NZ519065A - Method for the detection of mammalian carcinomas - Google Patents

Abstract

A method for detecting a cell proliferation disorder in the tissue of the large bowel of an individual, which comprises: (a) determining the presence or spatial distribution of at least one nuclear cell component in a sample from said tissue to be analyzed, wherein the nuclear cell component is selected from the group of p80-coilin bodies, PML, SC35, acetylated histone, CstF54, and jnRNP-1; (b) comparing said presence or spatial distribution with the presence or spatial distribution of the same nuclear cell component in a reference sample; and (c) characterizing said sample to be analyzed based on difference in its presence or spatial distribution upon comparison with said specific component in said reference sample. Also disclosed is the use in a method of a data processing system which comprises; (a) monitoring device generating data obtained from determining the presence or spatial distribution of at least one specific component in a cell nucleus of a sample from an 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 presence or spatial distribution of the same at least one specific component in various types of cancer; (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.

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 5 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 10 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 15 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 20 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 25 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, 30 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.

CONFIRMATION COPY 2 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 5 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 10 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 15 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 20 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 25 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 30 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 3 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-5 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 10 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 20 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 et 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 30 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 et 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 5 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 10 proteins. Everett et al., J. Virol. (1998) 72:6581-6591 describe observations on the interaction between viral gene products and PML bodies. Duprez et 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 15 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. 20 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 presence or spatial distribution of nuclear components 25 between normal and malignant cells which is useful and advantageous for the early detection and diagnosis of cancers or cell proliferation disorders in tissue of the large bowel in mammalians, and in particular humans.

It is an alternative or additional object to provide the public with a useful choice of method for detecting and grading such a cell proliferation disorder or 30 monitoring the treatment of such cell proliferation disorder.

Summary of the Invention In accordance with one aspect of the present invention as claimed, there is provided a method for detecting a cell proliferation disorder in the tissue of 5 the large bowel of an individual, which comprises: (a) determining the presence or spatial distribution of at least one nuclear cell component in a sample from said tissue to be analyzed, wherein the nuclear cell component is selected from the group of p80-coilin bodies, PML, SC35, acetylated histone, CstF64, and hnRNP-1; (b) comparing said presence or spatial distribution with the prescence or spatial distribution of the same nuclear cell component in a reference sample; and (c) characterizing said sample to be analyzed based on differences in its presence or spatial distribution upon comparison with said specific component in said reference sample.

Preferably, the differences in presence or spatial distribution of said nuclear cell component are selected from the group of increase in number of coiled bodies when p80-coilin is determined, increase in number of PML bodies when PML is determined, smaller speckled domains and/or more diffuse distribution when SC35 is determined, more cells labelled when acetylated histone 20 is determined, lumping together of antigen when CstF64 is determined, and more punctuate distribution when hnRNP-1 is determined.

Preferably, the reference sample is derived from normal cells of the same or similar source as the sample to be analyzed.

Preferably, the reference sample is derived from the same individual as 25 the sample to be analyzed.

Preferably, the reference sample is a statistic mean of data obtained from determining the presence or spatial distribution patterns of at least one nuclear cell component from the same or similar source as the sample to be analyzed, derived from a number of individuals, and selected by people skilled in 30 the art.

Preferably, the data are stored in a data base or on a carrier.

I. • ''X. ti:'- 5a Preferably, the proliferation disorder to be investigated is selected from the group consisting of colon cancer, rectum cancer, and sigmoid cancer.

Preferably, the presence or spatial distribution of said at least one specific component in the cell nucleus is determined by immunofluorescence 5 microscopy, or other techniques.

Preferably, the immunofluorescence microscopy involves the use of one or more antibodies raised against nuclear components from the type of cells to be analyzed.

Preferably, the one or more antibodies are monoclonals. 10 Preferably, the method further comprises assessing the type of proliferation disorder.

Preferably, the method further comprises grading the cell proliferation disorder.

In another aspect, the invention provides the use in a method of the 15 invention as hereinbefore described of a data processing system which comprises: (a) monitoring device generating data obtained from determining the presence or spatial distribution of at least one specific component in a cell nucleus of a sample from an individual; (b) first computer processor means for processing data obtained from 20 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 presence or spatial distribution of the same at least one specific component in various types of cancer; (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.

In a further aspect, the invention provides results generated from a 30 method of the invention as described hereinbefore.

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-5 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 10 CstF 64 [donor no. 2], (A & B) CstF 64 displayed a nucleoplasm^ 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 15 (A & B) and tumor (C & D) epthelium in tissue sections with the antibody 7G12 6 [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 5 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 10 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. 15 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 20 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 25 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 30 components, in particular proteins (antigens), are identified by suitable detection techniques, e.g. fluorescence microscopy using specific antibodies against these antigens. 7 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 5 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 10 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, 15 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 25 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 8 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 5 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 10 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 15 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 20 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, 25 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 30 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 9 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 10 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 15 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 20 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 25 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 5 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 10 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 15 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. 11 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 5 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 Donor No.

Age Sex Tissue Diagnosis Depth Penetration 1 52 M sigmoid moderately differentiated adenocarcinoma serosa 2 76 F colon moderately-well differentiated adenocarcinoma muscul. propia (-)lymphonodes 3 75 M sigmoid moderately differentiated adenocarcinoma muscul. propia (-)lymphonodes 4 69 M rectum moderately differentiated adenocarcinoma (mucinous type) muscul. propia (-)lymphonodes 89 F colon adenocarcinoma Dukes B 6 81 F colon villous adenoma 7 83 F colon poorly differentiated adenocarcinoma 8 71 M colon poorly differentiated adenocarcinoma 9 63 F rectum poorly-moderately differentiated adenocarcinoma perimuscular fat tissue 59 M colon well differentiated adenocarcinoma (villous adenoma type) Dukes B3 mesenterial fat tissue Samples were stored at -70°C. Cryostat sectioning was performed with a microtome at -20°C. The sections (5 pm 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 12 4% (w/v) paraformaldehyde diluted in phosphate-buffered saiine (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 5 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 10 permeabiiised 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.

PCT/EPOO/11050 13 Table 2 Antibody Subclass Animal Antigen Ref. 5E10 IgG mouse 126kD nucl body prot.- PML body 1 a-p80-coilin poly rabbit 80 kD coilin prot. - coiled bodies 2 a-CstF 64 mono mouse Cleavage Stimulation Factor 3 a-CPSF mono mouse Cleav. Polyadenyl. Spec. Factor 3 H5 IgM mouse subunit HO of RNA polymerase II 4 H14 IgM mouse subunit II0 and lla of RNA polymerase II 4 8WG16 lgG2a mouse RNA polymerase II 4 SC-35 lgG1 mouse kDa (non-snRNP) splicing factor 4B10 IgG mouse hnRNP A1 protein 4D11 IgG mouse hn RNP L protein 7G12 IgG mouse PTB (= hnRNP I protein) a-Oct1 - rabbit transcription factor 4 a-BRG1 - rabbit human brahma , 4 a-TF11F-rap74 poly rabbit transcription factor 4 a-TFIIH mono mouse transcription (p62) factor 4 1. N. Stuurman et al., J. Cell Sci. (1992) 101:773-784. 2. W. Schul etai, Mot. 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.

. K.A. Mattern et al., Exp. Cell Res. (1999) 246.

Tissue sections were subsequently washed 4x5 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 rnin with streptavidin coupled to Cy3 or FITC [Jackson Immuno Research Laboratories, PA, USA] diluted with PBG. 15 For single and double labeling, the following secondary antibodies were used: Donkey-anti Mouse IgG coupled to either FITC or Cy3 [Jackson], Donkey- 14 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 2x5 min in PBG and 2 x 5 min in PBS followed by incubation in PBS containing: 0.4 pg/ml Hoechst 33258 5 [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 10 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 20 (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 25 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 30 presentation. For the CLSM, image analysis was performed using the software package Scillmage (Van Balen etal., 1994).

RESULTS Labeling procedure with frozen material A distinct specific labeling was obtained with the following antibodies: 5 5E10, a-p80-coilin, a-CstF 64, a-CPSF 100, a-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), 15 and acetylated histone distributions are altered in the nuclei of colorectal cancer tissue.

The reference to any prior art in the specification is not, and should not be taken as, an acknowledgement, or any form of suggestion, that the prior art 20 forms part of the common general knowledge in New Zealand.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, 25 that is to say, in the sense of "including, but not limited to".

PCT/EP0O/11O5O 16 Table 3 Striking differences in nuclear distribution of specific antigens in human colorectal epithelial tissue antigen function difference in tumour in comparison to normal tissue info antigen p80-coilin unknown increase in number of coiled bodies 1 PML unknown increase in number of PML bodies 2 SC35 RNA splicing smaller speckled domains, more diffuse distribution 3 acetylated histone chromatin more cells labelled 4 CstF64 3' processing RNA lumping together of antigen 1 hnRNP-1 RNA packaging more punctuate distribution 1. W. Schul et al., J. Cell. Biochem. (1998) ZQ: 159-171. 2. G.G.Maui, 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 C.D. Allis, BioEssays (1998) 20:615-626. 5. G. Varani and K. Nagai, Annual Review of Biophysics and Biomolecular Structure (1998) 27:407-445.

Claims (16)

17 What we claim is:

1. A method for detecting a cell proliferation disorder in the tissue of the large bowel of an individual, which comprises: 5 (a) determining the presence or spatial distribution of at least one nuclear cell component in a sample from said tissue to be analyzed, wherein the nuclear cell component is selected from the group of p80-coilin bodies, PML, SC35, acetylated histone, CstF64, and hnRNP-1; (b) comparing said presence or spatial distribution with the prescence or 10 spatial distribution of the same nuclear cell component in a reference sample; and (c) characterizing said sample to be analyzed based on differences in its presence or spatial distribution upon comparison with said specific component in said reference sample. 15

2. The method as claimed in claim 1, wherein the differences in presence or spatial distribution of said nuclear cell component are selected from the group of increase in number of coiled bodies when p80-coilin is determined, increase in number of PML bodies when PML is determined, smaller speckled domains and/or more diffuse distribution when SC35 is determined, more cells labelled when 20 acetylated histone is determined, lumping together of antigen when CstF64 is determined, and more punctuate distribution when hnRNP-1 is determined.

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 25 analyzed.

4. The method as claimed in any one of claims 1 to 3 wherein the reference sample is derived from the same individual as the sample to be analyzed. 30

5. The method as claimed in claim 1 or 2, wherein the reference sample is a statistic mean of data obtained from determining the presence or spatial distribution patterns of at least one nuclear cell component from the same or 18 similar source as the sample to be analyzed, derived from a number of individuals, and selected by people skilled in the art.

6. The method as claimed in claim 5, wherein the data are stored in a data 5 base or on a carrier.

7. The method as claimed in any one of claims 1 to 6, wherein the proliferation disorder to be investigated is selected from the group consisting of colon cancer, rectum cancer, and sigmoid cancer. 10

8. The method as claimed in any one of Claims 1 to 7, wherein the presence or spatial distribution of said at least one specific component in the cell nucleus is determined by immunofluorescence microscopy. 15

9. The method as claimed in claim 8, wherein the immunofluorescence microscopy involves the use of one or more antibodies raised against nuclear components from the type of cells to be analyzed.

10. The method as claimed in claim 9, wherein the one or more antibodies 20 are monoclonals.

11. The method of any one of claims 1 to 10 wherein the method further comprises assessing the type of proliferation disorder. 25

12. The method of any one of claims 1 to 11 wherein the method further comprises grading the cell proliferation disorder.

13. The use in a method of any one of claims 1 to 12 of a data processing system which comprises: 30 (a) monitoring device generating data obtained from determining the presence or spatial distribution of at least one specific component in a cell nucleus of a sample from an individual; 19 (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 5 storage medium; (d) second storage means containing data from reference cytological material comprising the presence or spatial distribution of the same at least one specific component in various types of cancer; (e) second processor means for comparing data from first storage 10 means with second storage means, and determining if there are cell proliferation disorders present in the cytological material.

14. Use as claimed in claim 13 wherein the monitoring device is a video camera. 15

15. A method according to claim 1 substantially as herein described with particular reference to any one of the Examples and/or Figures. 20

16. Use according to claim 13 substantially as herein described with particular reference to any one of the Examples and/or Figures. UNIVERSITEIT VAN AMSTERDAM 25 By its attorneys BALDWIN SHELSTON WATERS 30 intellectual Pr Office ot f \ 9 DEC 2003 •l^ 35 1