WO2003025217A1 - Procede de pronostic - Google Patents

Procede de pronostic Download PDF

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WO2003025217A1
WO2003025217A1 PCT/AU2002/001293 AU0201293W WO03025217A1 WO 2003025217 A1 WO2003025217 A1 WO 2003025217A1 AU 0201293 W AU0201293 W AU 0201293W WO 03025217 A1 WO03025217 A1 WO 03025217A1
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kail
tissue
tumour
elevated
expression
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PCT/AU2002/001293
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WO2003025217A8 (fr
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Albert George Frauman
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The University Of Melbourne
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Publication of WO2003025217A8 publication Critical patent/WO2003025217A8/fr

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57434Specifically defined cancers of prostate
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to methods of cancer prognosis and detection, and in particular, but not exclusively, to methods of prostate cancer prognosis and detection.
  • the invention also relates to kits for use in such methods.
  • Prostate cancer is a leading cause of male morbidity and mortality.
  • current methods for predicting individual patient outcome that utilise standard histological staging are generally unreliable as guides to optimal therapeutic management.
  • Some prognostic indication is possible in men with clinically localised disease by correlation of histological grade and levels of prostate-specific antigen (PSA).
  • PSA prostate-specific antigen
  • the Gleason system of histological grading is generally adopted, particularly for patients with well and poorly differentiated cancers (29-32).
  • PSA prostate-specific antigen
  • more than 75% of patients with clinically localised prostate cancer have moderately differentiated tumours (33) in relation to which the PSA/grade correlation is not appropriate.
  • PSA levels cannot be relied upon to predict the extent and outcome of cancer in individual patients.
  • prostate cancer metastasis suppressor gene KAIl from human chromosome l lpll.2 (1). Sequencing of the KAIl cDNA clone revealed complete identity to the human lymphocyte surface antigen CD82, variously designated as R2, 14A, 4F9 or C33 in several laboratories, using different monoclonal antibodies (2-5).
  • the predicted protein encoded by KAI1/CD82 has been characterised as a member of the transmembrane 4 (TM4) family, a recently described group of structurally related cell surface proteins characterised by four highly conserved membrane spanning domains (6).
  • TM4 family The biological function of the TM4 family is unclear, although various data suggest a role in cell growth, adhesion and motility (6); consistent with these functions, other TM4 family molecules have been implicated in tumour progression (7) and in the negative regulation of tumour cell metastasis (8).
  • KAIl gene expression may serve as a marker of metastatic human prostate cancer.
  • Dong et al (1) showed by Northern analysis that KAIl RNA expression is high in normal human prostate tissue, but is reduced in human cell lines derived from prostate cancer metastases.
  • the study demonstrated that transfection of highly metastatic rat prostate cancer cells with KAIl suppresses the ability of the cells to form lung metastases in nude mice.
  • KAIl expression decreases as the cancer progresses and this has been shown in non-small cell lung (34), breast (35), hepatocellular (36) and bladder (37) cancers and invasive squamous cell carcinomas from the lung, head, neck and cervix (38).
  • the present inventors have now shown that not only do levels of KAIl expression vary inversely as a function of histological grade, but that within tumour grades for specific tissue types there is variation of KAIl expression. Furthermore, the inventors have determined that this variation of KAIl expression within a tumour of defined grade and tissue type is predictive of tumour metastasis. This understanding gives rise to far greater prognostic accuracy than was previously available and will allow clinicians to better manage individual patient therapies so that appropriate interventions may be taken when required, and more often avoided when not required. Furthermore, the present inventors have identified elevated KAIl expression in normal tissue surrounding cancerous lesions.
  • the determination of increased KAIl expression in normal tissues surrounding the tumour will offer a means of verifying negative biopsy results, to thereby eliminate or at least reduce false negative results.
  • the diagnostic and prognostic methods according to the present invention therefore provide powerful tools for clinicians in tumour detection, control of tumour metastasis and maintaining patient quality of life.
  • a method of prognosis of a cancer patient comprising subjecting a sample of primary tumour tissue of defined type and grade from the patient to quantitative analysis of KAIl gene expression and determining whether KAIl expression level is elevated or reduced relative to a threshold value for the defined tumour tissue type and grade; wherein elevated expression is indicative of restraint of future tumour progression and reduced expression is indicative of future tumour metastasis.
  • the threshold value may be determined by statistical analysis of KAIl gene expression levels in respect of samples of primary tumour tissue of defined type and grade previously taken from patients in relation to whom the status of tumour progression is known.
  • a method of prognosis of a cancer patient comprising subjecting a sample of primary tumour tissue of defined type and grade from the patient to quantitative analysis of KAIl gene expression and subjecting a sample of normal tissue of the defined type and from the same patient, that is not derived from adjacent to the tumour, to quantitative analysis of KAIl gene expression, determining the quotient of KAIl gene expression from the tumour tissue and KAI gene expression from the normal tissue and determining whether the quotient is elevated or reduced relative to a quotient threshold value for the defined tumour tissue type and grade; wherein an elevated quotient is indicative of restraint of future tumour progression and a reduced quotient is indicative of future tumour metastasis.
  • a method of prognosis of a cancer patient comprising subjecting a sample of normal tissue derived from adjacent to tumour tissue of the same type, to quantitative analysis of KAIl gene expression and determining whether KAIl expression level is elevated or reduced relative to a threshold value for tissue of that type located adjacent to tumour tissue; wherein elevated expression is indicative of restraint of future tumour progression and reduced expression is indicative of future tumour metastasis.
  • a method of detection of tumour presence within tissue of a patient comprising subjecting a sample of tissue of defined type to quantitative analysis of KAIl gene expression and determining whether KAIl expression level is elevated or reduced relative to a threshold value for tissue of the defined type; wherein elevated expression is indicative of tumour presence within the tissue.
  • the samples of tumour and normal tissue may be taken by biopsy or from biological fluids obtained from the patient.
  • biological fluids are urine, blood, lymph, saliva, tears or semen.
  • the tumour tissue is prostate, breast, lung, colon, liver, brain, kidney, trachea, rectum, stomach, pancreas, cervix, uterus, ovary, testicle, thyroid or epidermal tissue.
  • the tumour tissue is prostate, breast or lung tissue, particularly preferably it is prostate tissue.
  • KAIl gene expression is conducted by determining whether KAIl protein level is elevated or reduced relative to a threshold value for KAIl protein level.
  • an ELISA assay is conducted to determine whether KAIl protein levels are elevated or reduced.
  • immunohistochemical analysis is conducted to determine whether KAIl protein levels are elevated or reduced.
  • determination of whether KAIl protein level is elevated or reduced is by conducting an assay wherein labelled KAIl antibody is bound to a support and is contacted with protein from the tumour tissue, wherein following binding of KAIl protein to the labelled KAIl antibody and detection of the label, a quantitative measure of KAIl expression is obtained.
  • the label is radioactive, fluorescent or colourimetric.
  • KAIl gene expression is conducted by determining whether KAIl mRNA level is elevated or reduced relative to a threshold value for KAIl mRNA level.
  • RT-PCR analysis may be conducted to determine whether KAIl mRNA levels are elevated or reduced.
  • RNA from tumour tissue of defined type and grade is subjected to RT-PCR amplification using a primer or primers complementary to at least a fragment of KAIl cDNA; the amplified cDNAs are then separately hybridized to a labelled probe complementary to at least a fragment of KAIl cDNA; the hybridized labels are detected to give a quantitative measure of KAIl mRNA in the tumour tissue which is compared to a threshold value for the defined tumour tissue type and grade.
  • the label is radioactive, fluorescent or colourimetric.
  • kits for use in a method of prognosis as outlined above.
  • a kit may for example include ELISA, immunohistochemistry or RT-PCR compounds and reagents, such as antibodies, oligonucleotide sequences, solid supports, labels, enzymes, buffer solutions and the like.
  • the kit may additionally include appropriate instructions for conducting the prognostic methods according to the invention.
  • Figure 1 Immunohistochemical detection of KAIl in BPH not associated with cancer, BPH associated with prostate cancer and well, moderately and poorly differentiated prostate cancers.
  • panel a) represents the specimen incubated with primary anti-human KAIl antibody
  • panel b) represents the same specimen tested for non-specific staining (i.e. control slide).
  • Magnification x400 for all panels.
  • KAIl staining scores for BPH not associated with cancer and well, moderately and poorly differentiated prostate cancers The score is the product of two parameters: 'Stain density multiplied by scan area' and 'Ratio of target area to scan area', as measured using the MCID system (see Methods section).
  • the solid line represents the mean score within each specimen category.
  • the broken line represents the 95 % upper score limit for BPH not associated with cancer.
  • the present invention relates to a method of prognosis of a cancer patient. What is meant by this is that by following the method according to the invention it is possible to determine the likelihood of a primary tumour metastasising to form secondary tumours elsewhere within the patient's body. This is obviously of great importance in formulating a treatment plan that will serve to prolong the patient's length and quality of life.
  • the invention also provides a method for detection of a tumour in a tissue, particularly in the instance where this tumour is at a size such that it cannot be readily detected using conventional techniques.
  • This detection technique may conveniently be utilised in conjunction with other conventional techniques, such as biopsy. While the inventors have noted that for primary tumour tissue of defined type and grade the level of KAIl gene expression (when compared to a threshold value) is predictive of metastasis, and that the same can be said for normal tissue adjacent to tumour tissue, it is also the case that generally high KAIl expression levels (relative to a threshold value for a particular tissue) are predictive for the presence of tumour. Generally the analytical steps associated with the detection method correspond to those of the prognostic methods that are more specifically discussed herein.
  • the prognostic method may be conducted by taking a sample of normal tissue of the same type as the tumour tissue that is adjacent to the tumour tissue.
  • samples may be obtained by way of regular biopsy or operative techniques that are well known in the surgical field, and will of course vary depending upon the location of the tumour and the organ concerned. It may in fact be the case that the tissue sample is taken from the patient some time before conducting the prognostic method and that it is then stored in an appropriate manner until the prognostic method is conducted.
  • the methods according to the present invention can be applied to a wide range of human tumours, such as for example prostate, breast, lung, colon, liver, brain, kidney, trachea, rectum, stomach, pancreas, cervix, uterus, ovary, testicle, thyroid or epidermal tumours.
  • the primary tissue sample can be drawn from tumours within any of the tissues mentioned.
  • the tissue sample is taken from prostate, breast or lung tissue, most preferably from the prostate.
  • the sample of tumour tissue to be obtained from biological fluids including blood, lymph, saliva, tears and semen, in the case where cells from the defined tissue are likely to be present in a biological fluid.
  • normal tissue may be obtained in the same manner as the tumour tissue, and should be obtained from tissue of the same type which is of course not affected by a tumour and which is not directly adjacent to tumour.
  • the level of expression can be quantitated either directly or alternatively indirectly by, for example, detection of levels of KAIl mRNA, within the tissue concerned.
  • the quantitative analysis may also be conducted by contacting an extract obtained from the tumour or normal tissue sample (preferably an extract of isolated protein) against a solid support to which KAIl antibody has been applied.
  • Solid supports such as beads, tubes or polymer particles are well known in the chromatography art.
  • After binding of the protein to the antibody there are then many means of obtaining a signal through which the level of binding of protein to antibody can be measured as a means of assaying the level of protein expressed within the tumour or normal tissue cells.
  • a label attached to the antibody such as a radioactive, fluorescent or colourimetric label which can be arranged so that the signal emitted by the label is only detected in the situation where protein and antibody are bound together.
  • indicator molecules that will emit a signal (for example such as colour, fluorescence, etc.) when they come into contact with protein that is bound to antibody.
  • Similar detection means can be adopted in relation to assays that quantify relative amounts of mRNA in tumour and normal tissue of the same type.
  • Such techniques may involve the isolation of RNA from samples of tumour or normal tissue and amplification by RT-PCR to produce a KAIl cDNA sequence or fragment thereof using appropriate oligonucleotide primers.
  • Such techniques may involve the hybridisation of the KAIl cDNA or fragment to a labelled probe nucleotide sequence that is complementary to at least a fragment of the KAIl cDNA. The hybridized labelled probe can then be detected and quantified.
  • probes utilised in such techniques will be at least five nucleotides in length, particularly preferably at least ten nucleotides in length, still more preferably at leat 20 nucleotides in length, in order to substantially prevent non-specific binding of the probe to the KAIl cDNA sequence.
  • Threshold value may, for example, be determined if raw KAIl gene expression levels are being monitored, by use of the arithmetic mean and/or standard deviation of KAIl gene expression levels from samples of tumour tissue of the same type and grade. In the case of the more powerful quotient threshold, a quotient will be calculated from KAIl gene expression of tumour against normal tissue of the same type. KAIl expression or quotient can then be correlated against the appropriate threshold value and preferably statistical analysis, such as a Chi 2 test, will be conducted to determine significant variation above or below threshold value.
  • tumour tissue which is indicative of tumour progression
  • the threshold value determined in relation to the tumour tissue type will be specific for defined tumour grades.
  • the primary tumour tissue under analysis is to be categorised in a consistent manner to the population samples of tumour tissue from which the threshold value has been determined.
  • tumour tissue may be graded into the categories of well differentiated, moderately differentiated and poorly differentiated, as will be well understood by persons skilled in the art.
  • a more powerful grading approach will be adopted, such as for example in the case of prostatic tumours, the Gleason classification system (29).
  • Other well recognised grading systems may be adopted for tumours of other tissue types.
  • Numerous possible means of quantifying KAIl gene expression levels are available and would be well known to persons skilled in the art. Examples of such techniques include Northern analysis, Western analysis, immuno precipitation using a labelled probe and immunohistochemical quantification (immuno- image analysis).
  • the quantitated KAIl expression level for the tumour tissue of defined type and grade (or from adjacent normal tissue of the same type) will be divided by the quantitated KAIl expression level obtained for normal tissue of the same type that is located remote from the tumour, preferably from the same patient, to produce a quotient.
  • this value will be compared to a threshold value calculated by statistical analysis from samples of the same primary tumour tissue grade and type, which has been demonstrated to be indicative of tumour progression. This demonstration of tumour progression will be achieved by reviewing KAIl levels from samples obtained from patients whose tumour progression status is known.
  • KAIl levels may be plotted in respect of patients suffering from tumours of known type and grade who have been shown over the course of time to have had either metastatic or non- metastatic disease.
  • a separate "quotient threshold" can be determined from patient populations by statistically analysing KAIl expression levels from samples obtained from patients known to have had metastatic or non-metastatic disease and dividing this by KAIl expression levels for normal tissue of the same type surrounding (but not directly adjacent to) tumour in the same patient.
  • tumour of future tumour progression it is intended to mean that as a result of an elevated quotient or elevated expression of the KAIl gene within the tumour tissue concerned (or in adjacent normal tissue of the same type), relative to the appropriate threshold, it is unlikely for metastasis of the tumour to subsequently occur.
  • indicator of future tumour metastasis indicates that due to a reduced quotient or reduced expression of the KAIl gene within the tumour tissue concerned (or adjacent normal tissue of the same type), relative to the appropriate threshold, it is likely that the primary tumour will metastasise to form secondary tumours in other parts of the body.
  • Antibodies useful in methods according to the invention can be produced according to standard techniques known in the art, as for example explained in "Immunochemical methods in cell and molecular biology", R J Mayer and J H Walker, Academic Press, 24- 28 Oval Road, London, UK, 1987, the teaching of which is included herein by way of reference. Antibodies concerned may be monoclonal or polyclonal, preferably monoclonal. The invention will now be further described with reference to the following non-limiting examples.
  • H&E haematoxylin and eosin
  • RNA from fresh normal prostate tissue was extracted using the acid-guanidinium thiocyanate method described by Chomczynski and Sacchi (12).
  • 40 x 5 ⁇ m serial sections containing the microdissected tissue were cut from each paraffin block with a microtome and collected into 50 ml polypropylene conical tubes (Greiner). Tissue sections were deparaffinized by two 10-min washes in 1.5 ml of xylene at room temperature and then pelleted by centrifugation at 13000g for 5 min. The tissues were rehydrated and xylene was removed by washing twice with 1.5ml of 100 per cent ethanol at room temperature and centrifugation at 13000g for 5 min.
  • Total cellular RNA was isolated by homogenizing the precipitate in 500 ⁇ l of 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.1 M -mercaptoethanol, and 0.05 per cent N-lauroylsarcosine. 0.1 volume of 2 M sodium acetate (pH 4.0), 1 volume of water-saturated phenol, and 0.4 volume of chloroform- isoamyl alcohol (in a ratio of 24:1) were then added sequentially and the suspension was centrifuged at lOOOOg for 30 min at 4°C.
  • the upper aqueous phase was transferred to a fresh 1.5 ml micro fuge tube, mixed with an equal volume of isopropanol in the presence of 20 ⁇ g of glycogen (Boehringer Mannheim, Victoria, Australia) as a carrier and placed at -70°C for at least 30 min to precipitate the RNA.
  • glycogen Boehringer Mannheim, Victoria, Australia
  • the pelleted RNA was redissolved in 300 ⁇ l of denaturing solution and reprecipitated with an equal volume of isopropanol for 30 min at -70°C.
  • the RNA was centrifuged again, resuspended in 75 per cent ethanol, and incubated at room temperature for 10 min.
  • RNA was vacuum-dried for 10 min, dissolved in 10 ⁇ l of DEPC-treated water, and then stored at -70°C until ready for further use. Due to the small amounts of archived tissue used for RNA extraction, direct RNA qualification was not possible, although microdissected tissue section sizes were comparable and treated identically for RNA extraction.
  • Extracted RNA 100 ng from frozen normal prostate and 3 ⁇ l from formalin-fixed, paraffin-embedded tissue was diluted to a total volume of 11 ⁇ l with DEPC-treated water, heated to 70°C for 2 min, and then chilled on ice.
  • the reaction mixture was then brought to a final volume of 20 ⁇ l containing 40 U of rRNasin (Promega, Victoria, Australia), 10 ⁇ M random hexamers, 1 mM dNTPs (dGTP, dATP, dTTP, and dCTP) (Clontech, NSW, Australia), 200 U of Moloney murine leukaemia virus reverse transcriptase (M- MuLV, Promega, Victoria, Australia), 25 mM Tris-HCl 37.5 mM KC1, 1.5 mM MgC12, and 5 mM dithiothreitol (pH 8.3) and incubated at 42°C for 1 h, followed by 5 min at 70°C to inactivate the reverse transcriptase.
  • rRNasin Promega, Victoria, Australia
  • 10 ⁇ M random hexamers 1 mM dNTPs (dGTP, dATP, dTTP, and dCTP) (Clon
  • the reverse transcription product (cDNA) was then amplified by PCR using two different oligonucleotide primer pairs designed to detect the KAIl gene and that of the housekeeping gene ⁇ -actin. PCR amplification of the cytoplasmic /3-actin gene was used to assess cDNA integrity and as a reference to allow semi-quantitation of KAIl mRNA.
  • the forward primer was 5'TGGCATTGCCGACAGGATGCAGAA-3' SEQ ID NO:5 and the reverse was 5'- CTCGTCATACTCCTGCTTGCTGAT-3 ' SEQ LD NO:6, designed to span intron "E" (172 bp for cDNA size).
  • cDNA was amplified in a total volume of 20 ⁇ l containing the cDNA was amplified in a total volume of 20 ⁇ l containing the cDNA preparation (1 ⁇ l for frozen tissue and 3 ⁇ l for formalin-fixed, paraffin-embedded tissue); 200 ⁇ M of each dGTP, dATP, dTTP, and dCTP (Clontech, NSW, Australia); 0.5 ⁇ M of each of the appropriate forward and reverse primers for KAIl or B-actin; 1.5 mM MgC12, and 0.5 unit of "Red Hot" Taq polymerase (Advanced Biotechnologies, Victoria, Australia).
  • cDNA prepared from fresh tissue was performed using 25 cycles of PCR, whereas 40 cycles were required to detect PCR products from archived tissue.
  • a modification of a high stringency PCR protocol adapted for the specific detection of low-level mRNA, as previously described in our laboratory (13) was used. Briefly, the reaction mixture was initially heated to 94°C for 3 min, followed by 5 cycles at 94°C for 30 s, 70°C for 30 s, and 74°C for 1 min, followed by 25-40 cycles at 94°C for 30 s, 55°C for 30 s, and 74°C for 1 min, and a final extension time of 10 min at 74°C.
  • Negative control reactions lacking cDNA were included to monitor contamination and preparations of fresh normal prostate tissue were amplified as a positive reaction control.
  • 10 ⁇ l of each PCR product was combined with 1 ⁇ l of loading dye (6 x, being 0.25 per cent bromophenol blue, 0.25 per cent xylene cyanol, and 15 per cent Ficoll 400) and analysed by electrophoresis on 2.5 per cent agarose gels (DNA grade agarose, Progen Industries Limited, Australia) containing 0.5 mg/ml ethidium bromide for 1.5 h at 65 V in 1 x TAE buffer (367 mM Tris, 360 mM boric acid, 8 mM EDTA, pH 8.0).
  • Radioactive oligonucleotide DNA probes for hybridization to KAIl and /3-actin were designed to sequences internal to the PCR primers used.
  • the probes for detecting the PCR amplification products for KAIl-(l), KAIl-(2), and 3-actin corresponded to bases 864-893 (5'CGTGGGCGTGGGTGTGGCCATCATCGAGCT3' SEQ ID NO:7), 751-780 (5'CCCGGCAACAGGACCCAG AGTGGCAACCAC3 ' SEQ ID NO:8), and 2872-2901 (5'GAGCGCAAGTACTCCGTGTGGATCGGCGGC3' SEQ LD NO: 9), respectively.
  • the probes were dissolved in distilled water at 75 ng/ ⁇ l and 5' end labelled at 37°C for 30 min in a reaction containing 7.5 ng of DNA probe, 10 mM Tris-HCl, 10 mM MgC12, 5 mM DTT, 0.1 mM spermidine (pH 1.6), and 5 ⁇ l of ⁇ -[32P]ATP (3000-5000 Ci/mmol) (Progen, Australia) and 0.5 unit of T4 polynucleotide kinase (Progen Industries Ltd., Australia).
  • Membranes were washed once in 2 x SSC and pre-hybridized for 15 min at 42°C in rapid- hyb buffer (Amersham Lifesciences, NSW, Australia). Hybridization was performed at 42°C for 1.5 h in the same buffer with radioactively labelled jS-actin and KAIl probes at final concentrations of not less than 1.87 ng/ml. Following hybridization, the membrane was rinsed at 42°C, once in 2 x SSC, 0.1 per cent SDS for 10 min and once in 1 x SSC , 0.1 per cent SDS for 15 min.
  • Membranes were then imaged with autoradiography by exposure to X-ray film (BioMax, Kodak, Integrated Sciences, Victoria, Australia) for at least 2 h at -70°C. Autoradiographs were quantitated by computer-assisted densitometry using the MCLD software (Imaging Research Inc., Ontario, Canada). The hybridizing intensity of each KAIl band was normalized to its respective /3-actin band.
  • Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide.
  • 'DAKO Protein Block Serum-Free' was applied for 5 minutes to reduce background staining prior to specimens being incubated with 1:250 preparation (in PBS) of polyclonal rabbit anti-human KAIl (C-16) antibody (Santa Cruz Biotechnology, Santa Cruz/California) for 2 hours. This was followed by sequential incubation of specimens in an anti-goat/rabbit/mouse biotinylated link antibody and peroxidase-labelled streptavidin.
  • Protein expression was visualised after incubation with 3,3XDiaminobenzidine (DAB) substrate-chromogen solution, yielding a brown end-product, and counterstaining with haematoxylin.
  • DAB 3,3XDiaminobenzidine
  • DAB staining target area
  • Intensity and Saturation 0.00-1.00 Study selection: Grain count. The following data was measured for each digitised image: Sample selection: 'Density (D) x Area (A)' and 'Proportional Area 1 .
  • DxA (units: intensity x pixel) is the density of the measured grains multiplied by the area of the sample window. Proportional area is the ratio of the total target area to the scanned area.
  • Each sample window was the size of the default measurement box used by the program; the size of the box allowed for measurements within a region half the size of an average epithelial cell. Only cytoplasmic regions of the cell were measured. Twenty windows within ten adjacent fields (200x magnification) in each specimen were measured, hi samples with different cancer patterns, such as moderately differentiated specimens which may contain regions of both well and poorly differentiated tissue, equal numbers of fields in each area were measured (to a total of 10 fields).
  • KAIl staining score for each specimen, which is representative of KAIl protein expression within the sample. Scores were averaged for each category of cancer and the standard deviation (SD), standard error of the mean (SEM) and 95% confidence interval were also calculated.
  • SD standard deviation
  • SEM standard error of the mean
  • prostate cancers exhibited significantly higher KAIl protein expression than that seen in BPH, with 27.5% exhibiting KAIl expression beyond the 95% upper limit observed in BPH (Table 1). BPH not associated with cancer versus individual categories of prostate cancer (well, moderately and poorly differentiated specimens).
  • KAIl expression in most solid cancers decreases as the cancer progresses, with metastatic deposits having the lowest KAIl levels. This is true of non- small cell lung (35), breast (36), hepatocellular (37) and bladder (38) cancers and invasive squamous cell carcinomas from the lung, head and neck and cervix (39). Despite this general observation, however, changes in KAIl expression in a number of different primary cancers is not so straightforward.
  • KAIl expression in prostate cancer is also somewhat unclear. Some studies have shown an inverse relationship between KAIl expression and tumour grade (Gleason score) (20), sage (20) and clinical metastatic behaviour (19). In contrast, recent findings from our own laboratory have shown a biphasic pattern of KAIl mRNA expression in primary prostate cancers according to histological grade, compared to non-malignant prostate tissues (BPH not associated with cancer); KAIl was significantly elevated in low grade prostate tumours and reduced in high grade tumours (21). The current immunohistochemical study, comparing BPH not associated with cancer with well, moderately and poorly differentiated cancers, appears to mirror and therefore complements our mRNA findings.
  • KAIl exhibits metastasis suppressor ability
  • the increase in KAIl expression seen in BPH associated with prostate cancers may serve as a protective mechanism to restrain further cancer progression and, potentially, subsequent metastasis development.
  • this effect may be as a result of changes in the microenvironment due to the presence of cancer in the prostate or the by-product of a local field effect, where alterations in KAIl expression in the cancerous region affects surrounding normal tissue. This so-called 'field
  • prostate cancer the findings that prostatic intraepithelial neoplasia, a likely prostate cancer precursor lesion (69-76), and prostate cancer can arise independently within the same prostate (65-67) supports the notion that a field effect may underlie prostate carcinogenesis.
  • KAIl protein levels were seen in BPH associated with prostate cancer lesions and across all grades of primary prostate cancers, compared to BPH not associated with cancer, though KAIl levels in poorly differentiated cancers appeared to fall towards levels in BPH not associated with cancer. It is conceivable that differential KAIl expression in primary prostate cancers might have important prognostic implications for the development of subsequent metastases. Thus, patients with the highest levels of KAIl expression may be afforded greater protection from development of metastases than those patients with low-level KAIl expression. This might allow clinicians to target therapies more intensively in those patients with primary prostate cancer who are considered to be at greatest risk of subsequent metastases, thus favourably impacting upon morbidity and mortality associated with prostate cancer.
  • chromosome 11 which contains the KAIl gene, plays an important role in metastasis suppressor activity via other genes, such as the cell adhesion molecule CD44, at lip 13 (77), and the new breast caner metastasis suppressor BRMS1, at llql3.1-13.2 (78).
  • CD44 is also downregulated during prostate cancer progression in humans (81). The mechanism by which KAIl expressions downregulated during progression is still unknown, though it is known that allelic loss (19, 82) and mutations (19) are not involved.
  • CD44 is a metastasis suppressor gene for prostatic cancer located on human chromosome l lpl3, Cancer Res, 57(5), 846, 1997.

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Abstract

Cette invention se rapporte à un procédé de pronostic du cancer chez un patient, ce procédé consistant à soumettre un échantillon de tissu tumoral primaire de type et de qualité définis prélevé sur le patient à une analyse quantitative d'expression de gène KAI1 et à déterminer si le niveau d'expression de KAI1 est élevé ou réduit par rapport à une valeur seuil pour ce type et cette qualité définis de tissu tumoral; une expression élevée indiquant un recul de la progression future de la tumeur et une expression réduite indiquant une métastase future de la tumeur. Cette invention se rapporte à un procédé servant à détecter la présence d'une tumeur dans un tissu d'un patient, ce procédé consistant à soumettre un échantillon de tissu d'un type défini à une analyse quantitative d'expression de gène KAI1 et à déterminer si le niveau d'expression de KAI1 est élevé ou réduit par rapport à une valeur seuil pour ce type défini de tissu; une expression élevée indiquant la présence d'une tumeur dans ce tissu.
PCT/AU2002/001293 2001-09-20 2002-09-20 Procede de pronostic WO2003025217A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007009755A2 (fr) * 2005-07-18 2007-01-25 Epigenomics Ag Compositions et methodes de diagnostic du cancer contenant des marqueurs pantumoraux
US11118182B2 (en) 2016-11-17 2021-09-14 Ramot At Tel-Aviv University Ltd. Modulators of human KAI1 metastasis suppressor gene, methods and uses thereof

Citations (2)

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WO1996034117A1 (fr) * 1995-04-28 1996-10-31 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF HEALT Procedes de diagnostic et therapie genique faisant appel a des reactifs derives du gene suppresseur de metastases humain kai1
WO2000014234A1 (fr) * 1998-09-08 2000-03-16 Urocor, Inc. Promoteur prostatique specifique et regulation de l'expression genetique

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1996034117A1 (fr) * 1995-04-28 1996-10-31 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF HEALT Procedes de diagnostic et therapie genique faisant appel a des reactifs derives du gene suppresseur de metastases humain kai1
WO2000014234A1 (fr) * 1998-09-08 2000-03-16 Urocor, Inc. Promoteur prostatique specifique et regulation de l'expression genetique

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DONG J. ET AL.: "KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2", SCIENCE, vol. 268, 1995, pages 884 - 886 *
GUO X. ET AL.: "KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases", CANCER RESEARCH, vol. 56, 1996, pages 4876 - 4880 *
HIGASHIYAMA M. ET AL.: "KAI1/CD82 expression in nonsmall cell ling carcinoma is a novel, favorable prognostic factor", CANCER, vol. 83, no. 3, 1998, pages 466 - 474 *
HUANG C. ET AL.: "Correlation of reduction in MRP-1/CD9 and KAI1/CD82 expression with recurrences in breast cancer patients", AMERICAN JOURNAL OF PATHOLOGY, vol. 153, no. 3, 1998, pages 973 - 983 *
JOHANSSON J. ET AL.: "Fifteen-year survival in prostate cancer", JAMA, vol. 277, no. 6, 1997, pages 467 - 471 *
LIU F. ET AL.: "Frequent down-regulation and lack of mutation of the KAI1 metastasis suppressor gene in epithelial ovarian carcinoma", GYNECOLOGIC ONCOLOGY, vol. 78, no. 1, 2000, pages 10 - 15 *
LIU F. ET AL.: "KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma", AMERICAN JOURNAL OF PATHOLOGY, vol. 159, no. 5, 2001, pages 1629 - 1634 *
MIYAZAKI T. ET AL.: "Mutation and expression of the metastasis suppressor gene KAI1 in esophageal squamous cell carcinoma", CANCER, vol. 89, no. 5, 2000, pages 955 - 962 *
WHITE A. ET AL.: "Frequent downregulation of the KAI1(CD82) metastasis suppressor protein in human cancer cell lines", ONCOGENE, vol. 16, 1998, pages 3143 - 3149 *
YANG X. ET AL.: "KAI1, a putative marker for metastatic potential in human breast cancer", CANCER LETTERS, vol. 119, 1997, pages 149 - 155 *
YU Y. ET AL.: "Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers", CLINICAL CANCER RESEARCH, vol. 3, 1997, pages 1045 - 1049 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007009755A2 (fr) * 2005-07-18 2007-01-25 Epigenomics Ag Compositions et methodes de diagnostic du cancer contenant des marqueurs pantumoraux
WO2007009755A3 (fr) * 2005-07-18 2007-05-24 Epigenomics Ag Compositions et methodes de diagnostic du cancer contenant des marqueurs pantumoraux
US11118182B2 (en) 2016-11-17 2021-09-14 Ramot At Tel-Aviv University Ltd. Modulators of human KAI1 metastasis suppressor gene, methods and uses thereof

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