution patterns from the same or similar source as the sample to be analyzed, derived from a number of individuals, and selected by people skilled in the art.
5. The method as claimed in claim 4, wherein said spatial distribution patterns are stored in a data base or on a carrier.
6. The method as claimed in any one of claim 1 to 5, wherein the proliferation disorder to be investigated is selected from the group consisting of colon cancer, lung cancer, breast cancer, cervix cancer, ovary cancer, prostate cancer, skin cancer, Hodgkin and non-Hodgkin disease.
7. The method as claimed in any one of claims 1 to 6, wherein said at least 5 one specific component in the cell nucleus is selected from the group consisting of the antigens p80-coilin, PML, SC35, acetylated histones, CstF64, hnRNP-l, and NuMA protein.
8. The method as claimed in any one of Claims 1 to 7, wherein the spatial 10 distribution of said at least one specific component in the cell nucleus is determined by immunofluorescence microscopy.
9. The method as claimed in claim 8, wherein said immunofluorescence microscopy involves the use of one or more antibodies raised against nuclear
15 components from the type of cells to be analyzed.
10. The method as claimed in claim 9, wherein said one or more antibodies are monoclonals.
20 11. A data processing system for analyzing cytological material for the presence of a cell proliferation disorder in an individual, which comprises:
(a) monitoring device (e.g. video camera) generating data obtained from the spatial distribution of at least one specific component in the cell nucleus of a sample from said individual; 25 (b) first computer processor means for processing data obtained from the monitoring device;
(c) first storage means for storing data from the first computer on a first storage medium;
(d) second storage means containing data from reference cytological 30 material comprising the spatial distribution of the same at least one specific component in various types of cancer, the grade of cancer development, etc. (e) second processor means for comparing data from first storage means with second storage means, and determining if there are cell proliferation disorders present in the cytological material, and assessing the type of proliferation disorder and the grading of the disorder.
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