CA2389364A1 - Method for the detection of mammalian carcinomas - Google Patents
Method for the detection of mammalian carcinomas Download PDFInfo
- Publication number
- CA2389364A1 CA2389364A1 CA002389364A CA2389364A CA2389364A1 CA 2389364 A1 CA2389364 A1 CA 2389364A1 CA 002389364 A CA002389364 A CA 002389364A CA 2389364 A CA2389364 A CA 2389364A CA 2389364 A1 CA2389364 A1 CA 2389364A1
- Authority
- CA
- Canada
- Prior art keywords
- spatial distribution
- sample
- cell
- cancer
- nuclear
- 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.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000001514 detection method Methods 0.000 title abstract description 17
- 201000009030 Carcinoma Diseases 0.000 title abstract description 4
- 210000004027 cell Anatomy 0.000 claims abstract description 75
- 238000009826 distribution Methods 0.000 claims abstract description 51
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 49
- 239000000523 sample Substances 0.000 claims abstract description 25
- 210000003855 cell nucleus Anatomy 0.000 claims abstract description 16
- 239000013074 reference sample Substances 0.000 claims abstract description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 230000035755 proliferation Effects 0.000 claims abstract description 10
- 208000035269 cancer or benign tumor Diseases 0.000 claims abstract description 7
- 210000001519 tissue Anatomy 0.000 claims description 44
- 201000011510 cancer Diseases 0.000 claims description 28
- 239000000427 antigen Substances 0.000 claims description 12
- 108091007433 antigens Proteins 0.000 claims description 12
- 102000036639 antigens Human genes 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 10
- 238000012545 processing Methods 0.000 claims description 10
- 238000003860 storage Methods 0.000 claims description 10
- 206010009944 Colon cancer Diseases 0.000 claims description 8
- 238000011161 development Methods 0.000 claims description 8
- 102100031144 Coilin Human genes 0.000 claims description 7
- 210000003092 coiled body Anatomy 0.000 claims description 7
- 108010051876 p80-coilin Proteins 0.000 claims description 7
- 102100029666 Serine/arginine-rich splicing factor 2 Human genes 0.000 claims description 6
- 230000002380 cytological effect Effects 0.000 claims description 6
- 208000029742 colonic neoplasm Diseases 0.000 claims description 5
- 108010033040 Histones Proteins 0.000 claims description 4
- 101000587430 Homo sapiens Serine/arginine-rich splicing factor 2 Proteins 0.000 claims description 4
- 238000012806 monitoring device Methods 0.000 claims description 4
- 210000003850 cellular structure Anatomy 0.000 claims 5
- 238000010820 immunofluorescence microscopy Methods 0.000 claims 2
- 208000015634 Rectal Neoplasms Diseases 0.000 claims 1
- 208000009125 Sigmoid Neoplasms Diseases 0.000 claims 1
- 206010038038 rectal cancer Diseases 0.000 claims 1
- 201000001275 rectum cancer Diseases 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 27
- 102000004169 proteins and genes Human genes 0.000 abstract description 20
- 210000004940 nucleus Anatomy 0.000 abstract description 16
- 230000009466 transformation Effects 0.000 abstract description 4
- 210000004962 mammalian cell Anatomy 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 208000009956 adenocarcinoma Diseases 0.000 description 9
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 241000282414 Homo sapiens Species 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 238000002372 labelling Methods 0.000 description 7
- 208000035475 disorder Diseases 0.000 description 6
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 6
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 5
- 210000000981 epithelium Anatomy 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 206010006187 Breast cancer Diseases 0.000 description 4
- 208000026310 Breast neoplasm Diseases 0.000 description 4
- 102000005221 Cleavage Stimulation Factor Human genes 0.000 description 4
- 108010081236 Cleavage Stimulation Factor Proteins 0.000 description 4
- 108010035916 Nuclear Matrix-Associated Proteins Proteins 0.000 description 4
- 102000008297 Nuclear Matrix-Associated Proteins Human genes 0.000 description 4
- 102000007999 Nuclear Proteins Human genes 0.000 description 4
- 108010089610 Nuclear Proteins Proteins 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 210000001072 colon Anatomy 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 101710132817 Polypyrimidine tract-binding protein 1 Proteins 0.000 description 3
- 102100033073 Polypyrimidine tract-binding protein 1 Human genes 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 238000003125 immunofluorescent labeling Methods 0.000 description 3
- 230000003211 malignant effect Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 206010000830 Acute leukaemia Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- -1 PML Proteins 0.000 description 2
- 102000015097 RNA Splicing Factors Human genes 0.000 description 2
- 108010039259 RNA Splicing Factors Proteins 0.000 description 2
- 201000000582 Retinoblastoma Diseases 0.000 description 2
- 101710123513 Serine/arginine-rich splicing factor 2 Proteins 0.000 description 2
- 108010048992 Transcription Factor 4 Proteins 0.000 description 2
- 102100023489 Transcription factor 4 Human genes 0.000 description 2
- 230000001594 aberrant effect Effects 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000010191 image analysis Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000001926 lymphatic effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000001613 neoplastic effect Effects 0.000 description 2
- 210000000299 nuclear matrix Anatomy 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 210000000664 rectum Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 208000003200 Adenoma Diseases 0.000 description 1
- 206010001233 Adenoma benign Diseases 0.000 description 1
- 208000000058 Anaplasia Diseases 0.000 description 1
- 101710145634 Antigen 1 Proteins 0.000 description 1
- 101000668428 Arabidopsis thaliana Heterogeneous nuclear ribonucleoprotein 1 Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 102000036365 BRCA1 Human genes 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 108700020462 BRCA2 Proteins 0.000 description 1
- 102000052609 BRCA2 Human genes 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 101150008921 Brca2 gene Proteins 0.000 description 1
- 102000013701 Cyclin-Dependent Kinase 4 Human genes 0.000 description 1
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 1
- 102100034157 DNA mismatch repair protein Msh2 Human genes 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010034791 Heterochromatin Proteins 0.000 description 1
- 102000017013 Heterogeneous Nuclear Ribonucleoprotein A1 Human genes 0.000 description 1
- 108010019372 Heterogeneous-Nuclear Ribonucleoproteins Proteins 0.000 description 1
- 102000006479 Heterogeneous-Nuclear Ribonucleoproteins Human genes 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101001134036 Homo sapiens DNA mismatch repair protein Msh2 Proteins 0.000 description 1
- 101000702559 Homo sapiens Probable global transcription activator SNF2L2 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 229910015837 MSH2 Inorganic materials 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 101000854012 Mus musculus Heterogeneous nuclear ribonucleoprotein A1 Proteins 0.000 description 1
- 101001135343 Mus musculus Polypyrimidine tract-binding protein 1 Proteins 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 102100036961 Nuclear mitotic apparatus protein 1 Human genes 0.000 description 1
- 101710104794 Nuclear mitotic apparatus protein 1 Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 102000004598 Small Nuclear Ribonucleoproteins Human genes 0.000 description 1
- 108010003165 Small Nuclear Ribonucleoproteins Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000009134 cell regulation Effects 0.000 description 1
- 210000002230 centromere Anatomy 0.000 description 1
- 102000005352 centromere protein F Human genes 0.000 description 1
- 108010031377 centromere protein F Proteins 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 201000010897 colon adenocarcinoma Diseases 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 230000009395 genetic defect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004458 heterochromatin Anatomy 0.000 description 1
- 102000048439 human SMARCA2 Human genes 0.000 description 1
- 208000013403 hyperactivity Diseases 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 208000030776 invasive breast carcinoma Diseases 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 201000010453 lymph node cancer Diseases 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 210000000479 mitotic spindle apparatus Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000003562 morphometric effect Effects 0.000 description 1
- 238000013425 morphometry Methods 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 150000001282 organosilanes Chemical class 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 208000024051 villous adenoma of colon Diseases 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
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
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)
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 s 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 ~o 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 ~5 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 2o 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 2s 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, so 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.
CONFIRi~~IATION COPY
Background of the Invention and Prior Art Normal cells in a healthy body live in a complex, interdependent ~o 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 ~5 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 2o 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 2s 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, so 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.
CONFIRi~~IATION COPY
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 s 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 ~5 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 2o 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 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 s 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 o 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.
~5 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 2o 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.
25 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 3o 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.
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 ~5 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 2o 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 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 s 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 o 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.
~5 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 2o 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.
25 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 3o 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 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 ~o proteins. Everett et al., J. Virol. (1998) 72:6581-6591 describe observations on the inetraction 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 ~5 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.
2o 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 25 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 so 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.
o 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-I, 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-~5 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 2o 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.
25 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
displayed a complex, interconnected domain-like pattern in epithelial nuclei of 30 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 7612 [donor no. 2]. (A & B): 7612 strongly stained few bright foci in nuclei of healthy epithelium. (C & D): hnRNP1 displayed a weak and puntated labeling pattern in nuclei of tumor epithelium.
Figure 5 shows fluorescence microscopy images of monolabeled nuclei s 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 o 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.
~5 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 2o 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 3o 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 o 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, ~5 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-I, mitotic apparatus protein), a change in spatial distribution.
2o 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.
3o 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 2o 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 3o 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 o 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 ~5 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 2o 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.
3o 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 o 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 s 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.
T~hlo ~
Donor Age Sex Tissue Diagnosis Depth No. Penetration 1 52 M sigmoidmoderately differentiatedserosa adenocarcinoma 2 76 F colon moderately-well differentiatedmuscul. propia adenocarcinoma (-)lymphonodes 3 75 M sigmoidmoderately differentitatedmuscul. propia adenocarcinoma (-)lymphonodes 4 69 M rectum moderately differentitatedmuscul. propia adenocarcinoma (-)lymphonodes (mutinous type) 5 89 F colon adenocarcinoma Dukes B
(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 ~5 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.
2o 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 25 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 so 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.
o 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-I, 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-~5 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 2o 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.
25 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
displayed a complex, interconnected domain-like pattern in epithelial nuclei of 30 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 7612 [donor no. 2]. (A & B): 7612 strongly stained few bright foci in nuclei of healthy epithelium. (C & D): hnRNP1 displayed a weak and puntated labeling pattern in nuclei of tumor epithelium.
Figure 5 shows fluorescence microscopy images of monolabeled nuclei s 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 o 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.
~5 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 2o 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 3o 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 o 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, ~5 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-I, mitotic apparatus protein), a change in spatial distribution.
2o 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.
3o 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 2o 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 3o 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 o 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 ~5 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 2o 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.
3o 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 o 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 s 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.
T~hlo ~
Donor Age Sex Tissue Diagnosis Depth No. Penetration 1 52 M sigmoidmoderately differentiatedserosa adenocarcinoma 2 76 F colon moderately-well differentiatedmuscul. propia adenocarcinoma (-)lymphonodes 3 75 M sigmoidmoderately differentitatedmuscul. propia adenocarcinoma (-)lymphonodes 4 69 M rectum moderately differentitatedmuscul. propia adenocarcinoma (-)lymphonodes (mutinous type) 5 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 different-perimuscular fated adenocarcinoma fat tissue 59 M colon well differentiated Dukes B3 adeno-carcinoma (villous mesenterial fat adenoma tissue type) 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 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)).
~5 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 Antibody SubclassAnimal Antigen Ref.
I
5E10 IgG mouse 126kD nucl body prot.- PML 1 body a-p80-coilinpoly rabbit 80 kD coilin prot. - coiled 2 bodies a-CstF 64 mono mouse Cleavage Stimulation Factor 3 a-CPSF mono mouse Cleav. Polyadenyl. Spec. Factor3 H5 IgM mouse subunit I10 of RNA polymerise4 II
H14 IgM mouse subunit I10 and Ila of RNA 4 polymerise II
8WG16 IgG2a mouse RNA polymerise II 4 SC-35 IgG1 mouse 35 kDa (non-snRNP) splicing 5 factor 4810 IgG mouse hnRNP A1 protein 5 4D11 IgG mouse hn RNP L protein 5 7612 IgG mouse PTB (= hnRNP I protein) 5 a-Oct1 - rabbit transcription factor 4 a-BRG1 - rabbit human brahma 4 a-TF11 F-rap74poly 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 et al., Mol. Biol. Cell (1998) 9:1025-1036.
3. W. Schul et al., EM80 J. (1996) 15:2883-2892.
4. M.A. Grande et al., J. Cell Sci. (1997) 110:1781-1791.
5. K.A. Mattern et al., Exp. Cell Res. (1999) 246.
1o 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 ~g/ml Hoechst [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 1o 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 3o 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, a-p80-coilin, a-CstF 64, a-CPSF 100, a-CPSF 160, SC-35, 4G3, 4810, 4D11, 5 7612, 2D3, 8232, 8252. 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 1o 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 15 tissue.
Table 3 Striking differences in nuclear distribution of specific antigens in human colorectal epithelial tissue antigen function difference in tumour info in antigen comparison to normal tissue p80-coilin unknown increase in number of 1 coiled bodies PML unknown increase in number of 2 PML
bodies SC35 RNA splicing smaller speckled domains,3 more diffuse distribution acetylated histonechromatin more cells labelled 4 CstF64 3' processing lumping together of antigen1 RNA
hnRNP-1 RNA packaging more punctuate distribution5 I' 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 C.D. Allis, BioEssays (1998) 20:615-626.
0 5. G. Varani and K. Nagai, Annual Review of Biophysics and Biomolecular Structure (1998) 27:407-445.
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)).
~5 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 Antibody SubclassAnimal Antigen Ref.
I
5E10 IgG mouse 126kD nucl body prot.- PML 1 body a-p80-coilinpoly rabbit 80 kD coilin prot. - coiled 2 bodies a-CstF 64 mono mouse Cleavage Stimulation Factor 3 a-CPSF mono mouse Cleav. Polyadenyl. Spec. Factor3 H5 IgM mouse subunit I10 of RNA polymerise4 II
H14 IgM mouse subunit I10 and Ila of RNA 4 polymerise II
8WG16 IgG2a mouse RNA polymerise II 4 SC-35 IgG1 mouse 35 kDa (non-snRNP) splicing 5 factor 4810 IgG mouse hnRNP A1 protein 5 4D11 IgG mouse hn RNP L protein 5 7612 IgG mouse PTB (= hnRNP I protein) 5 a-Oct1 - rabbit transcription factor 4 a-BRG1 - rabbit human brahma 4 a-TF11 F-rap74poly 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 et al., Mol. Biol. Cell (1998) 9:1025-1036.
3. W. Schul et al., EM80 J. (1996) 15:2883-2892.
4. M.A. Grande et al., J. Cell Sci. (1997) 110:1781-1791.
5. K.A. Mattern et al., Exp. Cell Res. (1999) 246.
1o 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 ~g/ml Hoechst [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 1o 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 3o 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, a-p80-coilin, a-CstF 64, a-CPSF 100, a-CPSF 160, SC-35, 4G3, 4810, 4D11, 5 7612, 2D3, 8232, 8252. 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 1o 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 15 tissue.
Table 3 Striking differences in nuclear distribution of specific antigens in human colorectal epithelial tissue antigen function difference in tumour info in antigen comparison to normal tissue p80-coilin unknown increase in number of 1 coiled bodies PML unknown increase in number of 2 PML
bodies SC35 RNA splicing smaller speckled domains,3 more diffuse distribution acetylated histonechromatin more cells labelled 4 CstF64 3' processing lumping together of antigen1 RNA
hnRNP-1 RNA packaging more punctuate distribution5 I' 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 C.D. Allis, BioEssays (1998) 20:615-626.
0 5. G. Varani and K. Nagai, Annual Review of Biophysics and Biomolecular Structure (1998) 27:407-445.
Claims (10)
1. 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, CstF64, and hnRNP-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 nuclear cell component of said sample to be analyzed based on differences in its presence or spatial distribution upon comparison with said specific component in said reference sample.
(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 presence or spatial distribution of the same nuclear cell component in a reference sample;
and (c) characterizing said nuclear cell component of said sample to be analyzed based on differences in its presence or spatial distribution upon comparison with said specific component in said reference sample.
2. The method as claimed in claim 1, wherein said 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 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 said 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 said 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, rectum cancer, and sigmoid cancer.
7. The method as claimed in any one of Claims 1 to 8, wherein the presence or spatial distribution of said at least one specific component in the cell nucleus is determined by immunofluorescence microscopy.
8. The method as claimed in claim 7, wherein said immunofluorescence microscopy involves the use of one or more antibodies raised against nuclear components from the type of cells to be analyzed.
9. The method as claimed in claim 8, wherein said one or more antibodies are monoclonals.
10. 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;
(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.
(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.
(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.
(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.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP99203582 | 1999-11-01 | ||
| EP99203582.4 | 1999-11-01 | ||
| PCT/EP2000/011050 WO2001033229A1 (en) | 1999-11-01 | 2000-11-01 | Method for the detection of mammalian carcinomas |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2389364A1 true CA2389364A1 (en) | 2001-05-10 |
Family
ID=8240801
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002389364A Abandoned CA2389364A1 (en) | 1999-11-01 | 2000-11-01 | Method for the detection of mammalian carcinomas |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1226439A1 (en) |
| JP (1) | JP2003513282A (en) |
| AU (1) | AU2156501A (en) |
| CA (1) | CA2389364A1 (en) |
| HU (1) | HUP0203269A2 (en) |
| NZ (1) | NZ519065A (en) |
| WO (1) | WO2001033229A1 (en) |
| ZA (1) | ZA200203401B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1599226B1 (en) * | 2003-02-21 | 2012-09-19 | Medvet Science Pty. Ltd. | A method of diagnosis and treatment |
| US20090029408A1 (en) * | 2007-07-25 | 2009-01-29 | Wyeth | Methods for Characterizing Cell Proximity |
| JP2013502201A (en) * | 2009-08-21 | 2013-01-24 | オンコセラピー・サイエンス株式会社 | CSTF2 as a target gene for the treatment and diagnosis of lung cancer |
| US11551360B2 (en) | 2020-04-28 | 2023-01-10 | QATAR UNIVERSITY, Office of Academic Research | Device and method for cancer detection |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5264343A (en) * | 1990-08-31 | 1993-11-23 | Eleanor Roosevelt Institute | Method for distinguishing normal and cancer cells |
| US5599919A (en) * | 1994-12-09 | 1997-02-04 | Fox Chase Cancer Center | Nucleic acid encoding a transiently-expressed kinetochore protein, and methods of use |
| US5891857A (en) * | 1996-02-20 | 1999-04-06 | Vanderbilt University | Characterized BRCA1 and BRCA2 proteins and screening and therapeutic methods based on characterized BRCA1 and BRCA2 proteins |
-
2000
- 2000-11-01 HU HU0203269A patent/HUP0203269A2/en unknown
- 2000-11-01 JP JP2001535063A patent/JP2003513282A/en active Pending
- 2000-11-01 AU AU21565/01A patent/AU2156501A/en not_active Abandoned
- 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 NZ NZ519065A patent/NZ519065A/en unknown
-
2002
- 2002-04-29 ZA ZA200203401A patent/ZA200203401B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NZ519065A (en) | 2004-03-26 |
| JP2003513282A (en) | 2003-04-08 |
| HUP0203269A2 (en) | 2003-02-28 |
| EP1226439A1 (en) | 2002-07-31 |
| AU2156501A (en) | 2001-05-14 |
| ZA200203401B (en) | 2003-04-29 |
| WO2001033229A1 (en) | 2001-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2010241288B2 (en) | Method and quantification assay for determining c-kit/SCF/pAKT status | |
| DE69929185T2 (en) | TUMOR MARKER | |
| EP1388734B1 (en) | Method for solution based diagnosis | |
| Onsum et al. | Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients | |
| US20020172987A1 (en) | Methods and reagents for the rapid and efficient isolation of circulating cancer cells | |
| CN103392009B (en) | The presence that ERG gene rearrangements and protein are overexpressed in rudimentary PIN (LG PIN) in biopsy of prostate | |
| CA2118015A1 (en) | Method of detecting tumors containing complexes of p53 and hsp70 | |
| US20100075341A1 (en) | Standardized evaluation of therapeutic efficacy based on cellular biomarkers | |
| CA2623445A1 (en) | Comprehensive diagnostic testing procedures for personalized anticancer chemotherapy (pac) | |
| KR20030080002A (en) | Methods and reagents for the rapid and efficient isolation of circulating cancer cells | |
| Pauwels et al. | Clear cell sarcoma of the stomach | |
| EP2661628B1 (en) | Diagnostic method | |
| WO2013102757A1 (en) | Diagnostic method and system | |
| CA2389364A1 (en) | Method for the detection of mammalian carcinomas | |
| US11054426B2 (en) | Diagnostic method | |
| Polak et al. | Immunocytochemistry today: Techniques and practice | |
| Fischer et al. | Method for procuring specific populations of viable human prostate cells for research | |
| Yigzaw et al. | Review of Immunohistochemistry Techniques: Applications, Current Status, and Future Perspectives | |
| Rosolem et al. | Paulo Henrique Leal Bertolo et al.(2019) Relationship Between the Immunodetection of Alpha-Smooth Muscle Actin and the Aggressiveness of Mammary Papillar Tumors in Female Dog | |
| Carr et al. | Will a tumor metastasize? Quantitate, semi-quantitate or pseudo-quantitate? A brief review of the microsopic prediction of tumor metastasis | |
| Feng et al. | A Sensitive Immunofluorescence Assay for Detection of p53 Protein in the Sputum | |
| Konishi et al. | S phase determination in intact colonic crypts by histone H3 messenger RNA in situ hybridization and confocal microscopy. | |
| Wefers et al. | T Cell Infiltration in Serous Tubal Intraepithelial Carcinoma (STIC) | |
| AU2002313797A1 (en) | Method and quantification assay for determining c-kit/SCF/pAKT status |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 20061101 |