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.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Urology & Nephrology (AREA)
• Chemical & Material Sciences (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Hematology (AREA)
• Medicinal Chemistry (AREA)
• Analytical Chemistry (AREA)
• Biotechnology (AREA)
• Hospice & Palliative Care (AREA)
• Oncology (AREA)
• Food Science & Technology (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Cell Biology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Publications (1)

Family

ID=8240801

Family Applications (1)

Country Status (8)

Cited By (4)

Citations (3)

• 2000
  • 2000-11-01 JP JP2001535063A patent/JP2003513282A/ja active Pending
  • 2000-11-01 HU HU0203269A patent/HUP0203269A2/hu unknown
  • 2000-11-01 NZ NZ519065A patent/NZ519065A/en unknown
  • 2000-11-01 CA CA002389364A patent/CA2389364A1/en not_active Abandoned
  • 2000-11-01 EP EP00984990A patent/EP1226439A1/en not_active Withdrawn
  • 2000-11-01 WO PCT/EP2000/011050 patent/WO2001033229A1/en not_active Application Discontinuation
  • 2000-11-01 AU AU21565/01A patent/AU2156501A/en not_active Abandoned
• 2002
  • 2002-04-29 ZA ZA200203401A patent/ZA200203401B/en unknown

Patent Citations (3)

Non-Patent Citations (10)

Also Published As

Similar Documents

Legal Events